BACKGROUND OF THE INVENTION

[0001] The conversion of biomass, such as corn, sugarcane or other energy crops, as well as simple sugars, to ethanol is routinely completed through the use of yeast fermentation. However, during yeast metabolism a major byproduct of fermentation is glycerol. Glycerol is formed during anaerobic growth as a way for the yeast to balance its redox state and regenerate NAD ⁺ used as a co factor during glycolysis. It has been shown that the function of glycerol is likely not as a metabolite itself but rather as an electron sink capturing electrons allowing further growth-linked metabolism to continue. As glycerol is a byproduct with low value, it can be an undesirable by-product of fermentation. It would be beneficial to reduce or eliminate this by-product and further direct more carbon towards desired end-products, such as ethanol.

[0002] Several strategies are available in the art for the conversion of glycerol to higher value products though biochemical or other means, but relatively little has been demonstrated for the removal or reduction of glycerol and improvement of overall sugar yield to ethanol or other desired end-products of metabolism. Through engineering of alternate pathways, potentially with the simultaneous reduction or deletion of the glycerol pathway, alternate or replacement electron acceptors for the regeneration of NAD ⁺ can be used during yeast metabolism. Such alternate or replacement electron acceptors could be molecules such as formate or hydrogen.

[0003] The elimination of glycerol synthesis genes has been demonstrated but removal of this pathway completely blocked anaerobic growth of the yeast, preventing useful application during an industrial process. Ansell, R., et al., EMBO J. 7(5:2179-87 (1997); Pahlman, A-K., et al, J. Biol. Chem. 27(5:3555-63 (2001); Guo, ZP., et al, Metab. Eng. 13:49-59 (2011). Other methods to bypass glycerol formation require the co-utilization of additional carbon sources, such as xylose or acetate, to serve as electron acceptors. Liden, G., et al, Appl. Env. Microbiol. (52:3894-96 (1996); Medina, V.G., et al, Appl. Env. Microbiol. 7(5:190-195 (2010). By incorporating a formate pathway as an alternate electron acceptor, glycerol formation can be bypassed and ethanol yield can be increased. The engineering of a pyruvate formate lyase from E. coli, which is capable of converting

Arty. Dkt. No. 2608.0550000 pyruvate to formate, has been done to increase formate production. Waks, Z., and Silver, P.A., Appl. Env. Microbiol. 75: 1867-1875 (2009). Formate engineering in Waks and Silver was done, however, to provide a source of formate in S. cerevisiae for the production of hydrogen by a secondary microorganism, E. coli. Waks and Silver did not combine formate production with the removal of glycerol formation, and the use of formate as an alternate electron acceptor for the reduction of glycerol was not proposed or evaluated. Thus, despite prior efforts to bypass and/or eliminate glycerol production, there exists a need for the engineering of alternate or replacement electron acceptors in a cell to direct more carbon towards desired end-products, such as ethanol.

[0004] The importance of engineering alternate or replacement electron acceptors is exemplified in the process of corn mash fermentation. About 16 billion gallons of corn- based ethanol are produced annually, so even small increases in ethanol yield, such as 5- 10%, can translate into an extra billion or so gallons of ethanol over current yields. Ethanol production from corn mash typically results in glycerol yields ranging from 10- 12 g/L. See Yang, R.D., et ah, "Pilot plant studies of ethanol production from whole ground corn, corn flour, and starch," Fuel Alcohol U.S.A., February 13-16, 1982 (reported glycerol levels to be as high as 7.2% w/w of initial sugar consumed in normal corn mash fermentations or approximately 1.4 g/100 mL using 20% sugar). By reducing or eliminating the glycerol yield in the production of ethanol from corn and re- engineering metabolic processes, increased ethanol yields can be achieved. Additional benefits may be gained in the production of ethanol from corn. Corn mash is a nutrient rich medium, in some cases containing lipid and protein content that can be >3% of the total fermentation volume. As a result of the energy contained in these components, even higher ethanol yields may be achieved than what is predicted using, for example, pure sugar. The additional increases can come from the metabolism of lipids or amino acids in the corn mash medium. The recombinant cells and methods of the invention enable increasing ethanol yields from biomass fermentation by reducing or eliminating glycerol.

BRIEF SUMMARY OF THE INVENTION

[0005] The invention is generally directed to the reduction or removal of glycerol production in a host cell and to the engineering of an alternate electron acceptor for the regeneration of NAD ⁺ . [0006] One aspect of the invention relates to a recombinant microorganism comprising: a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis; and one or more native and/or heterologous enzymes that function in one or more engineered metabolic pathways to convert a carbohydrate source to ethanol, wherein said one or more native and/or heterologous enzymes is activated, upregulated or downregulated. In some embodiments, the recombinant microorganism produces less glycerol than a control recombinant microorganism without deletion of said one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis. In some embodiments, the carbohydrate source is biomass. In some embodiments, the biomass comprises a lignocellulosic material selected from the group consisting of grass, switch grass, cord grass, rye grass, reed canary grass, mixed prairie grass, miscanthus, sugar-processing residues, sugarcane bagasse, sugarcane straw, agricultural wastes, rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, corn fiber, stover, soybean stover, corn stover, forestry wastes, recycled wood pulp fiber, paper sludge, sawdust, hardwood, softwood, agave, and combinations thereof. In some embodiments, the biomass is corn mash or corn starch.

[0007] In particular aspects, the one or more native enzymes that function to produce glycerol are encoded by a gpdl polynucleotide, a gpd2 polynucleotide, or both a gpdl polynucleotide and a gpd2 polynucleotide. In certain embodiments, the recombinant microorganism further comprises a native and/or heterologous gpdl polynucleotide operably linked to a native gpd2 promoter polynucleotide. In other aspects, the one or more native enzymes that function to produce glycerol are encoded by a gppl polynucleotide, a gpp2 polynucleotide, or both a gppl polynucleotide and a gpp2 polynucleotide.

[0008] In particular aspects, the one or more native enzymes that function to regulate glycerol synthesis are encoded by an fpsl polynucleotide.

[0009] In further aspects, the engineered metabolic pathways comprise conversion of pyruvate to acetyl-CoA and formate. In certain embodiments, pyruvate is converted to acetyl-CoA and formate by a pyruvate formate lyase (PFL). In some embodiments, the PFL is of prokaryotic or eukaryotic origin. In some embodiments, PFL is from one or more of a Bifidobacteria, an Escherichia, a Thermoanaerobacter, a Clostridia, a Streptococcus, a Lactobacillus, a Chlamydomonas, a Piromyces, a Neocallimastix, or a Bacillus species. In some embodiemtns, PFL is from one or more of a Bacillus licheniformis, a Streptococcus thermophilus, a Lactobacillus plantarum, a Lactobacillus casei, a Bifidobacterium adolescentis, a Clostridium cellulolyticum, a Escherichia coli, a Chlamydomonas reinhardtii PflA, a Piromyces sp. E2, or a Neocallimastix frontalis. In one embodiment, PFL is from a Bifidobacterium adolescentis.

[0010] In additional aspects, the engineered metabolic pathways comprise conversion of acetyl-CoA to ethanol. In certain embodiments, acetyl-CoA is converted to acetaldehyde by an acetaldehyde dehydrogenase and acetaldehyde is converted to ethanol by an alcohol dehydrogenase. In certain embodiments, acetyl-CoA is converted to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase. In some embodiments, the acetaldehyde dehydrogenase, alcohol dehydrogenase, or bifuntional acetaldehyde/alcohol dehydrogenase is of prokaryotic or eukaryotic origin. In one embodiment, acetaldehyde dehydrogenase is from C. phytofermentans . In some embodiments, bifunctional acetaldehyde/alcohol dehydrogenase is from an Escherichia, a Clostridia, a Chlamydomonas, a Piromyces, or a Bifidobacteria species. In some embodiments, bifunctional acetaldehyde/alcohol dehydrogenase is from Escherichia coli, Clostridium phytofermentans, Chlamydomonas reinhardtii, Piromyces sp. E2, or Bifidobacterium adolescentis. In one embodiment, bifunctional acetaldehyde/alcohol dehydrogenase is from a Bifidobacterium adolescentis or Piromyces sp. E2.

[0011] In further aspects, the recombinant microorganism comprises a deletion of one or more native enzymes encoded by an fdhl polynucleotide, an fdh2 polynucleotide, or both an fdhl polynucleotide and an fdh2 polynucleotide.

[0012] In certain embodiments, the carbohydrate source for the recombainant microorganism is lignocellulose. In certain embodiments, the recombinant microorganism produces ethanol. In certain embodiments, the recombinant microorganism produces formate.

[0013] In certain embodiments, the recombinant microorganism is selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Yarrowia lipolytica, Hansenula polymorpha, Phajfia rhodozyma, Candida utliis, Arxula adeninivorans, Pichia stipitis, Debaryomyces hansenii, Debaryomyces polymorphus, Schizo saccharomyces pombe, Candida albicans, and Schwanniomyces occidentalis . In one embodiment, the recombinant microorganism is Saccharomyces cerevisiae.

[0014] In certain embodiments, the recombinant microorganism comprises one or more native enzymes that function to produce glycerol encoded by both a gpdl polynucleotide and a gpd2 polynucleotide, an engineered metabolic pathway that comprises conversion of pyruvate to acetyl-CoA and formate by a pyruvate formate lyase and an engineered metabolic pathway that comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, and the recombinant microorganism further comprises a deletion of one or more native enzymes encoded by both an fdhl polynucleotide and an fdh2 polynucleotide.

[0015] In certain embodiments, the recombinant microorganism comprises one or more native enzymes that function to produce glycerol encoded by both a gppl polynucleotide and a gpp2 polynucleotide, an engineered metabolic pathway that comprises conversion of pyruvate to acetyl-CoA and formate by a pyruvate formate lyase. In further embodiments, one engineered metabolic pathway of the recombinant microorganism converts acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase and the recombinant microorganism further comprises a deletion of one or more native enzymes encoded by both an fdhl polynucleotide and an fdh2 polynucleotide.

[0016] In certain embodiments, the recombinant microorganism comprises one or more native enzymes that function to regulate glycerol synthesis encoded by an fpsl polynucleotide, an engineered metabolic pathway that comprises conversion of pyruvate to acetyl-CoA and formate by a pyruvate formate lyase. In further embodiments, one engineered metabolic pathway of the recombinant microorganism converts acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase and the recombinant microorganism further comprises a deletion of one or more native enzymes encoded by both an fdhl polynucleotide and an fdh2 polynucleotide.

[0017] In certain embodiments, the recombinant microorganism comprises one or more native enzymes that function to regulate glycerol synthesis encoded by an fpsl polynucleotide and one or more native enzymes that function to produce glycerol encoded by both a gpdl polynucleotide and a gpd2 polynucleotide, and an engineered metabolic pathway that comprises conversion of pyruvate to acetyl-CoA and formate by a pyruvate formate lyase and an engineered metabolic pathway that comprises conversion of acetyl- Co A to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, and the recombinant microorganism further comprises a deletion of one or more native enzymes encoded by both an fdhl polynucleotide and an fdh2 polynucleotide.

[0018] In certain embodiments, the recombinant microorganism comprises one or more native enzymes that function to regulate glycerol synthesis encoded by an fpsl polynucleotide, an engineered metabolic pathway that comprises conversion of pyruvate to acetyl-CoA and formate by a pyruvate formate lyase and an engineered metabolic pathway that comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, and the recombinant microorganism further comprises a deletion of one or more native enzymes encoded by both an fdhl polynucleotide and an fdh2 polynucleotide.

[0019] In certain embodiments, the recombinant microorganism comprises one or more native enzymes that function to produce glycerol encoded by both a gpdl polynucleotide and a gpd2 polynucleotide, an engineered metabolic pathway that comprises conversion of pyruvate to acetyl-CoA and formate by a pyruvate formate lyase and an engineered metabolic pathway that comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, and the recombinant microorganism further comprises a native and/or heterologous gpdl polynucleotide operably linked to a native gpd2 promoter polynucleotide.

[0020] In certain embodiments, the recombinant microorganism comprises one or more native enzymes that function to produce glycerol encoded by both a gpdl polynucleotide and a gpd2 polynucleotide, and an engineered metabolic pathway that comprises conversion of pyruvate to acetyl-CoA and formate by a pyruvate formate lyase and an engineered metabolic pathway that comprises conversion of acetyl-CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, further comprising a native and/or heterologous gpdl polynucleotide operably linked to a native gpd2 promoter polynucleotide and a deletion of one or more native enzymes encoded by both an fdhl polynucleotide and an fdh2 polynucleotide.

[0021] In certain embodiments, the recombinant microorganism comprises one or more native enzymes that function to produce glycerol encoded by both a gpdl polynucleotide and a gpd2 polynucleotide and one or more native enzymes that function to regulate glycerol synthesis encoded by an fpsl polynucleotide, and an engineered metabolic pathway that comprises conversion of pyruvate to acetyl-CoA and formate by a pyruvate formate lyase and an engineered metabolic pathway that comprises conversion of acetyl- CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, further comprising a native and/or heterologous gpdl polynucleotide operably linked to a native gpd2 promoter polynucleotide.

[0022] In certain embodiments, the recombinant microorganism comprises one or more native enzymes that function to produce glycerol encoded by both a gpdl polynucleotide and a gpd2 polynucleotide and one or more native enzymes that function to regulate glycerol synthesis encoded by an fpsl polynucleotide, and an engineered metabolic pathway that comprises conversion of pyruvate to acetyl-CoA and formate by a pyruvate formate lyase and an engineered metabolic pathway that comprises conversion of acetyl- CoA to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase, further comprising a native and/or heterologous gpdl polynucleotide operably linked to a native gpd2 promoter polynucleotide and a deletion of one or more native enzymes encoded by both an fdhl polynucleotide and an fdh2 polynucleotide.

[0023] In some embodiments, the deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis in the recombinant microorganism reduces glycerol formation by: more than about 10% of the glycerol produced by a recombinant microorganism without a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis; more than about 20%> of the glycerol produced by a recombinant microorganism without a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis; more than about 30% of the glycerol produced by a recombinant microorganism without a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis; more than about 40% of the glycerol produced by a recombinant microorganism without a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis; more than about 50%> of the glycerol produced by a recombinant microorganism without a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis; more than about 60% of the glycerol produced by a recombinant microorganism without a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis; more than about 70% of the glycerol produced by a recombinant microorganism without a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis; more than about 80% of the glycerol produced by a recombinant microorganism without a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis; more than about 90% of the glycerol produced by a recombinant microorganism without a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis; more than about 95% of the glycerol produced by a recombinant microorganism without a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis; or more than about 99% of the glycerol produced by a recombinant microorganism without a deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis.

[0024] In some embodiments, the recombinant microorganism produces an amount of formate selected from: at least about 0.012 g/L in 24 hours; at least about 0.022 g/L in 48 hours; or at least about 2.5 g/L in 142 hours.

[0025] In some embodiments, the recombinant microorganism produces a formate yield selected from: at least about 0.05 -fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 0.1 -fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 0.5 -fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 1.0- fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 5.0-fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 10.0-fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 20.0-fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 30.0- fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 40.0-fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 50.0-fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 75.0-fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; or at least about 100- fold more formate than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes.

In some embodiments, the recombinant microorganism produces an ethanol yield selected from: at least about 1% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 2% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 3% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 4% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 5% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 10% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 20% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 30% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 40% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 50% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 60% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 70% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 80% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 90% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 95% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; or at least about 99% more ethanol than is produced by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes.

[0027] In some embodiments, the conversion of the carbohydrate source to ethanol by the recombinant microorganism, or the enzymes engineered therein, is under anaerobic conditions.

[0028] In some embodiments, the recombinant microorganism has an acetate uptake

(g/L) under anaerobic conditions selected from: at least about 1% more acetate uptake than that taken up by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 10% more acetate uptake than that taken up by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 20% more acetate uptake than that taken up by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 30% more acetate uptake than that taken up by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 40% more acetate uptake than that taken up by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 50% more acetate uptake than that taken up by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 60% more acetate uptake than that taken up by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 70% more acetate uptake than that taken up by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 80% more acetate uptake than that taken up by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; and at least about 90% more acetate uptake than that taken up by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes.

In some embodiments, the recombinant microorganism produces more ethanol at a slower glucose utilization rate compared to a recombinant microorganism without deletion of one or more native enzymes that function to produce glycerol and/or regulate glycerol synthesis, wherein the glucose utilization rate is selected from: at least about 1% less glucose used per hour than that used by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 5% less glucose used per hour than that used by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 10%> less glucose used per hour than that used by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 20% less glucose used per hour than that used by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 30%> less glucose used per hour than that used by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 40%> less glucose used per hour than that used by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 50% less glucose used per hour than that used by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 60%> less glucose used per hour than that used by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 70% less glucose used per hour than that used by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; at least about 80% less glucose used per hour than that used by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes; and at least about 90%> less glucose used per hour than that used by a recombinant microorganism without activation, upregulation, or downregulation of one or more native and/or heterologous enzymes.

[0030] Another aspect of the invention relates to a recombinant microorganism comprising: one or more heterologous enzymes that function to regulate glycerol synthesis, wherein said one or more heterologous enzymes is activated, upregulated or downregulated; and one or more native and/or heterologous enzymes that function in one or more engineered metabolic pathways to convert a carbohydrate source to ethanol, wherein said one or more native and/or heterologous enzymes is activated, upregulated or downregulated. In certain embodiments, the one or more heterologous enzymes that function to regulate glycerol synthesis are encoded by an fpsl polynucleotide. In one embodiment, the fpsl polynucleotide is from Escherichia coli.

[0031] In some embodiments, one of the engineered metabolic pathways of the above recombinant microorganism comprises conversion of pyruvate to acetyl-CoA and formate. In certain embodiments, pyruvate is converted to acetyl-CoA and formate by a pyruvate formate lyase (PFL). In some embodiments, PFL is of prokaryotic or eukaryotic origin. In some embodiments, PFL is from one or more of a Bifidobacteria, an Escherichia, a Thermoanaerobacter, a Clostridia, a Streptococcus, a Lactobacillus, a Chlamydomonas, a Piromyces, a Neocallimastix, or a Bacillus species. In some embodiments, the PFL is from one or more of a Bacillus licheniformis, a Streptococcus thermophilus, a Lactobacillus plantarum, a Lactobacillus casei, a Bifidobacterium adolescentis, a Clostridium cellulolyticum, a Escherichia coli, a Chlamydomonas reinhardtii PflA, a Piromyces sp. E2, or a Neocallimastix frontalis. In one embodiment, PFL is from a Bifidobacterium adolescentis.

[0032] In some embodiments, one of said engineered metabolic pathways of the above recombinant microorganism comprises conversion of acetyl-CoA to ethanol. In some embodiments, acetyl-CoA is converted to acetaldehyde by an acetaldehyde dehydrogenase and acetaldehyde is converted to ethanol by an alcohol dehydrogenase. In other embodiments, acetyl-CoA is converted to ethanol by a bifunctional acetaldehyde/alcohol dehydrogenase. In some embodiments, the acetaldehyde dehydrogenase, alcohol dehydrogenase, or bifuntional acetaldehyde/alcohol dehydrogenase is of prokaryotic or eukaryotic origin. In one embodiment, acetaldehyde dehydrogenase is from C. phytofermentans. In certain embodiments, the bifunctional acetaldehyde/alcohol dehydrogenase is from an Escherichia, a Clostridia, a Chlamydomonas, a Piromyces, or a Bifidobacteria species. In some embodiments, the bifunctional acetaldehyde/alcohol dehydrogenase is from Escherichia coli, Clostridium phytofermentans, Chlamydomonas reinhardtii, Piromyces sp. E2, or Bifidobacterium adolescentis . In one embodiment, the bifunctional acetaldehyde/alcohol dehydrogenase is from a Bifidobacterium adolescentis or Piromyces sp. E2.

[0033] In further aspects, the recombinant microorganism comprises a deletion of one or more native enzymes encoded by an fdhl polynucleotide, an fdh2 polynucleotide, or both an fdhl polynucleotide and an fdh2 polynucleotide.

[0034] In some embodiments, the recombinant microorganism produces ethanol. In other embodiments, the recombinant microorganism produces formate. In some embodiments, the recombinant microorganism is selected from the group consisting of Saccharomyces cerevisiae, Kluyveromyces lactis, Kluyveromyces marxianus, Pichia pastoris, Yarrowia lipolytica, Hansenula polymorpha, Phajfia rhodozyma, Candida utliis, Arxula adeninivorans, Pichia stipitis, Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomyces pombe, Candida albicans, and Schwanniomyces occidentalis . In one embodiment, the recombinant microorganism is Saccharomyces cerevisiae.

[0035] In some embodiments, the recombinant microorganisms of the invention further comprise one or more native and/or heterologous enzymes that function in one or more engineered metabolic pathways to convert xylose to xylulose-5 -phosphate and/or arabinose to xylulose-5 -phosphate, wherein the one or more native and/or heterologous enzymes are activated, upregulated or downregulated.

[0036] In some embodiments, the recombinant microorganisms of the invention further comprise one or more native and/or heterologous enzymes which encodes a saccharolytic enzyme, including amylases, cellulases, hemicellulases, cellulolytic and amylolytic accessory enzymes, inulinases, levanases, and pentose sugar utilizing enzymes. In one aspect, the saccharolytic enzyme is an amylase, where the amylase is selected from H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. luckowense R. speratus, Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum or Arabidopsis thaliana. In another aspect, the saccharolytic enzyme is an amylase from S. fibuligera glucoamylase (glu-0111-CO).

[0037] Another aspect of the invention relates to a method for decreasing cellular glycerol comprising contacting biomass with a recombinant microorganism of the invention. A further aspect of the invention relates to a method for increasing cytosolic formate comprising contacting biomass with a recombinant microorganism of the invention. Another aspect of the invention relates to a process for converting biomass to ethanol comprising contacting biomass with a recombinant microorganism of the invention. In some embodiments, the biomass comprises lignocellulosic biomass. In some embodiments, the lignocellulosic biomass is selected from the group consisting of grass, switch grass, cord grass, rye grass, reed canary grass, mixed prairie grass, miscanthus, sugar-processing residues, sugarcane bagasse, sugarcane straw, agricultural wastes, rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, corn fiber, stover, soybean stover, corn stover, forestry wastes, recycled wood pulp fiber, paper sludge, sawdust, hardwood, softwood, agave, and combinations thereof. In some embodiments, the biomass is corn mash or corn starch.

[0038] In another aspect, the present invention also describes industrial yeast strains that express enzymes for the production of fuel ethanol from corn starch.

BRIEF DESCRIPTION OF THE DRAWINGS/FIGURES

[0039] Figure 1 shows a schematic of high yield metabolism.

[0040] Figure 2 depicts the glycolysis pathway.

[0041] Figure 3 shows a schematic of the glycolysis/fermentation pathway.

[0042] Figure 4 shows a map depicting location of primers used to make marked deletion of GPDl . [0043] Figure 5 shows a map depicting location of primers used to remove marker from

GPD1 locus.

[0044] Figure 6 shows a map depicting location of primers used to make marked deletion of GPD2.

[0045] Figure 7 shows a map depicting location of primers used to remove marker from

GPD2 locus.

[0046] Figure 8 shows a map depicting location of primers used to make marked deletion of FDHl .

[0047] Figure 9 shows a map depicting location of primers used to remove marker from

FDH1 locus.

[0048] Figure 10 shows a map depicting location of primers used to make marked deletion of FDH2.

[0049] Figure 11 shows a map depicting location of primers used to remove marker from

FDH2 locus.

[0050] Figure 12 shows an alignment of PFL enzymes from various organisms.

[0051] Figure 13 shows a graph of formate production over 48 hours.

[0052] Figure 14 shows a graph of formate production at the end of 142 hours in microaerobic and anaerobic conditions.

[0053] Figure 15 shows a graph of the growth of strains of the invention over 72 hours as measured by OD.

[0054] Figure 16 shows a graph of glycerol production (g/L) of strains of the invention over 72 hours.

[0055] Figure 17 shows a graph of ethanol production (g/L) of strains of the invention over 72 hours.

[0056] Figure 18 shows a graph of glucose utilization (g/L) of strains of the invention over 72 hours.

[0057] Figure 19 shows a graph of the growth of a strain of the invention over 142 hours as measured by OD.

[0058] Figure 20 shows a graph of the relative growth rate (mOD/min) of strains of the invention.

[0059] Figure 21 shows a graph of ethanol production (g/L) of strains of the invention after 50 hours of fermentation. [0060] Figure 22 shows a graph of ethanol production (g/L) of strains of the invention after 50 hours of fermentation.

[0061] Figure 23 shows a graph of glycerol production (g/L) of strains of the invention after 50 hours of fermentation.

[0062] Figure 24 shows a graph of glycerol production (g/L) of strains of the invention after 50 hours of fermentation.

[0063] Figure 25 shows a graph of glucose utilization (g/L), glycerol production (g/L), and ethanol production (g/L) after 72 hours of fermentation.

[0064] Figure 26 shows a diagram depicting integration of E. coli AADHs at the GPD1 locus.

[0065] Figure 27 shows a diagram depicting integration of E. coli AADHs at the FCY1 locus

[0066] Figure 28 shows a schematic diagram of a strategy for PCR construction and integration of KT-MX and NT-MX integration cassettes into both chromosomes of a target loci.

[0067] Figure 29 shows a schematic diagram of strategy used to replace integrated KT-

MX and NT-MX selection cassettes with a Mascoma Assembly on both chromosomes of a target loci.

[0068] Figure 30 shows a molecular map and genotyping of MA0370 integrated at the

FDH1 site of M3625.

[0069] Figure 31 shows an image of an agarose gel containing PCR products used to genotype and sequence the MA0370 site.

[0070] Figure 32 shows a molecular map and genotyping of MA0280 integrated at the

FDH2 site of M3625.

[0071] Figure 33 shows an image of an agarose gel containing PCR products used to genotype and sequence the MA0280 site.

[0072] Figure 34 shows a molecular map and genotyping of MA0289 integrated at the

GPD2 site of M3625.

[0073] Figure 35 shows an image of an agarose gel containing PCR products used to genotype and sequence the MA0370 site.

[0074] Figure 36 shows a molecular map and genotyping of MA0317 integrated at the

FCY1 site of M3625. [0075] Figure 37 shows an image of an agarose gel containing PCR products used to genotype and sequence the MA0317 site.

[0076] Figure 38 shows a graph depicting the results of a starch assay performed with strains M2390, M2519, M2691, M3498, and M3625.

[0077] Figure 39 shows an anti-peptide Western blot analysis of cell extracts (PflA, PflB,

AdhE) and aerobic culture supernatants (AE9).

[0078] Figure 40 shows a graph depicting the results of a formate lyase assay performed with engineered strains M3465, M3625, M3679, M3680, and M2390.

[0079] Figure 41 shows a graph depicting the results of an alcohol dehydrogenase assay performed with engineered strains M3465, M3625, M3679, and M3680.

[0080] Figure 42 shows a graph depicting the results of a glucoamylase activity assay performed with engineered strains M3625 and M3680 using 5(^g/mL AE9 on corn starch at room temperature (~25°C).

[0081] Figure 43 A is a schematic showing insertion of promoters and terminators used to express, GPD2, and B. adolescentis pflA, pflB and adhE at the GPDl locus in M3624 and

M3515.

[0082] Figure 43B is a schematic showing insertion of promoters and terminators used to express GPDl and B. adolescentis pflA, pflB and adhE at the GPD2 locus in M3624 and M3515.

[0083] Figure 43C is a schematic showing deletion of the FDH1 gene in M3624 and

M3515.

[0084] Figure 43D is a schematic showing insertions of promoters and terminators used to express B. adolescentis pflA, pflB and adhE at the FDH2 locus in M3624 and M3515.

[0085] Figure 44A shows HPLC analysis of formate produced by glycerol reduction strains during fermentation of 28% solids corn mash.

[0086] Figure 44B shows HPLC analysis of glycerol produced by glycerol reduction strains during fermentation of 28% solids corn mash.

[0087] Figure 44C shows HPLC analysis of ethanol produced by glycerol reduction strains during fermentation of 28% solids corn mash.

[0088] Figure 45A is a schematic showing insertion of promoters and terminators used to express B. adolescentis pflA, pflB and adhE at the GPD2 locus in M3465.

[0089] Figure 45B is a schematic showing deletion of the FDH1 gene in M3465. [0090] Figure 45 C is a schematic showing insertion of promoters and terminators used to express B. adolescentis pflA, pflB and adhE at the FDH2 locus in M3465.

[0091] Figure 46 A is a schematic showing insertion of promoters and terminators used to express B. adolescentis pflA, pflB and adhE at the GPD1 locus in M3469.

[0092] Figure 46B is a schematic showing deletion of the FDH1 gene in M3469.

[0093] Figure 46C is a schematic showing insertion of promoters and terminators used to express B. adolescentis pflA, pflB and adhE at the FDH2 locus in M3469.

[0094] Figure 47 shows HPLC analysis of ethanol titers produced by glycerol reduction strains during fermentation of 30% solids corn mash.

[0095] Figure 48 shows HPLC analysis of glycerol titers produced by glycerol reduction strains during fermentation of 30% solids corn mash.

[0096] Figure 49 shows the reverse reaction catalyzed by Bifidobacterium adolecentis bifunctional alcohol dehydrogenase (AdhE) in which ethanol is converted to acetaldehyde.

[0097] Figure 50 shows a diagram of the reaction for the conversion of acetaldehyde to acetyl Co A by AdhE.

[0098] Figure 51 is a schematic showing insertion of promoters and terminators used to express B. adolescentis pflA, pflB and adhE at the GPP1 locus in TB655.

[0100] Figure 52 is a schematic showing insertion of promoters and terminators used to express B. adolescentis pflA, pflB and adhE at the GPP2 locus in TB656.

[0101] Figure 53 is a graph depicting decreased glycerol formation in strains TB655 and

TB656 compared to strain M3297.

[0102] Figure 54 is a graph depicting increased ethanol yield in strains TB655 and TB656 compared to strain M3297.

[0103] Figure 55 is a graph depicting formate production in strains TB655, TB656, and

M3297.

DETAILED DESCRIPTION OF THE INVENTION Definitions

[0104] The term "heterologous" when used in reference to a polynucleotide, a gene, a polypeptide, or an enzyme refers to a polynucleotide, gene, polypeptide, or an enzyme not normally found in the host organism. "Heterologous" also includes a native coding region, or portion thereof, that is reintroduced into the source organism in a form that is different from the corresponding native gene, e.g., not in its natural location in the organism's genome. The heterologous polynucleotide or gene may be introduced into the host organism by, e.g. , gene transfer. A heterologous gene may include a native coding region that is a portion of a chimeric gene including non-native regulatory regions that is reintroduced into the native host. Foreign genes can comprise native genes inserted into a non-native organism, or chimeric genes.

[0105] The term "heterologous polynucleotide" is intended to include a polynucleotide that encodes one or more polypeptides or portions or fragments of polypeptides. A heterologous polynucleotide may be derived from any source, e.g., eukaryotes, prokaryotes, viruses, or synthetic polynucleotide fragments.

[0106] The terms "promoter" or "surrogate promoter" is intended to include a polynucleotide that can transcriptionally control a gene-of-interest that it does not transcriptionally control in nature. In certain embodiments, the transcriptional control of a surrogate promoter results in an increase in expression of the gene-of-interest. In certain embodiments, a surrogate promoter is placed 5' to the gene-of-interest. A surrogate promoter may be used to replace the natural promoter, or may be used in addition to the natural promoter. A surrogate promoter may be endogenous with regard to the host cell in which it is used, or it may be a heterologous polynucleotide sequence introduced into the host cell, e.g., exogenous with regard to the host cell in which it is used.

[0107] The terms "gene(s)" or "polynucleotide" or "polynucleotide sequence(s)" are intended to include nucleic acid molecules, e.g., polynucleotides which include an open reading frame encoding a polypeptide, and can further include non-coding regulatory sequences, and introns. In addition, the terms are intended to include one or more genes that map to a functional locus. In addition, the terms are intended to include a specific gene for a selected purpose. The gene may be endogenous to the host cell or may be recombinantly introduced into the host cell, e.g., as a plasmid maintained episomally or a plasmid (or fragment thereof) that is stably integrated into the genome. In addition to the plasmid form, a gene may, for example, be in the form of linear DNA. In certain embodiments, the gene or polynucleotide is involved in at least one step in the bioconversion of biomass to, e.g., ethanol. Accordingly, the term is intended to include any gene encoding a polypeptide, such as the enzymes acetate kinase (ACK), phosphotransacetylase (PTA), lactate dehydrogenase (LDH), pyruvate formate lyase (PFL), aldehyde dehydrogenase (ADH) and/or alcohol dehydrogenase (ADH), acetyl- CoA transferase (ACS), acetaldehyde dehydrogenase (ACDH), acetaldehyde/alcohol dehydrogenase (AADH), glycerol-3 -phosphate dehydrogenase (GPD), glycerol 3- phosphatase (GPP), acetyl-CoA synthetase, thiolase, CoA transferase, acetoacetate decarboxylase, alcohol acetyltransferase enzymes in the D-xylose pathway, such as xylose isomerase and xylulokinase, enzymes in the L-arabinose pathway, such as L- arabinose isomerase and L-ribulose-5 -phosphate 4-epimerase. The term gene is also intended to cover all copies of a particular gene, e.g., all of the DNA sequences in a cell encoding a particular gene product.

[0108] The term "transcriptional control" is intended to include the ability to modulate gene expression at the level of transcription. In certain embodiments, transcription, and thus gene expression, is modulated by replacing or adding a surrogate promoter near the 5' end of the coding region of a gene-of-interest, thereby resulting in altered gene expression. In certain embodiments, the transcriptional control of one or more genes is engineered to result in the optimal expression of such genes, e.g., in a desired ratio. The term also includes inducible transcriptional control as recognized in the art.

[0109] The term "expression" is intended to include the expression of a gene at least at the level of mRNA production.

[0110] The term "expression product" is intended to include the resultant product, e.g., a polypeptide, of an expressed gene.

[0111] The term "increased expression" is intended to include an alteration in gene expression at least at the level of increased mRNA production and, preferably, at the level of polypeptide expression. The term "increased production" is intended to include an increase in the amount of a polypeptide expressed, in the level of the enzymatic activity of the polypeptide, or a combination thereof, as compared to the native production of, or the enzymatic activity, of the polypeptide.

[0112] The terms "activity," "activities," "enzymatic activity," and "enzymatic activities" are used interchangeably and are intended to include any functional activity normally attributed to a selected polypeptide when produced under favorable conditions. Typically, the activity of a selected polypeptide encompasses the total enzymatic activity associated with the produced polypeptide. The polypeptide produced by a host cell and having enzymatic activity may be located in the intracellular space of the cell, cell- associated, secreted into the extracellular milieu, or a combination thereof. Techniques for determining total activity as compared to secreted activity are described herein and are known in the art.

[0113] The term "xylanolytic activity" is intended to include the ability to hydro lyze glycosidic linkages in oligopentoses and polypentoses.

[0114] The term "arabinolytic activity" is intended to include the ability to hydrolyze glycosidic linkages in oligopentoses and polypentoses.

[0115] The term "cellulolytic activity" is intended to include the ability to hydrolyze glycosidic linkages in oligohexoses and polyhexoses. Cellulolytic activity may also include the ability to depolymerize or debranch cellulose and hemicellulose.

[0116] As used herein, the term "lactate dehydrogenase" or "LDH" is intended to include the enzymes capable of converting pyruvate into lactate. It is understood that LDH can also catalyze the oxidation of hydroxybutyrate. LDH includes those enzymes that correspond to Enzyme Commission Number 1.1.1.27.

[0117] As used herein the term "alcohol dehydrogenase" or "ADH" is intended to include the enzymes capable of converting acetaldehyde into an alcohol, such as ethanol. ADH also includes the enzymes capable of converting acetone to isopropanol. ADH includes those enzymes that correspond to Enzyme Commission Number 1.1.1.1.

[0118] As used herein, the term "phosphotransacetylase" or "PTA" is intended to include the enzymes capable of converting acetyl-phosphate into acetyl-CoA. PTA includes those enzymes that correspond to Enzyme Commission Number 2.3.1.8.

[0119] As used herein, the term "acetate kinase" or "ACK" is intended to include the enzymes capable of converting acetate into acetyl-phosphate. ACK includes those enzymes that correspond to Enzyme Commission Number 2.7.2.1.

[0120] As used herein, the term "pyruvate formate lyase" or "PFL" is intended to include the enzymes capable of converting pyruvate into acetyl-CoA and formate. PFL includes those enzymes that correspond to Enzyme Commission Number 2.3.1.54.

[0121] As used herein, the term "formate dehydrogenase" or "FDH" is intended to include the enzymes capable of converting formate and NAD ⁺ to NADH and C0 ₂ . FDH includes those enzymes that correspond to Enzyme Commission Number 1.2.1.2. [0122] As used herein, the term "acetaldehyde dehydrogenase" or "ACDH" is intended to include the enzymes capable of converting acetyl-CoA to acetaldehyde. ACDH includes those enzymes that correspond to Enzyme Commission Number 1.2.1.3.

[0123] As used herein, the term "acetaldehyde/alcohol dehydrogenase" is intended to include the enzymes capable of converting acetyl-CoA to ethanol. Acetaldehyde/alcohol dehydrogenase includes those enzymes that correspond to Enzyme Commission Numbers 1.2.1.10 and 1.1.1.1.

[0124] As used herein, the term "glycerol-3 -phosphate dehydrogenase" or "GPD" is intended to include the enzymes capable of converting dihydroxyacetone phosphate to glycerol-3-phosphate. GPD includes those enzymes that correspond to Enzyme Commission Number 1.1.1.8.

[0125] As used herein, the term "glycerol 3 -phosphatase" or "GPP" is intended to include the enzymes capable of converting glycerol 3-phosphate to glycerol. GPP includes those enzymes that correspond to Enzyme Commission Number 3.1.3.21.

[0126] As used herein, the term "acetyl-CoA synthetase" or "ACS" is intended to include the enzymes capable of converting acetate to acetyl-CoA. Acetyl-CoA synthetase includes those enzymes that correspond to Enzyme Commission Number 6.2.1.1.

[0127] As used herein, the term "thiolase" is intended to include the enzymes capable of converting acetyl-CoA to acetoacetyl-CoA. Thiolase includes those enzymes that correspond to Enzyme Commission Number 2.3.1.9.

[0128] As used herein, the term "CoA transferase" is intended to include the enzymes capable of converting acetate and acetoacetyl-CoA to acetoacetate and acetyl-CoA. CoA transferase includes those enzymes that correspond to Enzyme Commission Number 2.8.3.8.

[0129] As used herein, the term "acetoacetate decarboxylase" is intended to include the enzymes capable of converting acetoacetate to acetone and carbon dioxide. Acetoacetate decarboxylase includes those enzymes that correspond to Enzyme Commission Number 4.1.1.4.

[0130] As used herein, the term "alcohol acetyltransferase" is intended to include the enzymes capable of converting acetyl-CoA and ethanol to ethyl acetate. Alcohol acetyltransferase includes those enzymes that correspond to Enzyme Commission Number 2.3.1.84. [0131] The term "pyruvate decarboxylase activity" is intended to include the ability of a polypeptide to enzymatically convert pyruvate into acetaldehyde and carbon dioxide (e.g. , "pyruvate decarboxylase" or "PDC"). Typically, the activity of a selected polypeptide encompasses the total enzymatic activity associated with the produced polypeptide, comprising, e.g., the superior substrate affinity of the enzyme, thermostability, stability at different pHs, or a combination of these attributes. PDC includes those enzymes that correspond to Enzyme Commission Number 4.1.1.1.

[0132] The term "ethanologenic" is intended to include the ability of a microorganism to produce ethanol from a carbohydrate as a fermentation product. The term is intended to include, but is not limited to, naturally occurring ethanologenic organisms, ethanologenic organisms with naturally occurring or induced mutations, and ethanologenic organisms which have been genetically modified.

[0133] The terms "fermenting" and "fermentation" are intended to include the enzymatic process (e.g., cellular or acellular, e.g., a lysate or purified polypeptide mixture) by which ethanol is produced from a carbohydrate, in particular, as a product of fermentation.

[0134] The term "secreted" is intended to include the movement of polypeptides to the periplasmic space or extracellular milieu. The term "increased secretion" is intended to include situations in which a given polypeptide is secreted at an increased level (i.e., in excess of the naturally-occurring amount of secretion). In certain embodiments, the term "increased secretion" refers to an increase in secretion of a given polypeptide that is at least about 10% or at least about 100%, 200%, 300%, 400%, 500%, 600%, 700%, 800%, 900%), 1000%), or more, as compared to the naturally-occurring level of secretion.

[0135] The term "secretory polypeptide" is intended to include any polypeptide(s), alone or in combination with other polypeptides, that facilitate the transport of another polypeptide from the intracellular space of a cell to the extracellular milieu. In certain embodiments, the secretory polypeptide(s) encompass all the necessary secretory polypeptides sufficient to impart secretory activity to a Gram-negative or Gram-positive host cell or to a yeast host cell. Typically, secretory proteins are encoded in a single region or locus that may be isolated from one host cell and transferred to another host cell using genetic engineering. In certain embodiments, the secretory polypeptide(s) are derived from any bacterial cell having secretory activity or any yeast cell having secretory activity. In certain embodiments, the secretory polypeptide(s) are derived from a host cell having Type II secretory activity. In certain embodiments, the host cell is a thermophilic bacterial cell. In certain embodiments, the host cell is a yeast cell.

[0136] The term "derived from" is intended to include the isolation (in whole or in part) of a polynucleotide segment from an indicated source or the purification of a polypeptide from an indicated source. The term is intended to include, for example, direct cloning, PCR amplification, or artificial synthesis from or based on a sequence associated with the indicated polynucleotide source.

[0137] The term "recombinant microorganism" or "recombinant host cell" is intended to include progeny or derivatives of the recombinant microorganisms of the invention. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny or derivatives may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0138] By "thermophilic" is meant an organism that thrives at a temperature of about

45°C or higher.

[0139] By "mesophilic" is meant an organism that thrives at a temperature of about 20-

45°C.

[0140] The term "organic acid" is art-recognized. "Organic acid," as used herein, also includes certain organic solvents such as ethanol. The term "lactic acid" refers to the organic acid 2-hydroxypropionic acid in either the free acid or salt form. The salt form of lactic acid is referred to as "lactate" regardless of the neutralizing agent, i.e., calcium carbonate or ammonium hydroxide. The term "acetic acid" refers to the organic acid methanecarboxylic acid, also known as ethanoic acid, in either free acid or salt form. The salt form of acetic acid is referred to as "acetate."

[0141] Certain embodiments of the present invention provide for the "insertion," {e.g., the addition, integration, incorporation, or introduction) of certain genes or particular polynucleotide sequences within thermophilic or mesophilic microorganisms, which insertion of genes or particular polynucleotide sequences may be understood to encompass "genetic modification(s)" or "transformation(s)" such that the resulting strains of said thermophilic or mesophilic microorganisms may be understood to be "genetically modified" or "transformed." In certain embodiments, strains may be of bacterial, fungal, or yeast origin. [0142] Certain embodiments of the present invention provide for the "inactivation" or

"deletion" of certain genes or particular polynucleotide sequences within thermophilic or mesophilic microorganisms, which "inactivation" or "deletion" of genes or particular polynucleotide sequences may be understood to encompass "genetic modification(s)" or "transformation(s)" such that the resulting strains of said thermophilic or mesophilic microorganisms may be understood to be "genetically modified" or "transformed." In certain embodiments, strains may be of bacterial, fungal, or yeast origin.

[0143] The term "consolidated bioprocessing" or "CBP" refers to biomass processing schemes involving enzymatic or microbial hydrolysis that commonly involve four biologically mediated transformations: (1) the production of saccharo lytic enzymes (amylases, cellulases, and hemicellulases); (2) the hydrolysis of carbohydrate components present in pretreated biomass to sugars; (3) the fermentation of hexose sugars (e.g., glucose, mannose, and galactose); and (4) the fermentation of pentose sugars (e.g., xylose and arabinose). These four transformations occur in a single step in a process configuration called CBP, which is distinguished from other less highly integrated configurations in that it does not involve a dedicated process step for cellulase and/or hemicellulase production.

[0144] The term "CBP organism" is intended to include microorganisms of the invention, e.g., microorganisms that have properties suitable for CBP.

[0145] In one aspect of the invention, the genes or particular polynucleotide sequences are inserted to activate the activity for which they encode, such as the expression of an enzyme. In certain embodiments, genes encoding enzymes in the metabolic production of ethanol, e.g., enzymes that metabolize pentose and/or hexose sugars, may be added to a mesophilic or thermophilic organism. In certain embodiments of the invention, the enzyme may confer the ability to metabolize a pentose sugar and be involved, for example, in the D-xylose pathway and/or L-arabinose pathway.

[0146] In one aspect of the invention, the genes or particular polynucleotide sequences are partially, substantially, or completely deleted, silenced, inactivated, or down-regulated in order to inactivate the activity for which they encode, such as the expression of an enzyme. Deletions provide maximum stability because there is no opportunity for a reverse mutation to restore function. Alternatively, genes can be partially, substantially, or completely deleted, silenced, inactivated, or down-regulated by insertion of nucleic acid sequences that disrupt the function and/or expression of the gene (e.g., PI transduction or other methods known in the art). The terms "eliminate," "elimination," and "knockout" are used interchangeably with the terms "deletion," "partial deletion," "substantial deletion," or "complete deletion." In certain embodiments, strains of thermophilic or mesophilic microorganisms of interest may be engineered by site directed homologous recombination to knockout the production of organic acids. In still other embodiments, RNAi or antisense DNA (asDNA) may be used to partially, substantially, or completely silence, inactivate, or down-regulate a particular gene of interest.

[0147] In certain embodiments, the genes targeted for deletion or inactivation as described herein may be endogenous to the native strain of the microorganism, and may thus be understood to be referred to as "native gene(s)" or "endogenous gene(s)." An organism is in "a native state" if it has not been genetically engineered or otherwise manipulated by the hand of man in a manner that intentionally alters the genetic and/or phenotypic constitution of the organism. For example, wild-type organisms may be considered to be in a native state. In other embodiments, the gene(s) targeted for deletion or inactivation may be non-native to the organism.

[0148] Similarly, the enzymes of the invention as described herein can be endogenous to the native strain of the microorganism, and can thus be understood to be referred to as "native" or "endogenous."

[0149] The term "upregulated" means increased in activity, e.g., increase in enzymatic activity of the enzyme as compared to activity in a native host organism.

[0150] The term "downregulated" means decreased in activity, e.g., decrease in enzymatic activity of the enzyme as compared to activity in a native host organism.

[0151] The term "activated" means expressed or metabolically functional.

[0152] The term "adapted for growing" means selection of an organism for growth under conditions in which the organism does not otherwise grow or in which the organism grows slowly or minimally. Thus, an organism that is said to be adapted for growing under the selected condition, grows better than an organism that has not been adapted for growing under the selected conditions. Growth can be measured by any methods known in the art, including, but not limited to, measurement of optical density or specific growth rate. [0153] The term "carbohydrate source" is intended to include any source of carbohydrate including, but not limited to, biomass or carbohydrates, such as a sugar or a sugar alcohol. "Carbohydrates" include, but are not limited to, monosaccharides (e.g., glucose, fructose, galactose, xylose, arabinose, or ribose), sugar derivatives (e.g., sorbitol, glycerol, galacturonic acid, rhamnose, xylitol), disaccharides (e.g., sucrose, cellobiose, maltose, or lactose), oligosaccharides (e.g., xylooligomers, cellodextrins, or maltodextrins), and polysaccharides (e.g., xylan, cellulose, starch, mannan, alginate, or pectin).

[0154] As used herein, an "amylolytic enzyme" can be any enzyme involved in amylase digestion, metabolism and/or hydrolysis. The term "amylase" refers to an enzyme that breaks starch down into sugar. Amylase is present in human saliva, where it begins the chemical process of digestion. Foods that contain much starch but little sugar, such as rice and potato, taste slightly sweet as they are chewed because amylase turns some of their starch into sugar in the mouth. The pancreas also makes amylase (a-amylase) to hydrolyse dietary starch into disaccharides and trisaccharides which are converted by other enzymes to glucose to supply the body with energy. Plants and some bacteria also produce amylase. All amylases are glycoside hydrolases and act on a-l ,4-glycosidic bonds. Some amylases, such as γ-amylase (glucoamylase), also act on a-l ,6-glycosidic bonds. Amylase enzymes include a-amylase (EC 3.2.1.1), β-amylase (EC 3.2.1.2), and γ- amylase (EC 3.2.1.3). The a-amylases are calcium metalloenzymes, unable to function in the absence of calcium. By acting at random locations along the starch chain, a-amylase breaks down long-chain carbohydrates, ultimately yielding maltotriose and maltose from amylose, or maltose, glucose and "limit dextrin" from amylopectin. Because it can act anywhere on the substrate, α-amylase tends to be faster-acting than β-amylase. In animals, it is a major digestive enzyme and its optimum pH is about 6.7-7.0. Another form of amylase, β-amylase is also synthesized by bacteria, fungi, and plants. Working from the non-reducing end, β-amylase catalyzes the hydrolysis of the second a- 1 ,4 glycosidic bond, cleaving off two glucose units (maltose) at a time. Many microbes produce amylase to degrade extracellular starches. In addition to cleaving the last a(l- 4)glycosidic linkages at the nonreducing end of amylose and amylopectin, yielding glucose, γ-amylase will cleave a(l-6) glycosidic linkages. Another amylolytic enzyme is alpha-glucosidase that acts on maltose and other short malto-oligosaccharides produced by alpha-, beta- , and gamma-amylases, converting them to glucose. Another amylolytic enzyme is pullulanase. Pullulanase is a specific kind of glucanase, an amylolytic exoenzyme, that degrades pullulan. Pullulan is regarded as a chain of maltotriose units linked by alpha- 1 ,6-glycosidic bonds. Pullulanase (EC 3.2.1.41) is also known as pullulan-6-glucanohydrolase (Debranching enzyme). Another amylolytic enzyme, isopullulanase, hydro lyses pullulan to isopanose (6-alpha-maltosylglucose). Isopullulanase (EC 3.2.1.57) is also known as pullulan 4-glucanohydrolase. An "amylase" can be any enzyme involved in amylase digestion, metabolism and/or hydrolysis, including a-amylase, β -amylase, glucoamylase, pullulanase, isopullulanase, and alpha-glucosidase.

[0155] As used herein, a "saccharo lytic enzyme" can be any enzyme involved in carbohydrate digestion, metabolism and/or hydrolysis, including amylases, cellulases, hemicellulases, cellulolytic and amylolytic accessory enzymes, inulinases, levanases, and pentose sugar utilizing enzymes.

Biomass

[0156] Biomass can include any type of biomass known in the art or described herein.

For example, biomass can include, but is not limited to, starch, sugar, and lignocellulosic materials. Starch materials can include, but are not limited to, mashes such as corn, wheat, rye, barley, rice, or milo. Sugar materials can include, but are not limited to, sugar beets, artichoke tubers, sweet sorghum, or cane. The terms "lignocellulosic material," "lignocellulosic substrate," and "cellulosic biomass" mean any type of biomass comprising cellulose, hemicellulose, lignin, or combinations thereof, such as but not limited to woody biomass, forage grasses, herbaceous energy crops, non-woody-plant biomass, agricultural wastes and/or agricultural residues, forestry residues and/or forestry wastes, paper-production sludge and/or waste paper sludge, waste-water-treatment sludge, municipal solid waste, corn fiber from wet and dry mill corn ethanol plants, and sugar-processing residues. The terms "hemicellulosics," "hemicellulosic portions," and "hemicellulosic fractions" mean the non-lignin, non-cellulose elements of lignocellulosic material, such as but not limited to hemicellulose (i.e., comprising xyloglucan, xylan, glucuronoxylan, arabinoxylan, mannan, glucomannan, and galactoglucomannan, inter alia), pectins (e.g., homogalacturonans, rhamnogalacturonan I and II, and xylogalacturonan), and proteoglycans (e.g., arabinogalactan-protein, extensin, and pro line -rich proteins). [0157] In a non-limiting example, the lignocellulosic material can include, but is not limited to, woody biomass, such as recycled wood pulp fiber, sawdust, hardwood, softwood, and combinations thereof; grasses, such as switch grass, cord grass, rye grass, reed canary grass, miscanthus, or a combination thereof; sugar-processing residues, such as but not limited to sugar cane bagasse; agricultural wastes, such as but not limited to rice straw, rice hulls, barley straw, corn cobs, cereal straw, wheat straw, canola straw, oat straw, oat hulls, and corn fiber; stover, such as but not limited to soybean stover, corn stover; succulents, such as but not limited to, Agave; and forestry wastes, such as but not limited to, recycled wood pulp fiber, sawdust, hardwood (e.g., poplar, oak, maple, birch, willow), softwood, or any combination thereof. Lignocellulosic material may comprise one species of fiber; alternatively, lignocellulosic material may comprise a mixture of fibers that originate from different lignocellulosic materials. Other lignocellulosic materials are agricultural wastes, such as cereal straws, including wheat straw, barley straw, canola straw and oat straw; corn fiber; stovers, such as corn stover and soybean stover; grasses, such as switch grass, reed canary grass, cord grass, and miscanthus; or combinations thereof.

[0158] Paper sludge is also a viable feedstock for lactate or acetate production. Paper sludge is solid residue arising from pulping and paper-making, and is typically removed from process wastewater in a primary clarifier. At a disposal cost of $30/wet ton, the cost of sludge disposal equates to $5/ton of paper that is produced for sale. The cost of disposing of wet sludge is a significant incentive to convert the material for other uses, such as conversion to ethanol. Processes provided by the present invention are widely applicable. Moreover, the saccharification and/or fermentation products may be used to produce ethanol or higher value added chemicals, such as organic acids, aromatics, esters, acetone and polymer intermediates.

Glycerol Reduction

[0159] Anaerobic growth conditions require the production of endogenouse electron acceptors, such as the coenzyme nicotinamide adenine dinucleotide (NAD ⁺ ). In cellular redox reactions, the NAD /NADH couple plays a vital role as a reservoir and carrier of reducing equivalents. Ansell, R., et al, EMBO J. 7(5:2179-87 (1997). Cellular glycerol production, which generates an NAD ⁺ , serves as a redox valve to remove excess reducing power during anaerobic fermentation in yeast. Glycerol production is, however, an energetically wasteful process that expends ATP and results in the loss of a reduced three- carbon compound. Ansell, R., et al, EMBO J. 7(5:2179-87 (1997). To generate glycerol from a starting glucose molecule, glycerol 3 -phosphate dehydrogenase (GPD) reduces dihydroxyacetone phosphate to glycerol 3 -phosphate and glycerol 3 -phosphatase (GPP) dephosphorylates glycerol 3-phosphate to glycerol. Despite being energetically wasteful, glycerol production is a necessary metabolic process for anaerobic growth as deleting GPD activity completely inhibits growth under anaeroblic conditions. See Ansell, R., et al., EMBO J. 76:2179-87 (1997).

[0160] GPD is encoded by two isogenes, gpdl and gpd2. GPD1 encodes the major isoform in anaerobically growing cells, while GPD2 is required for glycerol production in the absence of oxygen, which stimulates its expression. Pahlman, A-K., et al, J. Biol. Chem. 27(5:3555-63 (2001). The first step in the conversion of dihydroxyacetone phosphate to glycerol by GPD is rate controlling. Guo, Z.P., et al, Metab. Eng. 75:49-59 (2011). GPP is also encoded by two isogenes, gppl and gpp2. The deletion of GPP genes arrests growth when shifted to anaerobic conditions, demonstrating that GPP is important for cellular tolerance to osmotic and anaerobic stress. See Pahlman, A-K., et al., J. Biol. Chem. 276:3555-63 (2001).

[0161] Because glycerol is a major by-product of anaerobic production of ethanol, many efforts have been made to delete cellular production of glycerol. However, because of the reducing equivalents produced by glycerol synthesis, deletion of the glycerol synthesis pathway cannot be done without compensating for this valuable metabolic function. Attempts to delete glycerol production and engineer alternate electron acceptors have been made. Liden, G., et al, Appl. Env. Microbiol. (52:3894-96 (1996); Medina, V.G., et al, Appl. Env. Microbiol. 7(5:190-195 (2010). Liden and Medina both deleted the gpdl and gpd2 genes and attempted to bypass glycerol formation using additional carbon sources. Liden engineered a xylose reductase from Pichia stipitis into an S. cerevisiae gpdl/2 deletion strain. The xylose reductase activity facilitated the anaerobic growth of the glycerol-deleted strain in the presence of xylose. See Liden, G., et al, Appl. Env. Microbiol. (52:3894-96 (1996). Medina engineered an acetylaldehyde dehydrogenase, mhpF, from E. coli into an S. cerevisiae gpdl/2 deletion strain to convert acetyl-CoA to acetaldehyde. The acetylaldehyde dehydrogenase activity facilitated the anaerobic growth of the glycerol-deletion strain in the presence of acetic acid but not in the presence of glucose as the sole source of carbon. Medina, V.G., et al, Appl. Env. Microbiol. 7(5: 190-195 (2010); see also EP 2277989. Medina noted several issues with the mhpF- containing strain that needed to be addressed before implementing industrially, including significantly reduced growth and product formation rates than yeast comprising GPD1 and GPD2.

[0162] Additional attempts to redirect flux from glycerol to ethanol have included the engineering of a non-phosphorylating NADP+-dependent glyceraldehydes-3 -phosphate dehydrogenase (GAPN) into yeast, either with or without the simultaneous knockout of GPD1. Bro, C, et al., Metab. Eng. 5:102-111 (2006); U.S. Patent Appl. Pub. No. US2006/0257983; Guo, Z.P., et al, Metab. Eng. 75:49-59 (2011). However, other cellular mechanisms exist to control the production and accumulation of glycerol, including glycerol exporters such as FPSl, that do not require the engineering of alternate NADP+/NADPH coupling or deletion of glycerol synthesis genes. Tamas, M.J., et al, Mol. Microbiol. 57: 1087-1004 (1999).

[0163] FPSl is a channel protein located in the plasma membrane that controls the accumulation and release of glycerol in yeast osmoregulation. Null mutants of this strain accumulate large amounts of intracellular glycerol, grow much slower than wild-type, and consume the sugar substrate at a slower rate. Tamas, M.J., et al, Mol. Microbiol. 57: 1087-1004 (1999). Despite slower growth under anaerobic conditions, an fpslA strain can serve as an alternative to eliminating NAD ⁺ -dependant glycerol activity. An fpslA strain has reduced glycerol formation yet has a completely functional NAD ⁺ -dependant glycerol synthesis pathway. Alternatively, rather than deleting endogenous FPSl, constitutively active mutants of FPS 1 or homo logs from other organisms can be used to regulate glycerol synthesis while keep the NAD ⁺ -dependant glycerol activity intact. In embodiments of the invention that modulate FPSl, the recombinant host cells can still synthesize and retain glycerol and achieve improved robustness relative to strains that are unable to make glycerol.

[0164] An example FPSl sequence from S. cerevisiae is shown below.

[0165] S. cerevisiae FPSl (nucleotide; coding sequence underlined; SEQ ID NO: 1):

[0166] ttgacggcagttctcatagcatctcaaagcaatagcagtgcaaaagtacataaccgtagg aaggtacgcggtaggtat ttgagttcgttggtggttatcctccgcaaggcgcttcggcggttatttgttgatagtcga agaacaccaaaaaaaatgctgttattgct ttctccgtaaacaataaaacccggtagcgggataacgcggctgatgcttttatttaggaa ggaatacttacattatcatgagaacatt gtcaagggcattctgatacgggccttccatcgcaagaaaaaggcagcaacggactgaggg acggagagagttacggcataag aagtagtaggagagcagagtgtcataaagttatattattctcgtcctaaagtcaattagt tctgttgcgcttgacaatatatgtcgtgta ataccgtcccttagcagaagaaagaaagacggatccatatatgttaaaatgcttcagaga tgtttctttaatgtgccgtccaacaaa ggtatcttctgtagcttcctctattttcgatcagatctcatagtgagaaggcgcaattca gtagttaaaagcggggaacagtgtgaat ccggagacggcaagattgcccggccctttttgcggaaaagataaaacaagatatattgca ctttttccaccaagaaaaacaggaa gtggattaaaaaatcaacaaagtataacgcctattgtcccaataagcgtcggttgttctt ctttattattttaccaagtacgctcgaggg tacattctaatgcattaaaagacatgagtaatcctcaaaaagctctaaacgactttctgt ccagtgaatctgttcatacacatgatagtt ctaggaaacaatctaataagcagtcatccgacgaaggacgctcttcatcacaaccttcac atcatcactctggtggtactaacaac aataataacaataataataataataataacagtaacaacaacaacaacggcaacgatggg ggaaatgatgacgactatgattatg aaatgcaagattatagaccttctccgcaaagtgcgcggcctactcccacgtatgttccac aatattctgtagaaagtgggactgcttt cccgattcaagaggttattcctagcgcatacattaacacacaagatataaaccataaaga taacggtccgccgagtgcaagcagt aatagagcattcaggcctagagggcagaccacagtgtcggccaacgtgcttaacattgaa gatttttacaaaaatgcagacgatg cgcataccatcccggagtcacatttatcgagaaggagaagtaggtcgagggctacgagta atgctgggcacagtgccaataca ggcgccacgaatggcaggactactggtgcccaaactaatatggaaagcaatgaatcacca cgtaacgtccccattatggtgaag ccaaagacattataccagaaccctcaaacacctacagtcttgccctccacataccatcca attaataaatggtcttccgtcaaaaac acttatttgaaggaatttttagccgagtttatgggaacaatggttatgattattttcggt agtgctgttgtttgtcaggtcaatgttgctgg gaaaatacagcaggacaatttcaacgtggctttggataaccttaacgttaccgggtcttc tgcagaaacgatagacgctatgaaga gtttaacatccttggtttcatccgttgcgggcggtacctttgatgatgtggcattgggct gggctgctgccgtggtgatgggctatttc tgcgctggtggtagtgccatctcaggtgctcatttgaatccgtctattacattagccaat ttggtgtatagaggttttcccctgaagaa agttccttattactttgctggacaattgatcggtgccttcacaggcgctttgatcttgtt tatttggtacaaaagggtgttacaagaggc atatagcgattggtggatgaatgaaagtgttgcgggaatgttttgcgtttttccaaagcc ttatctaagttcaggacggcaatttttttc cgaatttttatgtggagctatgttacaagcaggaacatttgcgctgaccgatccttatac gtgtttgtcctctgatgttttcccattgatg atgtttattttgattttcattatcaatgcttccatggcttatcagacaggtacagcaatg aatttggctcgtgatctgggcccacgtcttg cactatatgcagttggatttgatcataaaatgctttgggtgcatcatcatcatttctttt gggttcccatggtaggcccatttattggtgc gttaatgggggggttggtttacgatgtctgtatttatcagggtcatgaatctccagtcaa ctggtctttaccagtttataaggaaatgat tatgagagcctggtttagaaggcctggttggaagaagagaaatagagcaagaagaacatc ggacctgagtgacttctcatacaat aacgatgatgatgaggaatttggagaaagaatggctcttcaaaagacaaagaccaagtca tctatttcagacaacgaaaatgaag caggagaaaagaaagtgcaatttaaatctgttcagcgcggcaaaagaacgtttggtggta taccaacaattcttgaagaagaaga ttccattgaaactgcttcgctaggtgcgacgacgactgattctattgggttatccgacac atcatcagaagattcgcattatggtaat gctaagaaggtaacatgagaaaacagacaagaaaaagaaacaaataatatagactgatag aaaaaaatactgcttactaccgcc ggtataatatatatatatatatatatttacatagatgattgcatagtgttttaaaaagct ttcctaggttaagctatgaatcttcataaccta accaactaaatatgaaaatactgacccatcgtcttaagtaagttgacatgaactcagcct ggtcacctactatacatgatgtatcgca tggatggaaagaataccaaacgctaccttccaggttaatgatagtatccaaacctagttg gaatttgccttgaacatcaagcagcg attcgatatcagttgggagcatcaatttggtcattggaataccatctatgcttttctcct cccatattcgcaaaagtagtaagggctcgt tatatacttttgaatatgtaagatataattctatatgatttagtaatttattttctatac gctcagtatttttctgcagttgtcgagtaggtatta aacgcaaaagaagtccatccttttcatcattcaaatggacatcttggcaaagggcccagt tatggaaaatctgggagtcatacaac gattgcagttggctatgccactcctggtaaggaatcatcaagtctgataattctgttttt tagccctttttttttttttttcatggtgttctcttc tcattgcttttcaattttaagttcgttacctttcatatagagtttcttaacagaaatttc acaacgaaaatataattaactacaggca

[0167] S. cerevisiae FPS 1 (amino acid; SEQ ID N0:2):

Pyruvate Formate Lyase (PFL)

[0168] The conversion of the pyruvate to acetyl-CoA and formate is performed by pyruvate formate lyase (PFL). In E. coli, PFL is the primary enzyme responsible for the production of formate. PFL is a dimer of PflB that requires the activating enzyme PflAE, which is encoded by pflA, radical S-adenosylmethionine, and a single electron donor. See Waks, Z., and Silver, P. A., Appl. Env. Microbiol. 75: 1867-1875 (2009). Waks and Silver engineered strains of S. cerevisiae to secrete formate by the addition of PFL and AdhE from E. coli and deletion of endogenous formate dehydrogenases and to produce hydrogen in a two-step process using E. coli. Waks and Silver, however, did not combine formate production with the removal of glycerol formation, and the use of formate as an alternate electron acceptor for the reduction of glycerol was not proposed or evaluated.

[0169] PFL enzymes for use in the recombinant host cells of the invention can come from a bacterial or eukaryotic source. Examples of bacterial PFL include, but are not limited to, Bacillus licheniformis DSM13, Bacillus licheniformis ATCC 14580, Streptococcus thermophilus CNRZ1066, Streptococcus thermophilus LMG18311, Streptococcus thermophilus LMD-9, Lactobacillus plantarum WCFS1 (Gene Accession No. lp_2598), Lactobacillus plantarum WCFS1 (Gene Accession No. lp_3313), Lactobacillus plantarum JDMl (Gene Accession No. JDM1_2695), Lactobacillus plantarum JDMl (Gene Accession No. JDM1_2087), Lactobacillus casei bl23, Lactobacillus casei ATCC 334, Bifidobacterium adolescentis, Bifidobacterium longum NCC2705, Bifidobacterium longum DJO10A, Bifidobacterium animalis DSM 10140, Clostridium cellulolyticum, or Escherichia coli. Additional PFL enzymes may be from the PFLl family, the RNR pfl superfamily, or the PFL2 superfamily.

[0170] pfl A sequences from bacteria include:

[0171] Bacillus licheniformis DSM13 (nucleotide; SEQ ID NO:3):

[0172] Bacillus licheniformis DSM13 (amino acid; SEQ ID NO:4):

[0173] Bacillus licheniformis ATCC14580 (nucleotide; SEQ ID NO:5):

[0174] Bacillus licheniformis ATCC14580 (amino acid; SEQ ID NO:6):

[0175] Streptococcus thermophilus CNRZ1066 (nucleotide; SEQ ID NO:7):

[0176] Streptococcus thermophilus CNRZ1066 (amino acid; SEQ ID NO:8):

[0177] Streptococcus thermophilus LMG1831 1 (nucleotide; SEQ ID NO:9):

[0178] Streptococcus thermophilus LMG18311 (amino acid; SEQ ID NO: 10):

[0179] Streptococcus thermophilus LMD-9 (nucleotide; SEQ ID NO: 1 1):

[0180] Streptococcus thermophilus LMD-9 (amino acid; SEQ ID NO: 12):

[0181] Lactobacillus plantarum WCFSl (Gene Accession No: lp_2596) (nucleotide; SEQ ID NO: 13):

[0182] Lactobacillus plantarum WCFSl (Gene Accession No: lp_2596) (amino acid; SEQ ID NO: 14):

[0183] Lactobacillus plantarum WCFSl (Gene Accession No: lp_3314)

(nucleotide; SEQ ID NO: 15): [0184] Lactobacillus plantarum WCFSl (Gene Accession No: lp_3314) (amino acid; SEQ ID NO: 16):

[0185] Lactobacillus plantarum JDMl (Gene Accession No: JDMl 2660)

(nucleotide; SEQ ID NO: 17):

[0186] Lactobacillus plantarum JDMl (Gene Accession No: JDM1 2660) (amino acid; SEQ ID NO: 18):

[0187] Lactobacillus plantarum JDMl (Gene Accession No: JDM1 2085)

(nucleotide; SEQ ID NO: 19):

[0188] Lactobacillus plantarum JDMl (Gene Accession No: JDM1 2085) (amino acid; SEQ ID NO:20):

[0189] Lactobacillus casei bl23 (nucleotide; SEQ ID NO:21):

[0190] Lactobacillus casei bl23 (amino acid; SEQ ID NO:22):

[0191] Lactobacillus casei ATCC 334 (nucleotide; SEQ ID NO:23):

[0192] Lactobacillus casei ATCC 334 (amino acid; SEQ ID NO:24):

[0193] Bifidobacterium adolescentis (nucleotide; SEQ ID NO:25):

[0194] Bifidobacterium adolescentis (amino acid; SEQ ID NO:26):

[0195] Bifidobacterium longum NCC2705 (nucleotide; SEQ ID NO:27):

[0196] Bifidobacterium longum NCC2705 (amino acid; SEQ ID NO:28): [0197] Bifidobacterium longum DJO10A (nucleotide; SEQ ID NO:29):

[0198] Bifidobacterium longum DJO10A (amino acid; SEQ ID NO: 30):

[0199] Bifidobacterium animalis DSM 10140 (nucleotide; SEQ ID NO:31):

[0200] Bifidobacterium animalis DSM 10140 (amino acid; SEQ ID NO:32):

[0201] Clostridium cellulolyticum (nucleotide; SEQ ID NO:33):

[0202] Clostridium cellulolyticum (amino acid; SEQ ID NO:34):

[0203] Escherichia coli (nucleotide; SEQ ID NO:35):

[0204] Escherichia coli (amino acid; SEQ ID NO: 36):

[0205] pflB sequences from bacteria include:

[0206] Bacillus licheniformis DSM 13 (nucleotide; SEQ ID NO:37):

[0207] Bacillus licheniformis DSM 13 (amino acid; SEQ ID NO :38) :

[0208] Bacillus licheniformis ATCC14580 (nucleotide; SEQ ID NO:39):

[0209] Bacillus licheniformis ATCC14580 (amino acid; SEQ ID NO:40):

[0210] Streptococcus thermophilus CNRZ1066 (nucleotide; SEQ ID NO:41):

[0211] Streptococcus thermophilus CNRZ1066 (amino acid; SEQ ID NO:42):

[0212] Streptococcus thermophilus LMG18311 (nucleotide; SEQ ID NO:43): [0213] Streptococcus thermophilus LMG18311 (amino acid; SEQ ID NO:44):

[0214] Streptococcus thermophilus LMD-9 (nucleotide; SEQ ID NO:45):

[0215] Streptococcus thermophilus LMD-9 (amino acid; SEQ ID NO:46):

[0216] Lactobacillus plantarum WCFSl (Gene Accession No. lp_2598)

(nucleotide; SEQ ID NO:47):

[0217] Lactobacillus plantarum WCFSl (Gene Accession No. lp_2598) (amino acid; SEQ ID NO:48):

[0218] Lactobacillus plantarum WCFSl (Gene Accession No: lp_3313)

(nucleotide; SEQ ID NO:49):

[0219] Lactobacillus plantarum WCFSl (Gene Accession No: lp_3313) (amino acid; SEQ ID NO: 50):

[0220] Lactobacillus plantarum JDMl (Gene Accession No: JDM1 2695)

(nucleotide; SEQ ID NO:51):

[0221] Lactobacillus plantarum JDMl (Gene Accession No: JDM1 2695) (amino acid; SEQ ID NO:52):

[0222] Lactobacillus plantarum JDMl (Gene Accession No: JDM1_2087)

(nucleotide; SEQ ID NO:53):

[0223] Lactobacillus plantarum JDMl (Gene Accession No: JDM1 2087) (amino acid; SEQ ID NO:54):

[0224] Lactobacillus casei bl23 (nucleotide; SEQ ID NO:55): [0225] Lactobacillus casei bl23 (amino acid; SEQ ID NO: 56):

[0226] Lactobacillus casei ATCC 334 (nucleotide; SEQ ID NO:57):

[0227] Lactobacillus casei ATCC 334 (amino acid; SEQ ID NO:58):

[0228] Bifidobacterium adolescentis (nucleotide; SEQ ID NO: 59):

[0229] Bifidobacterium adolescentis (amino acid; SEQ ID NO: 60):

[0230] Bifidobacterium longum NCC2705 (nucleotide; SEQ ID NO:61):

[0231] Bifidobacterium longum NCC2705 (amino acid; SEQ ID NO:62):

[0232] Bifidobacterium longum DJO10A (nucleotide; SEQ ID NO:63):

[0233] Bifidobacterium longum DJO10A (amino acid; SEQ ID NO:64):

[0234] Bifidobacterium animalis DSM 10140 (nucleotide; SEQ ID NO:65):

[0235] Bifidobacterium animalis DSM 10140 (amino acid; SEQ ID NO:66):

[0236] Clostridium cellulolyticum (nucleotide; SEQ ID NO:67):

[0237] Clostridium cellulolyticum (amino acid; SEQ ID NO:68):

[0238] Escherichia coli (nucleotide; SEQ ID NO:69):

[0239] Escherichia coli (amino acid; SEQ ID NO:70):

[0240] Examples of eukaryotic PFL include, but are not limited to, Chlamydomonas reinhardtii PflAl, Piromyces sp. E2, or Neocallimastix frontalis, Acetabularia acetabulum, Haematococcus pluvialis, Volvox carteri, Ostreococcus tauri, Ostreococcus lucimarinus, Micromonas pusilla, Micromonas sp., Porphyra haitanensis, and Cyanophora paradoxa), an opisthokont (Amoebidium parasiticum), an amoebozoan (Mastigamoeba balamuthi), a stramenopile (Thalassiosira pseudonana (2)) and a haptophyte (Prymnesium parvum), M. pusilla, Micromonas sp. O. tauri and O. lucimarinus) an amoebozoan (M. balamuthi), and a stramenopile (T. pseudonana). See Stairs, C.W., et al, "Eukaryotic pyruvate formate lyase and its activating enzyme were acquired laterally from a firmicute," Mol. Biol, and Evol., published on-line on Feb. 3, 2011, at http://mbe.oxfordjournals.org/.

[0241] pflA sequences from eukaryotes include:

[0242] Chlamydomonas reinhardtii PflAl (nucleotide; SEQ ID NO:71):

[0243] Chlamydomonas reinhardtii PflAl (amino acid; SEQ ID NO:72):

[0244] Neocallimastix frontalis (nucleotide; SEQ ID NO:73):

[0245] Neocallimastix frontalis (amino acid; SEQ ID NO: 74):

[0246] pfll sequences from eukaryotes include:

[0247] Chlamydomonas reinhardtii PflA (nucleotide; SEQ ID NO:75):

[0248] Chlamydomonas reinhardtii PflA (amino acid; SEQ ID NO: 76):

[0249] Piromyces sp. E2 (nucleotide; SEQ ID NO:77):

[0250] Piromyces sp. E2 (amino acid; SEQ ID NO:78):

[0251] Neocallimastix frontalis (nucleotide - partial CDS, missing start; SEQ ID

NO:79): [0252] Neocallimastix frontalis (amino acid - partial CDS, missing start; SEQ ID

NO:80):

Acetaldehyde/ Alcohol Dehydrogenases

[0253] Engineering of acetaldehyde dehydrogenases, alcohol dehydrogenases, and/or bifunctional acetylaldehyde/alcohol dehydrogenases into a cell can increase the production of ethanol. However, because the production of ethanol is redox neutral, an acetaldehyde/alcohol dehydrogenase activity cannot serve as an alternative for the redox balancing that the production of glycerol provides to a cell in anaerobic metabolism. When Medina attempted to express an acetylaldehyde dehydrogenase, mhpF, from E. coli in an S. cerevisiae gpdl/2 deletion strain, the strain did not grow under anaerobic conditions in the presence of glucose as the sole source of carbon. Medina, V.G., et ah, Appl. Env. Microbiol. 7(5: 190-195 (2010); see also EP 2277989. Rather, the anaerobic growth of the glycerol-deletion strain required the presence of acetic acid. However, an acetylaldehyde dehydrogenase has not been expressed in combination with PFL or with the recombinant host cells of the invention. Additionally, replacing the endogenous acetylaldehyde dehydrogenase activity with either an improved acetaldehyde dehydrogenase or using a bifunctional acetaldehyde/alcohol dehydrogenase (AADH) can positively affect the in vivo kinetics of the reaction providing for improved growth of the host strain.

[0254] AADH enzymes for use in the recombinant host cells of the invention can come from a bacterial or eukaryotic source. Examples of bacterial AADH include, but are not limited to, Clostridium phytofermentans, Escherichia coli, Bacillus coagulans, Bacillus lentus, Bacillus licheniformis, Bacillus pumilus, Bacillus subtilis, Bacteroides amylophilus, Bacteroides capillosus, Bacteroides ruminocola, Bacteroides suis, Bifidobacterium adolescentis, Bifidobacterium animalis, Bifidobacterium bifidum, Bifidobacterium infantis, Bifidobacterium longum, Bifidobacterium thermophilum, Lactobacillus acidophilus, Lactobacillus brevis, Lactobacillus buchneri (cattle only), Lactobacillus bulgaricus, Lactobacillus casei, Lactobacillus cellobiosus, Lactobacillus curvatus, Lactobacillus delbruekii, Lactobacillus farciminis (swine only), Lactobacillus fermentum, Lactobacillus helveticus, Lactobacillus lactis, Lactobacillus plantarum, Lactobacillus reuterii, Leuconostoc mesenteroides, Pediococcus acidilacticii, Pediococcus pentosaceus, Propionibacterium acidpropionici (cattle only), Propionibacterium freudenreichii, Propionibacterium shermanii, Enterococcus cremoris, Enterococcus diacetylactis, Enterococcus faecium, Enterococcus intermedius, Enterococcus lactis, or Enterococcus thermophilus

[0255] AdhE bacterial sequences include:

[0256] Clostridium phytofermentans (nucleotide; SEQ ID NO:81):

[0257] Clostridium phytofermentans (amino acid; SEQ ID NO: 82):

[0258] Escherichia coli (nucleotide; SEQ ID NO:83):

[0259] Escherichia coli (amino acid; SEQ ID NO: 84):

[0260] Bifidobacterium adolescentis (amino acid; SEQ ID NO: 100):

[0261] Bacillus coagulans (amino acid; SEQ ID NO: 101):

[0262] Bacillus licheniformis (amino acid; SEQ ID NO: 102):

[0263] Enterococcus faecium TX1330 (amino acid; SEQ ID NO:103):

[0264] Examples of eukaryotic AdhE include, but are not limited to, Chlamydomonas reinhardtii AdhE, Piromyces sp. E2, or Neocallimastix frontalis.

[0265] AdhE sequences from eukaryotes include:

[0266] Chlamydomonas reinhardtii AdhE (nucleotide; SEQ ID NO:85):

[0267] Chlamydomonas reinhardtii AdhE (amino acid; SEQ ID NO: 86):

[0268] Piromyces sp. E2 (nucleotide; SEQ ID NO:87): [0269] Piromyces sp. E2 (amino acid; SEQ ID NO: 88):

Consolidated Bioprocessing

[0270] Consolidated bioprocessing (CBP) is a processing strategy for cellulosic biomass that involves consolidating into a single process step four biologically-mediated events: enzyme production, hydrolysis, hexose fermentation, and pentose fermentation. Implementing this strategy requires development of microorganisms that both utilize cellulose, hemicellulosics, and other biomass components while also producing a product of interest at sufficiently high yield and concentrations. The feasibility of CBP is supported by kinetic and bioenergetic analysis. See van Walsum and Lynd (1998) Biotech. Bioeng. 55:316.

[0271] CBP offers the potential for lower cost and higher efficiency than processes featuring dedicated saccharolytic enzyme production. The benefits result in part from avoided capital costs, substrate and other raw materials, and utilities associated with saccharolytic enzyme production. In addition, several factors support the realization of higher rates of hydrolysis, and hence reduced reactor volume and capital investment using CBP, including enzyme -microbe synergy and the use of thermophilic organisms and/or complexed saccharolytic systems. Moreover, cellulose-adherent cellulolytic microorganisms are likely to compete successfully for products of cellulose hydrolysis with non-adhered microbes, e.g., contaminants, which could increase the stability of industrial processes based on microbial cellulose utilization. Progress in developing CBP-enabling microorganisms is being made through two strategies: engineering naturally occurring saccharolytic microorganisms to improve product-related properties, such as yield and titer; and engineering non-saccharolytic organisms that exhibit high product yields and titers to express a heterologous saccharolytic enzyme system enabling starch, cellulose, and, hemicellulose utilization.

Starch and Cellulose Degradation

[0272] The degradation of starch into component sugar units proceeds via amylolytic enzymes. Amylase is an example of an amylolytic enzyme that is present in human saliva, where it begins the chemical process of digestion. The pancreas also makes amylase (alpha amylase) to hydrolyze dietary starch into disaccharides and trisaccharides which are converted by other enzymes to glucose to supply the body with energy. Plants and some bacteria also produce amylases. Amylases are glycoside hydrolases and act on a-l,4-glycosidic bonds.

[0273] Several amylolytic enzymes are implicated in starch hydrolysis. Alpha-amylases

(EC 3.2.1.1) (alternate names: 1 ,4-a-D-glucan glucanohydrolase; glycogenase) are calcium metalloenzymes, i.e., completely unable to function in the absence of calcium. By acting at random locations along the starch chain, alpha-amylase breaks down long- chain carbohydrates, ultimately yielding maltotriose and maltose from amylose, or maltose, glucose and "limit dextrin" from amylopectin. Because it can act anywhere on the substrate, alpha-amylase tends to be faster-acting than beta-amylase. Another form of amylase, beta-amylase (EC 3.2.1.2) (alternate names: 1 ,4-a-D-glucan maltohydrolase; glycogenase; saccharogen amylase) catalyzes the hydrolysis of the second a- 1,4 glycosidic bond, cleaving off two glucose units (maltose) at a time. The third amylase is gamma-amylase (EC 3.2.1.3) (alternate names: Glucan 1 ,4-a-glucosidase; amyloglucosidase; Exo-l,4-a-glucosidase; glucoamylase; lysosomal a-glucosidase; 1,4-a- D-glucan glucohydrolase). In addition to cleaving the last a(l-4)glycosidic linkages at the nonreducing end of amylose and amylopectin, yielding glucose, gamma-amylase will cleave a(l-6) glycosidic linkages.

[0274] A fourth enzyme, alpha-glucosidase, acts on maltose and other short malto- oligosaccharides produced by alpha-, beta-, and gamma-amylases, converting them to glucose.

[0275] Three major types of enzymatic activities degrade native cellulose. The first type is endoglucanases (l,4-P-D-glucan 4-glucanohydrolases; EC 3.2.1.4). Endoglucanases cut at random in the cellulose polysaccharide chain of amorphous cellulose, generating oligosaccharides of varying lengths and consequently new chain ends. The second type are exoglucanases, including cellodextrinases (l,4-P-D-glucan glucanohydrolases; EC 3.2.1.74) and cellobiohydrolases (l,4-P-D-glucan cellobiohydrolases; EC 3.2.1.91). Exoglucanases act in a processive manner on the reducing or non-reducing ends of cellulose polysaccharide chains, liberating either glucose (glucanohydrolases) or cellobiose (cellobiohydrolase) as major products. Exoglucanases can also act on microcrystallme cellulose, presumably peeling cellulose chains from the microcrystallme structure. The third type are β-glucosidases (β-glucoside glucohydrolases; EC 3.2.1.21). β-Glucosidases hydrolyze soluble cellodextrins and cellobiose to glucose units. [0276] Even though yeast strains expressing enzymes for the production of fuel ethanol from whole grain or starch have been previously disclosed, the application has not been commercialized in the grain-based fuel ethanol industry, due to the relatively poor ability of the resulting strains to produce/tolerate high levels of ethanol. For example, U.S. Pat. No. 7,226,776 discloses that a polysaccharase enzyme expressing ethanologen can make ethanol directly from carbohydrate polymers, but the maximal ethanol titer demonstrated is 3.9 g/1. U.S. Pat. No. 5,422,267 discloses the use of a glucoamylase in yeast for production of alcoholic beverages; however, no commercially relevant titers of ethanol are disclosed.

Heterologous Saccharolytic Enzymes

[0277] According to one aspect of the present invention, the expression of heterologous saccharolytic enzymes the recombinant microorganisms of the invention can be used advantageously to produce products such as ethanol from biomass sources. For example, cellulases from a variety of sources can be heterologously expressed to successfully increase efficiency of ethanol production. The saccharolytic enzymes can be from fungi, yeast, bacteria, plant, protozoan or termite sources. In some embodiments, the saccharolytic enzyme is from H. grisea, T. aurantiacus, T. emersonii, T. reesei, C. lacteus, C. formosanus, N. takasagoensis, C. acinaciformis, M. darwinensis, N. walkeri, S. fibuligera, C. luckowense R. speratus, Thermobfida fusca, Clostridum thermocellum, Clostridium cellulolyticum, Clostridum josui, Bacillus pumilis, Cellulomonas fimi, Saccharophagus degradans, Piromyces equii, Neocallimastix patricarum or Arabidopsis thaliana.

[0278] In some embodiments, the cellulase for expression in the recombinant microorganisms of the invention is any cellulase disclosed in Table 4 or Table 7 in copending International Appl. No. PCT/US2011/039192, incorporated by reference herein, or any cellulase suitable for expression in an appropriate host cell. In other embodiments, the amylase for expression in the recombinant microorganisms of the invention is any amylase such as alpha-amylases, beta-amylases, glucoamylases, alpha- glucosidases, pullulanase, or isopullulanase paralogues or orthologues, any amylase disclosed in Tables 15-19, preferably in Table 19, in copending International Appl. No. PCT/US2011/039192, incorporated by reference herein, or any amylase suitable for expression in an appropriate host cell. In some embodiments of the invention, multiple saccharolytic enzymes from a single organism are co-expressed in the same recombinant microorganism. In some embodiments of the invention, multiple saccharolytic enzymes from different organisms are co-expressed in the same recombinant microorganism. In particular, saccharolytic enzymes from two, three, four, five, six, seven, eight, nine or more organisms can be co-expressed in the same recombinant microorganism. Similarly, the invention can encompass co-cultures of yeast strains, wherein the yeast strains express different saccharolytic enzymes. Co-cultures can include yeast strains expressing heterologous saccharolytic enzymes from the same organisms or from different organisms. Co-cultures can include yeast strains expressing saccharolytic enzymes from two, three, four, five, six, seven, eight, nine or more organisms.

[0279] Lignocellulases for expression in the recombinant microorganisms of the present invention include both endoglucanases and exoglucanases. Other lignocellulases for expression in the recombinant microorganisms of the invention include accesory enzymes which can act on the lignocellulosic material. The lignocellulases can be, for example, endoglucanases, glucosidases, cellobiohydrolases, xylanases, glucanases, xylosidases, xylan esterases, arabinofuranosidases, galactosidases, cellobiose phosphorylases, cellodextrin phosphorylases, mannanases, mannosidases, xyloglucanases, endoxylanases, glucuronidases, acetylxylanesterases, arabinofuranohydrolases, swollenins, glucuronyl esterases, expansins, pectinases, and feruoyl esterases. In some embodiments, the lignocellulases of the invention can be any suitable enzyme for digesting the desired lignocellulosic material.

[0280] In certain embodiments of the invention, the lignocellulase can be an endoglucanase, glucosidase, cellobiohydrolase, xylanase, glucanase, xylosidase, xylan esterase, arabinofuranosidase, galactosidase, cellobiose phosphorylase, cellodextrin phosphorylase, mannanase, mannosidase, xyloglucanase, endoxylanase, glucuronidase, acetylxylanesterase, arabinofuranohydrolase, swollenin, glucuronyl esterase, expansin, pectinase, and feruoyl esterase paralogue or orthologue. In particular embodiments, the lignocellulase is derived from any species named in Tables 4 and 7, in copending International Appl. No. PCT/US2011/039192, incorporated by reference herein.

Xylose metabolism

[0281] Xylose is a five-carbon monosaccharide that can be metabolized into useful products by a variety of organisms. There are two main pathways of xylose metabolism, each unique in the characteristic enzymes they utilize. One pathway is called the "Xylose Reductase-Xylitol Dehydrogenase" or XR-XDH pathway. Xylose reductase (XR) and xylitol dehydrogenase (XDH) are the two main enzymes used in this method of xylose degradation. XR, encoded by the XYL1 gene, is responsible for the reduction of xylose to xylitol and is aided by cofactors NADH or NADPH. Xylitol is then oxidized to xylulose by XDH, which is expressed through the XYL2 gene, and accomplished exclusively with the cofactor NAD ⁺ . Because of the varying cofactors needed in this pathway and the degree to which they are available for usage, an imbalance can result in an overproduction of xylitol byproduct and an inefficient production of desirable ethanol. Varying expression of the XR and XDH enzyme levels have been tested in the laboratory in the attempt to optimize the efficiency of the xylose metabolism pathway.

[0282] The other pathway for xylose metabolism is called the "Xylose Isomerase" (XI) pathway. Enzyme XI is responsible for direct conversion of xylose into xylulose, and does not proceed via a xylitol intermediate. Both pathways create xylulose, although the enzymes utilized are different. After production of xylulose both the XR-XDH and XI pathways proceed through the enzyme xylulokinase (XK), encoded on gene XKS1, to further modify xylulose into xylulose-5 -phosphate where it then enters the pentose phosphate pathway for further catabolism.

[0283] Studies on flux through the pentose phosphate pathway during xylose metabolism have revealed that limiting the speed of this step may be beneficial to the efficiency of fermentation to ethanol. Modifications to this flux that may improve ethanol production include a) lowering phosphoglucose isomerase activity, b) deleting the GND1 gene, and c) deleting the ZWF1 gene (Jeppsson et al., Appl. Environ. Microbiol. 68: 1604-09 (2002)). Since the pentose phosphate pathway produces additional NADPH during metabolism, limiting this step will help to correct the already evident imbalance between NAD(P)H and NAD ⁺ cofactors and reduce xylitol byproduct. Another experiment comparing the two xylose metabolizing pathways revealed that the XI pathway was best able to metabolize xylose to produce the greatest ethanol yield, while the XR-XDH pathway reached a much faster rate of ethanol production (Karhumaa et al., Microb Cell Fact. 2007 Feb 5;6:5). See also International Publication No. WO2006/009434, incorporated herein by reference in its entirety. [0284] In some embodiments, the recombinant microorganisms of the invention have the ability to metabolize xylose using one or more of the above enzymes.

Arabinose Metabolism

[0285] Arabinose is a five-carbon monosaccharide that can be metabolized into useful products by a variety of organisms. L- Arabinose residues are found widely distributed among many heteropolysaccharides of different plant tissues, such as arabinans, arabinogalactans, xylans and arabinoxylans. Bacillus species in the soil participate in the early stages of plant material decomposition, and B. subtilis secretes three enzymes, an endo-arabanase and two arabinosidases, capable of releasing arabinosyl oligomers and L- arabinose from plant cell.

[0286] Three pathways for L-arabinose metabolism in microorganisms have been described. Many bacteria, including Escherichia coli, use arabinose isomerase (AraA; E.C. 5.3.1.4), ribulokinase (AraB; E.C. 2.7.1.16), and ribulose phosphate epimerase (AraD; E.C. 5.1.3.4) to sequentially convert L-arabinose to D-xylulose-5 -phosphate through L-ribulose and L-ribulose 5-phosphate. See, e.g., Sa-Nogueira I, et ah, Microbiology 143:957-69 (1997). The D-xylulose-5 -phosphate then enters the pentose phosphate pathway for further catabolism. In the second pathway, L-arabinose is converted to L-2-keto-3-deoxyarabonate (L-KDA) by the consecutive action of enzymes arabinose dehydrogenase (ADH), arabinolactone (AL), and arabinonate dehydratase (AraC). See, e.g., Watanabe, S, et al, J. Biol. Chem. 281: 2612-2623 (2006). L-KDA can be further metabolized in two alternative pathways: 1) L-KDA conversion to 2- ketoglutarate via 2-ketoglutaric semialdehyde (KGSA) by L-KDA dehydratase and KGSA dehydrogenase or 2) L-KDA conversion to pyruvate and glycolaldehyde by L- KDA aldolase. In the third, fungal pathway, L-arabinose is converted to D-xylulose-5- phosphate through L-arabinitol, L-xylulose, and xylitol, by enzymes such as NAD(P)H- dependent aldose reductase (AR), L-arabinitol 4-dehydrogenase (ALDH), L-xylulose reductase (LXR), xylitol dehydrogenase (XylD), and xylulokinase (XylB). These, and additional proteins involved in arabinose metabolism and regulation may be found at http://www.nmpdr.org/FIG/wiki/rest.cgi/NmpdrPlugin/SeedViewe r?page=Subsystems;su bsystem=L-Arabinose_utilization, visited March 21, 2011, which is incorporated by reference herein in its entirety. [0287] AraC protein regulates expression of its own synthesis and the other genes of the

Ara system. See Schleif, R., Trends Genet. i<5(12):559-65 (2000). In the E. coli, the AraC protein positively and negatively regulates expression of the proteins required for the uptake and catabolism of the sugar L-arabinose. Homologs of AraC, such as regulatory proteins RhaR and RhaS of the rhamnose operon, have been identified that contain regions homologous to the DNA-binding domain of AraC (Leal, T.F. and de Sa- Nogueira, I., FEMS Microbiol Lett. 24i(l):41-48 (2004)). Such arabinose regulatory proteins are referred to as the AraC/XylS family. See also, Mota, L.J., et ah, Mol. Microbiol. JJ(3):476-89 (1999); Mota, L.J., et al., J Bacteriol. 7SJ(14):4190-201 (2001).

[0288] In E. coli, the transport of L-arabinose across the E. coli cytoplasmic membrane requires the expression of either the high-affinity transport operon, araFGH, a binding protein-dependent system on the low-affinity transport operon, araE, a proton symporter. Additional arabinose transporters include those identified from K. marxianus and P. guilliermondii, disclosed in U.S. Patent No. 7,846,712, which is incorporated by reference herein.

[0289] In some embodiments, the recombinant microorganisms of the invention have the ability to metabolize arabinose using one or more of the above enzymes.

Microorganisms

[0290] The present invention includes multiple strategies for the development of microorganisms with the combination of substrate-utilization and product-formation properties required for CBP. The "native cellulo lytic strategy" involves engineering naturally occurring cellulolytic microorganisms to improve product-related properties, such as yield and titer. The "recombinant cellulolytic strategy" involves engineering natively non-cellulolytic organisms that exhibit high product yields and titers to express a heterologous cellulase system that enables cellulose utilization or hemicellulose utilization or both.

[0291] Many bacteria have the ability to ferment simple hexose sugars into a mixture of acidic and pH-neutral products via the process of glycolysis. The glycolytic pathway is abundant and comprises a series of enzymatic steps whereby a six carbon glucose molecule is broken down, via multiple intermediates, into two molecules of the three carbon compound pyruvate. This process results in the net generation of ATP (biological energy supply) and the reduced cofactor NADH. [0292] Pyruvate is an important intermediary compound of metabolism. For example, under aerobic conditions pyruvate may be oxidized to acetyl coenzyme A (acetyl-CoA), which then enters the tricarboxylic acid cycle (TCA), which in turn generates synthetic precursors, C0 ₂ , and reduced cofactors. The cofactors are then oxidized by donating hydrogen equivalents, via a series of enzymatic steps, to oxygen resulting in the formation of water and ATP. This process of energy formation is known as oxidative phosphorylation.

[0293] Under anaerobic conditions (no available oxygen), fermentation occurs in which the degradation products of organic compounds serve as hydrogen donors and acceptors. Excess NADH from glycolysis is oxidized in reactions involving the reduction of organic substrates to products, such as lactate and ethanol. In addition, ATP is regenerated from the production of organic acids, such as acetate, in a process known as substrate level phosphorylation. Therefore, the fermentation products of glycolysis and pyruvate metabolism include a variety of organic acids, alcohols and C0 ₂ .

[0294] Most facultative anaerobes metabolize pyruvate aerobically via pyruvate dehydrogenase (PDH) and the tricarboxylic acid cycle (TCA). Under anaerobic conditions, the main energy pathway for the metabolism of pyruvate is via pyruvate - formate-lyase (PFL) pathway to give formate and acetyl-CoA. Acetyl-CoA is then converted to acetate, via phosphotransacetylase (PTA) and acetate kinase (ACK) with the co-production of ATP, or reduced to ethanol via acetalaldehyde dehydrogenase (ACDH) and alcohol dehydrogenase (ADH). In order to maintain a balance of reducing equivalents, excess NADH produced from glycolysis is re -oxidized to NAD ⁺ by lactate dehydrogenase (LDH) during the reduction of pyruvate to lactate. NADH can also be re- oxidized by ACDH and ADH during the reduction of acetyl-CoA to ethanol, but this is a minor reaction in cells with a functional LDH.

Host Cells

[0295] Host cells useful in the present invention include any prokaryotic or eukaryotic cells; for example, microorganisms selected from bacterial, algal, and yeast cells. Among host cells thus suitable for the present invention are microorganisms, for example, of the genera Aeromonas, Aspergillus, Bacillus, Escherichia, Kluyveromyces, Pichia, Rhodococcus, Saccharomyces and Streptomyces. [0296] In some embodiments, the host cells are microorganisms. In one embodiment the microorganism is a yeast. According to the present invention the yeast host cell can be, for example, from the genera Saccharomyces, Kluyveromyces, Candida, Pichia, Schizosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, and Yarrowia. Yeast species as host cells may include, for example, S. cerevisiae, S. bulderi, S. barnetti, S. exiguus, S. uvarum, S. diastaticus, K. lactis, K. marxianus, or K. fragilis. In some embodiments, the yeast is selected from the group consisting of Saccharomyces cerevisiae, Schizzo saccharomyces pombe, Candida albicans, Pichia pastoris, Pichia stipitis, Yarrowia lipolytica, Hansenula polymorpha, Phaffia rhodozyma, Candida utilis, Arxula adeninivorans, Debaryomyces hansenii, Debaryomyces polymorphus, Schizosaccharomyces pombe and Schwanniomyces occidentalis . In one particular embodiment, the yeast is Saccharomyces cerevisiae. In another embodiment, the yeast is a thermotolerant Saccharomyces cerevisiae. The selection of an appropriate host is deemed to be within the scope of those skilled in the art from the teachings herein.

[0297] In some embodiments, the host cell is an oleaginous cell. The oleaginous host cell can be an oleaginous yeast cell. For example, the oleaginous yeast host cell can be from the genera Blakeslea, Candida, Cryptococcus, Cunninghamella, Lipomyces, Mortierella, Mucor, Phycomyces, Pythium, Rhodosporidum, Rhodotorula, Trichosporon or Yarrowia. According to the present invention, the oleaginous host cell can be an oleaginous microalgae host cell. For example, the oleaginous microalgea host cell can be from the genera Thraustochytrium or Schizochytrium. Biodiesel could then be produced from the triglyceride produced by the oleaginous organisms using conventional lipid transesterification processes. In some particular embodiments, the oleaginous host cells can be induced to secrete synthesized lipids. Embodiments using oleaginous host cells are advantegeous because they can produce biodiesel from lignocellulosic feedstocks which, relative to oilseed substrates, are cheaper, can be grown more densely, show lower life cycle carbon dioxide emissions, and can be cultivated on marginal lands.

[0298] In some embodiments, the host cell is a thermotolerant host cell. Thermotolerant host cells can be particularly useful in simultaneous saccharification and fermentation processes by allowing externally produced cellulases and ethanol-producing host cells to perform optimally in similar temperature ranges. [0299] Thermotolerant host cells can include, for example, Issatchenkia orientalis, Pichia mississippiensis, Pichia mexicana, Pichia farinosa, Clavispora opuntiae, Clavispora lusitaniae, Candida mexicana, Hansenula polymorpha and Kluyveromyces host cells. In some embodiments, the thermotolerant cell is an S. cerevisiae strain, or other yeast strain, that has been adapted to grow in high temperatures, for example, by selection for growth at high temperatures in a cytostat.

[0300] In some particular embodiments, the host cell is a Kluyveromyces host cell. For example, the Kluyveromyces host cell can be a K lactis, K marxianus, K blattae, K phqffii, K. yarrowii, K. aestuarii, K dobzhanskii, K wickerhamii K. thermotolerans, or K waltii host cell. In one embodiment, the host cell is a K. lactis, or K marxianus host cell. In another embodiment, the host cell is a K marxianus host cell.

[0301] In some embodiments, the thermotolerant host cell can grow at temperatures above about 30° C, about 31° C, about 32° C, about 33° C, about 34° C, about 35° C, about 36° C, about 37° C, about 38° C, about 39° C, about 40° C, about 41° C or about 42° C. In some embodiments of the present invention the thermotolerant host cell can produce ethanol from cellulose at temperatures above about 30° C, about 31° C, about 32° C, about 33° C, about 34° C, about 35° C, about 36° C, about 37° C, about 38° C, about 39° C, about 40° C, about 41° C, about 42° C, or about 43 °C, or about 44 °C, or about 45 °C, or about 50° C.

[0302] In some embodiments of the present invention, the thermotolerant host cell can grow at temperatures from about 30° C to 60° C, about 30° C to 55° C, about 30° C to 50° C, about 40° C to 60° C, about 40° C to 55° C or about 40° C to 50° C. In some embodiments of the present invention, the thermotolterant host cell can produce ethanol from cellulose at temperatures from about 30° C to 60° C, about 30° C to 55° C, about 30° C to 50° C, about 40° C to 60° C, about 40° C to 55° C or about 40° C to 50° C.

[0303] In some embodiments, the host cell has the ability to metabolize xylose. Detailed information regarding the development of the xylose-utilizing technology can be found in the following publications: Kuyper M et al. FEMS Yeast Res. 4: 655-64 (2004), Kuyper M et al. FEMS Yeast Res. 5:399-409 (2005), and Kuyper M et al. FEMS Yeast Res. 5:925-34 (2005), which are herein incorporated by reference in their entirety. For example, xylose-utilization can be accomplished in S. cerevisiae by heterologously expressing the xylose isomerase gene, XylA, e.g., from the anaerobic fungus Piromyces sp. E2, overexpressing five S. cerevisiae enzymes involved in the conversion of xylulose to glycolytic intermediates (xylulokinase, ribulose 5-phosphate isomerase, ribulose 5- phosphate epimerase, transketolase and transaldolase) and deleting the GRE3 gene encoding aldose reductase to minimise xylitol production.

[0304] In some embodiments, the host cell has the ability to metabolize arabinose. For example, arabinose-utilization can be accomplished by heterologously expressing, e.g., one or more of arabinose isomerase, ribulokinase, or ribulose phosphate epimerase.

[0305] The host cells can contain antibiotic markers or can contain no antibiotic markers.

[0306] In certain embodiments, the host cell is a microorganism that is a species of the genera Thermoanaerobacterium, Thermoanaerobacter, Clostridium, Geobacillus, Saccharococcus, Paenibacillus, Bacillus, Caldicellulosiruptor, Anaerocellum, or Anoxybacillus. In certain embodiments, the host cell is a bacterium selected from the group consisting of: Thermoanaerobacterium thermo sulfur igenes, Thermoanaerobacterium aotearoense, Thermoanaerobacterium polysaccharolyticum, Thermoanaerobacterium zeae, Thermoanaerobacterium xylanolyticum,

Thermoanaerobacterium saccharolyticum, Thermoanaerobium brockii,

Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacter thermohydrosulfuricus, Thermoanaerobacter ethanolicus, Thermoanaerobacter brocki, Clostridium thermocellum, Clostridium cellulolyticum, Clostridium phytofermentans, Clostridium straminosolvens, Geobacillus thermoglucosidasius, Geobacillus stearothermophilus, Saccharococcus caldoxylosilyticus, Saccharoccus thermophilus, Paenibacillus campinasensis, Bacillus flavothermus, Anoxybacillus kamchatkensis, Anoxybacillus gonensis, Caldicellulosiruptor acetigenus, Caldicellulosiruptor saccharolyticus, Caldicellulosiruptor kristjanssonii, Caldicellulosiruptor owensensis, Caldicellulosiruptor lactoaceticus, and Anaerocellum thermophilum. In certain embodiments, the host cell is Clostridium thermocellum, Clostridium cellulolyticum, or Thermoanaerobacterium saccharolyticum.

Codon Optimized Polynucleotides

[0307] The polynucleotides encoding heterologous cellulases can be codon-optimized.

As used herein the term "codon-optimized coding region" means a nucleic acid coding region that has been adapted for expression in the cells of a given organism by replacing at least one, or more than one, or a significant number, of codons with one or more codons that are more frequently used in the genes of that organism.

[0308] In general, highly expressed genes in an organism are biased towards codons that are recognized by the most abundant tRNA species in that organism. One measure of this bias is the "codon adaptation index" or "CAI," which measures the extent to which the codons used to encode each amino acid in a particular gene are those which occur most frequently in a reference set of highly expressed genes from an organism.

[0309] The CAI of codon optimized sequences of the present invention corresponds to between about 0.8 and 1.0, between about 0.8 and 0.9, or about 1.0. A codon optimized sequence may be further modified for expression in a particular organism, depending on that organism's biological constraints. For example, large runs of "As" or "Ts" (e.g., runs greater than 3, 4, 5, 6, 7, 8, 9, or 10 consecutive bases) can be removed from the sequences if these are known to effect transcription negatively. Furthermore, specific restriction enzyme sites may be removed for molecular cloning purposes. Examples of such restriction enzyme sites include Pad, Ascl, BamHI, Bglll, EcoRI and Xhol. Additionally, the DNA sequence can be checked for direct repeats, inverted repeats and mirror repeats with lengths of ten bases or longer, which can be modified manually by replacing codons with "second best" codons, i.e., codons that occur at the second highest frequency within the particular organism for which the sequence is being optimized.

[0310] Deviations in the nucleotide sequence that comprise the codons encoding the amino acids of any polypeptide chain allow for variations in the sequence coding for the gene. Since each codon consists of three nucleotides, and the nucleotides comprising DNA are restricted to four specific bases, there are 64 possible combinations of nucleotides, 61 of which encode amino acids (the remaining three codons encode signals ending translation). The "genetic code" which shows which codons encode which amino acids is reproduced herein as Table 1. As a result, many amino acids are designated by more than one codon. For example, the amino acids alanine and proline are coded for by four triplets, serine and arginine by six, whereas tryptophan and methionine are coded by just one triplet. This degeneracy allows for DNA base composition to vary over a wide range without altering the amino acid sequence of the proteins encoded by the DNA. TABLE 1 : The Standard Genetic Code

[0311] Many organisms display a bias for use of particular codons to code for insertion of a particular amino acid in a growing peptide chain. Codon preference or codon bias, differences in codon usage between organisms, is afforded by degeneracy of the genetic code, and is well documented among many organisms. Codon bias often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, inter alia, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization.

[0312] Given the large number of gene sequences available for a wide variety of animal, plant and microbial species, it is possible to calculate the relative frequencies of codon usage. Codon usage tables are readily available, for example, at http://www.kazusa.or.jp/codon/ (visited February 28, 201 1), and these tables can be adapted in a number of ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000," Nucl. Acids Res. 28:292 (2000). Codon usage tables for yeast, calculated from GenBank Release 128.0 [15 February 2002], are reproduced below as Table 2. This table uses mRNA nomenclature, and so instead of thymine (T) which is found in DNA, the tables use uracil (U) which is found in RNA. The table has been adapted so that frequencies are calculated for each amino acid, rather than for all 64 codons.

TABLE 2: Codon Usage Table for Saccharomyces cerevisiae Genes

Amino Acid Codon Number Frequency per

hundred

Phe UUU 170666 26.1

Phe UUC 120510 18.4

Leu UUA 170884 26.2

Leu UUG 177573 27.2

Leu CUU 80076 12.3

Leu CUC 35545 5.4

Leu CUA 87619 13.4

Leu CUG 68494 10.5

He AUU 196893 30.1

He AUC 112176 17.2

He AUA 116254 17.8

Met AUG 136805 20.9

Val GUU 144243 22.1

Val GUC 76947 11.8

Val GUA 76927 11.8

Val GUG 70337 10.8

Ser UCU 153557 23.5

Ser UCC 92923 14.2

Ser UCA 122028 18.7

Ser UCG 55951 8.6

Ser AGU 92466 14.2

Ser AGC 63726 9.8

Pro ecu 88263 13.5

Pro CCC 44309 6.8

Pro CCA 119641 18.3

[0313] By utilizing this or similar tables, one of ordinary skill in the art can apply the frequencies to any given polypeptide sequence, and produce a nucleic acid fragment of a codon-optimized coding region which encodes the polypeptide, but which uses codons optimal for a given species. Codon-optimized coding regions can be designed by various different methods.

[0314] In one method, a codon usage table is used to find the single most frequent codon used for any given amino acid, and that codon is used each time that particular amino acid appears in the polypeptide sequence. For example, referring to Table 2 above, for leucine, the most frequent codon is UUG, which is used 27.2% of the time. Thus all the leucine residues in a given amino acid sequence would be assigned the codon UUG.

[0315] In another method, the actual frequencies of the codons are distributed randomly throughout the coding sequence. Thus, using this method for optimization, if a hypothetical polypeptide sequence had 100 leucine residues, referring to Table 2 for frequency of usage in the S. cerevisiae, about 5, or 5% of the leucine codons would be CUC, about 11, or 11% of the leucine codons would be CUG, about 12, or 12% of the leucine codons would be CUU, about 13, or 13% of the leucine codons would be CUA, about 26, or 26% of the leucine codons would be UUA, and about 27, or 27% of the leucine codons would be UUG.

[0316] These frequencies would be distributed randomly throughout the leucine codons in the coding region encoding the hypothetical polypeptide. As will be understood by those of ordinary skill in the art, the distribution of codons in the sequence can vary significantly using this method; however, the sequence always encodes the same polypeptide.

[0317] When using the methods above, the term "about" is used precisely to account for fractional percentages of codon frequencies for a given amino acid. As used herein, "about" is defined as one amino acid more or one amino acid less than the value given. The whole number value of amino acids is rounded up if the fractional frequency of usage is 0.50 or greater, and is rounded down if the fractional frequency of use is 0.49 or less. Using again the example of the frequency of usage of leucine in human genes for a hypothetical polypeptide having 62 leucine residues, the fractional frequency of codon usage would be calculated by multiplying 62 by the frequencies for the various codons. Thus, 7.28 percent of 62 equals 4.51 UUA codons, or "about 5," i.e., 4, 5, or 6 UUA codons, 12.66 percent of 62 equals 7.85 UUG codons or "about 8," i.e., 7, 8, or 9 UUG codons, 12.87 percent of 62 equals 7.98 CUU codons, or "about 8," i.e., 7, 8, or 9 CUU codons, 19.56 percent of 62 equals 12.13 CUC codons or "about 12," i.e., 11, 12, or 13 CUC codons, 7.00 percent of 62 equals 4.34 CUA codons or "about 4," i.e., 3, 4, or 5 CUA codons, and 40.62 percent of 62 equals 25.19 CUG codons, or "about 25," i.e., 24, 25, or 26 CUG codons.

[0318] Randomly assigning codons at an optimized frequency to encode a given polypeptide sequence, can be done manually by calculating codon frequencies for each amino acid, and then assigning the codons to the polypeptide sequence randomly. Additionally, various algorithms and computer software programs are readily available to those of ordinary skill in the art. For example, the "EditSeq" function in the Lasergene Package, available from DNAstar, Inc., Madison, WI, the backtranslation function in the VectorNTI Suite, available from InforMax, Inc., Bethesda, MD, and the "backtranslate" function in the GCG~Wisconsin Package, available from Accelrys, Inc., San Diego, CA. In addition, various resources are publicly available to codon-optimize coding region sequences, e.g., the "backtranslation" function at www.entelechon.com/2008/10/backtranslation-tool/ (visited February 28, 2011) and the "backtranseq" function available at emboss.bioinformatics.nl/cgi-bin emboss/backtranseq (visited February 28, 2011). Constructing a rudimentary algorithm to assign codons based on a given frequency can also easily be accomplished with basic mathematical functions by one of ordinary skill in the art. [0319] A number of options are available for synthesizing codon optimized coding regions designed by any of the methods described above, using standard and routine molecular biological manipulations well known to those of ordinary skill in the art. In one approach, a series of complementary oligonucleotide pairs of 80-90 nucleotides each in length and spanning the length of the desired sequence is synthesized by standard methods. These oligonucleotide pairs are synthesized such that upon annealing, they form double stranded fragments of 80-90 base pairs, containing cohesive ends, e.g., each oligonucleotide in the pair is synthesized to extend 3, 4, 5, 6, 7, 8, 9, 10, or more bases beyond the region that is complementary to the other oligonucleotide in the pair. The single-stranded ends of each pair of oligonucleotides is designed to anneal with the single-stranded end of another pair of oligonucleotides. The oligonucleotide pairs are allowed to anneal, and approximately five to six of these double-stranded fragments are then allowed to anneal together via the cohesive single stranded ends, and then they ligated together and cloned into a standard bacterial cloning vector, for example, a TOPO ^® vector available from Invitrogen Corporation, Carlsbad, CA. The construct is then sequenced by standard methods. Several of these constructs consisting of 5 to 6 fragments of 80 to 90 base pair fragments ligated together, i.e., fragments of about 500 base pairs, are prepared, such that the entire desired sequence is represented in a series of plasmid constructs. The inserts of these plasmids are then cut with appropriate restriction enzymes and ligated together to form the final construct. The final construct is then cloned into a standard bacterial cloning vector, and sequenced. Additional methods would be immediately apparent to the skilled artisan. In addition, gene synthesis is readily available commercially.

[0320] In additional embodiments, a full-length polypeptide sequence is codon-optimized for a given species resulting in a codon-optimized coding region encoding the entire polypeptide, and then nucleic acid fragments of the codon-optimized coding region, which encode fragments, variants, and derivatives of the polypeptide are made from the original codon-optimized coding region. As would be well understood by those of ordinary skill in the art, if codons have been randomly assigned to the full-length coding region based on their frequency of use in a given species, nucleic acid fragments encoding fragments, variants, and derivatives would not necessarily be fully codon optimized for the given species. However, such sequences are still much closer to the codon usage of the desired species than the native codon usage. The advantage of this approach is that synthesizing codon-optimized nucleic acid fragments encoding each fragment, variant, and derivative of a given polypeptide, although routine, would be time consuming and would result in significant expense.

Transposons

[0321] To select for foreign DNA that has entered a host it is preferable that the DNA be stably maintained in the organism of interest. With regard to plasmids, there are two processes by which this can occur. One is through the use of replicative plasmids. These plasmids have origins of replication that are recognized by the host and allow the plasmids to replicate as stable, autonomous, extrachromosomal elements that are partitioned during cell division into daughter cells. The second process occurs through the integration of a plasmid onto the chromosome. This predominately happens by homologous recombination and results in the insertion of the entire plasmid, or parts of the plasmid, into the host chromosome. Thus, the plasmid and selectable marker(s) are replicated as an integral piece of the chromosome and segregated into daughter cells. Therefore, to ascertain if plasmid DNA is entering a cell during a transformation event through the use of selectable markers requires the use of a replicative plasmid or the ability to recombine the plasmid onto the chromosome. These qualifiers cannot always be met, especially when handling organisms that do not have a suite of genetic tools.

[0322] One way to avoid issues regarding plasmid-associated markers is through the use of transposons. A transposon is a mobile DNA element, defined by mosaic DNA sequences that are recognized by enzymatic machinery referred to as a transposase. The function of the transposase is to randomly insert the transposon DNA into host or target DNA. A selectable marker can be cloned onto a transposon by standard genetic engineering. The resulting DNA fragment can be coupled to the transposase machinery in an in vitro reaction and the complex can be introduced into target cells by electroporation. Stable insertion of the marker onto the chromosome requires only the function of the transposase machinery and alleviates the need for homologous recombination or replicative plasmids.

[0323] The random nature associated with the integration of transposons has the added advantage of acting as a form of mutagenesis. Libraries can be created that comprise amalgamations of transposon mutants. These libraries can be used in screens or selections to produce mutants with desired phenotypes. For instance, a transposon library of a CBP organism could be screened for the ability to produce more ethanol, or less lactic acid and/or more acetate.

Native cellulolytic strategy

[0324] Naturally occurring cellulolytic microorganisms are starting points for CBP organism development via the native strategy. Anaerobes and facultative anaerobes are of particular interest. The primary objective is to engineer the metabolization of biomass to solvents, including but not limited to, acetone, isopropanol, ethyl acetate, or ethanol. Metabolic engineering of mixed-acid fermentations in relation to, for example, ethanol production, has been successful in the case of mesophilic, non-cellulolytic, enteric bacteria. Recent developments in suitable gene-transfer techniques allow for this type of work to be undertaken with cellulolytic bacteria.

Recombinant cellulolytic strategy

[0325] Non-cellulolytic microorganisms with desired product-formation properties are starting points for CBP organism development by the recombinant cellulolytic strategy. The primary objective of such developments is to engineer a heterologous cellulase system that enables growth and fermentation on pretreated lignocellulose. The heterologous production of cellulases has been pursued primarily with bacterial hosts producing ethanol at high yield (engineered strains of E. coli, Klebsiella oxytoca, and Zymomonas mobilis) and the yeast Saccharomyces cerevisiae. Cellulase expression in strains of K oxytoca resulted in increased hydrolysis yields - but not growth without added cellulase - for microcrystalline cellulose, and anaerobic growth on amorphous cellulose. Although dozens of saccharolytic enzymes have been functionally expressed in S. cerevisiae, anaerobic growth on cellulose as the result of such expression has not been definitively demonstrated.

[0326] Aspects of the present invention relate to the use of thermophilic or mesophilic microorganisms as hosts for modification via the native cellulolytic strategy. Their potential in process applications in biotechnology stems from their ability to grow at relatively high temperatures with attendant high metabolic rates, production of physically and chemically stable enzymes, and elevated yields of end products. Major groups of thermophilic bacteria include eubacteria and archaebacteria. Thermophilic eubacteria include: phototropic bacteria, such as cyanobacteria, purple bacteria, and green bacteria; Gram-positive bacteria, such as Bacillus, Clostridium, Lactic acid bacteria, and Actinomyces; and other eubacteria, such as Thiobacillus, Spirochete, Desulfotomaculum, Gram-negative aerobes, Gram-negative anaerobes, and Thermotoga. Within archaebacteria are considered Methanogens, extreme thermophiles (an art-recognized term), and Thermoplasma. In certain embodiments, the present invention relates to Gram-negative organotrophic thermophiles of the genera Thermus, Gram-positive eubacteria, such as genera Clostridium, and also which comprise both rods and cocci, genera in group of eubacteria, such as Thermosipho and Thermotoga, genera of Archaebacteria, such as Thermococcus, Thermoproteus (rod-shaped), Thermofilum (rod- shaped), Pyrodictium, Acidianus, Sulfolobus, Pyrobaculum, Pyrococcus, Thermodiscus, Staphylothermus, Desulfurococcus, Archaeoglobus, and Methanopyrus. Some examples of thermophilic or mesophilic (including bacteria, procaryotic microorganism, and fungi), which may be suitable for the present invention include, but are not limited to: Clostridium thermosulfurogenes, Clostridium cellulolyticum, Clostridium thermocellum, Clostridium thermohydrosulfuricum, Clostridium thermoaceticum, Clostridium thermosaccharolyticum, Clostridium tartarivorum, Clostridium thermocellulaseum, Clostridium phytofermentans, Clostridium straminosolvens, Thermoanaerobacterium thermosaccarolyticum, Thermoanaerobacterium saccharolyticum, Thermobacteroides acetoethylicus, Thermoanaerobium brockii, Methanobacterium thermoautotrophicum, Anaerocellum thermophilium, Pyrodictium occultum, Thermoproteus neutrophilus, Thermofilum librum, Thermothrix thioparus, Desulfovibrio thermophilus, Thermoplasma acidophilum, Hydrogenomonas thermophilus, Thermomicrobium roseum, Thermus flavas, Thermus ruber, Pyrococcus furiosus, Thermus aquaticus, Thermus thermophilus, Chloroflexus aurantiacus, Thermococcus litoralis, Pyrodictium abyssi, Bacillus stearothermophilus, Cyanidium caldarium, Mastigocladus laminosus, Chlamydothrix calidissima, Chlamydothrix penicillata, Thiothrix carnea, Phormidium tenuissimum, Phormidium geysericola, Phormidium subterraneum, Phormidium bijahensi, Oscillatoria filiformis, Synechococcus lividus, Chloroflexus aurantiacus, Pyrodictium brockii, Thiobacillus thiooxidans, Sulfolobus acidocaldarius, Thiobacillus thermophilica, Bacillus stearothermophilus, Cercosulcifer hamathensis, Vahlkampfia reichi, Cyclidium citrullus, Dactylaria gallopava, Synechococcus lividus, Synechococcus elongatus, Synechococcus minervae, Synechocystis aquatilus, Aphanocapsa thermalis, Oscillatoria terebriformis, Oscillatoria amphibia, Oscillatoria germinata, Oscillatoria okenii, Phormidium laminosum, Phormidium parparasiens, Symploca thermalis, Bacillus acidocaldarias, Bacillus coagulans, Bacillus thermocatenalatus, Bacillus licheniformis, Bacillus pamilas, Bacillus macerans, Bacillus circulans, Bacillus laterosporus, Bacillus brevis, Bacillus subtilis, Bacillus sphaericus, Desulfotomaculum nigrificans, Streptococcus thermophilus, Lactobacillus thermophilus, Lactobacillus bulgaricus, Bifidobacterium thermophilum, Streptomyces fragmentosporus, Streptomyces thermonitrificans, Streptomyces thermovulgaris, Pseudonocardia thermophila, Thermoactinomyces vulgaris, Thermoactinomyces sacchari, Thermoactinomyces Candidas, Thermomonospora curvata, Thermomonospora viridis, Thermomonospora citrina, Microbispora thermodiastatica, Microbispora aerata, Microbispora bispora, Actinobifida dichotomica, Actinobifida chromogena, Micropolyspora caesia, Micropolyspora fiaeni, Micropolyspora cectivugida, Micropolyspora cabrobrunea, Micropolyspora thermovirida, Micropolyspora viridinigra, Methanobacterium thermoautothropicum, Caldicellulosiruptor acetigenus, Caldicellulosiruptor saccharolyticus, Caldicellulosiruptor kristjanssonii, Caldicellulosiruptor owensensis, Caldicellulosiruptor lactoaceticus, variants thereof, and/or progeny thereof.

[0327] In particular embodiments, the present invention relates to thermophilic bacteria selected from the group consisting of Clostridium cellulolyticum, Clostridium thermocellum, and Thermoanaerobacterium saccharolyticum.

[0328] In certain embodiments, the present invention relates to thermophilic bacteria selected from the group consisting of Fervidobacterium gondwanense, Clostridium thermolacticum, Moorella sp., and Rhodothermus marinus.

[0329] In certain embodiments, the present invention relates to thermophilic bacteria of the genera Thermoanaerobacterium or Thermoanaerobacter, including, but not limited to, species selected from the group consisting of: Thermoanaerobacterium thermo sulfur igenes, Thermoanaerobacterium aotearoense, Thermoanaerobacterium poly saccharolyticum, Thermoanaerobacterium zeae, Thermoanaerobacterium xylanolyticum, Thermoanaerobacterium saccharolyticum, Thermoanaerobium brockii, Thermoanaerobacterium thermosaccharolyticum, Thermoanaerobacter thermohydrosulfuricus, Thermoanaerobacter ethanolicus, Thermoanaerobacter brockii, variants thereof, and progeny thereof.

[0330] In certain embodiments, the present invention relates to microorganisms of the genera Geobacillus, Saccharococcus, Paenibacillus, Bacillus, and Anoxybacillus, including, but not limited to, species selected from the group consisting of: Geobacillus thermoglucosidasius, Geobacillus stearothermophilus, Saccharococcus caldoxylosilyticus, Saccharoccus thermophilus, Paenibacillus campinasensis, Bacillus flavothermus, Anoxybacillus kamchatkensis, Anoxybacillus gonensis, variants thereof, and progeny thereof.

[0331] In certain embodiments, the present invention relates to mesophilic bacteria selected from the group consisting of Saccharophagus degradans; Flavobacterium johnsoniae; Fibrobacter succinogenes; Clostridium hungatei; Clostridium phytofermentans; Clostridium cellulolyticum; Clostridium aldrichii; Clostridium termitididis; Acetivibrio cellulolyticus; Acetivibrio ethanolgignens; Acetivibrio multivorans; Bacteroides cellulosolvens; and Alkalibacter saccharofomentans, variants thereof and progeny thereof.

Organism development via the native cellulolytic strategy

[0332] One approach to organism development for CBP begins with organisms that naturally utilize cellulose, hemicellulose and/or other biomass components, which are then genetically engineered to enhance product yield and tolerance. For example, Clostridium thermocellum is a thermophilic bacterium that has among the highest rates of cellulose utilization reported. Other organisms of interest are xylose-utilizing thermophiles such as Thermoanaerobacterium saccharolyticum and Thermoanaerobacterium thermosaccharolyticum. Organic acid production may be responsible for the low concentrations of produced ethanol generally associated with these organisms. Thus, one objective is to eliminate production of acetic and lactic acid in these organisms via metabolic engineering. Substantial efforts have been devoted to developing gene transfer systems for the above-described target organisms and multiple C. thermocellum isolates from nature have been characterized. See McLaughlin et al. (2002) Environ. Sci. Technol. 36:2122. Metabolic engineering of thermophilic, saccharolytic bacteria is an active area of interest, and knockout of lactate dehydrogenase in T. saccharolyticum has recently been reported. See Desai et al. (2004) Appl. Microbiol. Biotechnol. 65:600. Knockout of acetate kinase and phosphotransacetylase in this organism is also possible.

Organism development via the recombinant cellulolytic strategy

[0333] An alternative approach to organism development for CBP involves conferring the ability to grow on lignocellulosic materials to microorganisms that naturally have high product yield and tolerance via expression of a heterologous cellulasic system and perhaps other features. For example, Saccharomyces cerevisiae has been engineered to express over two dozen different saccharo lytic enzymes. See Lynd et al. (2002) Microbiol. Mol. Biol. Rev. 66:506.

[0334] Whereas cellulosic hydrolysis has been approached in the literature primarily in the context of an enzymatically-oriented intellectual paradigm, the CBP processing strategy requires that cellulosic hydrolysis be viewed in terms of a microbial paradigm. This microbial paradigm naturally leads to an emphasis on different fundamental issues, organisms, cellulasic systems, and applied milestones compared to those of the enzymatic paradigm. In this context, C. thermocellum has been a model organism because of its high growth rate on cellulose together with its potential utility for CBP.

[0335] In certain embodiments, organisms useful in the present invention may be applicable to the process known as simultaneous saccharification and fermentation (SSF), which is intended to include the use of said microorganisms and/or one or more recombinant hosts (or extracts thereof, including purified or unpurified extracts) for the contemporaneous degradation or depolymerization of a complex sugar (i.e., cellulosic biomass) and bioconversion of that sugar residue into ethanol by fermentation.

Ethanol Production

[0336] According to the present invention, a recombinant microorganism can be used to produce ethanol from biomass, which is referred to herein as lignocellulosic material, lignocellulosic substrate, or cellulosic biomass. Methods of producing ethanol can be accomplished, for example, by contacting the biomass with a recombinant microorganism as described herein, and as described in commonly owned U.S. Patent Application Publication No. 2011/0189744 Al, U.S. Patent Application Publication No. 2011/0312054 Al, U.S. Patent Application Publication No. 2012/0003701, International Appl. No. PCT/US2009/065571, International Appl. No. PCT/US2009/069443, International Appl. No. PCT/US2009/064128, International Appl. No. PCT/IB2009/005881, and PCT/US2009/065571, the contents of each are incorporated by reference herein.

[0337] Numerous cellulosic substrates can be used in accordance with the present invention. Substrates for cellulose activity assays can be divided into two categories, soluble and insoluble, based on their solubility in water. Soluble substrates include cellodextrins or derivatives, carboxymethyl cellulose (CMC), or hydroxyethyl cellulose (HEC). Insoluble substrates include crystalline cellulose, microcrystalline cellulose (Avicel), amorphous cellulose, such as phosphoric acid swollen cellulose (PASC), dyed or fluorescent cellulose, and pretreated lignocellulosic biomass. These substrates are generally highly ordered cellulosic material and thus only sparingly soluble.

[0338] It will be appreciated that suitable lignocellulosic material may be any feedstock that contains soluble and/or insoluble cellulose, where the insoluble cellulose may be in a crystalline or non-crystalline form. In various embodiments, the lignocellulosic biomass comprises, for example, wood, corn, corn stover, sawdust, bark, leaves, agricultural and forestry residues, grasses such as switchgrass, ruminant digestion products, municipal wastes, paper mill effluent, newspaper, cardboard or combinations thereof.

[0339] In some embodiments, the invention is directed to a method for hydrolyzing a cellulosic substrate, for example a cellulosic substrate as described above, by contacting the cellulosic substrate with a recombinant microorganism of the invention. In some embodiments, the invention is directed to a method for hydrolyzing a cellulosic substrate, for example a cellulosic substrate as described above, by contacting the cellulosic substrate with a co-culture comprising yeast cells expressing heterologous cellulases.

[0340] In some embodiments, the invention is directed to a method for fermenting cellulose. Such methods can be accomplished, for example, by culturing a host cell or co- culture in a medium that contains insoluble cellulose to allow saccharification and fermentation of the cellulose.

[0341] The production of ethanol can, according to the present invention, be performed at temperatures of at least about 30° C, about 31° C, about 32° C, about 33° C, about 34° C, about 35° C, about 36° C, about 37° C, about 38° C, about 39° C, about 40° C, about 41° C, about 42° C, about 43 °C, about 44 °C, about 45 °C, about 46 °C, about 47 °C, about 48 °C, about 49 °C, or about 50° C. In some embodiments of the present invention the thermotolerant host cell can produce ethanol from cellulose at temperatures above about 30° C, about 31° C, about 32° C, about 33° C, about 34° C, about 35° C, about 36° C, about 37° C, about 38° C, about 39° C, about 40° C, about 41° C, about 42° C, or about 43 °C, or about 44 °C, or about 45 °C, or about 50° C. In some embodiments of the present invention, the thermotolterant host cell can produce ethanol from cellulose at temperatures from about 30° C to 60° C, about 30° C to 55° C, about 30° C to 50° C, about 40° C to 60° C, about 40° C to 55° C or about 40° C to 50° C.

[0342] In some embodiments, methods of producing ethanol can comprise contacting a cellulosic substrate with a recombinant microorganism or co-culture of the invention and additionally contacting the cellulosic substrate with externally produced cellulase enzymes. Exemplary externally produced cellulase enzymes are commercially available and are known to those of skill in the art.

[0343] In some embodiments, the methods comprise producing ethanol at a particular rate. For example, in some embodiments, ethanol is produced at a rate of at least about 0.1 mg per hour per liter, at least about 0.25 mg per hour per liter, at least about 0.5 mg per hour per liter, at least about 0.75 mg per hour per liter, at least about 1.0 mg per hour per liter, at least about 2.0 mg per hour per liter, at least about 5.0 mg per hour per liter, at least about 10 mg per hour per liter, at least about 15 mg per hour per liter, at least about 20.0 mg per hour per liter, at least about 25 mg per hour per liter, at least about 30 mg per hour per liter, at least about 50 mg per hour per liter, at least about 100 mg per hour per liter, at least about 200 mg per hour per liter, at least about 300 mg per hour per liter, at least about 400 mg per hour per liter, or at least about 500 mg per hour per liter.

[0344] In some embodiments, the host cells of the present invention can produce ethanol at a rate of at least about 0.1 mg per hour per liter, at least about 0.25 mg per hour per liter, at least about 0.5 mg per hour per liter, at least about 0.75 mg per hour per liter, at least about 1.0 mg per hour per liter, at least about 2.0 mg per hour per liter, at least about 5.0 mg per hour per liter, at least about 10 mg per hour per liter, at least about 15 mg per hour per liter, at least about 20.0 mg per hour per liter, at least about 25 mg per hour per liter, at least about 30 mg per hour per liter, at least about 50 mg per hour per liter, at least about 100 mg per hour per liter, at least about 200 mg per hour per liter, at least about 300 mg per hour per liter, at least about 400 mg per hour per liter, or at least about 500 mg per hour per liter more than a control strain (e.g., a wild-type strain) and grown under the same conditions. In some embodiments, the ethanol can be produced in the absence of any externally added cellulases.

[0345] Ethanol production can be measured using any method known in the art. For example, the quantity of ethanol in fermentation samples can be assessed using HPLC analysis. Many ethanol assay kits are commercially available that use, for example, alcohol oxidase enzyme based assays. Methods of determining ethanol production are within the scope of those skilled in the art from the teachings herein. The U.S. Department of Energy (DOE) provides a method for calculating theoretical ethanol yield. Accordingly, if the weight percentages are known of C6 sugars (i.e., glucan, galactan, mannan), the theoretical yield of ethanol in gallons per dry ton of total C6 polymers can be determined by applying a conversion factor as follows:

(1.11 pounds of C6 sugar/pound of polymeric sugar) x (0.51 pounds of ethanol/pound of sugar) x (2000 pounds of ethanol/ton of C6 polymeric sugar) x (1 gallon of ethanol/6.55 pounds of ethanol) x (1/100%), wherein the factor (1 gallon of ethanol/6.55 pounds of ethanol) is taken as the specific gravity of ethanol at 20°C.

[0346] And if the weight percentages are known of C5 sugars (i.e., xylan, arabinan), the theoretical yield of ethanol in gallons per dry ton of total C5 polymers can be determined by applying a conversion factor as follows:

(1.136 pounds of C5 sugar/pound of C5 polymeric sugar) x (0.51 pounds of ethanol/pound of sugar) x (2000 pounds of ethanol/ton of C5 polymeric sugar) x (1 gallon of ethanol/6.55 pounds of ethanol) x (1/100%), wherein the factor (1 gallon of ethanol/6.55 pounds of ethanol) is taken as the specific gravity of ethanol at 20°C.

[0347] It follows that by adding the theoretical yield of ethanol in gallons per dry ton of the total C6 polymers to the theoretical yield of ethanol in gallons per dry ton of the total C5 polymers gives the total theoretical yield of ethanol in gallons per dry ton of feedstock.

[0348] Applying this analysis, the DOE provides the following examples of theoretical yield of ethanol in gallons per dry ton of feedstock: corn grain, 124.4; corn stover, 1 13.0; rice straw, 109.9; cotton gin trash, 56.8; forest thinnings, 81.5; harwood sawdust, 100.8; bagasse, 1 1 1.5; and mixed paper, 1 16.2. It is important to note that these are theoretical yields. The DOE warns that depending on the nature of the feedstock and the process employed, actual yield could be anywhere from 60% to 90% of theoretical, and further states that "achieving high yield may be costly, however, so lower yield processes may often be more cost effective." (Ibid.) EXEMPLIFICATION

[0349] The invention now being generally described, it will be more readily understood by reference to the following examples, which are included merely for purposes of illustration of certain aspects and embodiments of the present invention, and are not intended to limit the invention.

EXAMPLE 1

Improving Ethanol Yield Through Engineering of Alternate Electron Acceptors

[0350] The present Example describes pathways to reduce or eliminate glycerol by engineering alternate electron acceptors in a yeast cell. Glycerol is an undesired byproduct of sugar metabolism during anaerobic growth in yeast. The amount of glycerol produced during anaeroblic growth on glucose has been empirically determined by Medina, VG, et al, Appl. Env. Microbiol. 76:190-95 (2010):

[0351] 56 mmol glucose 1 g biomass + 88 mmol ethanol + 95 mmol C0 ₂ + 11 mmol glycerol + 1.7 mmol acetate

[0352] Assuming glycerol production is primarily for the regeneration of NAD ⁺ for the continuation of glycolysis, a half reaction for glycerol production is (Medina, VG, et al. , Appl. Env. Microbiol. 76: 190-95 (2010)):

[0353] 0.5 glucose + NADH + H ⁺ +ATP -> glycerol + NAD ⁺ + ADP + P _;

[0354] The following pathways describe engineering an alternate electron acceptor for glycerol in the above half reaction, engineering an increase in ethanol yield during anaerobic growth on glucose by using improved enzyme activities for converting glucose to ethanol, and/or deleting endogenous glycerol-producing or glycerol-regulating genes.

[0355] 1.1 Engineering of a formate pathway in yeast

[0356] Production of formate from glucose can provide similar reducing equivalents as glycerol, as shown in the following half reaction: [0357] 0.5 glucose + NADH + H + ADP + P _; -» formate + NAD + ATP + ethanol

[0358] In addition to balancing the redox constraints of the cell, this pathway provides increased ATP yield and results in an overall anaerobic growth equation of:

[0359] 56 mmol glucose 1 g biomass + 99 mmol ethanol + 95 mmol C0 ₂ + 11 mmol formate + 1.7 mmol acetate

[0360] Engineering in a formate pathway as an alternate electron acceptor to glycerol results in an increase of 12.5% in the theoretical yield of ethanol. Enzymes than can be targeted to engineer such an increase include pyruvate formate lyase (PFL) and formate dehydrogenase (FDH). See Figure 1.

[0361] 1.1.1 Expression of PFL

[0362] The conversion of pyruvate to acetyl-CoA and formate is performed by PFL.

Thus, to produce formate in yeast, a PFL can be expressed. PFLs are common in bacteria from a variety of sources. Vigorous hydrogen producing bacteria, such as from a Clostridium, thermoanaerobacterium, or other anaerobic bacteria will likely result in an increased productivity. Examples of PFL include, but are not limited, Bacillus licheniformis, Streptococcus thermophilus, Lactobacillus plantarum, Lactobacillus casei, Bifidobacterium adolescentis, Clostridium cellulolyticum, Escherichia coli, Chlamydomonas reinhardtii PflA, Piromyces sp. E2, or Neocallimastix frontalis. See Example 4 and Table 1 below.

[0363] 1.1.2 Deletion of FDH

[0364] To prevent yeast from converting formate to C0 ₂ and NADH, endogeneous FDH genes can be deleted or downregulated. Deleting or dowmQgulatingfdh l, fdh2, or both genes can enhance the redox balance and ethanol yield of the recombinant microorganisms of the invention.

[0365] 1.2 Improving conversion of acetyl-CoA to ethanol [0366] To improve the conversion of acetyl-CoA to ethanol, endogenous yeast genes can be replaced or complimented with either an improved acetaldehyde dehydrogenase (e.g. , from C. phytofermentans or other source) to convert acetyl-CoA to acetaldehyde, or a bifunctional acetaldehyde/alcohol dehydrogenase (AADH) to convert acetyl-CoA to acetaldehyde and acetaldehyde to ethanol. By engineering in one or more such enzymes, the in vivo kinetics of the conversion of acetyl-CoA to ethanol can be increased, providing for improved growth of the host strain. The bi-functional alcohol/aldehyde dehydrogenase can come from a variety of microbial sources, including but not limited to E. coli, C. acetobutylicum, T. saccharolyticum, C. thermocellum, C. phytofermentans, Piromyces SP E2, or Bifidobacterium adolescentis.

[0367] 1.3 Deletion or downregulation of glycerol pathway

[0368] Deleting or altering expression of glycerol formation genes will reduce or block endogenous production of glycerol and may enhance acetate uptake. Deletion of gpdl, gpd2, or both genes and/or deletion of gppl, gpp2, or both genes may be used to eliminate glycerol formation and enhance ethanol yield. However, the complete elimination of glycerol may not be practical for an industrial process. See Guo, ZP., et al., Metab. Eng. 75:49-59 (2011). Thus, rather than the complete removal of any one, all, or some combination of these glycerol formation genes, one or more of these genes can be altered or downregulated to reduce glycerol formation and enhance ethanol yield.

EXAMPLE 2

Deletion or Downregulation of Glycerol-Regulating Gene FPS 1 to Improve

Ethanol Yield

[0369] Instead of, or in addition to, downregulating glycerol production through deletion or alteration of glycerol-forming genes, glycerol production can be downregulated by deletion or alteration of a glycerol-regulating gene. FPSl is an aquaglyceroporin responsible for efflux of glycerol. An fpslA strain has reduced glycerol formation yet has a completely functional NAD ⁺ -dependant glycerol synthesis pathway. In addition to deletion of FPSl, constitutively active mutants of FPSl or homo logs from other organisms can be expressed to alter glycerol production. Because such FPSl deletion or alteration strains can still synthesize and retain glycerol, improved robustness may be observed relative to strains that are unable to make glycerol.

[0370] Null mutants of an fpslA strain grow much slower anaerobically than wild-type due to intracellular glycerol accumulation. Tamas, M.J., et al., Molecular Microbiol. 31(4): 1087-1104 (1999). However, preliminary data indicates that expression of a B. adolescentis bifunctional AADH in conjunction with B. adolescentis PFL in an fpslA strain can enable anaerobic growth of fpslA strain (see Example 7 and Figures 22 and 24). Additionally, significantly improved osmotic tolerance was also observed when FPSl was deleted in glycerol mutant strains containing ADH and PFL alone. Increased resistance to osmotic stress was determined by observation of improved growth of the fpslA mutant on several different high osmolality media including 1M sodium chloride, 1M sorbitol, and 1M xylitol. The fpslA mutant was created by marker recycle resulting in a deletion of a large region of the FPSl coding sequence (sequence of native and deletion below).

[0371] Sequence of FPSl locus (coding sequence is underlined; SEQ ID NO: 104):

[0372] aacgcggctgatgcttttatttaggaaggaatacttacattatcatgagaacattgtcaa gggcattctgatacgggcctt ccatcgcaagaaaaaggcagcaacggactgagggacggagagagttacggcataagaagt agtaggagagcagagtgtcat aaagttatattattctcgtcctaaagtcaattagttctgttgcgcttgacaatatatgtc gtgtaataccgtcccttagcagaagaaaga aagacggatccatatatgttaaaatgcttcagagatgtttctttaatgtgccgtccaaca aaggtatcttctgtagcttcctctattttcg atcagatctcatagtgagaaggcgcaattcagtagttaaaagcggggaacagtgtgaatc cggagacggcaagattgcccggc cctttttgcggaaaagataaaacaagatatattgcactttttccaccaagaaaaacagga agtggattaaaaaatcaacaaagtata acgcctattgtcccaataagcgtcggttgttcttctttattattttaccaagtacgctcg agggtacattctaatgcattaaaagacatg agtaatcctcaaaaagctctaaacgactttctgtccagtgaatctgttcatacacatgat agttctaggaaacaatctaataagcagtc atccgacgaaggacgctcttcatcacaaccttcacatcatcactctggtggtactaacaa caataataacaataataataataataat aacagtaacaacaacaacaacggcaacgatgggggaaatgatgacgactatgattatgaa atgcaagattatagaccttctccgc aaagtgcgcggcctactcccacgtatgttccacaatattctgtagaaagtgggactgctt tcccgattcaagaggttattcctagcg catacattaacacacaagatataaaccataaagataacggtccgccgagtgcaagcagta atagagcattcaggcctagagggc agaccacagtgtcggccaacgtgcttaacattgaagatttttacaaaaatgcagacgatg cgcataccatcccggagtcacatttat cgagaaggagaagtaggtcgagggctacgagtaatgctgggcacagtgccaatacaggcg ccacgaatggcag gtgcccaaactaatatggaaagcaatgaatcaccacgtaacgtccccattatggtgaagc caaagacattataccagaaccctca aacacctacagtcttgccctccacataccatccaattaataaatggtcttccgtcaaaaa cacttatttgaaggaatttttagccgagtt tatgggaacaatggttatgattattttcggtagtgctgttgtttgtcaggtcaatgttgc tgggaaaatacagcaggacaatttcaacg tggctttggataaccttaacgttaccgggtcttctgcagaaacgatagacgctatgaaga gtttaacatccttggtttcatccgttgcg ggcggtacctttgatgatgtggcattgggctgggctgctgccgtggtgatgggctatttc tgcgctggtggtagtgccatctcaggt gctcatttgaatccgtctattacattagccaatttggtgtatagaggttttcccctgaag aaagttccttattactttgctggacaattgat cggtgccttcacaggcgctttgatcttgtttatttggtacaaaagggtgttacaagaggc atatagcgattggtggatgaatgaaagt gttgcgggaatgttttgcgtttttccaaagccttatctaagttcaggacggcaatttttt tccgaatttttatgtggagctatgttacaagc aggaacatttgcgctgaccgatccttatacgtgtttgtcctctgatgttttcccattgat gatgtttattttgattttcattatcaatgcttcc atggcttatcagacaggtacagcaatgaatttggctcgtgatctgggcccacgtcttgca ctatatgcagttggatttgatcataaaa tgctttgggtgcatcatcatcatttcttttgggttcccatggtaggcccatttattggtg cgttaatgggggggttggtttacgatgtctg tatttatcagggtcatgaatctccagtcaactggtctttaccagtttataaggaaatgat tatgagagcctggtttagaaggcctggtt ggaagaagagaaatagagcaagaagaacatcggacctgagtgacttctcatacaataacg atgatgatgaggaatttggagaaa gaatggctcttcaaaagacaaagaccaagtcatctatttcagacaacgaaaatgaagcag gagaaaagaaagtgcaatttaaatc tgttcagcgcggcaaaagaacgtttggtggtataccaacaattcttgaagaagaagattc cattgaaactgcttcgctaggtgcga cgacgactgattctattgggttatccgacacatcatcagaagattcgcattatggtaatg ctaagaaggtaacatgagaaaacaga caagaaaaagaaacaaataatatagactgatagaaaaaaatactgcttactaccgccggt ataatatatatatatatatatatttacat agatgattgcatagtgttttaaaaagctttcctaggttaagctatgaatcttcataacct aaccaactaaatatgaaaatactgacccat cgtcttaagtaagttgacatgaactcagcctggtcacctactatacatgatgtatcgcat ggatggaaagaataccaaacgctacct tccaggttaatgatagtatccaaacctagttggaatttgccttgaacatcaagcagcgat tcgatatcagttgggagcatcaatttgg tcattggaataccatctatgcttttctcctcccatattcgcaaaagtagtaagggctcgt tatatacttttgaatatgtaagatataattct atatgatttagtaatttattttctatacgctcagtatttttctgcagttgtcgagtaggt attaaacgcaaaagaagtccatccttttcatca ttcaaatggacatcttggcaaagggcccagttatggaaaatctgggagtcatacaacgat tgcagttggctatgccactcctggta aggaatcatcaagtctgataattctgttttttagccctttttttttttttttcatg

[0373] Sequence of fpslA mutation (part of the fpsl coding sequence was not deleted

(underlined) and the region that was deleted is represented by a Δ; SEQ ID NO: 105):

[0374] aacgcggctgatgcttttatttaggaaggaatacttacattatcatgagaacattgtcaa gggcattctgatacgggcctt ccatcgcaagaaaaaggcagcaacggactgagggacggagagagttacggcataagaagt agtaggagagcagagtgtcat aaagttatattattctcgtcctaaagtcaattagttctgttgcgcttgacaatatatgtc gtgtaataccgtcccttagcagaagaaaga aagacggatccatatatgttaaaatgcttcagagatgtttctttaatgtgccgtccaaca aaggtatcttctgtagcttcctctattttcg atcagatctcatagtgagaaggcgcaattcagtagttaaaagcggggaacagtgtgaatc cggagacggcaagattgcccggc cctttttgcggaaaagataaaacaagatatattgcactttttccaccaagaaaaacagga agtggattaaaaaatcaacaaagtata acgcctattgtcccaataagcgtcggttgttcttctttattattttaccaagtacgctcg agggtacattctaatgcattaaaagacAg attcgcattatggtaatgctaagaaggtaacatgagaaaacagacaagaaaaagaaacaa ataatatagactgatagaaaaaaat actgcttactaccgccggtataatatatatatatatatatatttacatagatgattgcat agtgttttaaaaagctttcctaggttaagctat gaatcttcataacctaaccaactaaatatgaaaatactgacccatcgtcttaagtaagtt gacatgaactcagcctggtcacctacta tacatgatgtatcgcatggatggaaagaataccaaacgctaccttccaggttaatgatag tatccaaacctagttggaatttgccttg aacatcaagcagcgattcgatatcagttgggagcatcaatttggtcattggaataccatc tatgcttttctcctcccatattcgcaaaa gtagtaagggctcgttatatacttttgaatatgtaagatataattctatatgatttagta atttattttctatacgctcagtatttttctgcagt tgtcgagtaggtattaaacgcaaaagaagtccatccttttcatcattcaaatggacatct tggcaaagggcccagttatggaaaatct gggagtcatacaacgattgcagttggctatgccactcctggtaaggaatcatcaagtctg ataattctgttttttagcccttttttttttttt ttcatg

EXAMPLE 3

Generating Yeast Strains with a Deleted or Downregulated Glycerol-Production

Pathway

[0375] To create yeast strains with altered glycerol production, endogenous glycerol- producing or regulating genes can either be deleted or downregulated, by generating the following genetic backgrounds:

Haploid Strains Diploid Strains

Glycerol • gpdlA gpd2A fdhlA fdh2A • gpdlA/gpdlA gpd2A/gpd2A fdhlA/fdhlA fdh2A/fdh2A

Elimination

• fdhlA fdh2A fpslA

Background • fdhlA/fdhlA fdh2A/fdh2A fpslA/ fpslA

• gpdlA gpd2A fdhlA fdh2A

fpslA • gpdlA/gpdlA gpd2A/gpd2A fdhlA/fdhlA fdh2A/fdh2A fpslA/ fpslA

• gpdlA gpd2A fpslA

• gpdlA/gpdlA gpd2A/gpd2A fpslA/ fpslA

• gpdlA gpd2A

• gpdlA/gpdlA gpd2A/gpd2A

Glycerol • gpdlA gpd2A::GPDl fdhlA • gpdlA/gpdlA

fdh2A gpd2A/gpd2A::GPDl/GPDl fdhlA/fdhlA

Reduction

fdh2A/fdh2A

Background • gpdlA gpd2A::GPDl

• gpdlA/gpdlA • gpdlA gpd2A::GPDl gpd2A/gpd2A::GPDl/GPDl

fdhlAfdh2A fpslA

• gpdlA/gpdlA

• gpdlA gpd2A::GPDl fpslA gpd2A/gpd2A::GPDl/GPDl fdhlA/fdhlA fdh2A/fdh2A fpslA/ fpslA

• gpdlA:GPD2 gpd2A

• gpdlA/gpdlA

• gpdl ::GPD2 gpd2A gpd2A/gpd2A::GPDl/GPDl fpslA/ fpslA fdhlAfdh2A

• gpdlA/gpdlA::GPD2/GPD2

gpd2A/gpd2A

• gpdlA/gpdlA::GPD2/GPD2

gpd2A/gpd2A fdhlA/fdhlA fdh2A/fdh2A

[0376] Strains in the glycerol elimination background were created by deleting one or more of the following genes: gpdl, gpd2,fdhl,fdh2, and/or fpsl. Strains in the glycerol reduction background have been created by by deleting one or more of the following genes: gpdl, gpd2, fdhl,fdh2, and/or fpsl, and by expessing GPDl under the control of the gpd2 promoter (designated gpd2A::GPDl). These strains in which GPDl is expressed from the gpd2 promoter make a smaller amount of glycerol relative to a wild- type strain.

[0377] 3.1 Generation of glycerol-elimination strain gpdl A gpd2A fdhlA fdh2A

[0378] To produce glycerol-elimination strain gpdlA gpd2A fdhlA fdh2A, the following methods were used. All genetic modications were generated using positive selections to insert genetic elements and negative selections to remove genetic elements. See Figures 4-11. The genetic elements were amplified by PCR and transformed into host strains, followed by selection and screening for the desired modification. The sequence of native gpdl, gpd2,fdhl,fdh2 from S. cerevisiae, and resulting loci following deletion, are listed below.

[0379] Sequence of GPDl locus (coding sequence is underlined; SEQ ID NO: 89):

[0380] tacaaacgcaacacgaaagaacaaaaaaagaagaaaacagaaggccaagacagggtcaat gagactgttgtcctc ctactgtccctatgtctctggccgatcacgcgccattgtccctcagaaacaaatcaaaca cccacaccccgggcacccaaagtcc ccacccacaccaccaatacgtaaacggggcgccccctgcaggccctcctgcgcgcggcct cccgccttgcttctctccccttcc ttttctttttccagttttccctattttgtccctttttccgcacaacaagtatcagaatgg gttcatcaaatctatccaacctaattcgcacgt agactggcttggtattggcagtttcgtagttatatatatactaccatgagtgaaactgtt acgttaccttaaattctttctccctttaattttc ttttatcttactctcctacataagacatcaagaaacaattgtatattgtacacccccccc ctccacaaacacaaatattgataatataaa gatgtctgctgctgctgatagattaaacttaacttccggccacttgaatgctggtagaaa gagaagttcctcttctgtttctttgaagg ctgccgaaaagcctttcaaggttactgtgattggatctggtaactggggtactactattg ccaaggtggttgccgaaaattg gatacccagaagttttcgctccaatagtacaaatgtgggtgttcgaagaagagatcaatg gtgaaaaattgactgaaatcataaata ctagacatcaaaacgtgaaatacttgcctggcatcactctacccgacaatttggttgcta atccagacttgattgattcagtcaagga tgtcgacatcatcgttttcaacattccacatcaatttttgccccgtatctgtagccaatt gaaaggtcatgttgattcacacgtcagagc tatctcctgtctaaagggttttgaagttggtgctaaaggtgtccaattgctatcctctta catcactgaggaactaggtattcaatgtgg tgctctatctggtgctaacattgccaccgaagtcgctcaagaacactggtctgaaacaac agttgcttaccacattccaaaggattt cagaggcgagggcaaggacgtcgaccataaggttctaaaggccttgttccacagacctta cttccacgttagtgtcatcgaagat gttgctggtatctccatctgtggtgctttgaagaacgttgttgccttaggttgtggtttc gtcgaaggtctaggctggggtaacaacg cttctgctgccatccaaagagtcggtttgggtgagatcatcagattcggtcaaatgtttt tcccagaatctagagaagaaacatacta ccaagagtctgctggtgttgctgatttgatcaccacctgcgctggtggtag

taaggacgcctgggaatgtgaaaaggagttgttgaatggccaatccgctcaaggtttaat tacctgcaaagaagttcacgaatggt tggaaacatgtggctctgtcgaagacttcccattatttgaagccgtataccaaatcgttt acaacaactacccaatgaagaacctgc cggacatgattgaagaattagatctacatgaagattagatttattggagaaagataacat atcatactttcccccacttttttcgaggct cttctatatcatattcataaattagcattatgtcatttctcataactactttatcacgtt agaaattacttattattattaaattaatacaaaatt tagtaaccaaataaatataaataaatatgtatatttaaattttaaaaaaaaaatcctata gagcaaaaggattttccattataatattagct gtacacctcttccgcattttttgagggtggttacaacaccactcattcagaggctgtcgg cacagttgcttctagcatctggcgtccg tatgtatgggtgtattttaaataataaacaaagtgccacaccttcaccaattatgtcttt aagaaatggacaagttccaaagagcttgc ccaaggctcgacaaggatgtactttggaatatctatattcaagtacgtggcgcgcatatg tttgagtgtgcacacaataaaggtt

[0381] Sequence of gpdlA mutation (part of the gpdl coding sequence was not deleted

(underlined) and the region that was deleted is represented by a Δ; SEQ ID NO: 90):

[0382] tacaaacgcaacacgaaagaacaaaaaaagaagaaaacagaaggccaagacagggtcaat gagactgttgtcctc ctactgtccctatgtctctggccgatcacgcgccattgtccctcagaaacaaatcaaaca cccacaccccgggcacccaaagtcc ccacccacaccaccaatacgtaaacggggcgccccctgcaggccctcctgcgcgcggcct cccgccttgcttctctccccttcc ttttctttttccagttttccctattttgtccctttttccgcacaacaagtatcagaatgg gttcatcaaatctatccaacctaattcgcacgt agactggcttggtattggcagtttcgtagttatatatatactaccatgagtgaaactgtt acgttaccttaaattctttctccctttaattttc ttttatcttactctcctacataagacatcaagaaacaattgtatattgtacacccccccc ctccacaaacacaaatattgataatataaa gatgtctgctgctgctgatagAtctacatgaagattagatttattggagaaagataacat atcatactttcccccacttttttcgaggc tcttctatatcatattcataaattagcattatgtcatttctcataactactttatcacgt tagaaattacttattattattaaattaatacaaaat ttagtaaccaaataaatataaataaatatgtatatttaaattttaaaaaaaaaatcctat agagcaaaaggattttccattataatattagc tgtacacctcttccgcattttttgagggtggttacaacaccactcattcagaggctgtcg gcacagttgcttctagcatctggcgtcc gtatgtatgggtgtattttaaataataaacaaagtgccacaccttcaccaattatgtctt taagaaatggacaagttccaaagagcttg cccaaggctcgacaaggatgtactttggaatatctatattcaagtacgtggcgcgcatat gtttgagtgtgcacacaataaaggtt

[0383] Sequence of GPD2 locus (coding sequence is underlined; SEQ ID NO:91):

[0384] atagccatcatgcaagcgtgtatcttctaagattcagtcatcatcattaccgagtttgtt ttccttcacatgatgaagaaggt ttgagtatgctcgaaacaataagacgacgatggctctgccattgttatattacgcttttg cggcgaggtgccgatgggttgctgagg ggaagagtgtttagcttacggacctattgccattgttattccgattaatctattgttcag cagctcttctctaccctgtcattctagtatttt ttttttttttttttggttttacttttttttcttcttgcctttttttcttgttactttttt tctagttttttttccttccactaagctttttcc^^ ggttcttctttctactcctttagattttttttttatatattaatttttaagtttatgtat tttggtagattcaattctctttccctttccttttccttcgct ccccttccttatcaatgcttgctgtcagaagattaacaagatacacattccttaagcgaa cgcatccggtgttatatactcgtcgtgca tataaaattttgccttcaagatctactttcctaagaagatcattattacaaacacaactg cactcaaagatgactgctcatactaatatc aaacagcacaaacactgtcatgaggaccatcctatcagaagatcggactctgccgtgtca attgtacatttgaaacgtgcgccctt caaggttacagtgattggttctggtaactgggggaccaccatcgccaaagtcattgcgga aaacacagaattgcattcccatatctt cgagccagaggtgagaatgtgggtttttgatgaaaagatcggcgacgaaaatctgacgga tatcataaatacaagacaccagaa cgttaaatatctacccaatattgacctgccccataatctagtggccgatcctgatctttt acactccatcaagggtgctgacatccttgt tttcaacatccctcatcaatttttaccaaacatagtcaaacaattgcaaggccacgtggc ccctcatgtaagggccatctcgtgtcta aaagggttcgagttgggctccaagggtgtgcaattgctatcctcctatgttactgatgag ttaggaatccaatgtggcgca ggtgcaaacttggcaccggaagtggccaaggagcattggtccgaaaccaccgtggcttac caactaccaaaggattatcaaggt gatggcaaggatgtagatcataagattttgaaattgctgttccacagaccttacttccac gtcaatgtcatcgatgatgttgctggtat atccattgccggtgccttgaagaacgtcgtggcacttgcatgtggtttcgtagaaggtat gggatggggtaacaatg ccattcaaaggctgggtttaggtgaaattatcaagttcggtagaatgtttttcccagaat ccaaagtcgagacctactatcaagaatc cgctggtgttgcagatctgatcaccacctgctcaggcggtagaaacgtcaaggttgccac atacatggccaagaccggtaagtc agccttggaagcagaaaaggaattgcttaacggtcaatccgcccaagggataatcacatg cagagaagttcacgagtggctaca aacatgtgagttgacccaagaattcccattattcgaggcagtctaccagatagtctacaa caacgtccgcatggaagacctaccg gagatgattgaagagctagacatcgatgacgaatagacactctccccccccctccccctc tgatctttcctgttgcctctttttcccc caaccaatttatcattatacacaagttctacaactactactagtaacattactacagtta ttataattttctattctctttttctttaagaatct atcattaacgttaatttctatatatacataactaccattatacacgctattatcgtttac atatcacatcaccgttaatgaaagatacgaca ccctgtacactaacacaattaaataatcgccataaccttttctgttatctatagccctta aagctgtttcttcgagctttttcactgcagta attctccacatgggcccagccactgagataagagcgctatgttagtcactactgacggct ctccagtcatttatgtgattttttagtga ctcatgtcgcatttggcccgtttttttccgctgtcgcaacctatttccattaacggtgcc gtatggaagagtcatttaaaggcaggaga gagagattactcatcttcattggatcagattgatgactgcgtacggcagat

[0385] Sequence of gpd2A mutation (the entire coding sequence was deleted, which is represented by a Δ; SEQ ID NO:92):

[0386] atagccatcatgcaagcgtgtatcttctaagattcagtcatcatcattaccgagtttgtt ttccttcacatgatgaagaaggt ttgagtatgctcgaaacaataagacgacgatggctctgccattgttatattacgcttttg cggcgaggtgccgatgggttgctgagg ggaagagtgtttagcttacggacctattgccattgttattccgattaatctattgttcag cagctcttctctaccctgtcattctagtatttt ttttttttttttttggttttacttttttttcttrt^

ggttcttctttctactcctttagattttttttttatatattaatttttaagtttatgtat tttggtagattcaattctctttccctttccttttccttcgct ccccttccttatcActctgatctttcctgttgcctctttttcccccaaccaatttatcat tatacacaagttctacaactactactagtaac attactacagttattataattttctattctctttttctttaagaatctatcattaacgtt aatttctatatatacataactaccattatacacgcta ttatcgtttacatatcacatcaccgttaatgaaagatacgacaccctgtacactaacaca attaaataatcgccataaccttttctgttat ctatagcccttaaagctgtttcttcgagctttttcactgcagtaattctccacatgggcc cagccactgagataagagcgctatgttag tcactactgacggctctccagtcatttatgtgattttttagtgactcatgtcgcatttgg cccgtttttttccgctgtcgcaacctatttcca ttaacggtgccgtatggaagagtcatttaaaggcaggagagagagattactcatcttcat tggatcagattgatgactgcgtacgg cagat

[0387] Sequence of FDH1 locus (coding sequence is underlined; SEQ ID NO:93):

[0388] tatttttctatagatatttacactccgcaagtgcaaaaaaaaagcattatcgctaacgat caagaggaactgagaccttatt agttgtctttgttggcgtaacataaatttcttaggaaaagagaaaattatctcgaaggca aaaataaaccaagcctcgagtttaatgg ttttctaaaaaacactttaaaaacagatcgccataaaaggagaagctccgtaggagaccg ttttcgaaacctatgtagaaataaag ggaaagctccaacggtttggataaatctttagaagcatagagtttatacaacattcagta cgaaatgtactctcgaaacgttctctttt cacggtgcttagtagcagaaaaaagtgtcggaaattacctattttgtcaccactcgagga taggcttgaaagagagttttaacccca acttttctattttgcacttgtttggctatggtttaaaacattctgtttggaccaacagcc caagcggcttatcccttttctttttttcccttata atcgggaatttccttactaggaaggcaccgatactagaactccgaatgaaaaagacatgc cagtaataaaactattttgatgttatg cggaatatactattcttggattattcactgttaactaaaagttggagaaatcactctgca ctgtcaatcattgaaaaaaagaacatata aaagggcacaaaattgagtcttttttaatgagttcttgctgaggaaagtttagttaatat atcatttacgtaaaatatgcatattcttgtatt gtgctttttttattcattttaagcaggaacaatttacaagtattgcaacgctaatcaaat caaaataacagctgaaaattaatatgtc^aa gggaaaggttttgctggttctttacgaaggtggtaagcatgctgaagagcaggaaaagtt

gtatcagaaatttcattgaagaacagggatacgagttggttactaccattgacaaggacc ctgagccaacctcaacggtagacag ggagttgaaagacgctgaaattgtcattactacgccctttttccccgcctacatctcgag aaacaggattgcagaagctcctaacct gaagctctgtgtaaccgctggcgtcggttcagaccatgtcgatttagaagctgcaa

actggttctaacgtcgtttctgtcgcagagcacgttatggccacaattttggttttgata agaaactataatggtggtcatcaacaagc aattaatggtgagtgggatattgccggcgtggctaaaaatgagtatgatctggaagacaa aataatttcaacggtaggtgccggta gaattggatatagggttctggaaagattggtcgcatttaatccgaagaagttactgtact acgactaccaggaactacctgcggaa gcaatcaatagattgaacgaggccagcaagcttttcaatggcagaggtgatattgtt^

ttgctcagtcagatgttgttaccatcaactgtccattgcacaaggactcaaggggtttat tcaataaaaagcttatttcccacatgaaa gatggtgcatacttggtgaataccgctagaggtgctatttgtgtcgcagaagatgttgcc gaggcagtcaagtctggtaaattggct ggctatggtggtgatgtctgggataagcaaccagcaccaaaagaccatccctggaggact atggacaataaggaccacgtg aaacgcaatgactgttcatatcagtggcacatctctggatgctcaaaagaggtacgctca gggagtaaagaacatcctaaatagtt acttttccaaaaagtttgattaccgtccacaggatattattgtgcagaatggttcttatg ccaccagagcttatggacagaagaaata agagtgattatgagtatttgtgagcagaagttttccggtctccttttgttcttgttttgg cgtattctccactattcgtccatagcacattta taccttagctaaatattttgtaaagcaaaattttcgttatctcttaaaaaatagaagagc ggtttattaatatcaaataattgaaactgctg atatggtagctatatacaaaatctgctgtcaaaatttggcagtaaacgatcttcacggta gcggttcaaataaagaggaaaagtcttt ctcccttactgtttttctggaatttggctcgtcgttaataacagaactaaagatacagta aaaggagagatcgcaatcaacttcattaa ttgtaacagtagcataatcacaactgatcatctacactataaacagtttttatttctaat tatgggcgcctggccggctcaaacattgtg cttttaagactccaaaagtatctgctgcagaaaagagccatataatgttaagtgttcagg gataggttatcgcttactacttcaaacgt ttcgaaggaaagccagggaagcctatatctgattccctgtttcataatccaatgcagcca ctagcttataattatttgaactatttgtcg aacatcacagtaataaaatccccagaaagttccacttgctgcatattggcacctgttgat tcactctccatcacttttttgttagccgcc cagcctagaaagtctttaaatacatctgaaatttttttttttttaacagtgcacccgtgc atcatacctcatgcaaggtacctttttttctca aaggtattgtcttccattgaagtggcactatggcatgatgaaccctgagcatttctgaat tcaacagaaccaaattgtccagaaataa atctgtccgacatgaattatgaaactttttttcaattaagtgaagagaattttgcagcgt cttaccattattttgacccattggtcgcatgt ttgcgctttgacttcgagaaccatgttaaagcttacttgtacgacaaccaatgaagtata ttacggcagtttttttggactgggtcaaa aaaagtgttgcataatcaaatcaggaacacattaaaatgttgtaaaatttgtcttagtat cacctgagtggttattcattacgtacta

[0389] Sequence of fdhl/S. mutation (the entire coding sequence was deleted, which is represented by a Δ; SEQ ID NO:94):

[0390] tatttttctatagatatttacactccgcaagtgcaaaaaaaaagcattatcgctaacgat caagaggaactgagaccttatt agttgtctttgttggcgtaacataaatttcttaggaaaagagaaaattatctcgaaggca aaaataaaccaagcctcgagtttaatgg ttttctaaaaaacactttaaaaacagatcgccataaaaggagaagctccgtaggagaccg ttttcgaaacctatgtagaaataaag ggaaagctccaacggtttggataaatctttagaagcatagagtttatacaacattcagta cgaaatgtactctcgaaacgttctctttt cacggtgcttagtagcagaaaaaagtgtcggaaattacctattttgtcaccactcgagga taggcttgaaagagagttttaacccca acttttctattttgcacttgtttggctatggtttaaaacattctgtttggaccaacagcc caagcggcttatcccttttctttttttcccttata atcgggaatttccttactaggaaggcaccgatactagaactccgaatgaaaaagacatgc cagtaataaaactattttgatgttatg cggaatatactattcttggattattcactgttaactaaaagttggagaaatcactctgca ctgtcaAtggcagtaaacgatcttcacg gtagcggttcaaataaagaggaaaagtctttctcccttactgtttttctggaatttggct cgtcgttaataacagaactaaagatacag taaaaggagagatcgcaatcaacttcattaattgtaacagtagcataatcacaactgatc atctacactataaacagtttttatttctaa ttatgggcgcctggccggctcaaacattgtgcttttaagactccaaaagtatctgctgca gaaaagagccatataatgttaagtgttc agggataggttatcgcttactacttcaaacgtttcgaaggaaagccagggaagcctatat ctgattccctgtttcataatccaatgca gccactagcttataattatttgaactatttgtcgaacatcacagtaataaaatccccaga aagttccacttgctgcatattggcacctgt tgattcactctccatcacttttttgttagccgcccagcctagaaagtctttaaatacatc tgaaatttttttttttttaacagtgcacccgtg catcatacctcatgcaaggtacctttttttctcaaaggtattgtcttccattgaagtggc actatggcatgatgaaccctgagcatttct gaattcaacagaaccaaattgtccagaaataaatctgtccgacatgaattatgaaacttt ttttcaattaagtgaagagaattttgcag cgtcttaccattattttgacccattggtcgcatgtttgcgctttgacttcgagaaccatg ttaaagcttacttgtacgacaaccaatgaa gtatattacggcagtttttttggactgggtcaaaaaaagtgttgcataatcaaatcagga acacattaaaatgttgtaaaatttgtctta gtatcacctgagtggttattcattacgtacta

[0391] Sequence of FDH2 locus (coding sequence is underlined; SEQ ID NO:95):

[0392] tgtcgagacaatgtcattgcaagttatataaacattgtaatacatcacctcgatgaaaga gaaactggaatgatagatct ctttttctcaaaatttcgttaatatgtaataataaggttcctgatgtaatttgtttttgt acaaattattttagattctggaggttcaaataaaa tatatattacagccaacgattaggggggacaagacttgattacacatttttcgttggtaa cttgactcttttatgaaaagaaaacattaa gttgaaggtgcacgcttgaggcgctcctttttcatggtgcttagcagcagatgaaagtgt cagaagttacctattttgtcaccatttga gaataagcttgaaagaaagttgtaaccccaacttttctatcttgcacttgtttggaccaa cagccaaacggcttatcccttttcttttcc cttataatcgggaatttccttactaggaaggcaccgatactataactccgaatgaaaaag acatgccagtaataaaaataattgatgt tatgcggaatatactattcttggattattcactgttaactaaaagttggagaaatcactc tgcactgtcaatcattgaaaaaaagaacat ataaaagggcacaaaatcgagtcttttttaatgagttcttgctgaggaaaatttagttaa tatatcatttacataaaacatgcatattatt gtgttgtactttctttattcattttaagcaggaataattacaagtattgcaacgctaatc aaatcgaaataacagctgaaaattaatatgt cgaagggaaaggttttgctggttctttatgaaggtggtaagcatgctgaagagcaggaaa agttattggggtgtattgaaaatgaa cttggtatcagaaatttcattgaagaacagggatacgagttggttactaccattgacaag gaccctgagccaacctcaacggtaga cagggagttgaaagacgctgaaattgtcattactacgccctttttccccgcctacatctc gagaaacaggattgcagaagctccta acctgaagctctgtgtaaccgctggcgtcggttcagaccatgtcgatttagaagctgcaa atgaacggaaaatcacggtcaccga agttactggttctaacgtcgtttctgtcgcagagcacgttatggccacaattttggtttt gataagaaactataatggtggtcatcaata agcaattaatggtgagtgggatattgccggcgtggctaaaaaatgagtatgatctggaag acaaaataatttcaacggtaggtgcc ggtagaattggatatagggttctggaaagattggtcgcatttaatccgaagaagttactg tactacgactaccaggaactacctgcg gaagcaatcaatagattgaacgaggccagcaagcttttcaatggcagaggtgatattgtt cagagagtagagaaattggag tggttgctcagtcagatgttgttaccatcaactgtccattgcacaaggactcaaggggtt tattcaataaaaagcttatttcccacatg aaagatggtgcatacttggtgaataccgctagaggtgctatttgtgtcgcagaagatgtt gccgaggcagtcaagtctggtaaatt ggctggctatggtggtgatgtctgggataagcaaccagcaccaaaagaccatccctggag gactatggacaataaggaccacg tgggaaacgcaatgactgttcatatcagtggcacatctctgcatgctcaaaagaggtacg ctcagggagtaaagaacatcctaaa tagttacttttccaaaaagtttgattaccgtccacaggatattattgtgcagaatggttc ttatgccaccagagcttatggacagaaga aataagagtgattatgagtatttgtgagcagaagttttccggtctccttttgttcttgtt ttggcgtattctccactattcgtccatagcac atttataccttagctaaatattttgtaaagcaaaattttcgttatctcttaaaaaataga agagcggtttattaatatcaaataattgaaact gctgatatggtagctatatacaaaatctgctgtcaaaatttggcagtaaacgatcttcac ggtagcggttcaaataaagaggaaaag tccttctcccttactgtttttctggaatttggctcgtcgttaataacagaactaaagata cagtaaaaggagagatcgcaatcaacttc attaattgtaacagtagcataatcacaactggttatctgcgttatagacaattcttactc acaatgatgggcgcttagttggctgtaaac gtcgctttttaaaactccgaaaagttaccgctacagaaaaaaaccataaatgtatgctag ttgcgcagagaggtttagggtccaaa atttactaccctgtcgctcactacagcgactgtcccgaattaagcccgaagagacgcaga actgttgtatgaacctcatgaaacca ctgatcttgaagatttagaccttcagaatcgttttcaattagaagtatacaagaagtctt tgtacaataatgtcaagacagagctctga attatagttcagccttgttattttttttt

[0393] Sequence of fdh2A mutation (the entire coding sequence was deleted, which is represented by a Δ; SEQ ID NO:96):

[0394] tgtcgagacaatgtcattgcaagttatataaacattgtaatacatcacctcgatgaaaga gaaactggaatgatagatct ctttttctcaaaatttcgttaatatgtaataataaggttcctgatgtaatttgtttttgt acaaattattttagattctggaggttcaaataaaa tatatattacagccaacgattaggggggacaagacttgattacacatttttcgttggtaa cttgactcttttatgaaaagaaaacattaa gttgaaggtgcacgcttgaggcgctcctttttcatggtgcttagcagcagatgaaagtgt cagaagttacctattttgtcaccatttga gaataagcttgaaagaaagttgtaaccccaacttttctatcttgcacttgtttggaccaa cagccaaacggcttatcccttttcttttcc cttataatcgggaatttccttactaggaaggcaccgatactataactccgaatgaaaaag acatgccagtaataaaaataattgatgt tatgcggaatatactattcttggattattcactgttaactaaaagttggagaaatcactc tgcactgtcaatcattgaaaaaaagaacat ataaaagggcacaaaatcgagtcttttttaatgagttcttgctgaggaaaatttagttaa tatatcatttacataaaacatgcatattatt gtgttgtactttctttattcattttaagcaggaataattacaagtattgcaacgctaatc aaatcgaaataacagctgaaaattaatAta agagtgattatgagtatttgtgagcagaagttttccggtctccttttgttcttgttttgg cgtattctccactattcgtccatagcacattta taccttagctaaatattttgtaaagcaaaattttcgttatctcttaaaaaatagaagagc ggtttattaatatcaaataattgaaactgctg atatggtagctatatacaaaatctgctgtcaaaatttggcagtaaacgatcttcacggta gcggttcaaataaagaggaaaagtcctt ctcccttactgtttttctggaatttggctcgtcgttaataacagaactaaagatacagta aaaggagagatcgcaatcaacttcattaa ttgtaacagtagcataatcacaactggttatctgcgttatagacaattcttactcacaat gatgggcgcttagttggctgtaaacgtcg ctttttaaaactccgaaaagttaccgctacagaaaaaaaccataaatgtatgctagttgc gcagagaggtttagggtccaaaattta ctaccctgtcgctcactacagcgactgtcccgaattaagcccgaagagacgcagaactgt tgtatgaacctcatgaaaccactga tcttgaagatttagaccttcagaatcgttttcaattagaagtatacaagaagtctttgta caataatgtcaagacagagctctgaattat agttcagccttgttattttttttt

[0395] 3.2 Generation of glycerol-reduced strain comprising gpd2A::GPDl

[0396] Glycerol-reduction strain gpd2A::GPDl, was constructed as described above. The sequence of gpdlA/gpdlA gpd2A/gpd2A::GPDl/GPDl is provided below.

[0397] Sequence of GPD1 at GPD2 locus (inserted GPD1 is underlined; SEQ ID NO:97): agtaactgtgacgatatcaactctttttttattatgtaataagcaaacaagcacgaatgg ggaaagcctatgtgcaatcaccaaggtc gtcccttttttcccatttgctaatttagaatttaaagaaaccaaaagaatgaagaaagaa aacaaatactagccctaaccctgacttc gtttctatgataataccctgctttaatgaacggtatgccctagggtatatctcactctgt acgttacaaactccggttattttatcggaac atccgagcacccgcgccttcctcaacccaggcaccgcccccaggtaaccgtgcgcgatga gctaatcctgagccatcacccac cccacccgttgatgacagcaattcgggagggcgaaaaataaaaactggagcaaggaatta ccatcaccgtcaccatcaccatca tatcgccttagcctctagccatagccatcatgcaagcgtgtatcttctaagattcagtca tcatcattaccgagtttgttttccttcacat gatgaagaaggtttgagtatgctcgaaacaataagacgacgatggctctgccattgttat attacgcttttgcggcgaggtgccgat gggttgctgaggggaagagtgtttagcttacggacctattgccattgttattccgattaa tctattgttcagcagctcttctctaccctg tcattctagtattttttttttttttttttggttttacttt

ttgatttatccttgggttcttctttctactcctttagattttttttttatatattaattt ttaagtttatgtattttggtagattc

ccttttccttcgctccccttccttatcaatgtctgctgctgctgatagattaaacttaac ttccggccacttgaatgctggtagaaagag aagttcctcttctgtttctttgaaggctgccgaaaagcctttcaaggttactgtgattgg atctggtaactggggtactactattgccaa ggtggttgccgaaaattgtaagggatacccagaagttttcgctccaatagtacaaatgtg ggtgttcgaagaagagatcaatgg aaaaattgactgaaatcataaatactagacatcaaaacgtgaaatacttgcctggcatca ctctacccgacaatttggttgctaatcc agacttgattgattcagtcaaggatgtcgacatcatcgttttcaacattccacatcaatt tttgccccgtatctgtagccaattgaaagg tcatgttgattcacacgtcagagctatctcctgtctaaagggttttgaagttggtgctaa aggtgtccaattgctatcctcttacatcact gaggaactaggtattcaatgtggtgctctatctggtgctaacattgccaccgaagtcgct caagaacactggtctgaaacaacagt tgcttaccacattccaaaggatttcagaggcgagggcaaggacgtcgaccataaggttct aaaggccttgttccacagaccttact tccacgttagtgtcatcgaagatgttgctggtatctccatctgtggtgctttgaagaacg ttgttgccttaggttgtggtttcgtcgaag gtctaggctggggtaacaacgcttctgctgccatccaaagagtcggtttgggtgagatca tcagattcggtcaaatgtttttcccag aatctagagaagaaacatactaccaagagtctgctggtgttgctgatttgatcaccacct gcgctggtggtagaaacgtcaaggtt gctaggctaatggctacttctggtaaggacgcctgggaatgtgaaaaggagttgttgaat ggccaatccgctcaaggtttaat^ ctgcaaagaagttcacgaatggttggaaacatgtggctctgtcgaagacttcccattatt tgaagccgtataccaaatcgtttacaac aactacccaatgaagaacctgccggacatgattgaagaattagatctacatgaagattag acactctccccccccctccccctctg atctttcctgttgcctctttttcccccaaccaatttatcattatacacaagttctacaac tactactagtaacattactacagttattataatt ttctattctctttttctttaagaatctatcattaacgttaatttctatatatacataact accattatacacgctattatcgtttacatatcacat caccgttaatgaaagatacgacaccctgtacactaacacaattaaataatcgccataacc ttttctgttatctatagcccttaaagctg tttcttcgagctttttcactgcagtaattctccacatgggcccagccactgagataagag cgctatgttagtcactactgacggctctc cagtcatttatgtgattttttagtgactcatgtcgcatttggcccgtttttttccgctgt cgcaacctatttccattaacggtgccgtatgg aagagtcatttaaaggcaggagagagagattactcatcttcattggatcagattgatgac tgcgtacggcagatagtgtaatctga gcagttgcgagacccagactggcactgtctcaatagtatattaatgggcatacattcgta ctcccttgttcttgcccacagttctctct ctctttacttcttgtatcttgtctccccattgtgcagcgataaggaacattgttctaata tacacggatacaaaagaaatacacat

EXAMPLE 4

Cloning and Characterization of PFL and AADH Enzymes

[0398] To identify PFL enzymes for use in the strains of the invention, several PFL enzymes were identified for cloning and functional analysis. See Table 1. Functionality was determined by plasmid based expression of each PFL in the fcyA::ADHE gpdlA::ADHE gpd2AfdhlAfdh2A (M2158) background. Figure 22 shows fermentation performance in 20% corn mash. A PFL was determined to be functional based on the presence or absence of a yield increase over M2085. The C. cellulolyticum PFL was determined to be non- functional based on data shown in Figure 13. The strain listed as M1992 +pMU2481 is M2085 plus a plasmid expressing the C. cellulolyticum PFL. This strain does not appear to make formate.

Table 1. Analysis of PFL Enzymes

Escherichia coli yes 36 and 70

Chlamydomonas reinhardtii PflA yes 72 and 76

Piromyces sp. E2 yes 78

Neocallimastix frontalis yes 74 and 80

[0399] As shown in Example 7, eight of nine PFL enzymes that were tested can enable the glycerol elimination and glycerol reduction technologies described herein. An alignment of six of these PFL enzymes is shown in Figure 12. Four of the residues are absent in C. cellulolyticum yet conserved among the other PFL enzymes (indicated with asterisks in Figure 12). There is an insertion of 18 amino acids at position 640 of the B. adolescentis PFL, which is not present in the other PFL enzymes. The eukaryotic PFLs {Piromyces and Chlamydomonas) have an N-terminal extension which has been reported to be involved in mitochondrial targeting. Deletion of this sequence may improve the performance of these enzymes in S. cerevisiae. These differences may provide insights into identifying additional PFL enzymes for use in the strains of the invention.

[0400] To identify AADH enzymes for use in the strains of the invention, several AADH enzymes were identified for cloning and functional analysis. See Table 2. Functionality was determined though analysis of the data listed in Table 3 below and shown in Figure 20. An AADH enzyme was determined to be functional if a strain containing the genotype gpdlAgpd2A plus a given AADH, had a faster anaerobic growth rate than strain gpdlAgpd2A (Figure 20) and there was evidence for acetate consumption (Table 3).

Table 2. Analysis of AADH Enzymes

[0401] When glycerol deletion strains are grown anaerobically, they are not capable of growth or fermentation and cannot consume sugar during glycolysis. However, if these glycerol deletion strains are complemented with an AADH, the strains are able to grow with the supplementation of acetate in the media. Figure 20 shows the growth rates of the parental strain, the glycerol deletion strain, and four glycerol deletion strains expressing AADHs from Escherichia coli (Eco), Clostridium phytofermentans (Cph), Chlamydomonas reinhardtii (Chi), and Piromyces sp. E2 (Pir). As shown in Figure 20, all four genes can restore growth levels above the glycerol deletion strain (as noted by the dotted line) indicating a functional AADH.

[0402] The product yields and conversion of acetate by the strains above, as well as additional strains, are shown in Table 3. The glycerol deletion strain was unable to consume sugar or produce ethanol. The parent strain produced glycerol and ethanol but was unable to convert the acetate in the media, initially present at ~2 g/L, giving an ethanol yield of 0.41 g/g glucose, consistent with anaerobic ethanol yields. The glycerol deletion strains complemented with AADHs, however, were able to consume glucose and produce ethanol without producing glycerol, or the glycerol production was significantly decreased compared to the parent strain (Chi AADH). See Table 3. In these glycerol deletion mutants, the acetate levels were also reduced, resulting in higher ethanol yields (calculated as grams ethanol produced per gram consumed glucose) than was achieved by the parent strain.

Table 3. Product Yields and Acetate Conversion of Glycerol Deletion Strains Expressing AADH

Strain Glycerol ³ Acetate Uptake ⁸ Ethanol* Ethanol Yield" Growth

rate (hr ¹ )

Parent Ml 39 1.37 0.14 10.41 0.42 0.27

AgpdlAgpd2 0.00 0.00 0.00 0.00 0.01

M2032

Eco AADH 0.00 0.62 11.39 0.46 0.17

M1991

Cph AADH 0.00 0.66 11.21 0.45 0.18

M1991

Chi AADH 0.00 0.32 9.04 0.47 0.04

M1991

Pir AADH 0.00 0.68 11.17 0.45 0.17 M1991

Bad AADH 0.00 0.67 10.95 0.44 0.18

M1991

Eco mhpF 0.00 0.03 0.50 0.02 0.06

M1991

Cph ADH 0.00 0.60 11.29 0.45 0.19

(1428)

M1991

Cph ADH 0.00 0.74 11.22 0.45 0.20

(2642)

M1991

Tsac AADH 0.00 0.59 11.89 0.48 0.16

M1991

a grams per liter

b gram ethanol produced per gram sugar consumed

EXAMPLE 5

Expression of PFL and AADH and Detection of Formate

[0403] To examine the expression of formate in a yeast strain of the invention, E. coli

PFL was cloned and expressed in an FDH deletion strain. Strain M1992+pMU2483 has deletions of FDHl and FDH2 and a plasmid expressing the E. coli PflA and PflB. This strain was constructed by transforming strain M1992 (fdhlAfdh2A) with plasmids expressing either C. cellulolyticum PFL (pMU2481) or E. coli PFL (pMU2483).

[0404] The strains were grown in YNB medium buffered with HEPES at pH 6.5, and formate was measured using a formate detection kit from Megazymes (Cat. No. K- FORM), according to manufacturer's specifications. As shown in Figure 13, approximately 0.0125 g/L and 0.023 g/L formate was measured after 24 hours and 48 hours of growth, respectively. Similar results have been achieved by overexpressing an E. coli PFL in S. cerevesiae. Waks and Silver, Appl. Env. Microbiol. 75: 1867-75 (2009).

[0405] PFL was also co-expressed in a strain expressing AADH. See Figure 14. M2085 is a background strain with the genotype gpdlAgpd2AfdhlAfdh2A and M2032 has the genotype gpdlAgpdlA. Both of these strains are unable to grow anaerobically even in the presence of acetate. M2158 was created by integrating multiple copies of E. coli AADHs at the GPD1 and FCY1 locus in the M2085 background. The integration schemes are shown in Figures 26 and 27 and the corresponding nucleotide sequences for M2158 are listed below. One copy of AADH is driven by the native GPD1 promoter. See Figure 26. The second copy is oriented in the reverse direction and is driven by the phosphofructokinase promoter. See Figure 26. For AADH integration in FCY1, one copy is driven by the ENOl promoter and a second copy is driven by the PFK1 promoter. See Figure 27. M2182 was created by transforming a vector expressing the B. adolescentis PFLs into the M2158 background.

[0406] Sequence of M2158 AADH integrations at the GPD1 locus (nucleotide; SEQ ID

NO:98):

[0407] tagattcttttcgaatttgtggtgaagataggaaagttggtacagttctccatcaatttt ccatattttgctaaaaactcccttg catgtctctttgcattcatttctcctgtatacgggttcaacacatcaatcgaattttgca aagttgtctccatttctagaagactttcatcg ggaataaaaaattcatatccattattcaaaaacgataatgatccctcgtacttacctgtg taattggatattttataccatacttcaaaaa tatccttggcctcacttctggtaggatacctttcgccatgtctgccaatcatttgaactt gcgttaatctacaaccttcaggaatatcagt gggtataccgtagttagcgggaaaggagaaatatggcgcagaccctccaagaaagggaaa cagactcttctgagagccaatta gttcaatatccgcaaaacttctgagtgggatggagagtgccttagataatagaacaccta aacaaatggcaaaaataacgggcttc accattgttcctgtatggtgtattagaacatagctgaaaatacttctgcctcaaaaaagt gttaaaaaaaagaggcattatatagagg taaagcctacaggcgcaagataacacatcaccgctctcccccctctcatgaaaagtcatc gctaaagaggaacactgaaggttcc cgtaggttgtctttggcacaaggtagtacatggtaaaaactcaggatggaataattcaaa ttcaccaatttcaacgtcccttgtttaaa aagaaaagaatttttctctttaaggtagcactaatgcattatcgatgatgtaaccattca cacaggttatttagcttttgatccttgaacc attaattaacccagaaatagaaattacccaagtggggctctccaacacaatgagaggaaa ggtgactttttaagggggccagacc ctgttaaaaacctttgatggctatgtaataatagtaaattaagtgcaaacatgtaagaaa gattctcggtaacgaccatacaaatattg ggcgtgtggcgtagtcggtagcgcgctcccttagcatgggagaggtctccggttcgattc cggactcgtccaaattattttttacttt ccgcggtgccgagatgcagacgtggccaactgtgtctgccgtcgcaaaatgatttgaatt ttgcgtcgcgcacgtttctcacgtac ataataagtattttcatacagttctagcaagacgaggtggtcaaaatagaagcgtcctat gttttacagtacaagacagtccatactg aaatgacaacgtacttgacttttcagtattttctttttctcacagtctggttatttttga aagcgcacgaaatatatgtaggcaagcatttt ctgagtctgctgacctctaaaattaatgctattgtgcaccttagtaacccaaggcaggac agttaccttgcgtggtgttactatggcc ggaagcccgaaagagttatcgttactccgattattttgtacagctgatgggaccttgccg tcttcattttttttttttttcacctatagagc cgggcagagctgcccggcttaactaagggccggaaaaaaaacggaaaaaagaaagccaag cgtgtagacgtagtataacagt atatctgacacgcacgtgatgaccacgtaatcgcatcgcccctcacctctcacctctcac cgctgactcagcttcactaaaaagga aaatatatactctttcccaggcaaggtgacagcggtccccgtctcctccacaaaggcctc tcctggggtttgagcaagtctaagttt acgtagcataaaaattctcggattgcgtcaaataataaaaaaagtaaccccacttctact tctacatcggaaaaacattccattcaca tatcgtctttggcctatcttgttttgtcctcggtagatcaggtcagtacaaacgcaacac gaaagaacaaaaaaagaagaaaacag aaggccaagacagggtcaatgagactgttgtcctcctactgtccctatgtctctggccga tcacgcgccattgtccctcagaaaca aatcaaacacccacaccccgggcacccaaagtccccacccacaccaccaatacgtaaacg gggcgccccctgcaggccctc ctgcgcgcggcctcccgccttgcttctctccccttccttttctttttccagttttcccta ttttgtccctttttccgcacaacaagtatcaga atgggttcatcaaatctatccaacctaattcgcacgtagactggcttggtattggcagtt tcgtagttatatatatactaccatgagtga aactgttacgttaccttaaattctttctccctttaattttcttttatcttactctcctac ataagacatcaagaaacaattgtatattgtacacc ccccccctccacaaacacaaatattgataatataaagatggctgttactaatgtcgctga acttaacgcactcgtagagcgtgtaaa aaaagcccagcgtgaatatgccagtttcactcaagagcaagtagacaaaatcttccgcgc cgccgctctggctgctgcagatgct cgaatcccactcgcgaaaatggccgttgccgaatccggcatgggtatcgtcgaagataaa gtgatcaaaaaccactttgcttctg aatatatctacaacgcctataaagatgaaaaaacctgtggtgttctgtctgaagacgaca cttttggtaccatcactatcgctgaacc aatcggtattatttgcggtatcgttccgaccactaacccgacttcaactgctatcttcaa atcgctgatcagtctgaagacccgtaac gccattatcttctccccgcacccgcgtgcaaaagatgccaccaacaaagcggctgatatc gttctgcaggctgctatcgctgccg gtgctccgaaagatctgatcggctggatcgatcaaccttctgttgaactgtctaacgcac tgatgcaccacccagacatcaacctg atcctcgcgactggtggtccgggcatggttaaagccgcatacagctccggtaaaccagct atcggtgtaggcgcgggcaacact ccagttgttatcgatgaaactgctgatatcaaacgtgcagttgcatctgtactgatgtcc aaaaccttcgacaacggcgtaatctgtg cttctgaacagtctgttgttgttgttgactctgtttatgacgctgtacgtgaacgttttg caacccacggcggctatctgttgcagggta aagagctgaaagctgttcaggatgttatcctgaaaaacggtgcgctgaacgcggctatcg ttggtcagccagcctataaaattgct gaactggcaggcttctctgtaccagaaaacaccaagattctgatcggtgaagtgaccgtt gttgatgaaagcgaaccgttcgcac atgaaaaactgtccccgactctggcaatgtaccgcgctaaagatttcgaagacgcggtag aaaaagcagagaaactggttgctat gggcggtatcggtcatacctcttgcctgtacactgaccaggataaccaaccggctcgcgt ttcttacttcggtcagaaaatgaaaa cggcgcgtatcctgattaacaccccagcgtctcagggtggtatcggtgacctgtataact tcaaactcgcaccttccctgactctgg gttgtggttcttggggtggtaactccatctctgaaaacgttggtccgaaacacctgatca acaagaaaaccgttgctaagcgagct gaaaacatgttgtggcacaaacttccgaaatctatctacttccgccgtggctccctgcca atcgcgctggatgaagtgattactgat ggccacaaacgtgcgctcatcgtgactgaccgcttcctgttcaacaatggttatgctgat cagatcacttccgtactgaaagcagc aggcgttgaaactgaagtcttcttcgaagtagaagcggacccgaccctgagcatcgttcg taaaggtgcagaactggcaaactc cttcaaaccagacgtgattatcgcgctgggtggtggttccccgatggacgccgcgaagat catgtgggttatgtacgaacatccg gaaactcacttcgaagagctggcgctgcgctttatggatatccgtaaacgtatctacaag ttcccgaaaatgggcgtgaaagcga aaatgatcgctgtcaccaccacttctggtacaggttctgaagtcactccgtttgcggttg taactgacgacgctactggtcagaaat atccgctggcagactatgcgctgactccggatatggcgattgtcgacgccaacctggtta tggacatgccgaagtccctgtgtgct ttcggtggtctggacgcagtaactcacgccatggaagcttatgtttctgtactggcatct gagttctctgatggtcaggctctgcagg cactgaaactgctgaaagaatatctgccagcgtcctaccacgaagggtctaaaaatccgg tagcgcgtgaacgtgttcacagtgc agcgactatcgcgggtatcgcgtttgcgaacgccttcctgggtgtatgtcactcaatggc gcacaaactgggttcccagttccatat tccgcacggtctggcaaacgccctgctgatttgtaacgttattcgctacaatgcgaacga caacccgaccaagcagactgcattc agccagtatgaccgtccgcaggctcgccgtcgttatgctgaaattgccgaccacttgggt ctgagcgcaccgggcgaccgtact gctgctaagatcgagaaactgctggcatggctggaaacgctgaaagctgaactgggtatt ccgaaatctatccgtgaagctggc gttcaggaagcagacttcctggcgaacgtggataaactgtctgaagatgcattcgatgac cagtgcaccggcgctaacccgcgtt acccgctgatctccgagctgaaacagattctgctggatacctactacggtcgtgattatg tagaaggtgaaactgcagcgaagaa agaagctgctccggctaaagctgagaaaaaagcgaaaaaatccgcttaagtcgagagctt ttgattaagccttctagtccaaaaa acacgtttttttgtcatttatttcattttcttagaatagtttagtttattcattttatag tcacgaatgttttatgattctatatagggttgcaaac aagcatttttcattttatgttaaaacaatttcaggtttaccttttattctgcttgtggtg acgcgtgtatccgcccgctcttttggtcaccca tgtatttaattgcataaataattcttaaaagtggagctagtctatttctatttacatacc tctcatttctcatttcctcctaatgtgtcaatgat catattcttaactggaccgatcttattcgtcagattcaaaccaaaagttcttagggctac cacaggaggaaaattagtgtgatataatt taaataatttatccgccattcctaatagaacgttgttcgacggatatctttctgcccaaa agggttctaagctcaatgaagagccaatg tctaaacctcgttacattgaaaatacagtaaatggttccaccattattatgttggtcttg tttagtatggccgatcggcgtgtgttttgttt gcaccttttatatagtagaagaatatttgtcttaattcttattagtactgcaacctaacc actaattatcaacaattattggattatataaag gaggtaaattgccggattaaaatcaaatatcattcatcaacaagtattcatattgtcggc atatttttacatgcggtgtaagtatttggat cgtattcttatagtgtcaatacctcgaagcagcgtttcaagtaccagacgtatgtaggaa ctttttaacgtcgagtccgtaagatttga tcagtattaaaaaaatctagataaatgagtggtacaaataaaaacatcattaaaaatcgt taaataaaaaagtatgaagatcatctatt aaagtattagtagccattagccttaaaaaaatcagtgctagtttaagtataatctcgggc gcgccggccgaggcggttaagcggat tttttcgcttttttctcagctttagccggagcagcttctttcttcgctgcagtttcacct tctacataatcacgaccgtagtaggtatccag cagaatctgtttcagctcggagatcagcgggtaacgcgggttagcgccggtgcactggtc atcgaatgcatcttcagacagtttat ccacgttcgccaggaagtctgcttcctgaacgccagcttcacggatagatttcggaatac ccagttcagctttcagcgtttccagcc atgccagcagtttctcgatcttagcagcagtacggtcgcccggtgcgctcagacccaagt ggtcggcaatttcagcataacgacg gcgagcctgcggacggtcatactggctgaatgcagtctgcttggtcgggttgtcgttcgc attgtagcgaataacgttacaaatca gcagggcgtttgccagaccgtgcggaatatggaactgggaacccagtttgtgcgccattg agtgacatacacccaggaaggcg ttcgcaaacgcgatacccgcgatagtcgctgcactgtgaacacgttcacgcgctaccgga tttttagacccttcgtggtaggacgc tggcagatattctttcagcagtttcagtgcctgcagagcctgaccatcagagaactcaga tgccagtacagaaacataagcttcca tggcgtgagttactgcgtccagaccaccgaaagcacacagggacttcggcatgtccataa ccaggttggcgtcgacaatcgcca tatccggagtcagcgcatagtctgccagcggatatttctgaccagtagcgtcgtcagtta caaccgcaaacggagtgacttcaga acctgtaccagaagtggtggtgacagcgatcattttcgctttcacgcccattttcgggaa cttgtagatacgtttacggatatccata aagcgcagcgccagctcttcgaagtgagtttccggatgttcgtacataacccacatgatc ttcgcggcgtccatcggggaaccac cacccagcgcgataatcacgtctggtttgaaggagtttgccagttctgcacctttacgaa cgatgctcagggtcgggtccgcttcta cttcgaagaagacttcagtttcaacgcctgctgctttcagtacggaagtgatctgatcag cataaccattgttgaacaggaagcggt cagtcacgatgagcgcacgtttgtggccatcagtaatcacttcatccagcgcgattggca gggagccacggcggaagtagatag atttcggaagtttgtgccacaacatgttttcagctcgcttagcaacggttttcttgttga tcaggtgtttcggaccaacgttttcagagat ggagttaccaccccaagaaccacaacccagagtcagggaaggtgcgagtttgaagttata caggtcaccgataccaccctgag acgctggggtgttaatcaggatacgcgccgttttcattttctgaccgaagtaagaaacgc gagccggttggttatcctggtcagtgt acaggcaagaggtatgaccgataccgcccatagcaaccagtttctctgctttttctaccg cgtcttcgaaatctttagcgcggtacat tgccagagtcggggacagtttttcatgtgcgaacggttcgctttcatcaacaacggtcac ttcaccgatcagaatcttggtgttttctg gtacagagaagcctgccagttcagcaattttataggctggctgaccaacgatagccgcgt tcagcgcaccgtttttcaggataaca tcctgaacagctttcagctctttaccctgcaacagatagccgccgtgggttgcaaaacgt tcacgtacagcgtcataaacagagtc aacaacaacaacagactgttcagaagcacagattacgccgttgtcgaaggttttggacat cagtacagatgcaactgcacgtttga tatcagcagtttcatcgataacaactggagtgttgcccgcgcctacaccgatagctggtt taccggagctgtatgcggctttaacca tgcccggaccaccagtcgcgaggatcaggttgatgtctgggtggtgcatcagtgcgttag acagttcaacagaaggttgatcgat ccagccgatcagatctttcggagcaccggcagcgatagcagcctgcagaacgatatcagc cgctttgttggtggcatcttttgca cgcgggtgcggggagaagataatggcgttacgggtcttcagactgatcagcgatttgaag atagcagttgaagtcgggttagtg gtcggaacgataccgcaaataataccgattggttcagcgatagtgatggtaccaaaagtg tcgtcttcagacagaacaccacagg ttttttcatctttataggcgttgtagatatattcagaagcaaagtggtttttgatcactt tatcttcgacgatacccatgccggattcggca acggccattttcgcgagtgggattcgagcatctgcagcagccagagcggcggcgcggaag attttgtctacttgctcttgagtga aactggcatattcacgctgggctttttttacacgctctacgagtgcgttaagttcagcga cattagtaacagccataattcttaattaac tttgatatgattttgtttcagattttttatataaaagctttcccaaatagtgctaaagtg aacttagattttttggtacctgtttcgaaattaaa aatagaaaaatttctctccctatattgttattcttacttcaaatttgtttatcgtttatt tactaggcgagacttgagtagacgacaatccaa atagaattaacagattttattggtagaaagcaataatattctttagatggttgagaataa agaagtaaaaaaaccagtaaagagaaa aagaaaaggaagaaaattaaagaaaaaggatgattacacaagaagataataaaaaaactc ctttattaagagcggaagaatttaa taatgaagatgggaataagcaaaacaaaaacaaagaagggaaaaaaaataaaaaatcgta tttatttatttaaaaaatcatgttgat gacgacaatggaaaaaaaaaaccgatttcactttctcatccttatatttttcaaaggttg atgcaagtcgatctcaaatcggataacg ctgccaactgggaaattccgcaattccgcaagaaaaaaaaaaatgtgaaaacgtgattgc attttttacaggtcctaaaggatttag cccacatatcaagagggtggcagtaattgcactgattaagcattcgtcagcattaggcga atgtgtgcatgaatattgccagtgtgc tcgatattagagagtacattgaagaatattgtaccggattatgtacaataactttgttaa tgagatattaattttcttttttactagccgcta tcccatgcacgatgctaaatttcaagaagaaactgagatttaaaaaattagtggaagctg ataaaacggactataatggtgtatgga ttgaggaatctcgacatgtttttccatcgttttcaacgatgactgtaacccgtagattga accaggcatgccaaagttagttagatcag ggtaaaaattatagatgaggtatttattggagaaagataacatatcatactttcccccac ttttttcgaggctcttctatatcatattcata aattagcattatgtcatttctcataactactttatcacgttagaaattacttattattat taaattaatacaaaatttagtaaccaaataaata taaataaatatgtatatttaaattttaaaaaaaaaatcctatagagcaaaaggattttcc attataatattagctgtacacctcttccgcat tttttgagggtggttacaacaccactcattcagaggctgtcggcacagttgcttctagca tctggcgtccgtatgtatgggtgtatttt aaataataaacaaagtgccacaccttcaccaattatgtctttaagaaatggacaagttcc aaagagcttgcccaaggctcgacaag gatgtactttggaatatctatattcaagtacgtggcgcgcatatgtttgagtgtgcacac aataaaggtttttagatattttgcggcgtc ctaagaaaataaggggtttcttaaaaaataacaatagcaaacaaagttccttacgatgat ttcagatgtgaatagcatggtcatgatg agtatatacgtttttataaataattaaaagttttcctcttgtctgtttttttgttggctc gtggttgttctcgaaaaaggagagttttcattttc gaaataggtgattatcatcatgttgttatcaccccacgacgaagataatacggagctcac cgttttctttttttttccctttggctgaaatt tcccaccagaacaaacgtgacaaaattatctttgaatccaaagtagcttatatatatacg tagaagtgtttcgagacacacatccaaa tacgaggttgttcaatttaaacccaagaatacataaaaaaaatatagatatattaactta gtaaacaatgactgcaagcacaccatcc aatgtcatgacattgttcttgttaaggcatggacaaagtgaattgaatcacgagaatata ttctgtggttggattgacgctaagctaac cgaaaaaggtaaagaacaagctcgtcattctgccgagctaatcgaacaatattgtaaagc taataatttgagattaccccagattgg ttacacctcacgtttaattaggacccaacagaccatagaaacgatgtgtgaagaatttaa gttaaagccacaactgcaggttgttta cgactttaataaaatcaaacttggagacgaatttggcagtgatgacaaggataatatgaa aatcccgattcttcaaacttggaggct aaatgaacgtcattacggttcctggcagggccagaggaaaccgaatgttttaaaagaata tggtaaggataaatatatgttcattag gagagattacgagggtaagccaccacctgtagatcttgaccgtgagatgattcaacaaga aaatgagaagggctcttctactggg tacgaattcaaggagccaaacagacaaataaaatatgaattggaatgcagcaatcatgac attgtattaccggattccgaatctctt cgtgaagtggtttatagattgaatccttttctacaaaatgtcatattaaaattagccaat caatatgatgaatcttcatgcctgattgtgg gccatggaagttcagtgagatcgctactgaaaattctggagggtatatcagatgatgaca tcaagaatgttgatattccaaatggta tccccttagtcgttgaattagataagaataatggtcttaagtttatcagaaaattctacc tagatcctgaatctgctaagatcaatgctg agaaagtccgtaatgagggtttcataaaaaatccttaaggggggtaagtatataatataa ttgtgtattttccgaagtataatgaaaac caatagaaaacttattataagtccaatgaggtactttaaaaatgtgatatttataagaac attcctgaatgcagatatatgatatatattg taaatatatatagatgtgtatatgtatttccattttgtgtgaggttttcttcttttatct cctatataatttgtaaccttaattaacccatgacat aaccaatattagcctttgcaaattttgtaacttcttgacgttgttctaacgacaaatctt catgcttcgattttatatgccttgttaaagcat ccagtctcgaaaacgtcttctgatagccctcagatccaagaatttttatacactccgagc aacggaagacaatcttccttttagcgtg aatggtattttggtgtctcgttaaatcataggaccttgaaaattgggcaccacacggttc atttgtaatgagattcattatctgacacgt aaatatttcgttattaccgtcagaagatgacgtatgggccgatggtgatgcagtcgaagg tttcgaattcgaatttgtagatgaatgt gaagataagtgcttc

[0408] Sequence of M2158 AADH integrations at the GPD1 locus (nucleotide; SEQ ID

NO:99):

[0409] cagaattggtgatattcttcattatcccctcgacatctttacttttgatcagctttgtgt atagcgggatatccgattggaact tggcttcagcaacaaacttgccaaagtggattctcctactcaagctttgcaaacattcta tatctctagtggcaacagaaccgaagtt attcttatcatcaccatctcttttcgaaattaatggtataatcttttcaatataaacttt ttttattttatcattgtaattaacttctggggcataa ggcgccaaaatttgtgggtagttaatgctcggtaagaatgatttctgaatcttgtcagga aagaagggagtttcatcaggtgattcg aatcttctgatgcgagaatgcgcaatttcaagatttgaaagagcccaatccaagaaagat cctttaaaattcggaatttctaaacctg gatggtttgcctcataaactgaaggacatgtggcgaaatgcgacctctcaataaatttga agatgatcgaatcctccattctaactaa ttcatctctaatattttgtagatttaaaacagtttctggttttgtgaaatccatatctta taccaattttatgcaggatgctgagtgctatttgt tagcaaaacggaatcgatgtttcaacttttcaatactttttttcttcttttcttcgaact tcgaagattatagagatccattgaaaatttacg ataaaggaaatgcaacacgaagtttgaaaaaaagttgatattgaaaaaaaaaaaaaaaga gaaccaaaaaaaaattaaaaaacg tgaaaacgatggtttaacaacttttttcgaatttggtatacgtggaaaaacgaatgtata gatgcatttttaaagaatatatataaaattt agtaattgtattccgcgagcggcgcaataggtgatttcatttagctgctttcagtttgcg tatcatttcttcattgatcttggcttctctatc taaatcctctttcgacttgtaaagtccccaagttctaaaccataagaaccgcctcaatct ggaaaatttgtcagtatcaagaccataat tcgtgtatgactgaatcaaatgtaatccactttcgtcatgagtaaattcggccttgctca gagactcctggattttggctaacaacgca gtcccttcgatgcatatagctaggccacaaattatgccaataacggtccatgggttgatg ttttcttgaattctttcgtttttcatgctattt gcgtcttcccaagtcccagcgttccagtattcatactgcgcgttagagtggtagccataa gagccggcatattggtaattttcagtat taacgttagaacgtggtgaatacgatgtggtccagccttgcctcgttgtgtcatatacga tctttttctttgggtcacaaagaatatcat atgcttgagagatgactttaaatctatgtagtttttcgcttgatgttagcagcagcggtg atttactatcactgttggtaaccttttctgag ctaaatatttgaatgttatcggaatggtcagggtggtacaattttacataacgatgatat tttttttttaacgacttcttgtccagtttagga tttccagatccggcctttggaatgccaaaaatatcatagggagttggatctgccaactca ggccattgttcatcccttatcgtaagttt tctattgccatttttatcgttcgctgtagcatacttagctataaaagtgatttgtggggg acacttttctacacatgataagtgccacttg aataaaaatgggtatacgaacttatggtgtagcataacaaatatattgcaagtagtgacc tatggtgtgtagatatacgtacagttag ttacgagcctaaagacacaacgtgtttgttaattatactgtcgctgtaatatcttctctt ccattatcaccggtcattccttgcaggggc ggtagtacccggagaccctgaacttttctttttttttttgcgaaattaaaaagttcattt tcaattcgacaatgagatctacaagccattgt tttatgttgatgagagccagcttaaagagttaaaaatttcatagctactagtcttctagg cgggttatctactgatccgagcttccacta ggatagcacccaaacacctgcatatttggacgacctttacttacaccaccaaaaaccact ttcgcctctcccgcccctgataacgtc cactaattgagcgattacctgagcggtcctcttttgtttgcagcatgagacttgcatact gcaaatcgtaagtagcaacgtctcaagg tcaaaactgtatggaaaccttgtcacctcacttaattctagctagcctaccctgcaagtc aagaggtctccgtgattcctagccacct caaggtatgcctctccccggaaactgtggccttttctggcacacatgatctccacgattt caacatataaatagcttttgataatggca atattaatcaaatttattttacttctttcttgtaacatctctcttgtaatcccttattcc ttctagctatttttcataaaaaaccaagcaactgct tatcaacacacaaacactaaatcaaaatggctgttactaatgtcgctgaacttaacgcac tcgtagagcgtgtaaaaaaagcccag cgtgaatatgccagtttcactcaagagcaagtagacaaaatcttccgcgccgccgctctg gctgctgcagatgctcgaatcccact cgcgaaaatggccgttgccgaatccggcatgggtatcgtcgaagataaagtgatcaaaaa ccactttgcttctgaatatatctaca acgcctataaagatgaaaaaacctgtggtgttctgtctgaagacgacacttttggtacca tcactatcgctgaaccaatcggtattat ttgcggtatcgttccgaccactaacccgacttcaactgctatcttcaaatcgctgatcag tctgaagacccgtaacgccattatcttct ccccgcacccgcgtgcaaaagatgccaccaacaaagcggctgatatcgttctgcaggctg ctatcgctgccggtgctccgaaa gatctgatcggctggatcgatcaaccttctgttgaactgtctaacgcactgatgcaccac ccagacatcaacctgatcctcgcgact ggtggtccgggcatggttaaagccgcatacagctccggtaaaccagctatcggtgtaggc gcgggcaacactccagttgttatc gatgaaactgctgatatcaaacgtgcagttgcatctgtactgatgtccaaaaccttcgac aacggcgtaatctgtgcttctgaacag tctgttgttgttgttgactctgtttatgacgctgtacgtgaacgttttgcaacccacggc ggctatctgttgcagggtaaagagctgaa agctgttcaggatgttatcctgaaaaacggtgcgctgaacgcggctatcgttggtcagcc agcctataaaattgctgaactggcag gcttctctgtaccagaaaacaccaagattctgatcggtgaagtgaccgttgttgatgaaa gcgaaccgttcgcacatgaaaaactg tccccgactctggcaatgtaccgcgctaaagatttcgaagacgcggtagaaaaagcagag aaactggttgctatgggcggtatc ggtcatacctcttgcctgtacactgaccaggataaccaaccggctcgcgtttcttacttc ggtcagaaaatgaaaacggcgcgtat cctgattaacaccccagcgtctcagggtggtatcggtgacctgtataacttcaaactcgc accttccctgactctgggttgtggttct tggggtggtaactccatctctgaaaacgttggtccgaaacacctgatcaacaagaaaacc gttgctaagcgagctgaaaacatgt tgtggcacaaacttccgaaatctatctacttccgccgtggctccctgccaatcgcgctgg atgaagtgattactgatggccacaaa cgtgcgctcatcgtgactgaccgcttcctgttcaacaatggttatgctgatcagatcact tccgtactgaaagcagcaggcgttgaa actgaagtcttcttcgaagtagaagcggacccgaccctgagcatcgttcgtaaaggtgca gaactggcaaactccttcaaaccag acgtgattatcgcgctgggtggtggttccccgatggacgccgcgaagatcatgtgggtta tgtacgaacatccggaaactcactt cgaagagctggcgctgcgctttatggatatccgtaaacgtatctacaagttcccgaaaat gggcgtgaaagcgaaaatgatcgct gtcaccaccacttctggtacaggttctgaagtcactccgtttgcggttgtaactgacgac gctactggtcagaaatatccgctggca gactatgcgctgactccggatatggcgattgtcgacgccaacctggttatggacatgccg aagtccctgtgtgctttcggtggtct ggacgcagtaactcacgccatggaagcttatgtttctgtactggcatctgagttctctga tggtcaggctctgcaggcactgaaact gctgaaagaatatctgccagcgtcctaccacgaagggtctaaaaatccggtagcgcgtga acgtgttcacagtgcagcgactat cgcgggtatcgcgtttgcgaacgccttcctgggtgtatgtcactcaatggcgcacaaact gggttcccagttccatattccgcacg gtctggcaaacgccctgctgatttgtaacgttattcgctacaatgcgaacgacaacccga ccaagcagactgcattcagccagtat gaccgtccgcaggctcgccgtcgttatgctgaaattgccgaccacttgggtctgagcgca ccgggcgaccgtactgctgctaag atcgagaaactgctggcatggctggaaacgctgaaagctgaactgggtattccgaaatct atccgtgaagctggcgttcaggaa gcagacttcctggcgaacgtggataaactgtctgaagatgcattcgatgaccagtgcacc ggcgctaacccgcgttacccgctg atctccgagctgaaacagattctgctggatacctactacggtcgtgattatgtagaaggt gaaactgcagcgaagaaagaagctg ctccggctaaagctgagaaaaaagcgaaaaaatccgcttaagtcgagagcttttgattaa gccttctagtccaaaaaacacgttttt ttgtcatttatttcattttcttagaatagtttagtttattcattttatagtcacgaatgt tttatgattctatatagggttgcaaacaagcattttt cattttatgttaaaacaatttcaggtttaccttttattctgcttgtggtgacgcgtgtat ccgcccgctcttttggtcacccatgtatttaatt gcataaataattcttaaaagtggagctagtctatttctatttacatacctctcatttctc atttcctcctaatgtgtcaatgatcatattctta actggaccgatcttattcgtcagattcaaaccaaaagttcttagggctaccacaggagga aaattagtgtgatataatttaaataattt atccgccattcctaatagaacgttgttcgacggatatctttctgcccaaaagggttctaa gctcaatgaagagccaatgtctaaacct cgttacattgaaaatacagtaaatggttccaccattattatgttggtcttgtttagtatg gccgatcggcgtgtgttttgtttgcacctttta tatagtagaagaatatttgtcttaattcttattagtactgcaacctaaccactaattatc aacaattattggattatataaaggaggtaaat tgccggattaaaatcaaatatcattcatcaacaagtattcatattgtcggcatattttta catgcggtgtaagtatttggatcgtattctta tagtgtcaatacctcgaagcagcgtttcaagtaccagacgtatgtaggaactttttaacg tcgagtccgtaagatttgatcagtattaa aaaaatctagataaatgagtggtacaaataaaaacatcattaaaaatcgttaaataaaaa agtatgaagatcatctattaaagtatta gtagccattagccttaaaaaaatcagtgctagtttaagtataatctcgggcgcgccggcc gaggcggttaagcggattttttcgcttt tttctcagctttagccggagcagcttctttcttcgctgcagtttcaccttctacataatc acgaccgtagtaggtatccagcagaatctg tttcagctcggagatcagcgggtaacgcgggttagcgccggtgcactggtcatcgaatgc atcttcagacagtttatccacgttcg ccaggaagtctgcttcctgaacgccagcttcacggatagatttcggaatacccagttcag ctttcagcgtttccagccatgccagc agtttctcgatcttagcagcagtacggtcgcccggtgcgctcagacccaagtggtcggca atttcagcataacgacggcgagcct gcggacggtcatactggctgaatgcagtctgcttggtcgggttgtcgttcgcattgtagc gaataacgttacaaatcagcagggcg tttgccagaccgtgcggaatatggaactgggaacccagtttgtgcgccattgagtgacat acacccaggaaggcgttcgcaaac gcgatacccgcgatagtcgctgcactgtgaacacgttcacgcgctaccggatttttagac ccttcgtggtaggacgctggcagat attctttcagcagtttcagtgcctgcagagcctgaccatcagagaactcagatgccagta cagaaacataagcttccatggcgtga gttactgcgtccagaccaccgaaagcacacagggacttcggcatgtccataaccaggttg gcgtcgacaatcgccatatccgga gtcagcgcatagtctgccagcggatatttctgaccagtagcgtcgtcagttacaaccgca aacggagtgacttcagaacctgtac cagaagtggtggtgacagcgatcattttcgctttcacgcccattttcgggaacttgtaga tacgtttacggatatccataaagcgca gcgccagctcttcgaagtgagtttccggatgttcgtacataacccacatgatcttcgcgg cgtccatcggggaaccaccacccag cgcgataatcacgtctggtttgaaggagtttgccagttctgcacctttacgaacgatgct cagggtcgggtccgcttctacttcgaa gaagacttcagtttcaacgcctgctgctttcagtacggaagtgatctgatcagcataacc attgttgaacaggaagcggtcagtca cgatgagcgcacgtttgtggccatcagtaatcacttcatccagcgcgattggcagggagc cacggcggaagtagatagatttcg gaagtttgtgccacaacatgttttcagctcgcttagcaacggttttcttgttgatcaggt gtttcggaccaacgttttcagagatggagt taccaccccaagaaccacaacccagagtcagggaaggtgcgagtttgaagttatacaggt caccgataccaccctgagacgct ggggtgttaatcaggatacgcgccgttttcattttctgaccgaagtaagaaacgcgagcc ggttggttatcctggtcagtgtacag gcaagaggtatgaccgataccgcccatagcaaccagtttctctgctttttctaccgcgtc ttcgaaatctttagcgcggtacattgcc agagtcggggacagtttttcatgtgcgaacggttcgctttcatcaacaacggtcacttca ccgatcagaatcttggtgttttctggtac agagaagcctgccagttcagcaattttataggctggctgaccaacgatagccgcgttcag cgcaccgtttttcaggataacatcct gaacagctttcagctctttaccctgcaacagatagccgccgtgggttgcaaaacgttcac gtacagcgtcataaacagagtcaac aacaacaacagactgttcagaagcacagattacgccgttgtcgaaggttttggacatcag tacagatgcaactgcacgtttgatat cagcagtttcatcgataacaactggagtgttgcccgcgcctacaccgatagctggtttac cggagctgtatgcggctttaaccatg cccggaccaccagtcgcgaggatcaggttgatgtctgggtggtgcatcagtgcgttagac agttcaacagaaggttgatcgatcc agccgatcagatctttcggagcaccggcagcgatagcagcctgcagaacgatatcagccg ctttgttggtggcatcttttgcacg cgggtgcggggagaagataatggcgttacgggtcttcagactgatcagcgatttgaagat agcagttgaagtcgggttagtggtc ggaacgataccgcaaataataccgattggttcagcgatagtgatggtaccaaaagtgtcg tcttcagacagaacaccacaggtttt ttcatctttataggcgttgtagatatattcagaagcaaagtggtttttgatcactttatc ttcgacgatacccatgccggattcggcaac ggccattttcgcgagtgggattcgagcatctgcagcagccagagcggcggcgcggaagat tttgtctacttgctcttgagtgaaa ctggcatattcacgctgggctttttttacacgctctacgagtgcgttaagttcagcgaca ttagtaacagccataattcttaattaacttt gatatgattttgtttcagattttttatataaaagctttcccaaatagtgctaaagtgaac ttagattttttggtacctgtttcgaaattaaaaa tagaaaaatttctctccctatattgttattcttacttcaaatttgtttatcgtttattta ctaggcgagacttgagtagacgacaatccaaat agaattaacagattttattggtagaaagcaataatattctttagatggttgagaataaag aagtaaaaaaaccagtaaagagaaaaa gaaaaggaagaaaattaaagaaaaaggatgattacacaagaagataataaaaaaactcct ttattaagagcggaagaatttaata atgaagatgggaataagcaaaacaaaaacaaagaagggaaaaaaaataaaaaatcgtatt tatttatttaaaaaatcatgttgatga cgacaatggaaaaaaaaaaccgatttcactttctcatccttatatttttcaaaggttgat gcaagtcgatctcaaatcggataacgctg ccaactgggaaattccgcaattccgcaagaaaaaaaaaaatgtgaaaacgtgattgcatt ttttacaggtcctaaaggatttagccc acatatcaagagggtggcagtaattgcactgattaagcattcgtcagcattaggcgaatg tgtgcatgaatattgccagtgtgctcg atattagagagtacattgaagaatattgtaccggattatgtacaataactttgttaatga gatattaattttcttttttactagccgctatcc catgcacgatgctaaatttcaagaagaaactgagatttaaaaaattagtggaagctgata aaacggactataatggtgtatggattg aggaatctcgacatgtttttccatcgttttcaacgatgactgtaacccgtagattgaacc aggcatgccaaagttagttagatcaggg taaaaattatagatgaggtttaattaaacaagcacgcagcacgctgtatttacgtattta attttatatatttgtgcatacactactaggg aagacttgaaaaaaacctaggaaatgaaaaaacgacacaggaagtcccgtatttactatt ttttccttccttttgatggggcagggc ggaaatagaggataggataagcctactgcttagctgtttccgtctctacttcggtagttg tctcaattgtcgtttcagtattacctttaga gccgctagacgatggttgagctatttgttgagggaaaactaagttcatgtaacacacgca taacccgattaaactcatgaatagctt gattgcaggaggctggtccattggagatggtgccttattttccttataggcaacgatgat gtcttcgtcggtgttcaggtagtagtgta cactctgaatcagggagaaccaggcaatgaacttgttcctcaagaaaatagcggccatag gcatggattggttaaccacaccag atatgcttggtgtggcagaatatagtccttttggtggcgcaattttcttgtacctgtggt agaaagggagcggttgaactgttagtata tattggcaatatcagcaaatttgaaagaaaattgtcggtgaaaaacatacgaaacacaaa ggtcgggccttgcaacgttattcaaa gtcattgtttagttgaggaggtagcagcggagtatatgtattccttttttttgcctatgg atgttgtaccatgcccattctgctcaagcttt tgttaaaattatttttcagtattttttcttccatgttgcgcgttacgagaacagaagcga cagataaccgcaatcatacaactagcgcta ctgcggggtgtaaaaagcacaagaactaagccaagatcacaacagttatcgataaaatag cagtgtttgcatggccattgagaag gacaacattggcgtgcgcgccaatgttgtctcaccatgtagctccaaacgagttgtaaga gacggaccgctcacgcttccgaag cggtcagaaaacgcttcccagtatgcagttgacctacattcaacctgcaaatattgcttt gcttcaagaaatgattacacagacgtct attttcttctacataatgcacgaaacttgggcatttagtcatgtagccgcctagcgagcc tgggtgccgtcctatctcctttgttcgtgc aaagagacaggaacacacactgcgttctcttgcggccggtctggcggactcaggggtgcg gcgtttgcttaaccggagggaat aataaaatcggggtgacgcaagtatgaagtcatgtgtgcttagcaattacgtagagggat tagaaataatagtgtgcggttatcgg aaccggctcttgttcccgtttagagcaacccaggtgcaggcgtactttaaagtattttct ttcttttttttcctgctacttacgctaggag ctgccgcagctgcaaagccgacgtcggagaggcaggtgatcttcggctcggccgacaaat cccctggatatcattggcctgtcg aggtatcggccgcgtggaactaccgggaattactatgcaaaacaattggaaatctggtag gaaaaccttgttctagaacttggcg attgctgacaaagaagaaaagggcctattgttgctgcctcttttgttgttcttcctcgta ttgtcttgccggtgttctttgtgtcttttgtgt gtaggttcttactattatagtgctctttgctattatattttcttcgttttcactttgcgt aatgtaacggtcttaaacaaagttttttttttttcgct cttgcattttccttttctgctctatcttatttgctaattgtagtttcagaagttttactt aaatatagcactattttccagttttaatgtttcttctc attgctttcttttataattttcgcatataattatacatttacggtgtcttaactctccct cttcacccctcattattccagaaaatactaatact tcttcacacaaaagaacgcagttagacaatcaacaatgaatcctaaatcctctacaccta agattccaagacccaagaacgcattt attctgttcagacagcactaccacaggatcttaatagacgaatggaccgctcaaggtgtg gaaataccccataattcaaacatttct aaaattattggtacgaagtggaagggcttacaaccggaagataaggcacactgggaaaat ctagcggagaaggagaaactaga acatgaaaggaagtatcctgaatacaaatacaagccggtaagaaagtctaagaagaagca actacttttgaaggaaatcgagca acagcagcagcaacaacagaaagaacagcagcagcagaaacagtcacaaccgcaattaca acagccctttaacaacaatata gttcttatgaaaagagcacattctctttcaccatcttcctcggtgtcaagctcgaacagc tatcagttccaattgaacaatgatcttaag aggttgcctattccttctgttaatacttctaactatatggtctccagatctttaagtgga ctacctttgacgcatgataagacggcaaga gacctaccacagctgtcatctcaactaaattctattccatattactcagctccacacgac ccttcaacgagacatcattacctcaacg tcgctcaagctcaaccaagggctaactcgacccctcaattgccctttatttcatccatta tcaacaacagcagtcaaacaccggtaa ctacaactaccacatccacaacaactgcgacatcttctcctgggaaattctcctcttctc cgaactcctctgtactggagaacaaca gattaaacagtatcaacaattcaaatcaatatttacctccccctctattaccttctctgc aagattttcaactggatcagtaccagcagc taaagcagatgggaccaacttatattgtcaaaccactgtctcacaccaggaacaatctat tgtccacaactacccctacgcatcatc acattcctcatataccaaaccaaaacattcctctacatcaaattataaactcaagcaaca ctgaggtcaccgctaaaactagcctag tttctccgaaatgattttttttttccatttcttctttccgttatattatattatactata ttccctttaactaaaaatttatgcatttggctcctgttt aataaaagtttaaatc These strains were grown in YPD containing 50 g/L glucose under anaerobic and microaerobic conditions, and formate was measured over 142 hours. As shown in Figure 14, at the end of 142 hours, strains containing the heterologous PFL and AADH made more formate than the wildtype strain Ml 39. EXAMPLE 6

Expression of PFL and AADH and Detection of Ethanol

[0411] The purpose of this Example is to determine whether formate production can confer anaerobic growth on fdh, gpd, and/or fps deletion strains. Yeast strains containing an fdhlAfdh2AgpdlAgpd2A genetic background (M2025) were transformed with vectors expressing PflA/B cassettes from C. cellulolyticum (TB274) and E. coli (TB275). Each of these strains also contained a second construct expressing the E. coli AdhE. YPD medium was prepared and added to hungate tubes, oxygen was purged with nitrogen, and the tubes were autoclaved for 20 minutes. A pre-culture of TB274 and TB275 was prepared overnight in YPD medium containing antibiotics which select for maintenance of both plasmids. A pre-culture of Ml 901, the parent strain of M2025, and M2025 itself were prepared in YPD and included as positive and negative controls, respectively. A strain referred to as TB267 was created which contains only the bifunctional ADH plasmid. This strain was prepared in YPD plus antibiotic to select for the plasmid. This strain controls for the potential effect of ADH or other electron acceptors that may be present in YPD medium.

[0412] All strains were inoculated to final OD's of about 0.05 or below. The OD of each culture was measured at 0, 24, 48, and 72 hours (Figure 15), and samples were prepared and submitted for HPLC determination of metabolite levels. As expected, the Ml 901 strain grew fairly quickly, consuming all the sugar substrate by 24 hours. The M2025 and TB267 strains, which are unable to make glycerol, did not show a significant increase in OD during this experiment. The TB274 and TB275 strains, which express both PFL and ADH, were able to grow after a 24 hour lag time. See Figure 15. These data indicate that the introduced metabolic pathway in TB274 and TB275 does not block cellular growth.

[0413] The production of glycerol in these strains is shown in Figure 16. The anaerobic growth of Ml 901 was accompanied by glycerol production as expected. A trace amount of glycerol was observed in the PflA/B containing strains TB274 and TB275, but this was at or below the level of the negative controls which did not grow. See Figure 16. These data, in conjunction with the OD data, indicate that the expression of PflA/B and AdhE allowed for anaerobic growth of M2025 without associated glycerol production. [0414] The production of ethanol and glucose concentration are shown in Figures 17 and

18, respectively. Both Ml 901 and TB274 consumed all the sugar, but about 5 g/L glucose remained in the TB275 fermentation. See Figure 18. TB274 had an 11% increase in ethanol yield in comparison to Ml 901. See Figure 17. The increase in yield was higher than expected and likely came partially at the expense of biomass, although this was not determined.

[0415] Strains containing PFL and AADH were compared to other strains engineered to express AADH. A description of these strains appears in Table 4.

Table 4. Genetic Backgrounds for PFL Expression

[0416] These five strains were tested in nitrogen purged bottles. As shown in Figure 19, strain M2182 which has both PFL and AADH expression cassettes in a gdplA gpd2A fdhIA fdhIA background, had a faster growth rate and reached a higher OD than M2158, which contains only the bifunctional ADH activity and did not appear to grow at all. See Figure 19. These data show that a strain expressing both PFL and AADH activities grows better than the other engineered functionalities. Similar improvements over these other engineered functionalities are also observed in corn mash fermentations {see Example 7 below).

EXAMPLE 7

Production of Ethanol from Corn Mash

[0417] The purpose of this experiment was to determine whether PFLs cloned from the organisms listed below in Table 5 could provide for increased ethanol yield when used in fermentation of 20% corn mash. A total of nine PFLs have been tested for function in yeast. Of these, only the C. cellulolyticum PFL had no positive effect on growth of glycerol synthesis mutants in corn mash fermentations. Additionally, no formate was observed in formate assays when using a strain containing C. cellulolyticum PFL. This strain was not tested for performance on corn mash fermentation.

[0418] Eight PFLs were tested for functionality in strain M2158 which has the E. coli

AADH integrated on the chromosome of a gpdIA gpd2A fdhIA fdh2A background (M2085) or M2275, which is identical to M2158 except that it also has the gpd2A::GPDl glycerol reduction mutation. Two separate corn mash fermentation experiments were performed using 20% solids in a baffled shake flask using the strains listed in Table 5. Performance of the strains was evaluated by HPLC analysis of metabolites.

Table 5. Genetic Backgrounds for Corn Mash Fermentations

[0419] As shown in Figures 21 and 22, the addition of PFL improves the ethanol yield of strains containing only AADH. Figures 23 and 24 show that these strains do not make glycerol as expected. The approximately 4 g/L glycerol observed is already in the industrial mash substrate used for this experiment. Figure 25 demonstrates a 9% increase in ethanol yield with strain M2326, which is a glycerol reduction strain.

EXAMPLE 8

[0420] The following example demonstrates the creation of the Saccharomyces cerevisiae strain M3625. The genotype of strain M3625 is: Agpd2:: B. adolescentis pflA/pflB/adhE Afdhl Afdh2::B. adolescentis pflA/pfiB/adhE fcylA::5. fibuligera glucoamylase (glu-0111-CO). Strain M2390 is referred to as Ethanol Red (new) from LaSaffre (pahc.com/Phibro/Performance-Products/Catalog/23/Ethanol-Red .html).

[0421] The genetic modification techniques utilized to develop Saccharomyces cerevisiae strain M3625 relied upon directed integration to insert the genes for Bifidobacterium adolescentis pflA, pflB , AdhE and S. fibuligera glu-0111-CO at specific and known sites within the yeast chromosome. The directed integration approach creates transgenic strains with integration events that are stable and easy to characterize. Chromosomal integration, by its very nature, reduces the probability of any mobilization of the heterologous DNA and enhances strain stability relative to other approaches.

[0422] The MX cassettes are the most commonly used engineering tool when an insertion or deletion of a genetic element is desired at a given chromosomal loci (Wach A, et al., Yeast 70(13): 1793-1808 (1994)). A recyclable MX cassette contains one or more markers which enable both dominant and negative selection (Goldstein, A.L. and McCusker, J.H., Yeast 75: 1541-1553 (1999); Ito-Harashima, S. and McCusker, J.H., Yeast 27:53-61 (2004)). The dominant marker enables selection for the modification and the counter selectable marker enables subsequent removal of the marker system via Cre-Lox mediated recombination (Guldener, Ulrich, et al, Nucleic Acids Research (1996) 24(13) 2519-2524) or recombination between duplicated homologous regions flanking the cassette. Since the markers are removed, they can be reused during subsequent engineering steps and ensures no undesirable foreign genetic material remains in the strain.

[0423] To create stable homozygous integrations in M3625, two new HSV-thymidine kinase (TDK) based MX cassettes were developed. Expression of thymidine kinase in S. cerevisiae results in sensitivity to the compound fluoro-deoxyuracil (FUDR). The cellular toxicity of FUDR is dependent on the presence of two enzymes involved in pyrimidine metabolism: thymidine kinase (Tdk) and thymidilate synthetase (ThyA). Tdk converts FUDR to fluoro-dUMP (F-dUMP) which is a covalent inhibitor of ThyA and the basis for counter selection in a variety of eukaryotic organisms (Czako, M., and L. Marton, (1994) Plant Physiol 104: 1067-1071; Gardiner, D. M., and B. J. Howlett, (2004) Curr Genet 45:249-255; Khang, C. H., et al, (2005) Fungal Genet Biol 42:483-492; Szybalski, W. (1992) Bioessays 14:495-500). [0424] The HSV-TDK expression cassette was independently fused to two commonly used dominant selectable markers which confer resistance to the drugs G418 (Kan) or nourseothricin (Nat) (Goldstein, A.L. and McCusker, J.H., Yeast 75:1541-1553 (1999)). Transformation of both double expression cassettes, referred to as KT-MX and NT-MX, enables positive selection for integration into both chromosomes as illustrated in Figure 28A. The transformed deletion assembly contains four PCR products, a 5'flank(pl) which is homologous upstream of the target site, KT-MX cassette(p2), NT-MX cassette(p3), and a 3' flank(p4) homologous downstream of the target site. Each component is amplified individually using primers which create homologous overlapping extensions of each PCR product. See Tables 7 and 8. The bent dashed lines in Figure 28A represent homology between the KT/NT-MX cassettes and the 5' flank and the bent solid lines represent homology with the 3' flank. For each round of engineering, PCR amplicons of upstream and downstream regions flanking the target site are designed to contain homologous tails for both the KT-MX and NT-MX cassettes. Both the flanks and the markers are transformed followed by selection on YPD medium containing both G418 and Nat. See Figure 28B. Figure 28B shows a schematic of the chromosome after replacement of the target site with KT-MX and NT-MX.

[0425] After each engineering step taken in the construction of M3625, all markers are subsequently deleted and/or replaced with a desired expression cassette (Mascoma Assembly) resulting in a strain free of antibiotic markers (Figure 29). Figure 29 demonstrates that the transformed Mascoma Assembly contains a quantity of PCR products which is dependent on the desired engineering event (pX), a 5'flank(pl) which homologous upstream of the target site and a 3' flank (p4) homologous downstream of the target site. Each component is amplified individually using primers which create homologous overlapping extensions. The overlapping bent lines in Figure 29 represent homology at the end of those PCR products. Figure 29B shows a schematic of a chromosome following selection on FUDR and replacement of genetic markers with the Mascoma assembly. Confirmation of marker removal was evaluated by southern blot, PCR and dilution plating onto selective medium as described below.

[0426] Four loci were modified during the construction of M3625. The integration procedure strategy described above was used at the FDHl, GPDl and GPD2 loci using the Mascoma Assemblies listed in Table 6. Detailed molecular maps depicting components of each Mascoma Assembly are provided in Figures 30-37.

Table 6. Genetic modifications contained in M3625.

Table 7. Primers used for the creation of strain M3625.

Table 8. Sequences of Primers used for creation of strain M3625

Primer Sequence SEQ ID

NO

XI 1824 aagcctacaggcgcaagataacacatcac 110

X15546 ggacgaggcaagctaaacagatctctagacctactttatattatcaatatttgtgtttg 111 X15380 taggtctagagatctgtttagcttgc 112

X15382 gagactacatgatagtccaaaga 113

X15547 ccgtttcttttctttggactatcatgtagtctcatttattggagaaagataacatatca 114

XI 1829 ctcagcattgatcttagcagattcaggatctaggt 115

X11816 gcagtcatcaggatcgtaggagataagca 116

X15548 ggacgaggcaagctaaacagatctctagacctatgataaggaaggggagcg 117

aaggaaaa

X15549 ccgtttcttttctttggactatcatgtagtctcctctgatctttcctgttgcctctttt 118

X11821 tcacaagagtgtgcagaaataggaggtgga 119

X16096 catggtgcttagcagcagatgaaagtgtca 120

X15554 ggacgaggcaagctaaacagatctctagacctaattaattttcagctgttatttcgatt 121

X15555 ccgtttcttttctttggactatcatgtagtctcgagtgattatgagtatttgtgagcag 122

XI 1845 ttacttgtgaaactgtctccgctatgtcag 123

X15559 ggaaggcaccgatactagaactccg 124

X15550 gggacgaggcaagctaaacagatctctagacctaattaattttcagctgttattttgat 125

X15552 ccgtttcttttctttggactatcatgtagtctcgagtgattatgagtatttgtgagcag 126

X15553 accagcgtctggtggacaaacggccttcaac 127

[0427] Genotyping and Sequencing of MA0370

[0428] To confirm that FDH1 was deleted after insertion of MA370, PCR products were amplified from M2390 and M3625 genomic DNA using primers X17826 and X16944. The expected results are listed in Table 10 and the sequences of the primers used are listed in Table 11. A molecular map depicting the MA0370 integration site is shown in Figure 30. The molecular map depicts the location of flanks used to remove the KT-MX and NT-MX markers and the position of primers used for genotyping. See Figure 30 (5' flank, S. cerevisiae FDH1 upstream flanking region; 3' flank - S. cerevisiae FDH1 downstream flanking region. Region AA - amplified and sequenced chromosomal DNA region). Primer pair X15556/X15871 was used for the FDH1 5' Flank and primer pair X15870/ X15553 was used for the FHDl 3' Flank to create the assembly shown in Figure 30. Sequences for the primers for assembly used are found in Table 9. An agarose gel image showing PCR products used to determine genotype is shown in Figure 31 (lane 1 : 1KB ladder; lane 2: M2390 (X17826 X 16944); lane 3: M3625 (X17826/X16944)) (see Table 11).

Table 9. Primers used to create the MA0370 integration site.

[0429] In order to determine the exact DNA sequence of the M3625 MA0370 site, region

AA was amplified from genomic DNA of M3625 strain in 5 independent PCR reactions. All PCR products were purified and sequenced by the Sanger method at the Dartmouth College Sequencing facility.

Table 10. Primers and summary of results of MA0370 genotyping.

Table 11. Sequence of primers used for MA0370 genotyping.

[0430] Genotyping and Sequencing of MA0280

[0431] To confirm that MA280 was inserted at the FDH2 site, PCR products were amplified from M3625 genomic DNA. The primers and expected genotyping results are listed in Table 13. Sequences of the primers used for genotyping and sequencing MA0280 are listed in Table 14. A molecular map depicting the MA0280 integration site is shown in Figure 32. The molecular map depicts the location of flanks used to replace the KT-MX and NT -MX markers and insert the MA0280 expression cassette. The position of primers used for genotyping for genotyping are indicated on the map. See Figure 32 (Feature description on map of MA0280 site of the M3625 strain; FDH2 5' flank - S. cerevisiae FDH2 upstream flanking region; PFKlp - S.cerevisiae PFK1 gene promoter; ADHE - Bifidobacterium adolescentis ADHE coding gene; HXT2t - S.cerevisiae HXT2 gene terminator; ENOlp - S.cerevisiae ENOl gene promoter; PFLB - Bifidobacterium adolescentis PFLB coding gene; ENOlt - S. cerevisiae ENOl gene terminator; ADHlp - S. cerevisiae ADH1 gene promoter; PFLA - Bifidobacterium adolescentis PFLA coding gene; PDClt - S. cerevisiae PDC1 gene terminator; FBAlt - S. cerevisiae FBA1 gene terminator; TPIlp - S. cerevisiae TPI1 gene promoter; FDH2 3' flank - S.cerevisiae FDH2 downstream flanking region. Regions BA-BE, amplified and sequenced chromosomal DNA regions). Primer pair X16096/X 17243 was used for the FDH2 5' Flank, primer pair X16738/X16620 was used for pPFKl-ADH-HXT2, primer pair X16621/X13208 was used for pENOl-PFL-ENOlt, primer pair X13209/X17242 was used for pADHl-PFL-PDClt, primer pair XI 7241 /XI 6744 was used for pTPI-ADH- FBAltrc, and primer pair X17244/X11845 was used for the FDH2 5' Flank to create the assembly shown in Figure 32. Sequences for the primers used to create the assembly shown in Figure 32 are found in Table 12. An agarose gel image showing PCR products used to genotype and sequence the MA0280 site is shown in Figure 33 (lane 1 : 1KB ladder; lane 2: M3625 (17413 /15810); lane 3: M3625 (17834 /14554); lane 4: M3625 (16291/15229); lane 5: M3625 (16503/11317); lane 6: (16241/16946) lane 7: 1KB ladder) (see Table 14).

Table 12. Primers used to create the MA0280 integration site.

Primer Sequence SEQ ID NO

X16096 catggtgcttagcagcagatgaaagtgtca 120

XI 7243 tagttagatcagggtaaaaattatagatgaggtattaattttcagct 131

gttatttcgatt

X16738 ctaatcaaatcgaaataacagctgaaaattaatacctcatctataat 132

ttttaccctgat X16620 tcggatcagtagataacccgcctagaagactaggttacattgaaa 133 atacagtaaatggt

X16621 tggtggaaccatttactgtattttcaatgtaacctagtcttctaggcg 134

ggttatctact

X13208 ccgaaatattccacggtttagaaaaaaatcggaggtttagacattg 135

gctcttcattgag

X13209 aagctcaatgaagagccaatgtctaaacctccgatttttttctaaac 136

cgtggaatattt

XI 7242 acatcatcttttaacttgaatttattctctagctttcaatcattggagc 137

aatcatttta

XI 7241 gtccatgtaaaatgattgctccaatgattgaaagctagagaataa 138

attcaagttaaaag

XI 6744 aaaaacttctgctcacaaatactcataatcactcctacttattcccttc 139

gagattatatc

XI 7244 gttcctagatataatctcgaagggaataagtaggagtgattatgagta 140

tttgtgagcag

XI 1845 ttacttgtgaaactgtctccgctatgtcag 141 2] In order to determine exact DNA sequence of the M3625 MA0280 site, regions

BA-BE were amplified from genomic DNA of M3625 strain in 5 independent PCR reactions. All PCR products were purified and sequenced by the Sanger method at the Dartmouth College Sequencing facility.

Table 13. Primers and summary of results of MA0280 genotyping.

Table 14. Sequence of primers used for genotyping MA0280. Primer Sequence SEQ ID NO

17413 ggattcttcgagagctaaga 154

15810 gacttgcagggtaggctagctagaatt 155

17834 gctgcttcgaggtattgaca 156

14554 ggctcttcattgagcttagaaccc 157

16291 aactggaccgatcttattcgt 158

15229 agtccactgcggagtcatttcaaag 159

16503 ctgccagcgaattcgactctgcaat 160

11317 cagtcgctgtagtgagcgacagggtagtaa 161

16241 ctttgcattagcatgcgta 162

16946 taggtcgagaccagaatgcatgt 163

[0433] Genotyping and Sequencing of MA0289

[0434] To confirm that MA0289 was inserted at the GPD2 site, PCR products were amplified from M3625 genomic DNA. The primers and expected genotyping results are listed in Table 16. Sequences for the primers used for genotyping MA0289 are listed in Table 17. A molecular map depicting the MA0289 integration site is shown in Figure 34. The molecular map depicts the location of flanks used to replace the KT-MX and NT -MX markers and insert the MA0280 expression cassette. The position of primers used for genotyping are indicated on the map. See Figure 34 (Feature description on map of MA0280 site of the M3625 strain; GPD2 5' flank - S. cerevisiae GPD2 upstream flanking region; ADHE - Bifidobacterium adolescentis ADHE coding gene; HXT2t - S. cerevisiae HXT2 gene terminator; PDClt - S. cerevisiae PDC1 gene terminator; PFLA - Bifidobacterium adolescentis PFLA coding gene; ADHlp - S. cerevisiae ADH1 gene promoter; ENOlt - S. cerevisiae ENOl gene terminator; PFLB - Bifidobacterium adolescentis PFLB coding gene; ENOlp - S. cerevisiae ENOl gene promoter; FBAlt - S. cerevisiae FBA1 gene terminator; TPIlp - S. cerevisiae TPI1 gene promoter; GPD2 3' flank - S. cerevisiae GPD2 downstream flanking region; Regions CA-CF - amplified and sequenced chromosomal DNA regions). Primer pair X15473 X17460 was used for the GPD2 5' Flank, primer pair X17459/X17289 was used for ADH-HXT2, primer pair X17290/X13209 was used for pADHl-PFL-PDCltrc, primer pair X13208/X15735 was used for pENOl-PFL-ENOltrc, primer pair X15736/X17457 was used for pTPI-ADH- FBAltrc, and primer pair X17458/X15476 was used for the GPD2 3' Flank to create the assembly shown in Figure 34. Sequences for the primers used to create the assembly shown in Figure 34 are found in Table 15. An agarose gel image showing PCR products used to genotype and sequence the MA0280 site is shown in Figure 35 (lane 1 : 1KB ladder; lane 2: M3625 (17413 /15810); lane 3: M3625 (17834 /14554); lane 4: M3625 (16291/15229); lane 5: M3625 (16503/11317); lane 6: (16241/16946); lane 7: 1KB ladder).

Table 15. Primers used to create the MA0289 integration site.

Primer Sequence SEQ ID NO

X15473 agtcatcaggatcgtaggagataagc 142

X17460 agaagataatatttttatataattatattaatcctaatcttcatgtag 143

atctaattctt

X17459 cctttccttttccttcgctccccttccttatcaatggcagacgcaa 144

agaagaaggaaga

X17289 gtccatgtaaaatgattgctccaatgattgaaagttacattgaaa 145

atacagtaaatggt

X17290 tggtggaaccatttactgtattttcaatgtaactttcaatcattgga 146

gcaatcatttta

X13208 ccgaaatattccacggtttagaaaaaaatcggaggtttagacattg 135

gctcttcattgag

X13209 aagctcaatgaagagccaatgtctaaacctccgatttttttctaaac 136

cgtggaatattt

X15735 catcttttaacttgaatttattctctagcctagtcttctaggcgggttat 147

ctactgat

X15736 agataacccgcctagaagactaggctagagaataaattcaagtta 148

aaagatgatgttga

X17457 tgggggaaaaagaggcaacaggaaagatcagagctacttattc 149

ccttcgagattatatc

X17458 gttcctagatataatctcgaagggaataagtagctctgatctttcct 150

gttgcctcttttt

X15476 gtagatctgcccagaatgatgacgtt 151 [0435] In order to determine exact DNA sequence of the M3625 MA0289 site, regions

CA-CF were amplified from genomic DNA of M3625 strain in 5 independent PCR reactions. All PCR products were purified and sequenced by the Sanger method at the Dartmouth College Sequencing facility.

Table 16. Primers and summary of results of MA0289 genotyping.

Table 17. Sequence of primers used for genotyping MA0289.

[0436] Genotyping and Sequencing of MA0317

[0437] To confirm that MA0317 was inserted at the FCY1 site, PCR products were amplified from M3625 genomic DNA. The primers and expected genotyping results are listed in Table 19. Sequences for the primers used to genotype MA0317 are listed in Table 20. A molecular map depicting the MA0317 integration site is shown in Figure 36. The molecular map depicts the location of flanks used to replace the FCYl gene with MA0317 and the position or primers used for genotyping. See Figure 36 (Feature description on the map of MA0371 site of M3625; FCYl 5' flank - S. cerevisiae FCYl upstream flanking region; ENOlp - S. cerevisiae ENOl gene promoter; AE9 - S. fibuligera glu 0111 coding gene; ENOlt - S. cerevisiae ENOl gene terminator; PDClt - S. cerevisiae PDC1 terminator; ADHlp - S. cerevisiae ADH1 gene promoter; FCYl 3' flank - S. cerevisiae FCYl downstream flanking region; Regions FA-FE, amplified and sequenced chromosomal DNA regions). Primer pair X18868 X18844 was used for the FCY 5' Flank, primer pair X18845/X15464 was used for pENO-AE9-EN01t, primer pair X15465/X11750 was used for pADHl-AE9-PDClt, and primer pair X15479/X18869 was used for the FCY 3' Flank to create the assembly shown in Figure 36. Sequences for the primers used to create the assembly shown in Figure 36 are found in Table 18. An agarose gel image showing PCR products used to determine genotype is shown in Figure 37 (lane 1 : 1KB ladder; lane 2: M3625 (13092/17586); lane 3: M3625 (10871/14554); lane 4: M3625 (16291/17887); lane 5: M3625 (16640/16509); lane 6: M3625 (13246/13095); lane 7: 1KB ladder).

Table 18. Primers used to create the MA0317 integration site.

Primer Sequence SEQ ID NO

X18868 gccaaagtggattctcctactcaagctttgc 184

X18844 tcggatcagtagataacccgcctagaagactagtagctatgaaattt 185

ttaactctttaa

X18845 agccagcttaaagagttaaaaatttcatagctactagtcttctaggcg 186

ggttatctact

X15464 gtccatgtaaaatgattgctccaatgattgaaagaggtttagacattg 187

gctcttcattg

X15465 ctaagctcaatgaagagccaatgtctaaacctctttcaatcattggag 188

caatcatttta

XI 1750 ataaaattaaatacgtaaatacagcgtgctgcgtgctcgatttttttcta 189

aaccgtgga

X15479 agcacgcagcacgctgtatttacgta 190

X18869 agatcctgtggtagtgctgtctgaacagaa 191 - I l l -

[0438] In order to determine exact DNA sequence of the M3625 MA0317 site, regions

FA-FE were amplified from genomic DNA of M3625 strain in 5 independent PCR reactions. All PCR products were purified and sequenced by the Sanger method at the Dartmouth College Sequencing facility.

Table 19. Primers and summary of results of MA0317 genotyping.

Table 20. Sequence of primers used for genotyping MA0317.

[0439] Figure 38 shows the results of starch assay demonstrating starch degrading activity in M3625. The assay was performed as described in copending International Appl. No. PCT/US2011/039192, incorporated by reference herein in its entirey.

Western blot protein detection: [0440] Anti-PflA, anti-PflB, anti-GA (AE9) and anti-AdhE antibodies:

[0441] In an effort to detect the presence of and help characterize a number of enzymes engineered into the yeast strain, polyclonal antibodies were produced in rabbits at Lampire Biological Products, PipersviUe, PA, against synthesized peptides with sequence similarity to the engineered proteins. Table 11 depicts the peptides that were used as immunogens for the rabbits:

Table 21. Immunogens used for antibody production

Protein Immunogen SEQ ID NO

Sf GA (AE9) intact purified protein 106

DNKN RYKI NGNYKAGC NSGKKHIVESPQLSSRGGC CDHI DDNGQLTEEIN RYTG

Ba pfIA I CQ.N PDTWKM RDGKP VYYE | 107

GLTSSEENVENVAKIC

Ba pfIB I WEGFTEGNWQKDIDVRDC | 108

I KQRDKDSI PYRN DFTECPEC

! CNTITPDGLGRDEEEERIGN

Ba AdhE DAKKKEEPTKPTPEEKLC 109

CKNLGVNPGKTPEEGVEN

CGSYGGNSVSGVNQAVN

[0442] For all of the synthesized peptides a terminal Cys was added for conjugation.

Both the peptides and the purified GA protein were conjugated to KLH prior to injection into the rabbit. A 50 day protocol was used for antibody production with ELISA monitoring of the various bleeds against the immunogen. After testing these polyclonal antibodies in a Western blot against the lysate from the engineered yeast strains, serum from the positive rabbits was purified using a Protein G column. The purified antibodies were dialyzed into PBS, concentration was determined by absorbance at 280 nm and the antibodies were used for further evaluation of the strains. Upon evaluation by SDS- PAGE, the antibodies appeared to be >90% pure. [0443] Antibodies raised against the synthesized peptides were used in Western blot detection of each engineered protein in cell extracts and culture supernatants as described below.

[0444] Strain growth conditions:

[0445] Cells were plated from freezer stock on YPD (20g/L peptone, lOg/L yeast extract,

20g/L glucose) agar for 48 hours and used to inoculate 25mL YPD (20g/L peptone, lOg/L yeast extract, 20g/L glucose) in a 50mL culture tube. Cells were grown aerobically for 8 hours at 35°C with shaking at 250rpm, then lmL was removed to inoculate a sealed, C02 purged serum bottle containing 50mL YPD (20g/L peptone, lOg/L yeast extract, 20g/L glucose) with 7mg/L ergosterol, 289mg/L ethanol and 544mg/L Tween 80. These cultures were then grown anaerobically overnight (~16h) at 35°C with shaking at 250rpm. Cells were harvested by centrifugation and washed with 25mL deionized water. The resulting wet cell pellets were used for Western blot detection of PflA, PflB and AdhE.

[0446] Aerobic cultures used to inoculate the serum bottles were returned to the shaking incubator for an additional 40 hours. At the end of incubation, cells were pelleted by centrifugation and the supernatant was recovered and concentrated ~10X using a lOkDa molecular weight cut-off (MWCO) filter membrane. The resulting concentrates were used for Western blot detection of extracellular AE9 glucoamylase.

[0447] Cell lysis and sample preparation:

[0448] For Western blots of PflB and AdhE, cells were homogenized by mechanical disruption with 0.5mm diameter beads and agitation at 4800rpm in a bead beater. ΙΟΟμί of wet cells were added to homogenization buffer containing ImM phenylmethanesulfonylfluoride (PMSF), 2mM dithiothreitol (DTT) and 1% dimethyl sulfoxide (DMSO) in lOOmM sodium phosphate buffer pH 7.4. Cells were agitated for 6 cycles of 10 seconds each, cooling on ice between cycles. Cell debris was pelleted by centrifugation and supernatant was recovered. 15μΙ, of the resulting supernatant was added ίο15μΕ 2X concentrated SDS-PAGE sample buffer with 50mM DTT and loaded onto a 4-20% Tris-Glycine SDS-PAGE gel.

[0449] For Western blot detection of PflA, cells were lysed by adding 40μΙ, wet cells to

40μΙ _^ 2X concentrated SDS-PAGE sample buffer with 50mM DTT. The mixture was then incubated at room temperature for 30 minutes, followed by heating at 100°C for 2 minutes. Cells were pelleted by centrifugation and 30μί of the supernatant was loaded onto a 4-20% Tris-Glycine SDS-PAGE gel.

[0450] For AE9 analysis, 15μί of concentrated aerobic culture supernatant was added to

15μί 2X concentrated SDS-PAGE sample buffer with 50mM DTT and loaded onto a 4- 20% Tris-Glycine SDS-PAGE gel.

[0451] Following gel electrophoresis, proteins were transferred to a polyvinylidine fluoride (PVDF) membrane and blocked overnight with Tris buffered saline (TBS; lOmM Tris, 150mM sodium chloride pH 7.5) containing 2% weight by volume (w/v) bovine serum albumin (BSA). The blocking solution was then removed, and primary peptide antibodies were diluted to approximately 2μg/mL in Tris buffered saline with Tween 20 (TBST; TBS with 0.1% v/v Tween 20) and added to each membrane. After a 1 hour incubation, the primary antibody was discarded and the membrane was washed for 3 periods of 5 minutes each in lOmM Tris, 500mM sodium chloride, 0.1 % Tween 20 pH 7.5 (THST). The secondary antibody, goat anti-rabbit with horseradish peroxidase label, was diluted 1 :7500 in TBST, added to the blot and incubated for 1 hour. The secondary antibody was then discarded and the blot was again washed with THST for 3 periods of 5 minutes each. The wash solution was then discarded, enhanced chemiluminescence (ECL) substrate was added, and the blot was read by a series of composite exposures using a gel imaging camera.

[0452] As shown in Figure 39, for anti- PflA, PflB and AdhE primary antibodies, bands of approximately the correct molecular weight were detected in each experimental strain, whereas no band was detected in the background control strain (M2390). For anti-AE9, bands were detected in strains engineered to express the protein (M3625 and M3680) but were absent in other strains. See Figure 39. There appeared to be two distinct bands for PflB, which may indicate oxygenic cleavage of the protein due to aerobic cell lysis conditions.

[0453] Pyruvate formate lyase activity assay:

[0454] Pyruvate formate lyase (PflB) is activated in the absence of oxygen by Pfl activase

(PflA) and catalyzes the reaction of pyruvate and CoA to formate and acetyl-CoA. The activity of PflB was measured in cell extracts by measuring formate production when extracts were added to a reaction mixture containing pyruvate, CoA and DTT. [0455] Strain growth conditions:

[0456] Cells were plated from freezer stock on YPD (20g/L peptone, lOg/L yeast extract,

20g/L dextrose) agar for 48 hours and used to inoculate 25mL YPD (20g/L peptone, lOg/L yeast extract, 20g/L dextrose) in a 50mL culture tube. Cells were grown aerobically for 8 hours at 35°C with shaking at 250rpm, then lmL was removed to inoculate a sealed, C02 purged serum bottle containing 50mL YPD (20g/L peptone, lOg/L yeast extract, 20g/L dextrose)with 7mg/L ergosterol, 289mg/L ethanol and 544mg/L Tween 80. These cultures were then grown anaerobically overnight (~16h) at 35°C with shaking at 250rpm. Cells were harvested by centrifugation and washed with 25mL deionized water in an anaerobic chamber.

[0457] Cell lysis and sample preparation:

[0458] Cells were homogenized in an anaerobic chamber by mechanical disruption with

0.5mm diameter beads and agitation at 4800rpm in a bead beater. ΙΟΟμί of wet cells were added to homogenization buffer containing ImM PMSF, 2mM DTT and 1% DMSO in lOOmM sodium phosphate buffer pH 7.4. Cells were agitated for 6 cycles of 10 seconds each, cooling on ice between cycles. Cell debris was pelleted by centrifugation at 14,100 x g for 10 minutes and supernatant was recovered and clarified by filtration through a 0.22μιη filter membrane. The resulting extract was used directly in the activity assay.

[0459] Pfl activity assay:

[0460] A 2X concentrated assay substrate mixture consisted of 20mM sodium pyruvate,

0.1 ImM Co A and 20mM DTT. Reagents were weighed out, brought into an anaerobic chamber and added to lOmL of lOOmM sodium phosphate buffer pH 7.4 which had been thoroughly degassed. ΙΟΟμί of cell extract was added to ΙΟΟμί of the concentrated assay mixture and incubated at ambient temperature (~29°C) for 30 minutes. Samples were then removed from the anaerobic chamber and heated in a heating block at 100°C for 90 seconds followed by cooling on ice to precipitate protein. Precipitate was removed by centrifugation at 15,000 x g for 10 minutes. The resulting supernatant was analyzed for formate concentration using the formic acid assay kit available from Megazyme International Ireland, Bray, Co. Wicklow, Ireland. [0461] Remaining cell extracts were diluted 1 :8 in lOOmM sodium phosphate buffer pH

7.4 and assayed for total protein content using the BCA total protein determination method. Formate concentrations of the Pfl assay samples were normalized to the total protein concentration of the sample.

[0462] As shown in Figure 40, experimental strains with engineered Pfl activity (M3465,

M3625, M3679, and M3680) showed significantly higher amounts of formate present after incubation with the reaction mixture than the background control strain (M2390).

[0463] Alcohol dehydrogenase E (AdhE) enzymatic activity assays

[0464] AdhE is an intracellular bi-functional enzyme catalyzing the formation of ethanol from acetyl-CoA by way of acetaldehyde as an intermediate. This is accomplished by an acetaldehyde dehydrogenase activity and an alcohol dehydrogenase activity working in series. Saccharomyces cerevisiae strains have native alcohol dehydrogenase (Adh) activity; the intent of these activity assays is to show that Adh activity is retained by the engineered strains, and an additional acetaldehyde dehydrogenase activity (from AdhE) is present.

[0465] Alcohol Dehydrogenase Activity:

[0466] As mentioned above, Bifidobacterium adolecentis bifunctional alcohol dehydrogenase (AdhE) has 2 primary functions. One function is the conversion of acetaldehyde to ethanol. This reversible reaction utilizes NADH as a cofactor. In order to evaluate the presence of this enzyme and ensure that it has the desired activity, an assay was developed to evaluate the reverse reaction in which ethanol is converted to acetaldehyde. See Figure 49. The rate of the reaction is monitored by NADH absorbance at 340 nm.

[0467] Strain growth conditions:

[0468] Strains were patched from freezer stock onto a YPD (20g/L peptone, lOg/L yeast extract, 20g/L dextrose) agar plate and incubated overnight at 35°C. From that plate, 50mL shake tubes with 25 mL YPD (20g/L peptone, lOg/L yeast extract, 20g/L dextrose) were inoculated and incubated at 35°C, 250 rpm overnight. The cultures were centrifuged at 5000 rpm x 5 min at 4C, washed with deionized (DI) water and centrifuged at 5000 rpm x 5 min at 4C, washed a second time with DI water and centrifuged at 5000 rpm x 5 min at 4C, and then put on ice.

[0469] Cell lysis and sample preparation:

[0470] ΙΟΟμί of wet cell pellet was pipetted into a Zymo Research BashingBead 0.5mm

Tubes along with 500μΙ. lOOmM Na2P04, 2.5mM MgC12, 0.5mM CaC12 pH 7.4 buffer and lOOmM phenylmethylsulfonyl fluoride (PMSF). The cells were lysed by mechanical disruption using a MP FastPrep-24 set to run at 4.0m/s for 10 seconds three times with cooling on ice for 10 seconds between each run. This was repeated three times with chilling on ice for one minute in between each run. Each tube was then centrifuged for 10 minutes at 15,000 rpm using an Eppendorf centrifuge 5424. The supernatant was removed and transferred to 2mL tubes. ΙμΙ, of New England Bio labs DNAse I was added to each tube. The tubes were inverted and placed into an incubator set at 37°C for 30 min. The tubes were removed from the incubator and the samples were transferred to 0.22μιη filter centrifuge tubes which were centrifuged for 2 min at 10,000 rpm. 50μΙ _^ of sample was pulled and diluted with 450μί lOOmM Na ₂ P0 ₄ pH 7.4 in separate sample tubes and then placed on ice.

[0471] Alcohol dehydrogenase activity assay:

[0472] The assay used to determine alcohol dehydrogenase activity of AdhE was adapted from the method of Vallee, B.L. and Hoch, F.L., Proc Natl Acad Sci USA (1955) 41(6): 327-338. ΙΟΟμΕ 0.1M Na4P207 pH 9.6 buffer, 32μΕ 2M ethanol, and 1.66 L 0.025M NAD ⁺ were added to each well in a 96 well plate. Once the lysate was added to the reaction mixture, the total volume of reaction was equivalent to 153.66μΕ resulting in final concentrations of 65mM Na ₄ P ₂ 0 ₇ pH 9.6 buffer, 416.5mM ethanol, and 0.27mM NAD ⁺ . To begin the reaction, 20μί of 1 : 10 diluted lysate was pipetted into each well and the absorbance at 340nm was observed and recorded over 1.7 min using Spectramax M2 and Softmax software. Each sample was done in duplicate to ensure reproducibility. Thermo Scientific's BCA Protein Assay Kit was used to measure total protein concentration from the lysate generated. This data was used to normalize the data generated from the actual reaction during analysis.

Table 22. Data for alcohol dehydrogenase activity assay Average activity

(μιηοΙ Standard % p-

NADH/min/mg) Deviation %CV Change value

M2390 599 15.6 2.6

M3465 659 16.2 2.5 10 0.1942

M3625 999.5 132 13.2 66.9 0.0108

M3679 755 82 10.9 26 0.0603

M3680 698 29.7 4.3 16.5 0.0996

Alcohol dehydrogenase activities of engineered strains, p-value was based on a one-tailed T-test.

[0473] The background strain, M2390, performed as expected in this assay. Although it did not have AdhE engineered into its genome, it still expressed wild-type alcohol dehydrogenase and thus was active in the alcohol dehydrogenase assay. Other strains with AdhE engineered into their genomes should have expressed the bi-functional enzyme and should have been more active given the total protein concentration was equal in each sample used in the assay. With a p-value of <0.05, M3625 demonstrated a statistically significant higher activity than the background strain. However, the other strains have a p-value >0.05 indicating that they are within error of the background strain even though there was an increase in activity as shown by the % change over the background activity. See Table 22. After normalizing the protein concentrations, a graphical representation of the data shows that each strain was more active than the background strain M2390 during a 1.7 minute reaction period. Figure 41 shows the activity of each strain plotted in μιηοΐ NADH/mg total protein vs. time during a 1.7 minute reaction.

[0474] Based on these results, the assay showed alcohol dehydrogenase activity in all strains. However, M2390 is less active and slower at converting NAD ⁺ to NADH than the other strains indicating that the engineered AdhE is present in each strain and it appears to be functioning properly.

[0475] Acetaldehyde Dehydrogenase Activity:

[0476] The second activity of AdhE is the reversible reaction converting acetaldehyde to acetyl coenzyme A. This activity is not native to Saccharomyces cerevisiae strains, and should only be present in the engineered strains. In order to evaluate the presence of this enzyme and ensure that it has the desired activity, an assay was developed to measure the conversion of acetaldehyde to acetyl CoA by AdhE. The rate of the reaction is monitored by NADH absorbance at 340 nm. A diagram of the reaction is provided in Figure 50. [0477] Strain growth conditions:

[0478] Strains were patched from a freezer stock onto a YPD (20g/L peptone, lOg/L yeast extract, 20g/L dextrose) agar plate and incubated overnight at 35°C. From that plate, 50mL shake tubes with 25 mL YPD (20g/L peptone, lOg/L yeast extract, 20g/L dextrose) were inoculated and incubated at 35°C, 250 rpm overnight. The cultures were centrifuged at 5000 rpm x 5 min at 4°C, washed with DI water and centrifuged at 5000 rpm x 5 min at 4C, washed a second time with DI water and centrifuged at 5000 rpm x 5 min at 4°C, and then put on ice.

[0479] Cell lysis and sample preparation:

[0480] ΙΟΟμί of wet cell pellet was pipetted into a Zymo Research BashingBead 0.5mm

Tubes along with 500μΙ. lOOmM Na ₂ P0 ₄ , 2.5mM MgCl ₂ , 0.5mM CaCl ₂ pH 7.4 buffer and lOOmM phenylmethylsulfonyl fluoride (PMSF). The cells were lysed by mechanical disruption using a MP FastPrep-24 set to run at 4.0m/s for 10 seconds three times with cooling on ice for 10 seconds between each run. This was repeated three times with chilling on ice for one minute in between each run. Each tube was then centrifuged for 10 minutes at 15,000 rpm using an Eppendorf centrifuge 5424. The supernatant was removed and transferred to 2mL tubes. Ι μΙ, of New England Bio labs DNAse I was added to each tube. The tubes were inverted and placed into an incubator set at 37°C for 30 min. The tubes were removed from the incubator and the samples were transferred to 0.22μιη filter centrifuge tubes which were centrifuged for 2 min at 10,000 rpm using the Eppendorf centrifuge 5424. 50μΙ, of sample was pulled and diluted with 450μί lOOmM Na ₂ P0 ₄ pH 7.4 in separate sample tubes and then were placed on ice.

[0481] Acetaldehyde dehydrogenase activity assay:

[0482] 800μΕ 50mM Na ₄ P ₂ 0 ₇ pH 9.6, 50μΕ 0.025M NAD+, 50μΕ 1M acetaldehyde, and

50μΙ, 1 : 10 diluted lysate were added to a Plastibrand micro UV-cuvette. The cuvette was placed into a Shimadzu UV-1700 set to read absorbance at 340nm. 50μΙ _^ of 2mM CoA were pipetted into the cuvette which was then mixed by gently pipetting the contents of the cuvette and the absorbance was monitored for 5 minutes. The resulting final concentrations of each reagent were 40mM Na ₄ P ₂ 0 ₇ pH 9.6, 1.25mM NAD ⁺ , 50mM acetaldehyde, and O. lmM CoA. Each sample was done in duplicate to ensure reproducibility. Thermo Scientific 's BCA Protein Assay Kit was used to measure total protein concentration from the lysate generated. This data was used to normalize the data generated from the actual reaction during analysis.

[0483] Data for alcohol dehydrogenase (acetaldehyde dehydrogenase activity) assay is shown in Table 23. Note the lysate used in this assay was the same lysate used in the alcohol dehydrogenase assay detailed in the previous section.

Table 23. Acetaldehyde Dehydrogenase Activity

Average activity

(μιηοΙ Standard

NADH/min/mg) Deviation %CV p-value

M2390 0 0 0.0

M3465 163 24 14.7 0.0054

M3625 115 7.07 6.2 0.0009

M3679 106 9.97 9.4 0.0022

M3680 177 7.07 4.0 0.0004

Acetaldehyde dehydrogenase activity. p-value was based on a one-tailed T-test.

[0484] The background strain, M2390, performed as expected in this assay. The wild- type strain should have no acetaldehyde dehydrogenase activity, as demonstrated by this assay. The other strains with AdhE engineered into their genomes should have expressed the protein and had acetaldehyde dehydrogenase activity. This activity was observed in all of the engineered strains (M3465, M3625, M3679, and M3680) with minimal error and a p-value of <0.05.

[0485] Formate Dehydrogenase Activity

[0486] In strains M3465, M3625, M3679, and M3680 formate dehydrogenase was knocked out of the genome in the hopes to balance redox with the various engineering steps that were undertaken. The background strain, M2390, should have the gene intact. To ensure that the native Saccharomyces cerevisiae formate dehydrogenase gene was removed, an enzymatic assay was developed. Formate dehydrogenase catalyzes the conversion of formate to carbon dioxide at the expense of NAD ⁺ .

Formate + NAD ⁺ → C02 + NADH

FDH

[0487] Enzymatic activity can be monitored by measuring NADH formation at 340nm. [0488] Strain growth conditions:

[0489] M2390, M3465, M3625, M3679, and M3680 were patched from a freezer stock onto a YPD (20g/L peptone, lOg/L yeast extract, 20g/L dextrose) agar plate and incubated overnight at 35°C. From that plate, 50mL shake tubes with 25 mL YPD + 24mM Sodium Formate were inoculated and incubated at 35°C, 250 rpm overnight (20g/L peptone, lOg/L yeast extract, 20g/L dextrose). The cultures were centrifuged at 5000 rpm x 5 min at 4°C, washed with DI water and centrifuged at 5000 rpm x 5 min at 4°C, washed a second time with DI water and centrifuged at 5000 rpm x 5 min at 4C, and then put on ice.

[0490] Cell lysis and sample preparation:

[0491] ΙΟΟμί of wet cell pellet was pipetted into a Zymo Research BashingBead 0.5mm

Tubes along with 500μΙ. lOOmM Na ₂ P0 ₄ , 2.5mM MgCl ₂ , 0.5mM CaCl ₂ pH 7.4 buffer and lOOmM phenylmethylsulfonyl fluoride (PMSF). The cells were lysed by mechanical disruption using a MP FastPrep-24 set to run at 4.0m/s for 10 seconds three times with cooling on ice for 10 seconds between each run. This was repeated three times with chilling on ice for one minute in between each run. Each tube was then centrifuged for 10 minutes at 15,000 rpm using an Eppendorf centrifuge 5424. The supernatant was removed and transferred to 2mL tubes. Ι μΙ, of New England Bio labs DNAse I was added to each tube. The tubes were inverted and placed into an incubator set at 37°C for 30 min. The tubes were removed from the incubator and the samples were transferred to 0.22μιη filter centrifuge tubes which were centrifuged for 2 min at 10,000 rpm using the Eppendorf centrifuge 5424.

[0492] Formate dehydrogenase activity assay:

[0493] 800μΕ 62.5mM K ₂ P0 ₄ pH 7.0, 50μΕ 40mM NAD+, and 50uL 1M Sodium

Formate were added to a Plastibrand micro UV-cuvette. The cuvette was placed into a Shimadzu UV-1700 set to read absorbance at 340nm and blanked. Ι ΟΟμί of undiluted lysate sample were pipetted into the cuvette which was then mixed by gently pipetting the contents of the cuvette and the absorbance was monitored for 2.5 minutes. The resulting final concentrations of each reagent were 50mM Potassium Phosphate, 2mM NAD+, and 0.05M Sodium Formate. Each sample was done in duplicate to ensure reproducibility. Table 24. Fdh activity of engineered strains

Average μιηοΙ Standard

NADH/min Deviation %CV

M3631 0.03 0.00269 0.865

M2390 0.008 0 0

M3465 0 0 0

M3625 0 0 0

M3679 0 0 0

M3680 0 0 0

[0494] As shown in Table 24, the FDH knockout strains (M3465, M3625, M3679, and

M3680) did not exhibit any formate dehydrogenase activity. The background strain, M2390, had minimal activity. The positive control strain, M3631, which overexpresses FDH was active and produced a significant amount of NADH that was observed and recorded.

[0495] AE9 glucoamylase activity assay:

[0496] Saccharomycopsis fibuligera GLU1 glucoamylase (AE9) produces glucose from starch.

[0497] Extracellular AE9 glucoamylase activity on raw corn starch was assayed to determine the presence of glucoamylase activity in aerobic culture supematants of engineered strains. Cells were grown aerobically, removed by centrifugation, and the resulting supernatant was assayed for activity and compared to supernatant from strain M2390, which does not contain AE9.

[0498] Cell growth conditions:

[0499] Cells were plated on YPD (20g/L peptone, lOg/L yeast extract, 20g/L dextrose) agar for 48 hours and used to inoculate 25mL YPD (20g/L peptone, lOg/L yeast extract, 20g/L glucose) in a 50mL culture tube. Cells were grown aerobically for 48 hours at 35°C with shaking at 250rpm. After 48 hours, cells were removed via centrifugation and the supernatant was recovered.

[0500] Sample preparation: [0501] The recovered aerobic culture supernatant was clarified by filtration through a

0.22μηι filter membrane and concentrated ~10X using a lOkDa molecular weight cut-off filter. The retained concentrate was then analyzed for AE9 concentration via a phenyl reverse phase (phenyl-RP) HPLC method developed in-house using purified AE9 as a standard. Samples were diluted to an AE9 concentration of 5(^g/mL and used directly in the activity assay.

[0502] Glucoamylase activity assay:

[0503] A 2.2% (weight by volume) corn starch solution was made up in 50mM sodium acetate buffer pH 5.0. In a 96-well assay plate, 50μΙ, of supernatant (adjusted to 50μg/mL AE9 concentration) was added to 450μί 2.2% starch. The plate was incubated at room temperature without shaking, and 50μΙ, of sample was taken at 1, 2, 5, 10, 30, 120 and 210 minutes. Wells were mixed by pipette aspiration after initial enzyme addition, as well as at each sampling thereafter. Samples were analyzed via 3,5-dinitrosalicylic acid (DNS) method to determine reducing sugars.

[0504] As shown in Figure 42, the aerobic culture supernatants of M3625 and M3680 showed similar activity on raw corn starch, as measured by DNS analysis (Somogyi, M., Notes on Sugar Determination, JBC (200)45 (1952)). Amylolytic activity of M2390 supernatant was negligible in this assay.

[0505] The above data show that pflA, pflB and AdhE are present in strain M3625 and have the proper activity. Fdh activity, seen in the background strain as well as in the positive control when fdh was overexpressed, was not present in the engineered strains indicating that this gene was knocked out successfully.

EXAMPLE 9

[0506] The following example demonstrates the ethanol yield of the Saccharomyces cerevisiae strain M3624. The genotype of strain M3624 is: Agpdl ::GPD2-B.adolescentis pflA/pFIB/ adhEAgpd2 : : GPD 1 -B . adolescentis pflA/pflB/adhE Afdhl

Afdh2::B.adolescentis pflA/pflB/adhE. Strain M3624 was created according to the same methods employed above in Example 8. Detailed molecular maps for strain M3624 are shown in Figures 43A-D. Figure 43 A shows insertion at the GPD1 locus; GPD2 expressed from the GPD1 promoter; PFK2t-PFK2 terminator; HXT2t-HXT2 terminator; pADHl-ADHl promoter; PDC1 term-PDCl terminator; FBA1 term-FBAl terminator; pTPIl-TPIl promoter; Seer ENOl ter-ENOl terminator. Figure 43B shows insertion at the GPD2 locus; GPD1 expressed from the GPD2 promoter; TDH3 term-TDFB terminator; pPFKl-PFKl promoter; HXT2t-HXT2 terminator; PDC1 term-PDCl terminator; pADHl-ADHl promoter; S.cer ENOl ter-ENOl terminator; FBA1 term- FBAl terminator; pTPIl-TPIl promoter. Figure 43 C shows deletion of the FDHl gene; flanking regions to create deletion of FDHl . Figure 43D shows insertion at the FDH2 locus; pPFKl-PFKl promoter, S.cer ENOl ter-ENOl terminator; pADHl-ADhl promoter; FBA1 term-FBAl terminator; PDC1 term-PDCl terminator; pTPI-TPIl promoter.

[0507] The data shown in Figure 44 demonstrates that a 3.4% ethanol yield increase is obtained through reduction of glycerol and production of formate. M2390 is the control strain, M3515 and M3624 are engineered with the genotype Agpdl::GPD2- B. adolescentis pflA/pflB/adhE Agpd2::GPDl-B. adolescentis pflA/PFlB/adhE fdhlAfdh2A::B. adolescentis pflA/pflB/adhE. M3027 is engineered with the genotype Agpdl Agpd2::GPDl-B. adolescentis pflA/PFlB/adhE fdhlAfdh2A::B. adolescentis pflA/pflB/adhE. Panel A shows measurement of formate concentration, panel B shows measurement of glycerol concentration, and panel C shows measurement of ethanol concentration.

[0508] Both M3515 and M3624 have been engineered at 4 separate loci. The GPD1 gene is expressed from the GPD2 promoter and the GPD2 gene is expressed from the GPD1 promoter, the FDHl and FDH2 genes have been deleted. Additionally, the B. adolescentis pflA, pflB and adhE genes are expressed as shown in Figures 43A, B, and D.

EXAMPLE 10

[0509] The following example demonstrates the ethanol yield of the Saccharomyces cerevisiae strains M3465 and M3469. The genotype of strain M3465 is: Agpd2:: B. adolescentis pflA/pflB/adhE AfdhlA fdh2:: B. adolescentis pflA/pflB/adhE. The genotype of strain M3469 is: Agpdl :: B. adolescentis pflA/pflB/adhE fdhlA fdh2A:: B. adolescentis pflA/pflB/adhE. Strains M3465 and M3469 were created according to the same methods employed above in Example 8. Detailed molecular maps of strains M3465 and M3469 are shown in Figures 45 A-C and 46 A-C, respectively. Figure 45A shows insertion at the GPD2 locus; pPFKl-PFKl promoter; HXT2t-HXT2 terminator; PDCl term-PDCl terminator; pADHl-ADHl promoter; S.cer ENOl ter-ENOl terminator; FBA1 term-FBAl terminator; pTPIl-TPIl promoter. Figure 45B shows deletion of the FDHl gene; flanking regions to create deletion of FDHl . Figure 45 C shows insertion at the FDH2 locus; pPFKl-PFKl promoter; S.cer ENOl ter-ENOl terminator; pADHl- ADhl promoter; FBA1 term-FBAl terminator; PDCl term-PDCl terminator; pTPI-TPIl promoter. Figure 46A shows insertion at the GPDl locus; pPFKl-PFKl promoter; HXT2t-HXT2 terminator; PDCl term-PDCl terminator; pADHl-ADHl promoter; S.cer ENOl ter-ENOl terminator; FBA1 term-FBAl terminator; pTPIl-TPIl promoter. Figure 46B shows deletion of the FDHl gene; flanking regions to create deletion of FDHl . Figure 46C shows insertion at the FDH2 locus; pPFKl-PFKl promoter, S.cer ENOl ter- ENOl terminator; pADHl-ADhl promoter; FBA1 term-FBAl terminator; PDCl term- PDCl terminator; pTPI-TPIl promoter.

[0510] This example demonstrates that the ethanol yield increase is dependent on the level of glycerol reduction. Fermentation of 30% solids corn mash by M3465, which contains a deletion of the GPD2, FDHl and FDH2 genes and expression of B. adolescentis pflA, pflB and adhE genes from the GPD2 and FDH2 loci, results in a 1.5% increase in ethanol titer. As shown in Figure 47, fermentation of 30% solids corn mash by M3469, which contains a deletion of the GPDl, FDHl and FDH2 genes and expression of B. adolescentis pflA, pflB and adhE genes from the GPDl and FDH2 loci, results in a 2.5% increase in ethanol titer. M2390 is the control parent strain. As shown in Figure 48, fermentation of corn mash by M3465 and M3469 results in -15% lower glycerol and 30%) lower glycerol levels respectively. M2390, represented in Figures 47 and 48, is the control strain.

EXAMPLE 11

[0511] An alternative way to reduce glycerol formation is through deletion of the glycerol

-3-phosphate phosphatase (GPP) genes. Saccharomyces contains two copies of these genes, GPP1 and GPP2. The data below demonstrates that expression of B. adolescentis pflA, pflB and adhE in backgrounds containing deletions of FDHl, FDH2 and either GPP1 or GPP2 results in decreased glycerol formation (Figure 53; strain comparison, min buff medium, glucose 40g/L, anaerobic fermentations, 35°C - 72 hr.) and increased ethanol yield (Figure 54; strain comparison, min buff medium, glucose 40 g/L, anaerobic fermentations, 35°C - 72 hr.). Production of formate was also observed. See Figure 55 (strain comparison, min buff medium, glucose 40 g/L, anaerobic fermentations, 35°C - 72 hr.).

[0512] The strains engineered to measure the glycerol formation, ethanol yield, and formate production were the Saccharomyces cerevisiae strains M3297, TB655, and TB656. Strains M3297, TB655, and TB656 were created according to the same methods employed above in Example 8. The genotype of strain M3297 is: AfdhlAfdh2::pflA/pflB/adhE. This strain contains only deletion in the FDH genes plus expression fo pflA, pflB and AdhE. The genotype of strain TB655 is: AfdhlAfdh2::pflA/pflB/adhEAgppl ::pflA/pflB/adhE. This strain contains deletion in the FDH genes, expressionof pflA, pflB and AdhE, and deletion of GPP1. See Figure 51. The genotype of strain TB656 is: AfdhlAfdh2::pflA/pflB/adhEAgpp2::pflA/pflB/adhE. This strain contains deletion in the FDH genes, expressionof pflA, pflB and AdhE and deletion of GPP1. See Figure 52.

[0513] The amount of ethanol, glycerol, and formate produced by strains TB655 and

TB656 was measured using the methods described above. Compared to the control strain M3297, strains TB655 and TB656 demonstrated statistically significant changes in the amount of ethanol, glycerol, and formate produced. Relative to strain M3297, strain TB655 (gppl mutant) demonstrated a 1.3% increase in ethanol titer, 10% reduction in glycerol, and 100%) more formate produced, whereas strain TB656 (gpp2 mutant) demonstrated a 0.95% increase in ethanol titer, 6.1% reduction in glycerol formation, and 100% more formate produced. These results demonstrate the novel combination of GPP mutation with a metabolic engineering solution to balance redox during anaerobic growth.

Incorporation by Reference

[0514] All of the U.S. patents and U.S. published patent applications cited herein are hereby incorporated by reference. Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the following claims.