POST-TRANSCRIPTIONAL REGULATION OF BIOSYNTHETIC PATHWAYS

Field of the Invention

[0001] The invention relates to the fields of industrial microbiology and alcohol production. Embodiments of the invention relate to the the use of suitable post-transcriptional strategies for differential expression of genes during propagation and production phases to achieve regulated production via an engineered pathway in microorganisms.

Reference To Sequence Listing Submitted Electronically Via Efs-Web

[0002] The content of the electronically submitted Sequence Listing, (Name:

20140701_CL5645WOPCT_SequenceListing_ascii.txt; Size: 1,324,132 bytes; Date of Creation: June 23, 2014) filed herewith, is herein incorporated by reference in its entirety.

Background

[0003] Butanol is an important industrial chemical, useful as a fuel additive, as a feedstock chemical in the plastics industry, and as a food grade extractant in the food and flavor industry. Each year 10 to 12 billion pounds of butanol are produced by petrochemical means and the need for this commodity chemical will likely increase in the future.

[0004] Methods for the chemical synthesis of the butanol isomer isobutanol are known, such as oxo synthesis, catalytic hydrogenation of carbon monoxide (Ullmann's Encyclopedia of Industrial Chemistry, Wiley-VCHVerlag GmbH and Co., Weinheim, Germany, 5:716-719 (6th ed. 2003)) and Guerbet condensation of methanol with n-propanol (Carlini et al., J. Molec. Catal. A:Chem. 220:215-220 (2004)). These processes use starting materials derived from

petrochemicals, are generally expensive, and are not environmentally friendly. The production of isobutanol from plant-derived raw materials would minimize green house gas emissions and there is a need in the art for methods and host cells for such production.

Brief Summary of the Invention

[0005] Provided herein are methods of producing fermentation products. In

embodiments, the methods comprise: A) contacting a recombinant host cell comprising a first heterologous polynucleotide and a second heterologous nucleotide, the first heterologous polynucleotide nucleic acid sequence encoding a biocatalyst polypeptide with a nonsense or frameshift allele and the second heterologous polynucleotide comprising i) a promoter nucleic acid sequence; and ii) a nucleic acid sequence encoding a suppressor tRNA specific to a nonsense or frameshift allele of the first heterologous polynucleotide, with a carbon substrate under a first set of conditions; and B) contacting the recombinant host cell with a carbon substrate under a second set of conditions; wherein the first set of conditions and second set of conditions differ and wherein the nucleic acid sequence encoding the suppressor tRNA is differentially expressed under the first set of conditions than under the second set of conditions. In embodiments, the methods comprise A) contacting a recombinant host cell comprising a first heterologous polynucleotide and a second heterologous polynucleotide, the first heterologous polynucleotide comprising a nucleic acid sequence encoding a biocatalyst polypeptide, and the second heterologous polynucleotide comprising i) a promoter nucleic acid sequence; and ii) a nucleic acid sequence encoding an anti-sense RNA specific to the mRNA encoded by the first heterologous polynucleotide,with a carbon substrate under a first set of conditions; and B) contacting the recombinant host cell with a carbon substrate under a second set of conditions; wherein the first set of conditions and second set of conditions differ and wherein the nucleic acid sequence encoding the anti-sense RNA is differentially expressed under the first set of conditions than under the second set of conditions; and wherein the host cell produces butanol or 2-butanone under at least one of the first set or the second set of conditions. In embodiments, the first and/or second set of conditions is an environmental or a process cue. In embodiments, the environmental cue or process cue activates a transcriptional activator for a promoter described herein. In embodiments, the environmental or process cue activates a transcriptional repressor for a promoter described herein. In embodiments, the transcriptional repressor is a sulfonylurea- responsive repressor. In some embodiments, the transcriptional repressor is a Tet (tetracycline) repressor. In embodiments, the host cell produces a fermentation product such as butanol or 2- butanone under at least one of the first set or the second set of conditions. In some embodiments, the biocatalyst polypeptide is expressed through the use of a constutive promoter.

[0006] In embodiments, the first set of conditions and the second set of conditions differ in at least one of source of carbon substrate, dissolved oxygen concentration, temperature, pH, glucose concentration, or fermentation product concentration such as butanol or 2-butanone concentration. In embodiments, the dissolved oxygen concentration is greater during the first set of conditions than during the second set of conditions. In embodiments, one set of conditions is anaerobic. In embodiments, the second set of conditions is anaerobic. In embodiments, the average glucose concentration is lower in the first set of conditions than during the second set of conditions. In embodiments, the average glucose concentration is at least about 5 times lower in the first set of conditions than during the second set of conditions, at least about 50 times lower in the first set of conditions than during the second set of conditions, at least about 100 times lower in the first set of conditions than in the second set of conditions, or at least about 1000 times lower in the first set of conditions than in the second set of conditions. In embodiments, the rate of butanol production is lower under the first set of conditions than under the second set of conditions.

[0007] In embodiments, the source of carbon substrate for the first set of conditions differs from that of the second set of conditions. In embodiments, the source of the carbon substrate for the first set of conditions is molasses. In embodiments the source of the carbon substrate for the second set of conditions is corn mash.

[0008] Accordingly, heterologous polynucleotides comprising i) a promoter nucleic acid sequences; and ii) a nucleic acid sequence encoding suppressor tR A molecules are provided herein and may be employed in the disclosed methods. Heterologous polynucleotides comprising i) a promoter nucleic acid sequences; and ii) a nucleic acid sequence encoding anti-sense R A molecules are provided herein and may be employed in the disclosed methods.

[0009] In embodiments, isolated polynucleotides provided herein comprise: (a) a promoter nucleic acid sequence; and (b) a nucleic acid sequence encoding a suppressor tRNA; wherein the nucleic acid sequence of (b) is operably associated to the nucleic acid sequence of (a) such that the suppressor tRNA is differentially expressed during the production phase and the propagation phase of a fermentation process. In embodiments, isolated polynucleotides provided herein comprise: (a) a promoter nucleic acid sequence; and (b) a nucleic acid sequence encoding an anti-sense RNA; wherein the nucleic acid sequence of (b) is operably associated to the nucleic acid sequence of (a) such that the anti-sense RNA is differentially expressed during the production phase and the propagation phase of a fermentation process. In some embodiments, differential expression of an anti-sense RNA or a suppressor tRNA results in differential expression of a biocatalyst polypeptide. In embodiments, the expression of the biocatalyst polypeptide is higher in the production phase than in the propagation phase of fermentation. In embodiments, the biocatalyst polypeptide catalyzes a substrate to product conversion in a butanol or 2-butanone biosynthetic pathway. In embodiments, the biocatalyst polypeptide is selected from a group of enzymes having the following Enzyme Commission Numbers: EC 2.2.1.6, EC 1.1.1.86, EC 4.2.1.9, EC 4.1.1.72, EC 1.1.1.1, EC 1.1.1.265, EC 1.1.1.2, EC 1.2.4.4, EC 1.3.99.2, EC 1.2.1.57, EC 1.2.1.10, EC 2.6.1.66, EC 2.6.1.42, EC 1.4.1.9, EC 1.4.1.8, EC

4.1.1.14, EC 2.6.1.18, EC 2.3.1.9, EC 2.3.1.16, EC 1.1.130, EC 1.1.1.35, EC 1.1.1.157, EC 1.1.136, EC 4.2.1.17, EC 4.2.1.55, EC 1.3.1.44, EC 1.3.1.38, EC 1.3.1.44, EC 1.3.1.38 , EC 5.4.99.13, EC 4.1.1.5, EC 1.1.1.1, 2.7.1.29, 1.1.1.76, 1.2.1.57, and 4.2.1.28. In embodiments, the biocatalyst polypeptide catalyzes a substrate to product conversion in an isobutanol biosynthetic pathway. In embodiments, the biocatalyst polypeptide is acetolactate synthase, acetohydroxy acid isomeroreductase, acetohydroxy acid dehydratase, branched-chain alpha-keto acid decarboxylase, branched-chain alcohol dehydrogenase, branched-chain keto acid

dehydrogenase, butyryl-CoA dehydrogenase, butyraldehyde dehydrogenase, transaminase, valine dehydrogenase, valine decarboxylase, omega transaminase, acetyl-CoA acetyltransferase, 3-hydroxybutyryl-CoA dehydrogenase, crotonase, butyryl-CoA dehydrogenase, isobutyryl-CoA mutase, acetolactate decarboxylase, acetonin aminase, butanol dehydrogenase, butyraldehyde dehydrogenase, acetoin kinase, acetoin phosphate aminase, aminobutanol phosphate

phospholyase, aminobutanol kinase, butanediol dehydrogenase, or butanediol dehydratase. In embodiments, the biocatalyst polypeptide is a GPI-anchored cell wall protein involved in acid resistance. In embodiments, the biocatalyst polypeptide is Sedl protein or Spil protein or a homolog thereof. The isolated polynucleotide of claim 28, 29, or 31 wherein the biocatalyst polypeptide is a GPI-anchored cell wall protein involved in acid resistance. In embodiments, the biocatalyst polypeptide comprises at least about 80%, at least about 85%>, at least about 90%>, at least about 95%, at least about 99%, or at least about 100% identity to SEQ ID NO: 397, 399, 401, 403, 405, 407, 409, 411, 413, 415, 417, 419, 421, 423, 425, 427, 429, 431, 433, or 435.

[0010] In embodiments, a biocatalyst polypeptide may be a biosynthetic pathway polypeptide, a cell integrity polypeptide, a propagation polypeptide, a glycerol biosynthesis pathway polypeptide, by-product producing polypeptide, or an NADPH-generating polypeptide. In embodiments, the biocatalyst polypeptide catalyzes the substrate to product conversion of pyruvate to acetolactate. In embodiments, the biocatalyst polypeptide catalyzes the substrate to product conversion of pyruvate to acetolactate and comprises at least about 80%>, at least about 85%, at least about 90%, at least about 95%, at least about 99%, or 100% identity to SEQ ID NO: 1 or a variant or active fragment thereof.

[0011] In embodiments, the promoter nucleic acid sequence comprises at least about

80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 170, 171, 172, 175, 176, 177, 186, 186, 188, 189, 190, 191, 192, 193, 194, 194, 195, 196, 197, 198, 199, 200, 201, 202, 203, 204, 206, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 244, 245, 246, 247, 248, 249, 250, 251, 252, 253, 254, 255, 256, 257, or 258 or an active fragment thereof. In embodiments, the promoter nucleic acid sequence comprises at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 268, 269, 270, 271, 272, 273, 274, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 294, 295, 296, 297, 298, 299, 300, 301, 302, 303, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 344, 345, 346, 347, 348, 349, 350, 351, 352, 353, 354, 355, 356, 357, 358, 359, 360, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 376, 377, 378, 379, 380, 381, 382, or 383 or an active fragment thereof. In

embodiments, the promoter nucleic acid sequence comprises at least about 80%>, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 775, 776, 777, or 778 or an active fragment thereof. In embodiments, the promoter nucleic acid sequence comprises at least about 80%>, at least about 85%, at least about 90%>, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 779 or an active fragment thereof. In embodiments, the promoter nucleic acid sequence comprises at least about 90%>, at least about 95%, at least about 99% or 100% identity identity to SEQ ID NO: 686. In embodiments, the promoter nucleic acid sequence comprises at least about 80%>, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 384, 360, 386, or 331 or an active fragment thereof. In embodiments, the promoter nucleic acid sequence comprises at least about 80%>, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 772 or 773 or an active fragment thereof. In embodiments, the promoter nucleic acid sequence comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 779 or an active fragment thereof. In embodiments, the promoter nucleic acid sequence comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 168, 169, 388, or 173 or an active fragment thereof. In embodiments, the promoter nucleic acid sequence comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 768 or 769. In embodiments, the promoter nucleic acid sequence comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 779 or an active fragment thereof. In embodiments, the promoter nucleic acid sequence comprises at least about 80%>, at least about 85%, at least about 90%>, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 711 or an active fragment thereof. In embodiments, the promoter nucleic acid sequence comprises at least about 80%>, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, or 151 or a fragment thereof.

[0012] In embodiments, the promoter nucleic acid sequence comprises at least about

80%, at least about 85% , at least about 90%, at least about 95%, at lest about 99% or 100% identity to SEQ ID NO: 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, 150, or 151 or a fragment thereof. In embodiments, the promoter nucleic acid sequence comprises at least about 80%, at least about 85%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 168, 169, 170, 171, 172, 173, 174, 175, 176, or 177 or a fragment thereof.

[0013] In embodiments, the promoter nucleic acid sequence comprises at least about

80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% to SEQ ID NO: 168, 169, 170, 171, 172, 173, 174, 175, 176, or 177 or a fragment thereof and wherein the biocatalyst polypeptide is acetolactate synthase or ketol-acid reductoisomerase. In embodiments, the acetolactate synthase has at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 1 or a variant or active fragment thereof. In embodiments, the acetolactate synthase is B. subtilis AlsS or a variant or active fragment thereof.

[0014] In embodiments, the expression of the biocatalyst polypeptide is higher in the propagation phase than in the production phase of fermentation. In embodiments, the biocatalyst polypeptide is a biosynthetic pathway polypeptide or a cell integrity polypeptide. In some embodiments, the biocatalyst polypeptide is a propagation polypeptide, an isobutanol pathway by-product polypeptide, a glycerol biosynthesis pathway, or a polypeptide of an NADPH generating pathway. In some embodiments, the biocatalyst polypeptide is a phosphoketolase. In some embodiments, the phosphoketolase is derived from Lactobacillus plantarum. In some embodiments, the biocatalyst polypeptide is a phosphotransacetylase. In some embodiments, the phosphotransacetylase is derived from Lactobacillus plantarum. In some embodiments, biocatalyst polypeptide is an acetolactate reductase. In some embodiments, the acetolactate reductase is YMR226C. In some embodiments, the biocatalyst polypeptide is an aldehyde dehydrogenase. In some embodiments, the biocatalyst polypeptide is ALD6. In some embodiments, the biocatalyst polypeptide is an enzyme of the oxidative pentose phosphate pathway. In some embodiments, the biocatalyst polypeptide is glucose-6-phosphate

dehydrogenase, 6-phosphoglucononolactonase, or 6-phosphogluconate dehydrogenase. In embodiments, the biocatalyst polypeptide is glycerol 3-phosphate dehydrogenase. In

embodiments, the promoter nucleic acid sequence comprises at least about 80%, at least about 85%, at least about 90%, at least about 95% at least about 99% or 100% identity to SEQ ID NO: 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, or 163, or a fragment thereof.

[0015] In embodiments, the nucleic acid sequence encoding a biocatalyst polypeptide is codon-optimized for expression in a specific host cell.

[0016] Also disclosed herein are recombinant host cells comprising isolated

polynucleotides disclosed herein. In some embodiments, the recombinant host cells comprise biocatalyst polypeptides., In embodiments, the host cell is a bacteria, cyanobacteria, filamentous fungi, or yeast cell. In embodiments, the host cell is a bacterial or cyanobacterial cell. In embodiments, the genus of the host cell is Salmonella, Arthrobacter, Bacillus, Brevibacterium, Clostridium, Corynebacterium, Gluconobacter, Nocardia, Pseudomonas, Rhodococcus,

Streptomyces, Zymomonas, Escherichia, Lactobacillus, Lactococcus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, or Xanthomonas. In embodiments, the host cell is a filamentous fungi or yeast cell. In embodiments, the genus of the host cell is Saccharomyces, Pichia, Hansenula, Yarrowia, Aspergillus, Kluyveromyces, Pachysolen, Rhodotorula, Zygosaccharomyces,

Schizosaccharomyces, Torulaspora, Debayomyces, Williopsis, Dekkera, Kloeckera,

Metschnikowia, Issatchenkia or Candida. In embodiments, the host cell is Saccharomyces cerevisiae. In embodiments, the host cell comprises reduced or eliminated expression of endogenous pyruvate decarboxylase. In embodiments, the reduced or eliminated expression of decarboxylase is caused by gene deletion, disruption, or mutation. In embodiments, the gene disrupted or deleted is PDCl, PDC5, PDC6, or a combination thereof. In some embodiments, the host cell comprises reduced or eliminated expression of an endogenous enzyme having aldehyde dehydrogenase activity, glycerol-3 -phosphate dehydrogenase activity, acetolactate reductase activity, or a polypeptide affecting Fe-S cluster biosynthesis. In some embodiments, the reduced or eliminated expression of an endogenous enzyme having aldehyde dehydrogenase activity, glycerol-3 -phosphate dehydrogenase activity, acetolactate reductase activity, or a polypeptide affecting Fe-S cluster biosynthesis is caused by gene deletion, disruption, or mutation.

[0017] Provided are recombinant yeast host cells comprising an isobutanol biosynthetic pathway and at least one modified expression construct that differentially expresses a polypeptide under conditions in which propagation of biomass is favored over production of isobutanol whereby isobutanol production under the conditions is substantially reduced as compared to a host cell without the modified expression construct under the same conditions. In embodiments, the modified expression construct comprises SEQ ID NO: 711. In embodiments, the modified expression construct comprises a polynucleotide encoding a polypeptide capable of catalyzing the substrate to product conversion pyruvate to acetolactate. In embodiments, the polynucleotide encoding a polypeptide capable of catalyzing the substrate to product conversion pyruvate to acetolactate comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 2. In embodiments, the polypeptide capable of catalyzing the substrate to product conversion pyruvate to acetolactate comprises at least about 80%>, at least about 85%, at least about 90%>, at least about 95%, at least about 99% or 100%) identity to SEQ ID NO: 1. In embodiments, the modified expression construct comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100%) identity to SEQ ID NO: 790. In embodiments, the yeast host cell is Saccharomyces cerevisiae.

[0018] Also disclosed herein are methods for the production of a fermentation product comprising: (a) providing a disclosed recombinant host cell; (b) contacting the host cell with fermentable carbon substrate in a fermentation medium under conditions whereby the

fermentation product is produced; and (c) optionally, recovering the fermentation product.

[0019] In embodiments, the methods further comprise propagating the host cell under conditions whereby the host cell propagates prior to the contacting of (b). In embodiments, the conditions whereby the fermentation product are produced are anaerobic. In embodiments, the conditions whereby the fermentation product are produced are microaerobic. In embodiments, the fermentation product is selected from the group consisting of: butanol, 2-butanone, propanol, isopropanol, and ethanol. In embodiments, the fermentation product is butanol or 2-butanone. In embodiments, the fermentation product is isobutanol. In embodiments, the fermentation product is isobutanol and the isolated polynucleotide comprises the promoter nucleic acid sequence comprises at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 99% or 100% identity to SEQ ID NO: 168, 169, 170, 171, 172, 173, 174, 175, 176, or 177 or a fragment thereof and the biocatalyst polypeptide is acetolactate synthase or ketol-acid reductoisomerase .

[0020] In other embodiments uncoupling growth and production occurs not necessarily from differential expression, but from changing conditions or activity of a cell pathway, protein, or other component by the addition or deletion of another component. The addition, deletion or action of the other component may be combined with differential expression or may result in control over growth and/or production independent of differential expression.

Brief Description of the Drawings

[0021] Figure 1 depicts different isobutanol biosynthetic pathways. The steps labeled

"a", "b", "c", "d", "e", "f", "g", "h", "i", "j", and "k" represent substrate to product conversions described below, "a" may be catalyzed, for example, by acetolactate synthase, "b" may be catalyzed, for example, by acetohydroxyacid reductoisomerase. "c" may be catalyzed, for example, by acetohydroxy acid dehydratase, "d" may be catalyzed, for example, by branched- chain keto acid decarboxylase, "e" may be catalyzed, for example, by branched chain alcohol dehydrogenase, "f ' may be catalyzed, for example, by branched chain keto acid dehydrogenase, "g" may be catalyzed, for example, by acylating aldehyde dehydrogenase, "h" may be catalyzed, for example, by transaminase or valine dehydrogenase, "i" may be catalyzed, for example, by valine decarboxylase, "j" may be catalyzed, for example, by omega transaminase.

[0022] Figure 2 depicts a genetic switch in which a suppressor tRNA is produced in response to a process or environmental cue, and wherein the suppressor tRNA rescues the translation of a pathway polypeptide containing a nonsense or a frameshift allele.

[0023] Figure 3 depicts a genetic switch in which an anti-sense RNA is produced in response to a process or environmental cue, and wherein the anti-sense RNA blocks the translation of a pathway polypeptide.

[0024] Figure 4 depicts a genetic switch in which a suppressor tRNA is produced in response to a process or environmental cue, and wherein the suppressor tRNA rescues the translation of a propagation polypeptide containing by a nonsense or a frameshift allele.

[0025] Figure 5 depicts a genetic switch in which an anti-sense RNA is produced in response to a process or environmental cue, and wherein the anti-sense RNA blocks the translation of a propagation polypeptide. [0026] Figure 6 depicts a genetic switch in which the production of a suppressor tR A is blocked in response to a process or environmental cue, and wherein the translation of a pathway polypeptide containing by a nonsense or a frameshift allele is blocked due to the absence of the suppressor tRNA.

[0027] Figure 7 depicts a genetic switch in which the production of an anti-sense RNA is blocked in response to a process or environmental cue, and wherein the lack of production of the anti-sense RNA results in the translation of a pathway polypeptide.

[0028] Figure 8 depicts a genetic switch in which the production of a suppressor tRNA is blocked in response to a process or environmental cue, and wherein the translation of a propagation polypeptide containing by a nonsense or a frameshift allele is blocked due to the absence of the suppressor tRNA.

[0029] Figure 9 depicts a genetic switch in which the production of an anti-sense RNA is blocked in response to a process or environmental cue, and wherein the lack of production of the anti-sense RNA results in the translation of a propagation polypeptide.

[0030] Figure 10 depicts that isobutanol is synthesized at 26 and 37 hrs and that by 50 hrs of cell culture, isobutanol accumulation ceases.

[0031] Figure 11 depicts rate of isobutanol production, in g/L/hr, over fermentation time

[0032] Figure 12 depicts carbon dioxide evolution rate over fermentation time.

[0033] Figure 13 depicts oxygen consumption over fermentation time.

Detailed Description

[0034] Industrial fermentation processes with yeast may employ a stage of biomass production in order to provide sufficient biocatalyst for the fermentation stage to have desired yield and production rate. Ethanologen S. cerevisiae for example is typically propagated using fed-batch technology, in which low sugar concentrations and non-limiting aeration favor respiratory metabolism with high biomass yields, e.g., Y _xs ~ 0.5 g biomass/g glucose. The maintenance of low sugar concentrations in a fed-batch regime may be particularly important for a Crabtree-positive yeast like S. cerevisiae, in which the fraction of respiratory metabolism on overall metabolism is negatively correlated with increasing extracellular glucose concentrations. Due to the low sugar concentrations, specific glucose uptake rate is limited and respiratory capacity is sufficient to completely metabolize pyruvic acid formed in catabolism of the carbohydrate substrates to C0 ₂ . Under fermentative conditions with no oxygen or at higher glucose concentrations under aerobic conditions with the Crabtree effect in action, ethanologen yeasts like, e.g., S. cerevisiae produce ethanol and only low biomass yields are achieved, e.g., Y _xs ~ 0.15g biomass/g glucose.

[0035] Considerations for the propagation of biocatalysts that produce lower alkyl alcohols, such as butanologenic biocatalysts include (i) the negative effect of toxic products, such as butanol or 2-butanone, (ii) the accumulation of inhibitory pathway byproducts or

intermediates, and (iii) the loss of substrate to the formation of fermentation byproducts resulting in lower yields of biocatalyst. For example, when a butanol production pathway functions constitutively in yeast, then the butanol produced may inhibit growth during the propagation phase of a production process and may add cost and inefficiency to either or both the

infrastructure and the operation of the biocatalyst production phase. Control, particularly reduction or elimination, of butanol production during the biomass-forming phase would represent an advance in the art.

[0036] Applicants have addressed the stated problems by identifying various promoter nucleic acid sequences which provide differential expression of a suppressor tR A or an anti- sense R A specific for a gene of interest, and, as a result, differential expression of genes of interest under different conditions, thus providing a strategy for differential expression during biocatalyst propagation and fermentation product production phases.

[0037] Accordingly, provided herein are recombinant host cells that produce fermentation products and comprise promoter sequences that provide differential expression in the propagation vs. production phases of a process, as well as methods for using the same. Promoters that can be used to provide such differential expression, and means of identifying such promoters, are described in Int'l Appl. No. PCT/US2012/072186, which is incorporated herein by reference in its entirety.

[0038] Also provided herein is a suitable screening strategy to identify and evaluate candidate nucleic acid sequences to govern the differential expression of genes of interest during biocatalyst propagation and fermentation product production phases. Furthermore, hybrid sequences comprising nucleic acid sequences derived from more than one promoter region are provided. The nucleic acid sequences described herein may be employed as promoters for the differential expression of various polypeptides relevant to propagation and/or production and are hereinafter referred to from time to time as "genetic switches." The sequences and methods disclosed herein thus allow (i) biocatalyst polypeptides such as polypeptides which catalyze the substrate to product conversions of a biosynthetic pathway such as a butanol or 2-butanone biosynthetic pathway to be preferentially expressed during the production phase of fermentation through the use of promoters controlling suppressor tRNAs and/or anti-sense RNAs specific for the biocatalyst polypeptides of interest, (ii) biocatalyst polypeptides beneficial for cell integrity to be preferentially expressed during the production phase of fermentation through the use of promoters controlling suppressor tRNAs and/or anti-sense RNAs specific for the biocatalyst polypeptides of interest, (iii) biocatalyst polypeptides such as propagation polypeptides to be preferentially expressed during the biocatalyst propagation phase through the use of promoters controlling suppressor tRNAs and/or anti-sense RNAs specific for the biocatalyst polypeptides of interest, (iv) biocatalyst polypeptides being part of a NADPH generating pathway to be preferentially expressed during the propagation phase through the use of promoters controlling suppressor tRNAs and/or anti-sense RNAs specific for the biocatalyst polypeptides of interest, or (v) expression of polypeptides being part of a NADH consuming product pathway other than butanol to be preferentially reduced during the fermentation product production phase through the use of promoters controlling suppressor tRNAs and/or anti-sense RNAs specific for the biocatalyst polypeptides of interest, or a combination thereof. Applicants have also provided recombinant host cells utilizing recombinant polynucleotide sequences comprising the identified promoters and methods for producing fermentation products using the same.

[0039] In embodiments, recombinant host cells described herein produce butanol or 2- butanone from plant derived carbon sources. Accordingly, provided herein are methods for the production of butanol or 2-butanone using recombinant host cells comprising isolated

polynucleotides comprising promoter nucleic acid sequences controlling suppressor tRNAs and/or anti-sense RNAs that differentially regulate the expression of associated genes during the propagation and production phases of a fermentation process. In one embodiment, a polypeptide catalyzing the first step in a butanol biosynthetic pathway can be preferentially expressed during the production phase. In one embodiment, a polypeptide catalyzing a substrate to product conversion in an isobutanol biosynthetic pathway can be preferentially expressed during the production phase. In one embodiment, acetolactate synthase can be preferentially expressed during the production phase. In one embodiment, ketol-acid reductoisomerase can be

preferentially expressed during the production phase. In one embodiment, dihydroxyacid dehydratase can be preferentially expressed during the production phase. [0040] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application including the definitions will control. Also, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents and other references mentioned herein are incorporated by reference in their entireties for all purposes.

[0041] A used herein, the terms "comprises," "comprising," "includes," "including,"

"has," "having," "contains," or "containing," or any other variation thereof, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers. For example, a composition, a mixture, a process, a method, an article, or an apparatus that comprises a list of elements not expressly listed or inherent to only those elements but can include other elements not expressly listed or inherent to such

composition, mixture, process, method, article, or apparatus. Further, unless expressly stated to the contrary, "or" refers to an inclusive or and not to exclusive or. For example, a condition A or B is satisfied by any one of the following: A is true (or present) and B is false (or not present), A is false (or not present) and B is true (or present), and both A and B are true (or present).

[0042] Also, the indefinite articles "a" and "an" preceding an element or component of the invention are intended to be nonrestrictive regarding the number of instances, i.e.,

occurrences of the element or component. Therefore, "a" or "an" should be read to include one or at least one, and the singular word form of the element or component also includes the plural unless the number is obviously meant to be singular.

[0043] The term "invention" or "present invention" is a non-limiting term and is not intended to refer to any single embodiment of the particular invention but encompasses all possible embodiments as described in the application.

[0044] As used herein, the term "about" modifying the quantity of an ingredient or reactant of the invention employed refers to variation in the numerical quantity that can occur, for example, through typical measuring and liquid handling procedures used for making concentrates or solutions in the real world; through inadvertent error in these procedures; through differences in the manufacture, source, or purity of the ingredients employed to make the compositions or to carry out the methods; and the like. The term "about" also encompasses amounts that differ due to different equilibrium conditions for a composition resulting from a particular initial mixture. Whether or not modified by the term "about," the claims include equivalents to the quantities. In one embodiment, the term "about" means within 10% of the reported numerical value, alternatively within 5% of the reported numerical value.

[0045] The term "suppressor tRNA" refers to a tRNA molecule with a mutation in a gene encoding a tRNA that suppresses mutations in polynucleotides encoding missense, nonsense, or frameshift alleles. See, e.g., Tucker, S.D., et ah, Biochimie 71(6): 729-39 (1989). The suppressor tRNA suppresses the effect of a mutation on the coding sequence, allowing transcription to proceed. For example, a tRNA that recognizes the codon "UAC" in the mRNA and inserts tyrosine into the growing polypeptide chain is encoded by the wild-type tyrT gene. However, a mutation in the tyrT gene can alter the specificity of the tRNA from recognizing "UAC" to recognizing the stop codon "UAG", and therefore, instead of terminating the transcript when the stop codon is reached, the suppressor tRNA inserts a tryrosine in the polypeptide chain at the position of the stop codon. Non-limiting examples of suppressor tRNA molecules include those that suppress the UAG stop codon (Amber suppressors), the UGA stop codon (Opal suppressors), the UAA stop codon (Ochre suppressors), and frameshift suppressors (that recognize a codon of a different number or base pairs than the usual three base pairs, e.g., a codon of 4 base pairs).

[0046] The term "anti-sense RNA" or "asRNA" refers to a single-stranded RNA molecule that is complementary to a messenger RNA strand that is transcribed within a cell. Anti-sense RNA can be introduced into a cell, where it can inhibit translation of its complementary mRNA strand by binding to the complementary mRNA and blocking the translation machinery.

[0047] The term "nonsense mutation" refers to a point mutation in a DNA sequence that creates a premature stop codon (a nonsense codon in the transcribed mRNA) in an open reading frame, and results in a truncation of the transcribed amino acid sequence. Non-limiting examples of nonsense mutations include the UAG stop codon, the UGA stop codon, and the UAA stop codon.

[0048] The term "missense mutation" refers to a point mutation in a DNA sequence that changes the amino acid specified for the corresponding position in the polypeptide encoded by a gene and its mRNA.

[0049] The term "frameshift mutation" is a mutation that is caused by insertions or deletions of one or more nucleotides in a DNA sequence, wherein the insertion or deletion is not evenly divisible by three. The insertion or deletion therefore changes how the codons are grouped, and results in a completely different translation from the original sequence. [0050] The term "environmental cue" or "process cue" refers to any change in cellular environmental conditions, including, but not limited to, changes in fermentable carbon substrates, oxygen levels, glucose levels, pH, temperature, and butanol levels. In embodiments, the environmental cue or process cue activates a transcriptional activator for a promoter described herein. In embodiments, the environmental or process cue activates a transcriptional repressor for a promoter described herein.

[0051] The term "transcriptional activator" refers to a protein that increases the transcription of one or more genes.

[0052] The term "transcriptional repressor" refers to a protein that prevents the binding of

RNA polymerase to a promoter, and therefore blocks transcription of a gene. In embodiments, the transcriptional repressor is a sulfonylurea-responsive repressor as described in U.S. Pat. No. 8,257,956, which is incorprated herein by reference in its entirety. In some embodiments, the transcriptional repressor is a Tet (tetracycline) repressor, as described in U.S. Pat. No. 8,257,956, which is incorprated herein by reference in its entirety.

[0053] The term "growth phase" or "propagation phase" refers to the process steps during which yeast biomass is produced and inoculum build-up occurs.

[0054] The term "production phase" refers to the fermentation process steps during which a desired fermentation product, including, but not limited to butanol, isobutanol, 1-butanol, 2- butanol and/or 2-butanone production, occurs.

[0055] In some instances, "biomass" refers to the cell biomass of the fermentation product-producing microorganism, typically provided in units g/L dry cell weight (dew).

[0056] The term "fermentation product" includes any desired product of interest, including lower alkyl alcohols including, but not limited to butanol, lactic acid, 3 -hydroxy- propionic acid, acrylic acid, acetic acid, succinic acid, citric acid, fumaric acid, malic acid, itaconic acid, 1,3-propane-diol, ethylene, glycerol, isobutyrate, etc.

[0057] The term "lower alkyl alcohol" refers to any straight-chain or branched, saturated or unsaturated, alcohol molecule with 1-10 carbon atoms.

[0058] The term "butanol" refers to 1-butanol, 2-butanol, isobutanol, or mixtures thereof.

Isobutanol is also known as 2-methyl-l-propanol.

[0059] The term "butanol biosynthetic pathway" as used herein refers to an enzyme pathway to produce 1-butanol, 2-butanol, or isobutanol. For example, isobutanol biosynthetic pathways are disclosed in U.S. Patent No. 7,851,188, which is incorporated by reference herein. Components of the pathways consist of all substrates, cofactors, byproducts, intermediates, end- products, and enzymes in the pathways.

[0060] The term "2-butanone biosynthetic pathway" as used herein refers to an enzyme pathway to produce 2-butanone.

[0061] The term "propagation polypeptide" includes polypeptides associated with the production of biomass, and polypeptides associated with the performance of an enzyme that is associated with the production of biomass.

[0062] The term "biocatalyst polypeptide" includes polypeptides associated with the substrate to product conversions of an indicated biosynthetic pathway, for example a butanol or 2-butanone biosynthetic pathway, and polypeptides associated with the propagation or performance of a biocatalyst that is associated with the indicated biosynthetic pathway, including, but not limited to, cell integrity polypeptides and propagation polypeptides. For example, a polypeptide that is a part of an NADPH generating pathway or a polypeptide that is part of a non-butanol NADH consuming product pathway may be biocatalyst polypeptides.

[0063] The term "biosynthetic pathway polypeptide" includes polypeptides that catalyze substrate to product conversions of a recited biosynthetic pathway.

[0064] The term "cell integrity polypeptide" includes polypeptides involved in cell integrity, including polypeptides required for constituting the cellular architecture.

[0065] A "recombinant microbial host cell" is defined as a host cell that has been genetically manipulated. In embodiments, recombinant microbial host cells have been genetically manipulated to express a biosynthetic production pathway, wherein the host cell either produces a biosynthetic product in greater quantities relative to an unmodified host cell or produces a biosynthetic product that is not ordinarily produced by an unmodified host cell.

[0066] The term "fermentable carbon substrate" refers to a carbon source capable of being metabolized by the microorganisms such as those disclosed herein. Suitable fermentable carbon substrates include, but are not limited to, monosaccharides, such as glucose or fructose; disaccharides, such as lactose or sucrose; oligosaccharides; polysaccharides, such as starch, cellulose, or lignocellulose, hemicellulose; one-carbon substrates, fatty acids; and a combination of these.

[0067] "Fermentation medium" means the mixture of water, sugars (fermentable carbon substrates), dissolved solids, microorganisms producing fermentation products, fermentation product and all other constituents of the material held in the fermentation vessel in which the fermentation product is being made by the reaction of fermentable carbon substrates to fermentation products, water and carbon dioxide (C0 ₂ ) by the microorganisms present. From time to time, as used herein the term "fermentation broth" and "fermentation mixture" can be used synonymously with "fermentation medium."

[0068] The term "aerobic conditions" means conditions in the presence of oxygen.

[0069] The term "microaerobic conditions" means conditions with low levels of dissolved oxygen. For example, the oxygen level may be less than about 1% of air-saturation.

[0070] The term "anaerobic conditions" means conditions in the absence of oxygen. It will be understood that in many fermentation processes, an initial amount of oxygen is present at the onset of the process, but such oxygen is depleted over the course of the fermentation such that the majority of the process takes place in the absence of detectable oxygen.

[0071] The term "yield" refers to the amount of product per amount of carbon source in g/g. The yield may be exemplified for glucose as the carbon source. It is understood unless otherwise noted that yield is expressed as a percentage of the theoretical yield. In reference to a microorganism or metabolic pathway, "theoretical yield" is defined as the maximum amount of product that can be generated per total amount of substrate as dictated by the stoichiometry of the metabolic pathway used to make the product. For example, the theoretical yield for one typical conversion of glucose to isopropanol is 0.33 g/g. As such, a yield of isopropanol from glucose of 0.297 g/g would be expressed as 90% of theoretical or 90% theoretical yield. It is understood that while in the present disclosure the yield is exemplified for glucose as a carbon source, the invention can be applied to other carbon sources and the yield may vary depending on the carbon source used. One skilled in the art can calculate yields on various carbon sources.

[0072] The terms "acetohydroxyacid synthase," "acetolactate synthase" and "acetolactate synthetase" (abbreviated "ALS", "AlsS", "alsS" and/or "AHAS" herein) are used interchangeably to refer to an enzyme that catalyzes the conversion of pyruvate to acetolactate and C02.

Example acetolactate synthases are known by the EC number 2.2.1.6 (Enzyme Nomenclature 1992, Academic Press, San Diego). These enzymes are available from a number of sources, including, but not limited to, Bacillus subtilis (GenBank Nos: CAB07802.1 (SEQ ID NO: 1), Z99122 (SEQ ID NO: 2), NCBI (National Center for Biotechnology Information) amino acid sequence, NCBI nucleotide sequence, respectively), CAB15618 (SEQ ID NO: 789), Klebsiella pneumoniae (GenBank Nos: AAA25079 (SEQ ID NO:3), M73842 (SEQ ID NO:4)), and

Lactococcus lactis (GenBank Nos: AAA25161 (SEQ ID NO:5), L16975 (SEQ ID NO:6)). [0073] The terms "ketol-acid reductoisomerase" ("KARI"), and "acetohydroxy acid isomeroreductase" will be used interchangeably and refer to enzymes capable of catalyzing the reaction of (S)-acetolactate to 2,3-dihydroxyisovalerate. Example KARI enzymes may be classified as EC number EC 1.1.1.86 (Enzyme Nomenclature 1992, Academic Press, San Diego), and are available from a vast array of microorganisms, including, but not limited to, Escherichia coli (GenBank Nos: NP 418222 (SEQ ID NO: 7), NC 000913 (SEQ ID NO: 8)),

Saccharomyces cerevisiae (GenBank Nos: NP 013459 (SEQ ID NO: 9), NC OOl 144 (SEQ ID NO: 10)), Methanococcus maripaludis (GenBank Nos: CAF30210 (SEQ ID NO: 11), BX957220 (SEQ ID NO: 12)), and Bacillus subtilis (GenBank Nos: CAB14789 (SEQ ID NO: 13), Z99118 (SEQ ID NO: 14)). KARIs include Anaerostipes caccae KARI variants "K9G9", "K9D3", and "K9JB4P" (SEQ ID NOs: 167, 166, and 791 respectively). In some embodiments, KARI utilizes NADH. In some embodiments, KARI utilizes NADPH.

[0074] The terms "acetohydroxy acid dehydratase" and "dihydroxyacid dehydratase"

("DHAD") refer to an enzyme that catalyzes the conversion of 2,3-dihydroxyisovalerate to a- ketoiso valerate. Example acetohydroxy acid dehydratases are known by the EC number 4.2.1.9. Such enzymes are available from a vast array of microorganisms, including, but not limited to, E. coli (GenBank Nos: YP 026248 (SEQ ID NO: 15), NC 000913 (SEQ ID NO: 16)), S. cerevisiae (GenBank Nos: NP_012550 (SEQ ID NO: 17), NC 001142 (SEQ ID NO: 18)), M. maripaludis (GenBank Nos: CAF29874 (SEQ ID NO: 19), BX957219 (SEQ ID NO: 20)), B. subtilis

(GenBank Nos: CAB14105 (SEQ ID NO: 21), Z99115 (SEQ ID NO: 22)), L. lactis, N. crassa, and S. mutans. DHADs include S. mutans variant "12 V5" (SEQ ID NO: 792)

[0075] The terms "branched-chain a-keto acid decarboxylase" or "a-ketoacid

decarboxylase" or "a-ketoiso valerate decarboxylase" or "2-ketoisovalerate decarboxylase" ("KIVD") refer to an enzyme that catalyzes the conversion of a-ketoisovalerate to

isobutyraldehyde and C0 ₂ . Example branched-chain a-keto acid decarboxylases are known by the EC number 4.1.1.72 and are available from a number of sources, including, but not limited to, Lactococcus lactis (GenBank Nos: AAS49166 (SEQ ID NO: 23), AY548760 (SEQ ID NO: 24); CAG34226 (SEQ ID NO: 25), AJ746364 (SEQ ID NO: 26), Salmonella typhimurium (GenBank Nos: NP_461346 (SEQ ID NO: 27), NC_003197 (SEQ ID NO: 28)), Clostridium acetobutylicum (GenBank Nos: NPJ49189 (SEQ ID NO: 29), NC_001988 (SEQ ID NO: 30)), M. caseolyticus (SEQ ID NO: 165), and L. grayi (SEQ ID NO: 164). [0076] The term "alcohol dehydrogenase" ("ADH") refers to an enzyme that catalyzes the conversion of isobutyraldehyde to isobutanol, 2-butanone to 2-butanol, and butyraldehyde to 1- butanol. Alcohol dehydrogenases may be "branched chain alcohol dehydrogenases" or may be referred to as "butanol dehydrogenases." Example alcohol dehydrogenases suitable for embodiments disclosed herein may be known by the EC number 1.1.1.265, but may also be classified under other alcohol dehydrogenases, for example, according to published preference for NADH (typically 1.1.1.1) or NADPH (typically 1.1.1.2) as cofactors. Such enzymes are available from a number of sources, including, but not limited to, S. cerevisiae (GenBank Nos: NP 010656 (SEQ ID NO: 31), NC OOl 136 (SEQ ID NO: 32); NP 014051 (SEQ ID NO: 33) NC_001145 (SEQ ID NO: 34)), E. coli (GenBank Nos: NP_417484 (SEQ ID NO: 35),

NC 000913 (SEQ ID NO: 36)), C. acetobutylicum (GenBank Nos: NP 349892 (SEQ ID NO: 37), NC 003030 (SEQ ID NO: 38); NP 349891 (SEQ ID NO: 39), NC 003030 (SEQ ID NO: 40)), Pyrococcus furiosus (GenBank Nos: AAC25556, AF013169), Acinetobacter sp. (GenBank Nos: AAG10026, AF282240), Rhodococcus ruber (GenBank Nos: CAD36475, AJ491307), A. xylosoxidans (SEQ ID NO: 798), horse liver ADH (SEQ ID NO: 799), and B. indica (SEQ ID NO: 800). Butanol dehydrogenase enzymes are available, for example, from Rhodococcus ruber (GenBank Nos: CAD36475, AJ491307). Additional NADP dependent enzymes are known, and are available, for example, from Pyrococcus furiosus (GenBank Nos: AAC25556, AF013169). Additionally, a butanol dehydrogenase is available from Escherichia coli (GenBank Nos: NP 417484, NC_000913) and a cyclohexanol dehydrogenase is available from Acinetobacter sp. (GenBank Nos: AAG10026, AF282240). Butanol dehydrogenases are additionally available from, for example, C. acetobutylicum (GenBank NOs: NP_149325, NC_001988; note: this enzyme possesses both aldehyde and alcohol dehydrogenase activity); NP 349891, NC 003030; and NP_349892, NC_003030) and E. coli (GenBank NOs: NP_417484, NC_000913).

[0077] The term "branched-chain keto acid dehydrogenase" refers to an enzyme that catalyzes the conversion of a-ketoisovalerate to isobutyryl-CoA (isobutyryl-coenzyme A), typically using NAD ⁺ (nicotinamide adenine dinucleotide) as an electron acceptor. Example branched-chain keto acid dehydrogenases are known by the EC number 1.2.4.4. Such branched- chain keto acid dehydrogenases are comprised of four subunits and sequences from all subunits are available from a vast array of microorganisms, including, but not limited to, B. subtilis (GenBank Nos: CAB14336 (SEQ ID NO: 41), Z99116 (SEQ ID NO: 42); CAB14335 (SEQ ID NO: 43), Z99116 (SEQ ID NO: 44); CAB14334 (SEQ ID NO: 45), Z99116 (SEQ ID NO: 46); and CAB14337 (SEQ ID NO: 47), Z99116 (SEQ ID NO: 48)) and Pseudomonas putida

(GenBank Nos: AAA65614 (SEQ ID NO: 49), M57613 (SEQ ID NO: 50); AAA65615 (SEQ ID NO: 51), M57613 (SEQ ID NO: 52); AAA65617 (SEQ ID NO: 53), M57613 (SEQ ID NO: 54); and AAA65618 (SEQ ID NO: 55), M57613 (SEQ ID NO: 56)).

[0078] The term "acylating aldehyde dehydrogenase" refers to an enzyme that catalyzes the conversion of isobutyryl-CoA to isobutyraldehyde, typically using either NADH or NADPH as an electron donor. Example acylating aldehyde dehydrogenases are known by the EC numbers 1.2.1.10 and 1.2.1.57. Such enzymes are available from multiple sources, including, but not limited to, Clostridium beijerinckii (GenBank Nos: AAD31841 (SEQ ID NO: 57), AF157306 (SEQ ID NO: 58)), C. acetobutylicum (GenBank Nos: NPJ49325 (SEQ ID NO: 59),

NC 001988 (SEQ ID NO: 60); NP 149199 (SEQ ID NO: 61), NC 001988 (SEQ ID NO: 62)), P. putida (GenBank Nos: AAA89106 (SEQ ID NO: 63), U13232 (SEQ ID NO: 64)), and Thermus thermophilus (GenBank Nos: YP 145486 (SEQ ID NO: 65), NC 006461 (SEQ ID NO: 66)).

[0079] The term "transaminase" refers to an enzyme that catalyzes the conversion of a-ketoisovalerate to L-valine, using either alanine or glutamate as an amine donor. Example transaminases are known by the EC numbers 2.6.1.42 and 2.6.1.66. Such enzymes are available from a number of sources. Examples of sources for alanine-dependent enzymes include, but are not limited to, E. coli (GenBank Nos: YP 026231 (SEQ ID NO: 67), NC 000913 (SEQ ID NO: 68)) and Bacillus licheniformis (GenBank Nos: YP_093743 (SEQ ID NO: 69), NC_006322 (SEQ ID NO: 70)). Examples of sources for glutamate-dependent enzymes include, but are not limited to, E. coli (GenBank Nos: YP 026247 (SEQ ID NO: 71), NC 000913 (SEQ ID NO: 72)), S. cerevisiae (GenBank Nos: NP_012682 (SEQ ID NO: 73), NC_001142 (SEQ ID NO:

74) ) and Methanobacterium thermoautotrophicum (GenBank Nos: NP_276546 (SEQ ID NO:

75) , NC 000916 (SEQ ID NO: 76)).

[0080] The term "valine dehydrogenase" refers to an enzyme that catalyzes the conversion of a-ketoisovalerate to L-valine, typically using NAD(P)H as an electron donor and ammonia as an amine donor. Example valine dehydrogenases are known by the EC numbers 1.4.1.8 and 1.4.1.9 and such enzymes are available from a number of sources, including, but not limited to, Streptomyces coelicolor (GenBank Nos: NP_628270 (SEQ ID NO: 77), NC_003888 (SEQ ID NO: 78)) and B. subtilis (GenBank Nos: CAB14339 (SEQ ID NO: 79), Z99116 (SEQ ID NO: 80)). [0081] The term "valine decarboxylase" refers to an enzyme that catalyzes the conversion of L-valine to isobutylamine and C0 ₂ . Example valine decarboxylases are known by the EC number 4.1.1.14. Such enzymes are found in Streptomyces, such as for example, Streptomyces viridifaciens (GenBank Nos: AAN10242 (SEQ ID NO: 81), AY116644 (SEQ ID NO: 82)).

[0082] The term "omega transaminase" refers to an enzyme that catalyzes the conversion of isobutylamine to isobutyraldehyde using a suitable amino acid as an amine donor. Example omega transaminases are known by the EC number 2.6.1.18 and are available from a number of sources, including, but not limited to, Alcaligenes denitrificans (AAP92672 (SEQ ID NO: 83), AY330220 (SEQ ID NO: 84)), Ralstonia eutropha (GenBank Nos: YP_294474 (SEQ ID NO: 85), NC_007347 (SEQ ID NO: 86)), Shewanella oneidensis (GenBank Nos: NP_719046 (SEQ ID NO: 87), NC_004347 (SEQ ID NO: 88)), and P. putida (GenBank Nos: AAN66223 (SEQ ID NO: 89), AE016776 (SEQ ID NO: 90)).

[0083] The term "acetyl-CoA acetyltransferase" refers to an enzyme that catalyzes the conversion of two molecules of acetyl-CoA to acetoacetyl-CoA and coenzyme A (Co A).

Example acetyl-CoA acetyltransferases are acetyl-CoA acetyltransferases with substrate preferences (reaction in the forward direction) for a short chain acyl-CoA and acetyl-CoA and are classified as E.C. 2.3.1.9 [Enzyme Nomenclature 1992, Academic Press, San Diego]; although, enzymes with a broader substrate range (E.C. 2.3.1.16) will be functional as well. Acetyl-CoA acetyltransferases are available from a number of sources, for example, Escherichia coli

(GenBank Nos: NP 416728 (SEQ ID NO: 91), NC 000913 (SEQ ID NO: 92); NCBI (National Center for Biotechnology Information) amino acid sequence, NCBI nucleotide sequence), Clostridium acetobutylicum (GenBank Nos: NP_349476.1 (SEQ ID NO: 93), NC_003030 (SEQ ID NO: 94); NPJ49242 (SEQ ID NO: 95), NC_001988 (SEQ ID NO: 96), Bacillus subtilis (GenBank Nos: NP 390297 (SEQ ID NO: 97), NC 000964 (SEQ ID NO: 98)), and

Saccharomyces cerevisiae (GenBank Nos: NP_015297 (SEQ ID NO: 99), NC_001148 (SEQ ID NO: 100)).

[0084] The term "3-hydroxybutyryl-CoA dehydrogenase" refers to an enzyme that catalyzes the conversion of acetoacetyl-CoA to 3-hydroxybutyryl-CoA. 3 -Example

hydroxybutyryl-CoA dehydrogenases may be reduced nicotinamide adenine dinucleotide (NADH)-dependent, with a substrate preference for (S)-3-hydroxybutyryl-CoA or (R)-3- hydroxybutyryl-CoA. Examples may be classified as E.C. 1.1.1.35 and E.C. 1.1.1.30, respectively. Additionally, 3-hydroxybutyryl-CoA dehydrogenases may be reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent, with a substrate preference for (S)-3- hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA and are classified as E.C. 1.1.1.157 and E.C. 1.1.1.36, respectively. 3-Hydroxybutyryl-CoA dehydrogenases are available from a number of sources, for example, C. acetobutylicum (GenBank NOs: NP 349314 (SEQ ID NO: 101), NC 003030 (SEQ ID NO: 102)), B. subtilis (GenBank NOs: AAB09614 (SEQ ID NO: 103), U29084 (SEQ ID NO: 104)), Ralstonia eutropha (GenBank NOs: YP 294481 (SEQ ID NO: 105), NC_007347 (SEQ ID NO: 106)), and Alcaligenes eutrophus (GenBank NOs: AAA21973 (SEQ ID NO: 107), J04987 (SEQ ID NO: 108)).

[0085] The term "crotonase" refers to an enzyme that catalyzes the conversion of 3- hydroxybutyryl-CoA to crotonyl-CoA and H ₂ 0. Example crotonases may have a substrate preference for (S)-3-hydroxybutyryl-CoA or (R)-3-hydroxybutyryl-CoA and may be classified as E.C. 4.2.1.17 and E.C. 4.2.1.55, respectively. Crotonases are available from a number of sources, for example, E. coli (GenBank NOs: NP 415911 (SEQ ID NO: 109), NC 000913 (SEQ ID NO: 110)), C. acetobutylicum (GenBank NOs: NP 349318 (SEQ ID NO: 111), NC 003030 (SEQ ID NO: 112)), B. subtilis (GenBank NOs: CAB13705 (SEQ ID NO: 113), Z99113 (SEQ ID NO: 114)), and Aeromonas caviae (GenBank NOs: BAA21816 (SEQ ID NO: 115), D88825 (SEQ ID NO: 116)).

[0086] The term "butyryl-CoA dehydrogenase" refers to an enzyme that catalyzes the conversion of crotonyl-CoA to butyryl-CoA. Example butyryl-CoA dehydrogenases may be NADH-dependent, NADPH-dependent, or flavin-dependent and may be classified as E.C.

1.3.1.44, E.C. 1.3.1.38, and E.C. 1.3.99.2, respectively. Butyryl-CoA dehydrogenases are available from a number of sources, for example, C. acetobutylicum (GenBank NOs: NP 347102 (SEQ ID NO: 117), NC_ 003030 (SEQ ID NO: 118))), Euglena gracilis (GenBank NOs:

Q5EU90 SEQ ID NO: 119), AY741582 SEQ ID NO: 120)), Streptomyces collinus (GenBank NOs: AAA92890 (SEQ ID NO: 121), U37135 (SEQ ID NO: 122)), and Streptomyces coelicolor (GenBank NOs: CAA22721 (SEQ ID NO: 123), AL939127 (SEQ ID NO: 124)).

[0087] The term "isobutyryl-CoA mutase" refers to an enzyme that catalyzes the conversion of butyryl-CoA to isobutyryl-CoA. This enzyme uses coenzyme Bi ₂ as cofactor. Example isobutyryl-CoA mutases are known by the EC number 5.4.99.13. These enzymes are found in a number of Streptomyces, including, but not limited to, Streptomyces cinnamonensis (GenBank Nos: AAC08713 (SEQ ID NO: 125), U67612 (SEQ ID NO: 126); CAB59633 (SEQ ID NO: 127), AJ246005 (SEQ ID NO: 128)), S coelicolor (GenBank Nos: CAB70645 (SEQ ID NO: 129), AL939123 (SEQ ID NO: 130); CAB92663 (SEQ ID NO: 131), AL939121 (SEQ ID NO: 132)), and Streptomyces avermitilis (GenBank Nos: NP_824008 (SEQ ID NO: 133), NC 003155 (SEQ ID NO: 134); NP 824637 (SEQ ID NO: 135), NC 003155 (SEQ ID NO: 136)).

[0088] The term "butyraldehyde dehydrogenase" refers to an enzyme that catalyzes the conversion of butyryl-CoA to butyraldehyde, using NADH or NADPH as co factor.

Butyraldehyde dehydrogenases with a preference for NADH are known as E.C. 1.2.1.57 and are available from, for example, Clostridium beijerinckii (GenBank NOs: AAD31841 , AF157306) and C. acetobutylicum (GenBank NOs: NPJ49325, NC_001988).

[0089] The term "acetolactate decarboxylase" refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of alpha-acetolactate to acetoin. Example acetolactate decarboxylases are known as EC 4.1.1.5 and are available, for example, from Bacillus subtilis (GenBank Nos: AAA22223, L04470), Klebsiella terrigena (GenBank Nos: AAA25054, L04507) and Klebsiella pneumoniae (GenBank Nos: AAU43774, AY722056).

[0090] The term "acetoin aminase" or "acetoin transaminase" refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of acetoin to 3-amino-2- butanol. Acetoin aminase may utilize the cofactor pyridoxal 5'-phosphate or NADH (reduced nicotinamide adenine dinucleotide) or NADPH (reduced nicotinamide adenine dinucleotide phosphate). The resulting product may have (R) or (S) stereochemistry at the 3 -position. The pyridoxal phosphate-dependent enzyme may use an amino acid such as alanine or glutamate as the amino donor. The NADH- and NADPH-dependent enzymes may use ammonia as a second substrate. A suitable example of an NADH dependent acetoin aminase, also known as amino alcohol dehydrogenase, is described by Ito et al. (U.S. Pat. No. 6,432,688). An example of a pyridoxal-dependent acetoin aminase is the amine :pyruvate aminotransferase (also called amine :pyruvate transaminase) described by Shin and Kim, J. Org. Chem. 67:2848-2853 (2002)).

[0091] The term "acetoin kinase" refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of acetoin to phosphoacetoin. Acetoin kinase may utilize ATP (adenosine triphosphate) or phosphoenolpyruvate as the phosphate donor in the reaction. Enzymes that catalyze the analogous reaction on the similar substrate

dihydroxy acetone, for example, include enzymes known as EC 2.7.1.29 (Garcia- Alles et al, Biochemistry 45: 13037-13046 (2004)). [0092] The term "acetoin phosphate aminase" refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of phosphoacetoin to 3-amino-2- butanol O-phosphate. Acetoin phosphate aminase may use the cofactor pyridoxal 5 '-phosphate, NADH or NADPH. The resulting product may have (R) or (S) stereochemistry at the 3-position. The pyridoxal phosphate-dependent enzyme may use an amino acid such as alanine or glutamate. The NADH and NADPH-dependent enzymes may use ammonia as a second substrate. Although there are no reports of enzymes catalyzing this reaction on phosphoacetoin, there is a pyridoxal phosphate-dependent enzyme that is proposed to carry out the analogous reaction on the similar substrate serinol phosphate (Yasuta et ah, Appl. Environ. Microbial. (57:4999-5009 (2001)).

[0093] The term "aminobutanol phosphate phospho lyase", also called "amino alcohol O- phosphate lyase", refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of 3 -amino-2 -butanol O-phosphate to 2-butanone. Amino butanol phosphate phospho-lyase may utilize the cofactor pyridoxal 5'-phosphate. There are reports of enzymes that catalyze the analogous reaction on the similar substrate l-amino-2-propanol phosphate (Jones et al., Biochem J. 754: 167-182 (1973)). U.S. Appl. Pub. No. 2007/0259410 describes an aminobutanol phosphate phospho-lyase from the organism Erwinia carotovora.

[0094] The term "aminobutanol kinase" refers to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of 3-amino-2 -butanol to 3-amino-2butanol O- phosphate. Amino butanol kinase may utilize ATP as the phosphate donor. Although there are no reports of enzymes catalyzing this reaction on 3-amino-2-butanol, there are reports of enzymes that catalyze the analogous reaction on the similar substrates ethanolamine and l-amino-2- propanol (Jones et al, supra). U.S. Appl. Pub. No. 2009/0155870 describes, in Example 14, an amino alcohol kinase of Erwinia carotovora subsp. Atroseptica.

[0095] The terms "butanediol dehydrogenase" and "acetoin reductase" refer to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of acetoin to 2,3-butanediol. Butanedial dehydrogenases are a subset of the broad family of alcohol dehydrogenases. Butanediol dehydrogenase enzymes may have specificity for production of (R)- or (S)-stereochemistry in the alcohol product. (S)-specific butanediol dehydrogenases are known as EC 1.1.1.76 and are available, for example, from Klebsiella pneumoniae (GenBank Nos: BBA13085, D86412). (R)-specific butanediol dehydrogenases are known as EC 1.1.1.4 and are available, for example, from Bacillus cereus (GenBank Nos. NP 830481, NC_004722;

AAP07682, AE017000), and Lactococcus lactis (GenBank Nos. AAK04995, AE006323). [0096] The terms "butanediol dehydratase," "dial dehydratase" or "propanediol dehydratase" refer to a polypeptide (or polypeptides) having an enzyme activity that catalyzes the conversion of 2,3-butanediol to 2-butanone. Butanediol dehydratase may utilize the cofactor adenosyl cobalamin (also known as coenzyme Bw or vitamin B12; although vitamin B12 may refer also to other forms of cobalamin that are not coenzyme B12). Adenosyl cobalamin- dependent enzymes are known as EC 4.2.1.28 and are available, for example, from Klebsiella oxytoca (GenBank Nos: AA08099 (alpha subunit), D45071; BAA08100 (beta subunit), D45071; and BBA08101 (gamma subunit), D45071 (Note all three subunits are required for activity)], and Klebsiella pneumonia (GenBank Nos: AAC98384 (alpha subunit), AF102064; GenBank Nos: AAC98385 (beta subunit), AF102064, GenBank Nos: AAC98386 (gamma subunit), AF102064). Other suitable dial dehydratases include, but are not limited to, B12-dependent dial dehydratases available from Salmonella typhimurium (GenBank Nos: AAB84102 (large subunit), AF026270; GenBank Nos: AAB84103 (medium subunit), AF026270; GenBank Nos: AAB84104 (small subunit), AF026270); and Lactobacillus collinoides (GenBank Nos: CAC82541 (large subunit), AJ297723; GenBank Nos: CAC82542 (medium subunit); AJ297723; GenBank Nos: CAD01091 (small subunit), AJ297723); and enzymes from Lactobacillus brevis (particularly strains CNRZ 734 and CNRZ 735, Speranza et al, J. Agric. Food Chem. 45:3476-3480 (1997)), and nucleotide sequences that encode the corresponding enzymes. Methods of dial dehydratase gene isolation are well known in the art (e.g., U.S. Pat. No. 5,686,276).

[0097] The term "pyruvate decarboxylase" refers to an enzyme that catalyzes the decarboxylation of pyruvic acid to acetaldehyde and carbon dioxide. Pyruvate dehydrogenases are known by the EC number 4.1.1.1. These enzymes are found in a number of yeast, including Saccharomyces cerevisiae (GenBank Nos: CAA97575 (SEQ ID NO: 137), CAA97705 (SEQ ID NO: 138), CAA97091 (SEQ ID NO: 139)).

[0098] The term "phosphoketolase" refers to an enzyme that catalyzes the conversion of xyulose 5 -phosphate to glyceraldehyde 3 -phosphate and acetyl phosphate. Example

phosphoketolases are known by the EC number 4.1.2.9. In some embodiments, the

phosphoketolase is xpk from Lactobacillus plantarum (nucleic acid SEQ ID NO: 180; amino acid SEQ ID NO: 181).

[0099] The term "phosphotransacetylase" refers to an enzyme that catalyzes the conversion of acetyl-CoA and phosphate to CoA and acetyl phosphate. Example

phosphotransacetylases are known by the EC number 2.3.1.8. In some embodiments, the phosphotransacetylase is eutD from Lactobacillus plantarum (nucleic acid SEQ ID NO: 178; amino acid SEQ ID NO: 179).

[0100] The term "polypeptide" is intended to encompass a singular "polypeptide" as well as plural "polypeptides," and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term "polypeptide" refers to any chain or chains of two or more amino acids, and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, "protein," "amino acid chain," or any other term used to refer to a chain or chains of two or more amino acids, are included within the definition of "polypeptide," and the term "polypeptide" may be used instead of, or

interchangeably with any of these terms. The term "polypeptide" is also intended to refer to the products of post-expression modifications of the polypeptide, including without limitation glycosylation, acetylation, phosphorylation, amidation, derivatization by known

protecting/blocking groups, proteolytic cleavage, or modification by non-naturally occurring amino acids. In embodiments, the polypeptides provided herein, including, but not limited to biosynthetic pathway polypeptides, cell integrity polypeptides, propagation polypeptides, and other enzymes comprise full-length polypeptides and active fragments thereof.

[0101] A polypeptide of the invention may be of a size of about 10 or more, 20 or more,

25 or more, 50 or more, 75 or more, 100 or more, 200 or more, 500 or more, 1,000 or more, or 2,000 or more amino acids. Polypeptides may have a defined three-dimensional structure, although they do not necessarily have such structure. Polypeptides with a defined three- dimensional structure are referred to as folded, and polypeptides which do not possess a defined three-dimensional structure, but rather can adopt a large number of different conformations, and are referred to as unfolded.

[0102] Also included as polypeptides of the present invention are derivatives, analogs, or variants of the foregoing polypeptides, and any combination thereof. The terms "active variant," "active fragment," "active derivative," and "analog" refer to polypeptides of the present invention and include any polypeptides that are capable of catalyzing the indicated substrate to product conversion. Variants of polypeptides of the present invention include polypeptides with altered amino acid sequences due to amino acid substitutions, deletions, and/or insertions. Variants may occur naturally or be non-naturally occurring. Non-naturally occurring variants may be produced using art-known mutagenesis techniques. Variant polypeptides may comprise conservative or non-conservative amino acid substitutions, deletions and/or additions. Derivatives of polypeptides of the present invention, are polypeptides which have been altered so as to exhibit additional features not found on the native polypeptide. Examples include fusion proteins.

Variant polypeptides may also be referred to herein as "polypeptide analogs." As used herein a "derivative" of a polypeptide refers to a subject polypeptide having one or more residues chemically derivatized by reaction of a functional side group. Also included as "derivatives" are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acids. For example, 4-hydroxyproline may be substituted for proline; 5- hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted for serine; and ornithine may be substituted for lysine.

[0103] A "fragment" is a unique portion of polypeptide used in the invention which is identical in sequence to but shorter in length than the parent full-length sequence. A fragment may comprise up to the entire length of the defined sequence, minus one amino acid residue. For example, a fragment may comprise from 5 to 1000 contiguous amino acid residues. A fragment may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule. For example, a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 100 or 200 amino acids of a polypeptide as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments. Similarly, "active fragment", when used in reference to a polypeptide, is a portion of a polypeptide which retains the functionality of the subject polypeptide, but comprises less than the entire sequence of the polypeptide.

[0104] Alternatively, recombinant variants encoding these same or similar polypeptides can be synthesized or selected by making use of the "redundancy" in the genetic code. Various codon substitutions, such as the silent changes which produce various restriction sites, may be introduced to optimize cloning into a plasmid or viral vector or expression in a host cell system.

[0105] Preferably, amino acid "substitutions" are the result of replacing one amino acid with another amino acid having similar structural and/or chemical properties, i.e., conservative amino acid replacements, or they can be result of replacing one amino acid with an amino acid having different structural and/or chemical properties, i.e., non-conservative amino acid replacements. "Conservative" amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues involved. For example, nonpolar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine; polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; positively charged (basic) amino acids include arginine, lysine, and histidine; and negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Alternatively, "non-conservative" amino acid substitutions can be made by selecting the differences in polarity, charge, solubility, hydrophobicity, hydrophilicity, or the amphipathic nature of any of these amino acids.

"Insertions" or "deletions" are preferably in the range of about 1 to about 20 amino acids, more preferably 1 to 10 amino acids. The variation allowed may be experimentally determined by systematically making insertions, deletions, or substitutions of amino acids in a polypeptide molecule using recombinant DNA techniques and assaying the resulting recombinant variants for activity.

[0106] Polypeptides suitable for use in the present invention and fragments thereof are encoded by polynucleotides. The term "polynucleotide" is intended to encompass a singular nucleic acid as well as plural nucleic acids, and refers to an isolated nucleic acid molecule or construct, e.g., messenger RNA (mRNA), virally-derived RNA, or plasmid DNA (pDNA). A polynucleotide may comprise a conventional phosphodiester bond or a non-conventional bond {e.g., an amide bond, such as found in peptide nucleic acids (PNA)). The term "nucleic acid" refers to any one or more nucleic acid segments, e.g., DNA or RNA fragments, present in a polynucleotide. Polynucleotides according to the present invention further include such molecules produced synthetically. Polynucleotides of the invention may be native to the host cell or heterologous. In addition, a polynucleotide or a nucleic acid may be or may include a regulatory element such as a promoter, ribosome binding site, or a transcription terminator.

[0107] A polynucleotide or polypeptide sequence can be referred to as "isolated," in which it has been placed in an environment other than its native environment or is produced synthetically or is a non-naturally occurring, or engineered, sequence. For example, a

heterologous polynucleotide encoding a polypeptide or polypeptide fragment having enzymatic activity (e.g., the ability to convert a substrate to xylulose) contained in a vector is considered isolated for the purposes of the present invention. Further examples of an isolated polynucleotide include recombinant polynucleotides maintained in heterologous host cells or purified (partially or substantially) polynucleotides in solution. An isolated polynucleotide fragment in the form of a polymer of DNA can be comprised of one or more segments of cDNA, genomic DNA, or synthetic DNA.

[0108] The term "gene" refers to a nucleic acid fragment that is capable of being expressed as a specific protein, optionally including regulatory sequences preceding (5' non- coding sequences) and following (3' non-coding sequences) the coding sequence.

[0109] A "coding region" or "ORF" is a portion of nucleic acid which consists of codons translated into amino acids. Although a "stop codon" (TAG, TGA, or TAA) is not translated into an amino acid, it may be considered to be part of a coding region, if present, but any flanking sequences, for example promoters, ribosome binding sites, transcriptional terminators, introns, 5' and 3' non-translated regions, and the like, are not part of a coding region. "Suitable regulatory sequences" refer to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence that influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences can include promoters, translation leader sequences, introns, polyadenylation recognition sequences, RNA processing sites, effector binding sites and stem- loop structures.

[0110] The term "promoter" refers to a DNA sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3' to a promoter sequence. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic DNA segments. It is understood by those skilled in the art that different promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters which cause a gene to be expressed in most cell types at most times are commonly referred to as "constitutive promoters." Example constitutive promoters are provided herein and/or are known to those of skill in the art. Constitutive promoters include, but are not limited to, the following constitutive promoters suitable for use in yeast: FBA1, TDH3 (GPD), ADH1, ILV5, and GPM1. Other constitutive promoters are known in the art It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths may have identical promoter activity.

[0111] Some promoter nucleic acid sequences disclosed herein, including those in Tables

1, 2, 7, and 8, were arbitrarily taken to be 1000 bp 5' of the start codon of each gene. However, the sequences may be retrieved from publicly available databases such as the Yeastract database, www.yeastract.com (visited December 21, 2012) or the Saccharomyces Genome Database www.yeastgenome.org (visited December 21, 2012). The gene name (where available) and the systematic name (cf. Saccharomyces Genome Database where available) is indicated. As described above, it will be appreciated by one of ordinary skill in the art that fragments of different lengths of the sequences provided may have identical promoter activity, thus, reference to, for example "HEM13 promoter", will be understood to encompass a sequence provided herein or any fragment of the promoter region of the HEM 13 gene which has identical promoter activity or a substantially similar effect on expression of a target polypeptide or production of an indicated product. Thus, the disclosed nucleic acid sequences should not be construed as limited solely to the provided sequence.

[0112] In certain embodiments, the polynucleotide or nucleic acid is DNA. In the case of

DNA, a polynucleotide comprising a nucleic acid, which encodes a polypeptide normally may include a promoter and/or other transcription or translation control elements operably associated with one or more coding regions. An operable association is when a coding region for a gene product, e.g., a polypeptide, is associated with one or more regulatory sequences in such a way as to place expression of the gene product under the influence or control of the regulatory sequence(s). Two DNA fragments (such as a polypeptide coding region and a promoter associated therewith) are "operably associated" or "operably linked" or "coupled" if induction of promoter function results in the transcription of mRNA and if the nature of the linkage between the two DNA fragments does not interfere with the ability of the expression regulatory sequences to direct the expression of the gene product or interfere with the ability of the DNA template to be transcribed. Thus, a promoter region would be operably associated with a nucleic acid encoding a polypeptide if the promoter was capable of effecting transcription of that nucleic acid. Other transcription control elements, besides a promoter, for example enhancers, operators, repressors, and transcription termination signals, can be operably associated with the

polynucleotide. Suitable promoters and other transcription control regions are disclosed herein. An "expression construct", as used herein, comprises a promoter nucleic acid sequence operably linked to a coding region for a polypeptide and, optionally, a terminator nucleic acid sequence.

[0113] A variety of translation control elements are known to those of ordinary skill in the art. These include, but are not limited to ribosome binding sites, translation initiation and termination codons, and elements derived from viral systems (particularly an internal ribosome entry site, or IRES). In other embodiments, a polynucleotide of the present invention is RNA, for example, in the form of messenger RNA (mRNA). R A of the present invention may be single stranded or double stranded.

[0114] Polynucleotide and nucleic acid coding regions of the present invention may be associated with additional coding regions which encode secretory or signal peptides, which direct the secretion of a polypeptide encoded by a polynucleotide of the present invention.

[0115] The term "transformation" refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "recombinant" or

"transformed" organisms.

[0116] The terms "expression," and "expressed" refer to the transcription and stable accumulation of sense (mRNA) or antisense RNA derived from the nucleic acid fragment of the invention. Expression may also refer to translation of mRNA into a polypeptide. "Differentially expressed" refers to the differential production of the mRNA transcribed from the gene or the protein product encoded by the gene depending on the environment of the host cell. A

differentially expressed gene may be overexpressed or underexpressed as compared to the expression level under other conditions. In one aspect, it refers to a differential that is 1, 2, 3, 4, 5, 10 , or 20 times higher or lower than the expression level detected in a reference environment. The term "differentially expressed" also refers to nucleotide sequences in a cell which are expressed where silent or not expressed in a control environment or not expressed where expressed in a control cell.

[0117] The terms "plasmid" and "vector" refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA fragments. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell. "Transformation cassette" refers to a specific vector containing a foreign gene and having elements in addition to the foreign gene that facilitates transformation of a particular host cell. "Expression cassette" refers to a specific construct containing a gene and having elements in addition to the gene that allow for expression of that gene. [0118] "Native" refers to the form of a polynucleotide, gene, or polypeptide as found in nature with its own regulatory sequences, if present.

[0119] "Endogenous" refers to the native form of a polynucleotide, gene or polypeptide in its natural location in the organism or in the genome of an organism. "Endogenous

polynucleotide" includes a native polynucleotide in its natural location in the genome of an organism. "Endogenous gene" includes a native gene in its natural location in the genome of an organism. "Endogenous polypeptide" includes a native polypeptide in its natural location in the organism.

[0120] "Heterologous" refers to a polynucleotide, gene, or polypeptide not normally found in the host organism but that is introduced into the host organism. "Heterologous polynucleotide" may include a native coding region, or portion thereof, that is reintroduced into the source organism in a form that is different from the corresponding native polynucleotide. "Heterologous gene" 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. For example, a heterologous gene can 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. "Heterologous polypeptide" includes a native polypeptide that is reintroduced into the source organism in a form that is different from the corresponding native polypeptide.

[0121] By a nucleic acid or polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence of the present invention, it is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five point mutations per each 100 nucleotides of the reference nucleotide sequence. In other words, to obtain a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence, up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.

[0122] As a practical matter, whether any particular nucleic acid molecule or polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to a nucleotide sequence or polypeptide sequence of the present invention can be determined conventionally using known computer programs. A preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, can be determined using the FASTDB computer program based on the algorithm of Brutlag et ah, Comp. Appl. Biosci. (5:237-245 (1990). In a sequence alignment the query and subject sequences are both DNA sequences. An R A sequence can be compared by converting U's to T's. The result of the global sequence alignment is in percent identity. Preferred parameters used in a FASTDB alignment of DNA sequences to calculate percent identity are: Matrix=Unitary, k-tuple=4, Mismatch Penalty=l, Joining Penalty-30,

Randomization Group Length=0, Cutoff Score=l, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or the length of the subject nucleotide sequences, whichever is shorter.

[0123] If the subject sequence is shorter than the query sequence because of 5' or 3' deletions, not because of internal deletions, a manual correction must be made to the results. This is because the FASTDB program does not account for 5' and 3' truncations of the subject sequence when calculating percent identity. For subject sequences truncated at the 5' or 3' ends, relative to the query sequence, the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not

matched/aligned, as a percent of the total bases of the query sequence. Whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of the present invention. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score.

[0124] For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at 5' end. The 10 unpaired bases represent 10% of the sequence (number of bases at the 5' and 3' ends not matched/total number of bases in the query sequence) so 10%> is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%. In another example, a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5 ' or 3' of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present invention.

[0125] Polypeptides used in the invention are encoded by nucleic acid sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequences described elsewhere in the specification, including active variants, fragments or derivatives thereof.

[0126] The terms "active variant," "active fragment," "active derivative," and "analog" refer to polynucleotides of the present invention and include any polynucleotides that encode biocatalyst polypeptides used in the invention that retain their respective enzymatic activities or structure. Variants of polynucleotides of the present invention include polynucleotides with altered nucleotide sequences due to base pair substitutions, deletions, and/or insertions. Variants may occur naturally or be non-naturally occurring. Non-naturally occurring variants may be produced using art-known mutagenesis techniques. Derivatives of polynucleotides of the present invention, are polynucleotides which have been altered so that the polypeptides they encode exhibit additional features not found on the native polypeptide. Examples include

polynucleotides that encode fusion proteins. Variant polynucleotides may also be referred to herein as "polynucleotide analogs." As used herein a "derivative" of a polynucleotide refers to a subject polynucleotide having one or more nucleotides chemically derivatized by reaction of a functional side group. Also included as "derivatives" are those polynucleotides which contain one or more naturally occurring nucleotide derivatives. For example, 3-methylcytidine may be substituted for cytosine; ribothymidine may be substituted for thymidine; and N4-acetylcytidine may be substituted for cytosine.

[0127] A "fragment" when used in reference to a promoter sequence is a unique portion of the promoter nucleic acid sequence or the nucleic acid sequence encoding the biocatalyst polypeptide used in the invention which is identical in sequence to but shorter in length than the parent nucleic acid sequence. For example, a fragment may comprise from 5 to 1000 contiguous nucleotides. A fragment used as a probe, primer, or for other purposes, may be at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides. A fragment may comprise up to the entire length of the defined sequence, minus one nucleotide or amino acid. Fragments may be preferentially selected from certain regions of a molecule. For example, a polynucleotide fragment may comprise a certain length of contiguous nucleotides selected from the first 100 or 200 nucleotides of a polynucleotide as shown in a certain defined sequence. Clearly these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.

[0128] The term "codon degeneracy" refers to the nature in the genetic code permitting variation of the nucleotide sequence without effecting the amino acid sequence of an encoded polypeptide. The skilled artisan is well aware of the "codon-bias" exhibited by a specific host cell in usage of nucleotide codons to specify a given amino acid. Therefore, when synthesizing a gene for improved expression in a host cell, it is desirable to design the gene such that its frequency of codon usage approaches the frequency of preferred codon usage of the host cell.

[0129] 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.

[0130] 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 A. 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 A: The Standard Genetic Code

[0131] 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.

[0132] 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 the "Codon Usage Database" available at http://www.kazusa.or.jp/codon/ (visited March 20, 2008), and these tables can be adapted in a number of ways. See Nakamura, Y., et al. 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 B. 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 B: Codon Usage Table for Saccharomyces cerevisiae Genes

[0133] 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.

[0134] 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 http://www.entelechon.corn/bioinformatics/backtranslation.ph p?lang=eng (visited April 15, 2008) and the "backtranseq" function available at

http://bioinfo.pbi.nrc.ca:8090/EMBOSS/index.html (visited July 9, 2002). 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.

[0135] Codon-optimized coding regions can be designed by various methods known to those skilled in the art including software packages such as "synthetic gene designer"

(userpages.umbc.edu/~vyugl/codon/sgd/, visited March 19, 2012).

[0136] In some embodiments of the invention, a gene encoding a biocatalyst polypeptide contains a frameshift mutation, such that the polypeptide is not properly translated in the absence of a suppressor tRNA molecule. In some embodiments of the invention, a gene encoding a biocatalyst polypeptide contains a nonsense mutation, such that the polypeptide is not fully translated in the absence of a suppressor tRNA molecule. In some embodiments the nonsense mutation in the gene coding the biocatalyst polypeptide is the the UAG stop codon, the UGA stop codon, and/or the UAA stop codon.

Promoter nucleic acid sequences - "Genetic switches"

[0137] In some embodiments, the promoter activity is sensitive to one or more physiochemical differences between propagation and production stages of fermentation. In embodiments, the promoter activity is sensitive to the dissolved oxygen concentration. In embodiments, the promoter activity is sensitive to the glucose concentration. In some

embodiments, the promoter activity is sensitive to the source of the fermentable carbon substrate. In some embodiments, the promoter activity is sensitive to the concentration of butanol in fermentation medium. In some embodiments, the promoter activity is sensitive to the pH in the fermentation medium. In some embodiments, the promoter activity is sensitive to the

temperature in the fermentation medium. In embodiments, the promoter activity provides for differential expression in propagation and production stages of fermentation. Production and Propagation

[0138] Promoter nucleic acid sequences useful in the invention include those identified using "promoter prospecting" described and exemplified herein including those that comprise nucleic acid sequences which are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%) identical to the nucleotide sequences of SEQ ID NOs: 141-163, including variants, fragments or derivatives thereof that confer or increase sensitivity to fermentation conditions, such as, the concentration of oxygen, butanol, isobutyraldehyde, isobutyric acid, acetic acid, or a fermentable carbon substrate in the fermentation medium. A subset of these suitable promoter nucleic acid sequences is given in Tables 1 and 2 below.

Table 1 : Promoters -Upregulated in Corn Mash Production Fermentor

Compared to Propagation Tank

TABLE 2: Promoters Strongly-Downregulated in Corn Mash Production

Fermentor Compared to Propagation Tank

**Descriptions for Tables 1 and 2 from Saccharomyces Genome Database

(www.yeastgenome.org).

[0139] In embodiments of the invention, the promoter nucleic acid sequence is sensitive to the concentration of fermentable carbon substrates in fermentation medium. In yet a further embodiment, the promoter nucleic acid sequence is sensitive to the concentration of a fermentable carbon substrate selected from the group consisting of: monosaccharides, oligosaccharides, polysaccharides, fatty acids, and mixtures thereof.

[0140] In embodiments of the invention, promoter nucleic acid sequences suitable for use in the invention comprise nucleotide sequences that are at least about 80%, 85%, 90%, 95%, 96%o, 97%o, 98%), 99%o or 100% identical to a sequence selected from the group consisting of: SEQ ID NO: 140 [IMA1/YGR287], [HXK1], SEQ ID NO: 141 [HXK2], SEQ ID NO: 142

[SLT2], SEQ ID NO: 143 [YHR210c], SEQ ID NO: 144 [YJL171c], SEQ ID NO: 145 [PUNl], SEQ ID NO: 146 [PRE8], SEQ ID NO: 147 [COS3], SEQ ID NO: 148 [DIAl], SEQ ID NO: 149 [YNR062C], SEQ ID NO: 150 [PREIO], SEQ ID NO: 151 [AIM45], SEQ ID NO: 152 [ZRT1], SEQ ID NO: 153 [ZRT2], SEQ ID NO: 154 [PH084], SEQ ID NO: 155 [PCLl], SEQ ID NO: 156 [ARGl], SEQ ID NO: 157 [ZPSl], SEQ ID NO: 158 [FIT2], SEQ ID NO: 159 [FIT3], SEQ ID NO: 160 [FRE5], SEQ ID NO: 161 [CSM4], SEQ ID NO: 160 [SAM3], SEQ ID NO: 163 [FDH2] or a variant, fragment or derivative thereof.

[0141] In embodiments, promoter nucleic acid sequences suitable for use in the invention are selected from the group consisting of: SEQ ID NO: 140 [IMA1/YGR287], [HXK1], SEQ ID NO: 141 [HXK2], SEQ ID NO: 142 [SLT2], SEQ ID NO: 143 [YHR210c], SEQ ID NO: 144 [YJL171c], SEQ ID NO: 145 [PUNl], SEQ ID NO: 146 [PRE8], SEQ ID NO: 147 [COS3], SEQ ID NO: 148 [DIAl], SEQ ID NO: 149 [YNR062C], SEQ ID NO: 150 [PRE10], SEQ ID NO: 151 [AIM45], SEQ ID NO: 152 [ZRT1], SEQ ID NO: 153 [ZRT2], SEQ ID NO: 154 [PH084], SEQ ID NO: 155 [PCLl], SEQ ID NO: 156 [ARGl], SEQ ID NO: 157 [ZPSl], SEQ ID NO: 158 [FIT2], SEQ ID NO: 159 [FIT3], SEQ ID NO: 160 [FRE5], SEQ ID NO: 161 [CSM4], SEQ ID NO: 162 [SAM3], SEQ ID NO: 163 [FDH2] or a variant, fragment or derivative thereof.

[0142] In some embodiments, the promoter nucleic acid sequence drives transcription of a suppressor tRNA. In some embodiments, the expression of the suppressor tRNA allows for the translation of the biocatalyst polypeptide that contains a nonsense, missense, or frameshift allele. See Figures 2 and 6.

[0143] In some embodiments, the promoter nucleic acid sequence drives transcription of a suppressor tRNA. In some embodiments, the expression of the suppressor tRNA allows for the translation of the propagation polypeptide propagation polypeptide that a nonsense, missense, or frameshift allele. See Figures 4 and 8. [0144] In some embodiments, the promoter nucleic acid sequence drives transcription of anti-sense RNA. In some embodiments, the transcription of the anti-sense RNA blocks the translation of a biocatalyst polypeptide. See Figures 3 and 7.

[0145] In some embodiments, the promoter nucleic acid sequence drives transcription of anti-sense RNA. In some embodiments, the transcription of the anti-sense RNA blocks the translation of a propagation polypeptide. See Figures 5 and 9.

[0146] In embodiments, the expression of the suppressor tRNA or anti-sense RNA molecule is increased by a transcriptional activator in response to an environmental cue. In embodiments, the expression of the suppressor tRNA or antisense RNA molecule is blocked by a transcriptional repressor in response to an environmental cue.

[0147] In some embodiments, a sulfonylurea-responsive tetracycline promoter and/or a sulfonylurea-responsive tetracycline activator is used in an ALS sulfonylurea-resistant background as described in, for example, Falco, S.C., et ah, Genetics. 109(1): 21-35 (1985). In some embodiments, an inactive sulfonylurea or an ALS inactive sulfonylurea that binds to a sulfonylurea responsive activator or repressor derived from TET genes is used.

Oxygen

[0148] In embodiments, the promoter nucleic acid sequence is sensitive to the

concentration of oxygen in fermentation medium.

[0149] In embodiments, a distinguishing characteristic between the propagation and production stages is the presence of high (for example, greater than about 5% of air-saturation) dissolved oxygen concentrations during most of the propagation phase, and low (for example, less than about 5% or less than about 3%) dissolved oxygen concentrations, frequently even anaerobic conditions, in most of the production phase. Consequently, in embodiments, "high" vs. "low" dissolved oxygen concentrations results in the increase or decrease of expression of suppressor tRNAs and/or anti-sense RNAs that result in an increase or decrease of expression of biocatalyst polypeptides of interest in the propagation vs. the production stage of the process. Examples of biocatalyst polypeptides include, but are not limited to, acetohydroxyacid synthase (AHAS), glucose-6-phosphate dehydrogenase (ZWFl), phosphoketolase (XPK), and glycerol-3- phosphate dehydrogenase (GPD), described elsewhere herein.

[0150] In embodiments of the invention, differential control of promoters and the suppressor tRNAs and/or anti-sense RNAs that they control result in lower expression of desired biocatalyst polypeptides and consequently the gene products (biocatalyst polypeptides) under aerobic than under anaerobic conditions. Such biocatalyst polypeptides comprise, but are not limited to the acetohydroxyacid synthase (AHAS) of the butanol biosynthesis pathway.

Saccharomyces cerevisiae promoter nucleic acid sequences affected by aerobic or anaerobic conditions are shown in Table 3. In embodiments, the promoter is a HEM 13 (SEQ ID NO: 176) promoter or active fragment thereof. In embodiments, the promoter is a HES1 (SEQ ID NO: 177) promoter or active fragment thereof. In embodiments, the promoter nucleic acid sequences and the suppressor tRNAs and/or anti-sense RNA they control result in lower expression of polynucleotides encoding biosynthetic pathway polypeptides during the propagation phase and increased expression during production phase, for example acetohydroxyacid synthase. Suitable promoter nucleic acid sequences are provided herein and/or are available in the art (Boles, E., W. Lehnert, et al, Eur. J. Biochem. 217(1): 469-77 (1993); Heux, S., A. Cadiere, et al, FEMS Yeast Res. 8(2): 217-24 (2008); Kresnowati, M. T., W. A. van Winden, et al., Mol Syst Biol 2: 49 (2006); Kundaje, A., X. Xin, et al, PLoS Comput. Biol 4(11): el000224 (2008); Lai, L. C, A. L. Kosorukoff, et αΙ., ΜοΙ. Cell Biol. 25(10): 4075-91 (2005); ter Linde, J. J., H. Liang, et al, J. Bacteriol. 181(24): 7409-13 (1999); van den Brink, J., P. Daran-Lapujade, et al, BMC

Genomics 9: 100 (2008); and Wang, Y., M. Pierce, et al, PLoS Biol 2(5): E128 (2004)).

Promoter nucleic acid sequences useful in the invention include those comprising sequences provided herein and those that comprise sequences which are at least about 80%, 85%>, 90%>, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequences given in Table 3, including variants, fragments or derivatives thereof that confer or increase sensitivity to the concentration of oxygen. In embodiments, the promoter nucleic acid sequence comprises at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 775

[ANB1], 776 [TIR1], 777 [HEM13], or 778 [HES1] or a variant, fragment, or derivative thereof. In embodiments, the biocatalyst polypeptide comprises at least about 90%>, at least about 95%>, at least about 99% or at 100% identity to B. subtilis AlsS (SEQ ID NO: 1) or an active fragment thereof. Table 3:Example Candidate Promoter Sequences for Higher Expression in Low Oxygen

Other

Gene ID SEQ ID NO: Gene ID Other name SEQ ID NO:

name

YJ 150C DAN1 186 YMR119W ASH 231

YOR237W HES1 177 YKL079W SMY1 232

YJR047C ANB1 188 YLR413W YLR413W 233

YAL068C YAL068C 189 ARE1 YCR048W 234

YLR461W PAU4 190 AUS1 YOR011W 235

YML058W-A HUG1 191 DAN1 YJR150C 171

YLL064C YLL064C 192 DAN4 YJR151C 236

YGR131W YGR131W 193 EUG1 YDR518W 237

YOR010C TIR2 | SRP2 194 FET4 YMR319C 238

YER011W TIR1 I SRP1 195 PAU6 YNR076W 239

YIL176C YIL176C 196 PMT5 YDL093W 240

YOR009W TIR4 197 TIR2 YOR010C 172

YOL101C IZH4 198 TIR4 YOR009W 241

YDR213W UPC2 199 YSR3 YKR053C 242

YNR075W COS10 200 YMR319C FET4 243

YGL039W YGL039W 201 YPR194C OPT2 244

YHR048W YHR048W 202 YIR019C STA1/FL011 245

YOR277W ATF1 203 YHL042W YHL042W 246

YGR286C BI02 204 YHR210C YHR210C 247

YDR044W HEM13 176 YGL162W SUT1/ST01 248

YKR003W OSH6 206 YHL044W YHL044W 249

YLR194C YLR194C 207 YOL015W IRC10 250

YIR0033W MGA2 208 YJR047C ANB1/TIF51B 170

YOR175C YOR175C 209 YJR150C DAN1 186

YOL002C IZH2 210 YML083C YML083C 251

YBL106C SNI2 211 YBR085W AAC3 252

YHR004C NEM1 212 YOR010C TIR2 194

YMR006C PLB2 213 YER011W TIR1 175

YJR116W YJR116W 214 YKR053C YSR3/LBP2 253

YGR0044C CSP1 215 YER188W YER188W 254

YGR032W FKS2 216 YCL025C AGP1 255

YPL170W DAP1 217 YPL265W DIP5 256

YNR065C YSN1 218 YDL241W YDL241W 257

YDR275W BSC2 219 YBL029W YBL029W 258

YBR066C NRG2 220 YER014W HEM14 226 YBL005W-

YBL005W-A 221 YLR099C ICT1 111

A

YAL005C SSA1 111 YDR085C AFR1 118

YL 256W HAP1 223 YGR177C ATF2 229

YDR186C YDR186C 224 YMR038C CCS1 230

YMR087W YMR087W 225

[0151] In other embodiments, promoter nucleic acid sequences and the suppressor tRNAs and/or anti-sense RNAs that they control result in a lower expression of desired polynucleotides and consequently the encoded products under anaerobic than under aerobic conditions. Such polynucleotides comprise, but are not limited to, polypeptides which may produce by-products of the butanol biosynthesis pathway such as isobutyric acid or DHMB. Saccharomyces cerevisiae promoter nucleic acid sequences affected by such conditions are shown in Table 4. In embodiments, the promoter nucleic acid sequences and the suppressor tRNAs and/or anti-sense RNAs that they control result in lower expression of genes encoding by-product producing polypeptides during the propagation phase and lower expression during production phase, for example YMR226C or aldehyde dehydrogenases, including, but not limited to, ALD6. Promoter nucleic acid sequences useful in the invention comprise those provided herein and those which comprise sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequences of Table 4, including variants, fragments or derivatives thereof that confer or increase sensitivity to the concentration of oxygen.

Table 4: Example Candidate Promoter Sequences for Lower Expression in Low Oxygen

Other

Gene ID name SEQ ID NO: Gene ID Other name SEQ ID NO:

YM 058W FET3 268 YPR124W CTR1 326

YLR205C HMX1 269 YJR077C MIR1 327

YNL173C MDG1 270 YJR122W IBA57 328

YOR348C PUT4 271 YLL028W TPOl 329

YOR065W CYT1 272 YDL004W ATP16 330

YGR035C 273 YDR342C HXT7 331

YKR046C PET10 274 YDR461W MFA1 332

YGL191W COX13 275 YDR298C ATP5 333

YHR001W-A QCR10 276 YMR215W GAS3 334

YU113C-A 277 YPL271W ATP15 335

YBR177C EHT1 278 YMR251W-A HOR7 336

YMR145C NDE1 279 YDL067C COX9 337

YLR038C COX12 280 YU103C GSM1 338

YPR061C JID1 281 YIR038C GTT1 339

YJL048C UBX6 282 YPR028W YOP1 340

YLR042C 283 YDR253C MET32 341

YNL052W COX5A 284 YBL099W ATP1 342

YLR395C COX8 285 YPL002C SNF8 343

YKL068W-A 286 YNL307C MCK1 344

YGL032C AGA2 287 YPR165W RHOl 345

YDR384C AT03 288 YGR063C SPT4 346

YDR185C UPS3 289 YMR009W ADI1 347

YHR051W COX6 290 YMR256C COX7 348

YBR047W FMP23 291 YBR185C MBA1 349

YPR191W QCR2 292 YPR047W MSF1 350

YPR149W NCE102 293 YMR302C YME2 351

YJL116W QCR8 294 YDL086W 352

YOL126C MDH2 295 YGL101W 353

YGR243W FMP43 296 YIR035C 354

YGR183C QCR9 297 YLR108C 355

YOR273C TP04 298 YOR388C FDH1 356

YPR1458W CUR1 299 YPL275W FDH2 357

YIL015W BAR1 300 YPL276W FDH2 358

YIL155C GUT2 301 YDR256C CTA1 359

YMR286W MRPL33 302 YHR096C HXT5 360

YDR529C QCR7 303 YNL195C 361

YGR055W MUP1 304 YGR110W CLD1 362

YPL004C LSP1 305 YCR010C ADY2 363 YO 072W-B 306 YDL218W 364

YLR411W CTR3 307 YPL223C GRE1 365

YOR100C CRC1 308 YJR095W SFC1 366

YDR078C SHU2 309 YMR303C ADH2 367

YGR053C 310 YGR236C SPG1 368

YCR061W 311 YHR139C SPS100 369

YOR084W LPX1 312 YRP151C SUE1 370

YDR313C PIB1 313 YMR107W SPG4 371

YBR039W ATP3 314 YMR118C SHH3 372

YPR002W PDH1 315 YLR174W IDP2 373

YJL173C RFA3 316 YPL201C YIG1 374

YDR173C ARG82 317 YDR380W ARO10 375

YPR159C-A 318 YML054C CYB2 376

YJL131C AIM23 319 YPL147W PXA1 377

YJL180C ATP12 320 YDR070C FMP16 378

YPR036W-A 321 YPR001W CIT3 379

YHR090C YNG2 322 YER065C ICL1 380

YPR161C PNS1 323 YKR009C F0X2 381

YOR390W 324 YLL053C 382

YBL030C PET9 325 YGR256W GND2 383

Glucose

[0152] In embodiments, a distinguishing condition between the propagation and production phases is the presence of low glucose concentrations during the propagation phase and the presence of excess glucose during the production phase. Consequently "high" vs. "low" glucose concentrations could be used to express/repress suppressor tNRA and/or anti-sense RNA expression, and therefore biocatalyst polypeptide expression, in the propagation vs. production phase. Examples of such biocatalyst polypeptides of interest to differentially control their expression include, but are not limited to, acetohydroxyacid synthase (AHAS), glucoses- phosphate dehydrogenase (ZWF1), phosphoketolase (XPK), glycerol-3 -phosphate

dehydrogenase (GPD).

[0153] The hexose transporter gene family in S. cerevisiae contains the sugar transporter genes HXT1 to HXT17, GAL2 and the glucose sensor genes SNF3 and RGT2. The proteins encoded by HXT1 to HXT4 and HXT6 to HXT7 are considered to be the major hexose transporters in S. cerevisiae. The expression of most of the HXT glucose transporter genes is known to depend on the glucose concentration (Ozcan, S. and M. Johnston, Microbiol. Mol. Biol. Rev. 63(3): 554-69 (1999)). Consequently their promoters are provided herein for differential expression of genes under "high" or "low" glucose concentrations. [0154] In embodiments, promoter nucleic acid sequences comprising sequences from the promoter region of HXT2 (SEQ ID NO: 384), HXT5 (SEQ ID NO: 360), HXT6 (SEQ ID NO: 386), or HXT7 (SEQ ID NO: 331) are employed for higher expression under glucose-limiting conditions, and lower expression under glucose-excess conditions. HXT5, HXT6 and HXT7 show also strong expression with growth on ethanol, in contrast to HXT2 (Diderich, J. A., Schepper, M., et al, J. Biol. Chem. 274(22): 15350-9 (1999). It has been reported that under different oxygen conditions, HXT5 and HXT6 expression showed variability (Rintala, E., M. G. Wiebe, et al, BMC Microbiol. 8: 53 (2008)), however, equipped with this disclosure, one of skill in the art is readily able to make and test such promoter constructs under conditions relevant for a desired production process. Promoter nucleic acid sequences useful in the invention comprise those provided herein and those that comprise nucleic acid sequences which are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequences of HXT2 (SEQ ID NO: 384), HXT5 (SEQ ID NO: 360), HXT6 (SEQ ID NO: 386), or HXT7 (SEQ ID NO: 331), including variants, fragments or derivatives thereof that confer or increase sensitivity to the concentration of oxygen. In embodiments, the promoter nucleic acid sequence comprises at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 772 [HXT5] or 773 [HXT7] or a fragment or derivative thereof. In embodiments, the biocatalyst polypeptide comprises at least about 90%>, at least about 95%, at least about 99% or at 100% identity to B. subtilis AlsS (SEQ ID NO: 1) or an active fragment thereof.

[0155] In embodiments, HXT1 is the promoter for glucose-based control of gene expression, providing high expression under conditions of high glucose. HXT3 may have promise in promoter strength, but may also show some low expression under very low glucose concentrations (Brauer, M. J., C. Huttenhower, et al. Mol Biol Cell 19(1): 352-67 (2008)).

Equipped with this disclosure, one of skill in the art will be able to make and test the suitability of promoter constructs under the conditions relevant for a desired process. Accordingly, promoter nucleic acid sequences useful in the invention comprise those provided herein and those that comprise nucleic acid sequences which are at least about 80%>, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequences of HXT1 (SEQ ID NO: 168), HXT3 (SEQ ID NO: 169), HXT4 (SEQ ID NO: 388), or EN02 (SEQ ID NO: 173), including variants, fragments or derivatives thereof that confer or increase sensitivity to the concentration of oxygen. In embodiments, the promoter nucleic acid sequence comprises at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO: 768 [HXT3], 769 [HXT1], or 711 [hybrid promoter comprising HXT1 promoter nucleic acid sequence] or a fragment or derivative thereof. In embodiments, the biocatalyst polypeptide comprises at least about 90%, at least about 95%, at least about 99% or at 100% identity to B. subtilis AlsS (SEQ ID NO: 1) or an active fragment thereof. ρϋ

[0156] In some embodiments, a distinguishing condition between the propagation and production phases is the pH. In embodiments, promoter nucleic acid sequences from the

Saccharomyces cerevisiae YKL096W-A (CWP2) promoter (SEQ ID NO: 389) or YER150W (SPI1) promoter (SEQ ID NO: 190) are employed to govern differential expression in processes where the pH is different in different phases. Equipped with this disclosure, one of skill in the art will be able to make and test the suitability of promoter constructs under the conditions relevant for a desired process. Accordingly, promoter nucleic acid sequences useful in the invention include those provided herein and those that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequences of SEQ ID NO: 389 or SEQ ID NO: 190), including variants, fragments or derivatives thereof that confer or increase sensitivity to pH.

Temperature

[0157] In some embodiments, a distinguishing condition between the propagation and production phases is the temperature. In embodiments, promoter nucleic acid sequences from the Saccharomyces cerevisiae YBR027W promoter (HSP26) (SEQ ID NO: 391) or from the

YLL026W (HSP104) promoter (SEQ ID NO: 392) are employed to govern differential expression in processes where the temperature is different in different phases. Temperature sensitive promoters and candidate temperature sensitive promoters are also available in the art (Becerra, M., et ah, Comp Func Genomics 4(4): 366-75 (2003) and Al-Fageeh, MB, et αί, Biochem J J 7(2):247-59 (2006), both incorporated by reference). Equipped with this disclosure, one of skill in the art will be able to make and test the suitability of promoter constructs under the conditions relevant for a desired process. Accordingly, promoter nucleic acid sequences useful in the invention include those provided herein and those that are at least about 80%>, 85%, 90%>, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequences of SEQ ID NO: 391 or SEQ ID NO: 392, including variants, fragments or derivatives thereof that confer or increase sensitivity to temperature. Butanol

[0158] In some embodiments, a distinguishing condition between the propagation and production phases is the concentration of fermentation product such as butanol or 2-butanone. In embodiments, promoter nucleic acid sequences from the Saccharomyces cerevisiae YOL151W (GRE2) promoter (SEQ ID NO: 393) or from the YOR153W (PDR5) promoter (SEQ ID NO: 394) are employed to govern differential expression in processes where the butanol level is different in different phases. Equipped with this disclosure, one of skill in the art will be able to make and test the suitability of promoter constructs under the conditions relevant for a desired process. Accordingly, promoter nucleic acid sequences useful in the invention include those provided herein and those that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleotide sequences of SEQ ID NO: 393 or SEQ ID NO: 394, including variants, fragments or derivatives thereof that confer or increase sensitivity to the concentration of butanol.

Hybrid promoters

[0159] It will be appreciated that "hybrid promoters" which comprise nucleic acid sequences from more than one promoter region can be constructed and employed in

embodiments herein.

[0160] For example, in order to add an additional negative control trigger through the level of dissolved oxygen in the culture medium, a Roxl binding site (eg. "ATTGT") or a sequence comprising a Rox 1 binding site (e.g., SEQ ID NO: 395) (Badis, G. Mol Cell (2008) 32(6):878-87; Balasubramanian, B., et al., Mol Cell Biol 75(10) 6071-8 (1993)) could be added to either the regulatory (e.g., promoter) or to the coding sequence of the gene of interest. A Roxl binding site may already be present for the HXT1 promoter based on bioinformatic analysis (Maclsaac, K. D., T. Wang, et al, (2006). BMC Bioinformatics 7: 113 (2006)) Roxl and other transcription motifs are described, for example, in Badis, et al., Mol Cell 32(6): 878-87 (2008). Transcription factor motifs can also be found in the TRANSFAC database (Matys V, et al. , Nucleic Acids Res. 3/(l):374-8 (2003)).

[0161] Hybrid promoter nucleic acid sequences may comprise FBAl promoter sequences

(SEQ ID NO: 779) or a variant, fragment or derivative thereof. In one embodiment, a hybrid promoter nucleic acid sequence comprises nucleic acid sequences that are at least about 80%>, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to FBAl (SEQ ID NO: 779 or SEQ ID NO: 770) or a variant, fragment or derivative thereof. In one embodiment, a hybrid promoter nucleic acid sequence comprises nucleic acid sequences that are at least about 80%, 85%, 90%>, 95%, 96%, 97%, 98%, 99% or 100% identical to HXT1 (SEQ ID NO: 168) or DAN1 (SEQ ID NO: 186) promoters or a variant, fragment or derivative thereof. In embodiments, the promoter nucleic acid sequence comprises at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to P[FBA1 ::DAN1_AR314) (SEQ ID NO: 686) or a variant, fragment or derivative thereof. In embodiments, the promoter nucleic acid sequence comprises at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identity to P[FBA1 ::HXT1_331] (SEQ ID NO: 711) or a variant, fragment or derivative thereof.

[0162] In embodiments, the biocatalyst polypeptide comprises at least about 90%>, at least about 95%, at least about 99% or at 100% identity to B. subtilis AlsS (SEQ ID NO: 1) or an active fragment thereof.

[0163] In embodiments, an expression construct comprises SEQ ID NO: 711 or a polynucleotide encoding a polypeptide having at least about 90%>, at least about 95%, at least about 99% or at 100% identity to SEQ ID NO: 1 or both. In embodiments, an expression construct comprises at least about 90%>, at least about 95%, at least about 99% or at 100%) identity to SEQ ID NO: 790. In embodiments, a recombinant host cell comprises such an expression construct, and may further comprise an isobutanol biosynthetic pathway. In embodiments, such a recombinant host cell may be employed in methods wherein it is contacted with a carbon substrate under conditions whereby isobutanol is produced and optionally recovered.

Identification and Isolation of Additional Suitable Genetic Switches: "Promoter Prospecting"

[0164] Provided herein are methods for identifying promoter nucleic acid sequences that are sensitive to changes in cellular environment, i.e., they preferentially increase or decrease gene expression under certain conditions. For some embodiments disclosed herein, the process for promoter selection involves RNA transcript comparison between microbial cells grown under selected propagation conditions and cells grown under selected production conditions. Promoters associated with RNA transcripts that are upregulated or downregulated during the propagation phase as compared to during the production phase are suitable for use in the invention.

Promoters associated with RNA transcripts that are upregulated or downregulated during the production phase as compared to the propagation phase are also suitable for use in the invention. [0165] Another embodiment of the invention is directed to a method for screening candidate promoter sequences that are preferentially expressed during the production phase of fermentation, comprising:

(a) incubating a microorganism under propagation conditions;

(b) isolating ribonucleic acid molecules from the microorganism incubated in (a);

(c) incubating a microorganism under production conditions;

(d) isolating ribonucleic acid molecules from the microorganism incubated in (c);

(e) selecting only those isolated ribonucleic acid molecules in (d) that are expressed at a higher level than the corresponding isolated ribonucleic acid molecules in (b); and

(f) determining the polynucleotide sequences of the promoters associated with the expression of the ribonucleic acid molecules selected in (e).

[0166] Another embodiment of the invention is directed to a method for screening candidate promoter sequences that are preferentially transcribed less during the production phase of fermentation, comprising:

(a) incubating a microorganism under propagation conditions;

(b) isolating ribonucleic acid molecules from the microorganism incubated in (a);

(c) incubating a microorganism under production conditions;

(d) isolating ribonucleic acid molecules from the microorganism incubated in (c);

(e) selecting only those isolated ribonucleic acid molecules in (d) that are expressed at a lower level than the corresponding isolated ribonucleic acid molecules in (b); and

(f) determining the polynucleotide sequences of the promoters associated with the expression of the ribonucleic acid molecules selected in (e).

[0167] Another embodiment of the invention is directed to a method for screening candidate promoter sequences that are preferentially inhibited during the biomass propagation phase, comprising:

(a) incubating a microorganism under propagation conditions;

(b) isolating ribonucleic acid molecules from the microorganism incubated in (a);

(c) incubating a microorganism under production conditions;

(d) isolating ribonucleic acid molecules from the microorganism incubated in (c);

(e) selecting only those isolated ribonucleic acid molecules in (d) that are expressed at a higher level than the corresponding isolated ribonucleic acid molecules in (b); and (f) determining the polynucleotide sequences of the promoters associated with the expression of the ribonucleic acid molecules selected in (e).

[0168] In one embodiment, the polynucleotide sequences of the promoters associated with the expression of the ribonucleic acid molecules selected are introduced into a reporter construct. In a specific embodiment, the polynucleotide sequences of the promoters associated with the expression of the ribonucleic acid molecules selected are introduced into a fluorescent reporter construct. In one embodiment, the fluorescent reporter construct expresses green fluorescent protein (GFP). In another embodiment, the propagation conditions comprise growing the microorganism comprising a reporter construct in fermentation medium comprising low concentrations of a fermentable carbon substrate and the production conditions comprise growing the microorganism in fermentation medium comprising high concentrations of the same fermentable carbon substrate. In a specific embodiment, the fermentable carbon substrate is selected from the group consisting of: monosaccharides, oligosaccharides, polysaccharides, fatty acids, and mixtures thereof. In another embodiment, the propagation conditions comprise growing the microorganism comprising a reporter construct in fermentation medium comprising a high concentration of dissolved oxygen and the production conditions comprise growing the microorganism in fermentation medium comprising a low concentration of dissolved oxygen.

[0169] In another embodiment, the methods for identifying promoter nucleic acid sequences for use in the invention further comprise performing a literature search for candidate nucleic acid sequences. For example, literature references such as Li B.-Z., et al., J. Ind.

Microbiol. Biotechnol. 57:43-55 (2010), provide information as to which genes are expressed during fed-batch fermentation. Li et al. examined the transcriptional profile of yeast taken from industrial ethanol fermentations (both continuous and fed-batch using 80% corn mash and 20% grain mash as feedstocks). They sampled in the seed stage, during early "main" fermentation, and late main fermentation. They found strong up-regulation of genes involved in reserve carbohydrate metabolism and protein folding (the unfolded protein response). They detected derepression of glucose-repressed genes (e.g., HXK1 and GLK1, encoding the other hexokinase isozymes; gluconeogenic genes; high-affinity glucose transporters) and down-regulation of HXK2, even at high residual glucose (15 and 23 g/L, respectively, in the continuous and batch processes). The HXK2 response observed by Li et al. differs than what was observed in the promoter prospecting experiments in the Examples discussed below. [0170] Understanding the genetic aspects of how yeast respond rapidly to shifts from aerobic to anaerobic conditions may offer some guidance as to which oxygen-sensitive promoter nucleic acid sequences could potentially be suitable in the invention. At the whole-genome level, fermentative functions are induced, and activities in respirofermentative metabolism, the cell cycle, and translation are down-regulated. The five major transcriptional regulatory networks involved include the Msn2/4, Hapl, Roxl, Hap2/3/4/5, and Upc2 networks. In one study, the genome-wide response to anaerobiosis and subsequent reoxygenation involved 1 ,603 genes in glucose-grown cells. See Kwast K.E., et al., J. Exp. Biol. 201:1177-1195 (1998); Kwast K.E., et al., J. Bacteriol. 184:250-256 (2002); and Lai L.C, et al., Mol. Cell. Biol. 25:4075-4091 (2005).

[0171] Some biomass production processes at scale may be described as microaerobic rather than aerobic, which may narrow the band of transcriptional response to fully anaerobic conditions. However, a few studies have been done in this range. In one study, transcript levels of a panel of 60 genes (mostly involved in carbon metabolism) were monitored in chemostats at sufficient and limiting levels of 02 provision; it was found that the overall transcript abundances decreased with decreasing oxygen availability. Only PYC1 (encoding pyruvate carboxylase) and GPP1 (encoding glycerol phosphate phosphatase) transcripts increased in the anaerobic culture compared to the micro-aerobic conditions. When (micro)-aerobic conditions were switched to anaerobic conditions, the expression of a few genes increased significantly, including 3 genes from the pentose phosphate pathway (TKLl, TALI, and YGR032C), the respiratory gene COX5b, and the gluconeogenic genes MDH2 and PCK1. See Wiebe M.G., et al, FEMS Yeast Res. 8: 140-154 (2008). No whole-genome microarray studies have been done on comparable cultures.

[0172] Other network responses to shifts from anaerobic to aerobic conditions have been described and include activiation of Yap 1 networks and activation of heme-regulated networks. (Lai LC, et al, Eukaryot Cell. 5(9): 1468-89 (2006))

[0173] In embodiments, promoter nucleic acid sequences suitable for use in the invention comprise nucleotide sequences that are at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%) or 100%) identical to a sequence selected from the group consisting of: [MSN2], [MSN4], [HAP1], [ROX1], [HAP2], [HAP3], [HAP4], [HAP5], [UPC2], [PYC1], [GPP1], [TKLl],

[TALI], [YGR032C], [COX5b], [MDH2], [YHR210], and [PCK1]. In embodiments, promoter nucleic acid sequences suitable for use in the invention can be selected from the group consisting of: [MSN2], [MSN4], [HAP1], [ROX1], [HAP2], [HAP3], [HAP4], [HAP5], [UPC2], [PYC1], [GPPl], [TKLl], [TALI], [YGR032C], [COX5b], [MDH2], [YHR210], and [PCKl] or a variant, fragment or derivative thereof.

[0174] One of skill in the art, equipped with this disclosure, will be able to carry out the screening methods described herein for any microorganism, such as the recombinant host cells disclosed elsewhere herein, using conditions relevant for propagation and production of such microorganism. In embodiments, the microorganism is a yeast microorganism.

[0175] For example, an isobutanologen yeast strain known in the art or disclosed herein may be used for an isobutanol production run using the fermentation process developed for that strain and for the scale of fermentors used. The process may include (i) a biomass production phase, during which biomass is formed with a high yield on a carbon source feedstock (eg. beet molasses), and (ii) a fermentation phase, in which a carbon source feedstock (eg. corn mash) is fermented to isobutanol. Culture broth harvested from the fermentors at intervals throughout the process would provide samples including early, middle, and late timepoints of both phases.

Harvested cells could be rapidly chilled, and recovered from the broth by centrifugation. RNA can then be extracted from the cell pellet by methods known in the art (eg. using Trizol reagent, Life Technologies, Grand Island NY), followed by RNA analysis using standard methods (for example with a BioAnalyzer 2100; Agilent Technologies, Inc., Santa Clara CA) to determine a rRNA peak ratio of 1.8 or higher.

[0176] Whole-transcriptome analysis could then be performed using methods known to one skilled in the art, e.g., RNA-Seq (also known as Whole Transcriptome Shotgun Sequencing). The RNA from each timepoint could be enriched for mRNA using an oligo(dT) technology. The enriched pool would then be reverse-transcribed to cDNA, which is then fragmented to the appropriate length for the sequence method to be used. The fragmented cDNA is then prepared for sequencing (e.g., amplified to create a library) and then the DNA sequences of the fragments would be determined. The resulting raw dataset may be composed of many (typically in the millions) short sequence reads for each timepoint. Bio-informatic analysis would then be erformed to align these sequences, for example by use of a reference genome sequence available in the art. With adequate sequence coverage, then, the read depth for all genes in the genome would be determined for each sample point across the fermentation process. Using bio-informatic methods known in the art, these are converted into numerical descriptions of gene expression levels. Genes with particular expression patterns are identified by methods such as cluster analysis, which reports the fold change in abundance of groups of transcripts at the various timepoints, relative to a reference point (for example, the reference could be the last timepoint in the production phase). Genes with properties of potential utility are identified, for example those with low abundance throughout the biomass production phase and high abundance during the fermentation phase (or at least during certain periods within the fermentation phase). Nucleic acid sequences corresponding to the promoters of those genes could then be tested as candidates for regulated and predictable expression of heterologous genes during the fermentation process. They could also be engineered further; for example, regulatory elements within them could be transferred to other promoters (e.g., strong glycolytic promoters) to confer on them the regulatory properties of the identified genes.

Biocatalyst Polypeptides

[0177] One desirable feature of the polynucleotides, recombinant host cells, and methods disclosed herein is that accumulation of inhibitory by-products due to flux via enzymes of the butanol production pathway can be avoided during the growth phase. A non-limiting example with regard to an inhibitory by-product produced via enzymes of the isobutanol biosynthetic pathway is isobutyric acid. Another non-limiting example with regard to an inhibitory byproduct produced via enzymes of the isobutanol biosynthetic pathway is isobutyraldehyde. Yet another non-limiting example with regard to an inhibitory by-product produced via enzymes of the isobutanol biosynthetic pathway is acetic acid. Some acetolactate synthase enzymes demonstrate a significant oxygenase side reaction in which molecular oxygen electrophilically attacks a highly reactive carbanion/enamine to form a peroxy-adduct that decomposes to ThDP and peracetic acid. See Tse, M.T. and Schloss, J.V., Biochemistry 52: 10398-10403 (1993). The peracetic acid can further react with pyruvate to form two moles of acetate. In addition to the growth inhibitory effects and the loss of metabolic energy for coping with the stress generated by the presence of by-products, carbon lost to the by-products adds to a lower yield of biocatalyst on the employed carbon substrate.

[0178] Another desirable feature of some embodiments is that with oxygen supply, reducing equivalents produced in metabolic pathways leading to pyruvate can be oxidized to a greater extent by the respiratory chain rather than by a biosynthetic pathway such as a butanol biosynthetic pathway. A higher fraction of pyruvate can transit the mitochondrial membrane and be further metabolized by pyruvate dehydrogenase and the tricarboxylic acid cycle. Another desirable feature of some embodiments is that without oxygen supply, more reducing equivalents produced in metabolic pathways leading to pyruvate may be oxidized by the butanol production pathway and fewer by the glycerol production pathway. This way a lower yield of glycerol and a higher yield of butanol may be achieved.

[0179] Yet another desirable feature of some embodiments is that in case of excess pyruvic acid production, pyruvic acid can be excreted into the medium. See van Maris, A.J., et ah, Appl. Environ. Micriobiol. 70: 159-166 (2004). However, pyruvic acid even at very high concentrations is not growth inhibiting.

[0180] Such desirable features can be achieved using compositions and methods provided herein. For example, as shown in the Examples, compositions and methods herein provide preferential expression of the acetolactate synthase of an isobutanol production pathway during the production phase.

[0181] In one embodiment, the biocatalyst polypeptide encoded by the isolated polynucleotide of the invention confers host cell tolerance to the fermentation product. In another embodiment, the biocatalyst polypeptide encoded by the isolated polynucleotide of the invention confers host cell tolerance to fermentation by-products. In one embodiment, the biocatalyst polypeptide encoded by the isolated polynucleotide confers host cell tolerance to butanol. In another embodiment, the biocatalyst polypeptide encoded by the isolated

polynucleotide confers host cell tolerance to isobutyraldehyde. In another embodiment, the biocatalyst polypeptide encoded by the isolated polynucleotide confers host cell tolerance to isobutyric acid. In another embodiment, the biocatalyst polypeptide encoded by the isolated polynucleotide confers host cell tolerance to acetic acid.

[0182] In some embodiments of the invention, a gene encoding a biocatalyst polypeptide contains a frameshift mutation, such that the polypeptide is not properly translated in the absence of a suppressor tRNA molecule. In some embodiments of the invention, a gene encoding a biocatalyst polypeptide contains a nonsense or missense mutation, such that the polypeptide is not fully translated in the absence of a suppressor tRNA molecule. In some embodiments the nonsense mutation in the gene coding the biocatalyst polypeptide is the the UAG stop codon, the UGA stop codon, and/or the UAA stop codon.

Biosynthetic pathways

[0183] Biosynthetic pathways for the production of isobutanol that may be used include those described in U.S. Pat. No. 7,851,188, which is incorporated herein by reference. In one embodiment, the isobutanol biosynthetic pathway comprises the following substrate to product conversions: - a) pyruvate to acetolactate, which may be catalyzed, for example, by acetolactate synthase;

- b) acetolactate to 2,3-dihydroxyisovalerate, which may be catalyzed, for example, by acetohydroxy acid reductoisomerase;

- c) 2,3-dihydroxyisovalerate to a-ketoisovalerate, which may be catalyzed, for example, by acetohydroxy acid dehydratase;

- d) a-ketoisovalerate to isobutyraldehyde, which may be catalyzed, for example, by a branched-chain keto acid decarboxylase; and,

- e) isobutyraldehyde to isobutanol, which may be catalyzed, for example, by a branched- chain alcohol dehydrogenase.

[0184] In another embodiment, the isobutanol biosynthetic pathway comprises the following substrate to product conversions:

- a) pyruvate to acetolactate, which may be catalyzed, for example, by acetolactate synthase;

- b) acetolactate to 2,3-dihydroxyisovalerate, which may be catalyzed, for example, by ketol- acid reductoisomerase;

- c) 2,3-dihydroxyisovalerate to a-ketoisovalerate, which may be catalyzed, for example, by dihydroxyacid dehydratase;

- d) α-ketoisovalerate to valine, which may be catalyzed, for example, by transaminase or valine dehydrogenase;

- e) valine to isobutylamine, which may be catalyzed, for example, by valine decarboxylase;

- f) isobutylamine to isobutyraldehyde, which may be catalyzed by, for example, omega transaminase; and,

- g) isobutyraldehyde to isobutanol, which may be catalyzed, for example, by a branched- chain alcohol dehydrogenase.

[0185] In another embodiment, the isobutanol biosynthetic pathway comprises the following substrate to product conversions:

- a) pyruvate to acetolactate, which may be catalyzed, for example, by acetolactate synthase;

- b) acetolactate to 2,3-dihydroxyisovalerate, which may be catalyzed, for example, by acetohydroxy acid reductoisomerase;

- c) 2,3-dihydroxyisovalerate to α-ketoisovalerate, which may be catalyzed, for example, by acetohydroxy acid dehydratase;

- d) α-ketoisovalerate to isobutyryl-CoA, which may be catalyzed, for example, by branched-chain keto acid dehydrogenase; -e) isobutyryl-CoA to isobutyraldehyde, which may be catalyzed, for example, by aclylating aldehyde dehydrogenase; and,

- f) isobutyraldehyde to isobutanol, which may be catalyzed, for example, by a branched- chain alcohol dehydrogenase.

[0186] In another embodiment, the isobutanol biosynthetic pathway comprises the substrate to product conversions shown as steps k, g, and e in Figure 1.

[0187] Biosynthetic pathways for the production of 1-butanol that may be used include those described in U.S. Appl. Pub. No. 2008/0182308, which is incorporated herein by reference. In one embodiment, the 1-butanol biosynthetic pathway comprises the following substrate to product conversions:

- a) acetyl-CoA to acetoacetyl-CoA, which may be catalyzed, for example, by acetyl-CoA acetyl transferase;

- b) acetoacetyl-CoA to 3-hydroxybutyryl-CoA, which may be catalyzed, for example, by 3- hydroxybutyryl-CoA dehydrogenase;

- c) 3-hydroxybutyryl-CoA to crotonyl-CoA, which may be catalyzed, for example, by crotonase;

- d) crotonyl-CoA to butyryl-CoA, which may be catalyzed, for example, by butyryl-CoA dehydrogenase;

- e) butyryl-CoA to butyraldehyde, which may be catalyzed, for example, by butyraldehyde dehydrogenase; and,

- f) butyraldehyde to 1-butanol, which may be catalyzed, for example, by butanol

dehydrogenase.

[0188] Biosynthetic pathways for the production of 2-butanol that may be used include those described in U.S. Appl. Pub. No. 2007/0259410 and U.S. Appl. Pub. No. 2009/0155870, which are incorporated herein by reference. In one embodiment, the 2-butanol biosynthetic pathway comprises the following substrate to product conversions:

- a) pyruvate to alpha-acetolactate, which may be catalyzed, for example, by acetolactate synthase;

- b) alpha-acetolactate to acetoin, which may be catalyzed, for example, by acetolactate decarboxylase;

- c) acetoin to 3 -amino-2 -butanol, which may be catalyzed, for example, acetonin aminase; - d) 3-amino-2-butanol to 3-amino-2-butanol phosphate, which may be catalyzed, for example, by aminobutanol kinase;

- e) 3-amino-2-butanol phosphate to 2-butanone, which may be catalyzed, for example, by aminobutanol phosphate phosphorylase; and,

- f) 2-butanone to 2-butanol, which may be catalyzed, for example, by butanol

dehydrogenase.

[0189] In another embodiment, the 2-butanol biosynthetic pathway comprises the following substrate to product conversions:

- a) pyruvate to alpha-acetolactate, which may be catalyzed, for example, by acetolactate synthase;

- b) alpha-acetolactate to acetoin, which may be catalyzed, for example, by acetolactate decarboxylase;

- c) acetoin to 2,3-butanediol, which may be catalyzed, for example, by butanediol dehydrogenase;

- d) 2,3-butanediol to 2-butanone, which may be catalyzed, for example, by dial dehydratase; and,

- e) 2-butanone to 2-butanol, which may be catalyzed, for example, by butanol

dehydrogenase.

[0190] Biosynthetic pathways for the production of 2-butanone that may be used include those described in U.S. Appl. Pub. No. 2007/0259410 and U.S. Appl. Pub. No. 2009/0155870, which are incorporated herein by reference. In one embodiment, the 2-butanone biosynthetic pathway comprises the following substrate to product conversions:

- a) pyruvate to alpha-acetolactate, which may be catalyzed, for example, by acetolactate synthase;

- b) alpha-acetolactate to acetoin, which may be catalyzed, for example, by acetolactate decarboxylase;

- c) acetoin to 3 -amino-2 -butanol, which may be catalyzed, for example, acetonin aminase;

- d) 3-amino-2-butanol to 3-amino-2-butanol phosphate, which may be catalyzed, for example, by aminobutanol kinase; and,

- e) 3-amino-2-butanol phosphate to 2-butanone, which may be catalyzed, for example, by aminobutanol phosphate phosphorylase. [0191] In another embodiment, the 2-butanone biosynthetic pathway comprises the following substrate to product conversions:

- a) pyruvate to alpha-acetolactate, which may be catalyzed, for example, by acetolactate synthase;

- b) alpha-acetolactate to acetoin which may be catalyzed, for example, by acetolactate decarboxylase;

- c) acetoin to 2,3-butanediol, which may be catalyzed, for example, by butanediol dehydrogenase;

- d) 2,3-butanediol to 2-butanone, which may be catalyzed, for example, by diol dehydratase.

[0192] Another embodiment of the invention is directed to an isolated polynucleotide comprising:

(a) a constitutive promoter nucleic acid sequence; and

(b) a nucleic acid sequence encoding a biocatalyst polypeptide; wherein the nucleic acid sequence of (b) is coupled to the nucleic acid sequence of (a).

[0193] In another embodiment, the biocatalyst polypeptide comprises or is selected from the group of enzymes having the following Enzyme Commission Numbers: EC 2.2.1.6, EC 1.1.1.86, EC 4.2.1.9, EC 4.1.1.72, EC 1.1.1.1, EC 1.1.1.265, EC 1.1.1.2, EC 1.2.4.4, EC

1.3.99.2, EC 1.2.1.57, EC 1.2.1.10, EC 2.6.1.66, EC 2.6.1.42, EC 1.4.1.9, EC 1.4.1.8, EC

4.1.1.14, EC 2.6.1.18, EC 2.3.1.9, EC 2.3.1.16, EC 1.1.130, EC 1.1.1.35, EC 1.1.1.157, EC 1.1.136, EC 4.2.1.17, EC 4.2.1.55, EC 1.3.1.44, EC 1.3.1.38, EC 1.3.1.44, EC 1.3.1.38, EC 5.4.99.13, , EC 4.1.1.5, EC 1.1.1.1, 2.7.1.29, 1.1.1.76, and 4.2.1.28, or the enzymes acetonin aminase, acetoin phosphate aminase, aminobutanol phosphate phospholyase, and aminobutanol kinase.

[0194] In some embodiments, the biocatalyst polypeptide which catalyzes the substrate to product conversions of acetolactate to 2,3-dihydroxyisovalerate and/or the polypeptide catalyzing the substrate to product conversion of isobutyraldehyde to isobutanol utilize NADH as a cofactor.

[0195] In some embodiments, enzymes from the biosynthetic pathway are localized to the cytosol. In some embodiments, enzymes from the biosynthetic pathway that are usually localized to the mitochondria are localized to the cytosol. In some embodiments, an enzyme from the biosynthetic pathway is localized to the cytosol by removing the mitochondrial targeting sequence. In some embodiments, mitochondrial targeting is eliminated by generating new start codons as described in U.S. Pat. No. 7,851,188, which is incorporated herein by reference in its entirety.

[0196] In some embodiments, the biocatalyst polypeptide is KARL In some

embodiments, KARI preferentially utilizes NADH as a cofactor. In some embodiments, the biocatalyst polypeptide is ADH. In some embodiments, ADH preferentially utilizes NADH as a cofactor.

[0197] In some embodiments, the biocatalyst polypeptide is KIVD, is some

embodiments, the biocatalyst polypeptide is DHAD. In some embodiments, the biocatalyst polypeptide is ALS.

[0198] Genes and polypeptides that can be used for the substrate to product conversions described above as well as those for additional isobutanol pathways, are described herein and in the art, for example, in U.S. Patent Appl. Pub. No. 20070092957, PCT Pub. No. WO

2011/019894, and in PCT App. No. WO2012/129555, all incorporated by reference herein. US Appl. Pub. Nos. 2011/019894, 20070092957, 20100081154, describe dihydroxyacid

dehydratases including those from Lactococcus lactis (SEQ ID NO: 794) and Streptococcus mutans (SEQ ID NO: 793) and variants thereof, eg. S. mutans I2V5 (SEQ ID NO: 792).

Ketoisovalerate decarboxylases include those derived from Lactococcus lactis (SEQ ID NO: 795), Macrococcus caseofyticus (SEQ ID NO: 797) and Listeria grayi (SEQ ID NO: 796). U.S. Patent Appl. Publ. No. 2009/0269823 and U.S. Appl. Publ. No. 20110269199, incorporated by reference, describe alcohol dehydrogenases. Alcohol dehydrogenases include SadB from Achromobacter xylosoxidans (SEQ ID NO: 798) disclosed in U.S. Patent 8,188,250, incorporated herein by reference. Additional alcohol dehydrogenases include horse liver ADH (SEQ ID NO: 799) and Beijerinkia indica ADH (SEQ ID NO: 800), and those that utilize NADH as a cofactor. KARI enzymes are described for example, in U.S. Patent Nos. 7,910, 342 and 8,129,162; U.S. Publication No. 2010/0197519; International Publication No. WO 2012/129555, all of which are incorporated by reference. KARIs include Pseudomonas fluorescens KARI (SEQ ID NO: 801) and variants thereof and Anaerostipes caccae KARI (SEQ ID NO: 802) and variants thereof (eg. "K9G9", "K9D3", and "K9JB4P"; SEQ ID NOs: 167, 166, and 791 respectively). In one embodiment a butanol biosynthetic pathway comprises a) a ketol-acid reductoisomerase that has a K _m for NADH less than about 300 μΜ, less than about 100 μΜ, less than about 50 μΜ, less than about 20 μΜ or less than about 10 μΜ; b) an alcohol dehydrogenase that utilizes NADH as a cofactor; or c) both a) and b). Cell Integrity Polypeptides

[0199] Another embodiment of the invention is directed to an isolated polynucleotide comprising: (a) a promoter nucleic acid sequence; and (b) a nucleic acid sequence encoding a biocatalyst polypeptide for cell integrity. In embodiments, the nucleic acid sequence of (b) is coupled to the nucleic acid sequence of (a).

[0200] In one embodiment, the biocatalyst polypeptide is a GPI-anchored cell wall protein involved in acid resistance. In one embodiment, the biocatalyst polypeptide is YER150W (SPIl) (nucleic acid SEQ ID NO: 190; amino acid SEQ ID NO: 397), or a homolog thereof. In another embodiment, the biocatalyst polypeptide is encoded by a cell wall integrity gene activated by Rlml such as a polypeptide listed in Table 5 or a homolog thereof.

Table 5 Biocatalyst polypeptides for cell integrity

[0201] In embodiments, cell integrity polypeptides are preferentially expressed during the production phase. While not wishing to be bound by theory, it is believed that expression of such polypeptides may contribute to improved tolerance of a host cell to a fermentation product (e.g., butanol), thus improving production.

Propagation Polypeptides

[0202] Another embodiment of the invention is directed to an isolated polynucleotide comprising:

(a) a promoter nucleic acid sequence; and

(b) a nucleic acid sequence encoding a biocatalyst polypeptide necessary for cell

propagation; wherein the nucleic acid sequence of (b) is coupled to the nucleic acid sequence of (a).

[0203] In some embodiments, the propagation polypeptide is phosphoketolase. In some embodiments, the propagation polypeptide is phosphotransacetylase. Example phosphoketolases and phosphotransacetylases are described in PCT Publication No. WO/2011/159853 and U.S. App. Pub. No. 20120156735 Al, incorporated by reference herein. In some embodiments, the phosphoketolase is xpk from Lactobacillus plantarum (nucleic acid SEQ ID NO: 180; amino acid SEQ ID NO: 181). In some embodiments, the phosphotransacetylase is eutD from

Lactobacillus plantarum (nucleic acid SEQ ID NO: 178; amino acid SEQ ID NO: 179).

[0204] In embodiments, host cells comprising such nucleic acid sequences encoding biocatalyst polypeptides for cell propagation have reduced or eliminated pyruvate decarboxylase activity.

Biosynthetic pathway by-product producing polypeptides

[0205] DHMB can be produced as a result of a side-reaction that occurs when host cells are genetically manipulated to include a biosynthetic pathway, e.g., a biosynthetic pathway that involves the production of acetolactate. The presence of DHMB indicates that not all of the pathway substrates are being converted to the desired product. Thus, yield may be lowered. In addition, DHMB present in the fermentation media may have inhibitory effects on product production. For example, DHMB can decrease the activity of enzymes in the biosynthetic pathway or have other inhibitory effects on cell growth and/or productivity during fermentation. Thus, described herein are isolated polynucleotides resulting in lower expression of DHMB during the production phase of fermentation than in the propagation phase. The ability of a host cell to convert acetolactate to DHMB can be reduced or eliminated, for example, by reducing the expression of a polypeptide having acetolactate reductase activity. In some embodiments, the polypeptide having acetolactate reductase activity is YMR226C (nucleic acid SEQ ID NO: 182, amino acid SEQ ID NO: 183) or a homo log thereof.

[0206] The last step in the biosynthesis of isobutanol via a pyruvate-utilizing biosynthetic pathway is the conversion of isobutyraldehyde to isobutanol (Figure 1). A side reaction in this pathway is the conversion of isobutyraldehyde to isobutyric acid which results in reduced amounts of isobutyraldehyde available to convert into isobutanol and reduced isobutanol yield. Reducing or eliminating the conversion of isobutyraldehyde to isobutyric acid may result in increased amounts of isobutyraldehyde available for conversion to isobutanol. The conversion of isobutyraldehyde to isobutanol can be reduced or eliminated, for example, by reducing the expression of an aldehyde dehydrogenase. Thus, provided herein are isolated polynucleotides resulting in lower expression of an aldehyde dehydrogenase during the production phase of fermentation than in the propagation phase. In embodiments, a recombinant host cell of the invention can be S. cerevisiae, and a polypeptide having aldehyde dehydrogenase activity can be ALD2 (nucleic acid SEQ ID NO: 436; amino acid SEQ ID NO: 437), ALD3 (nucleic acid SEQ ID NO: 438; amino acid SEQ ID NO: 439), ALD4 (nucleic acid SEQ ID NO: 440; amino acid SEQ ID NO: 441), ALD5 (nucleic acid SEQ ID NO: 442; amino acid SEQ ID NO: 443), ALD6 (nucleic acid SEQ ID NO: 184; amino acid SEQ ID NO: 185), or combinations thereof. In other embodiments, a recombinant host cell can be Kluyveromyces lactis, and a polypeptide having aldehyde dehydrogenase activity can be KLLA0F00440, KLLA0E23057, KLLA0D 10021, KLLA0D09999G, or combinations thereof. In other embodiments, a recombinant host cell can be Pichia stipitis, and a polypeptide having aldehyde dehydrogenase activity can be ALD2, ALD3, ALD4, ALD5, ALD7, or combinations thereof. In other embodiments, a recombinant host cell can be Lactobacillus plantarum, and a polypeptide having aldehyde dehydrogenase activity can be AldH. In other embodiments, a recombinant host cell can be E. coli, and a polypeptide having aldehyde dehydrogenase activity can be aldA, aldB, or combinations thereof.

Glycerol biosynthesis pathway polypeptides

[0207] Endogenous NAD-dependent glycerol-3 -phosphate dehydrogenase is a key enzyme in glycerol synthesis, converting dihydroxyacetone phosphate (DHAP) to glycerol-3 - phosphate and playing a role in cellular oxidation of NADH. Yeast strains may have one or more genes encoding NAD-dependent glycerol-3 -phosphate dehydrogenase (GPD). In some yeasts, such as S. cerevisiae, S. pombe, and P. stipitis, GPD1 and GPD2 are functional homologs for NAD-dependent glycerol-3 -phosphate dehydrogenase. Provided herein are isolated polynucleotides that resulting in lower expression of glycerol-3 -phosphate dehydrogenase activity during the production phase of fermentation than in the propagation phase. In one embodiment, the biocatalyst polypeptide is GPD1 (nucleic acid SEQ ID NO: 444; amino acid SEQ ID NO: 445) or GPD2 (nucleic acid SEQ ID NO: 446; amino acid SEQ ID NO: 447), or a homolog thereof.

Polypeptides of an NADPH generating pathway

[0208] In some embodiments, the biocatalyst polypeptide is an enzyme of the oxidative pentose phosphate pathway. In some embodiments, the biocatalyst polypeptide is glucoses- phosphate dehydrogenase (ZWF1, nucleic acid SEQ ID NO: 448; amino acid SEQ ID NO: 449), 6-phosphoglucononolactonase (SOL3: nucleic acid SEQ ID NO: 450; amino acid SEQ ID NO: 451; or SOL4: SEQ ID NO: 452; amino acid SEQ ID NO: 453), or 6-phosphogluconate dehydrogenase nucleic acid (GND1 : nucleic acid SEQ ID NO: 454; amino acid SEQ ID NO: 455; GND2: nucleic acid SEQ ID NO: 456; amino acid SEQ ID NO: 457). For example, in one embodiment, ZWF1 is preferentially expressed in propagation. In some embodiments, the reducing equivalents (such as NADH) produced in glycolysis are utilized by the biosynthetic production pathway during the production phase. For example, in an isobutanol biosynthetic pathway described herein, 2 molecules of NADH produced during glycolysis are consumed per molecule of isobutanol produced. While not wishing to be bound by theory, it is believed that reduced expression of ZWF1 during such production processes may result in decreased excess NADPH production and consequently decreased by-product production.

Recombinant Microbial Host Cells

[0209] Standard recombinant DNA and molecular cloning techniques are well known in the art and are described by Sambrook, J., Fritsch, E. F. and Maniatis, T., Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989) (hereinafter "Maniatis"); and by Silhavy, T. J., Bennan, M. L. and Enquist, L. W., Experiments with Gene Fusions, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1984); and by Ausubel, F. M. et al., Current Protocols in Molecular Biology, published by Greene Publishing Assoc. and Wiley-Interscience (1987). Additional methods are in Methods in Enzymology, Volume 194, Guide to Yeast Genetics and Molecular and Cell Biology (Part A, 2004, Christine Guthrie and Gerald R. Fink (Eds.), Elsevier Academic Press, San Diego, CA). Molecular tools and techniques are known in the art and include splicing by overlapping extension polymerase chain reaction (PCR) (Yu, et al., Fungal Genet. Biol. 47:973-981 (2004)), positive selection for mutations at the URA3 locus of Saccharomyces cerevisiae (Boeke, J.D. et αί, Μοί Gen. Genet. 197, 345-346 (1984); M A Romanos, et al, Nucleic Acids Res. 19(1): 187 (1991)), the cre-lox site-specific recombination system as well as mutant lox sites and FLP substrate mutations (Sauer, B.m Mol Cell Biol 7: 2087-2096 (1987); Senecoff, et al, Journal of Molecular Biology 20i(2):405-421 (1988); Albert, et al, The Plant Journal 7(4,): 649-659 (1995)), "seamless" gene deletion (Akada, et al., (2006) Yeast 2J(5):399-405 (2006)), and gap repair methodology (Ma, et al., Genetics 55:201-216 (1981)).

[0210] The genetic manipulations of a recombinant host cell disclosed herein can be performed using standard genetic techniques and screening and can be made in any host cell that is suitable to genetic manipulation (Methods in Yeast Genetics, 2005, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 201-202).

[0211] Non-limiting examples of host cells for use in the invention include bacteria, cyanobacteria, filamentous fungi and yeasts.

[0212] In one embodiment, the recombinant host cell of the invention is a bacterial or a cyanobacterial cell. In another embodiment, the recombinant host cell comprises or is selected from the group consisting of: Salmonella, Arthrobacter, Bacillus, Brevibacterium, Clostridium, Corynebacterium, Gluconobacter, Nocardia, Pseudomonas, Rhodococcus, Streptomyces, Zymomonas, Escherichia, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Serratia, Shigella, Alcaligenes, Erwinia, Paenibacillus, and Xanthomonas . In some embodiments, the recombinant host cell is E. coli, S. cerevisiae, or L. plantarum.

[0213] In another embodiment, the recombinant host cell of the invention is a filamentous fungi or yeast cell. In another embodiment, the recombinant host cell comprises or is selected from the group consisting of: Saccharomyces, Pichia, Hansenula, Yarrowia, Aspergillus, Kluyveromyces, Pachysolen, Rhodotorula, Zy go saccharomyces, Galactomyces,

Schizosaccharomyces, Torulaspora, Debayomyces, Williopsis, Dekkera, Kloeckera,

Metschnikowia, and Candida. In another embodiment, the host cell does not express an enzyme or has reduced expression of an enzyme having the following Enzyme Commission Number: EC 4.1.1.1.

[0214] In some embodiments, the yeast is crabtree-positive. Crabtree-positive yeast cells demonstrate the crabtree effect, which is a phenomenon whereby cellular respiration is inhibited when a high concentration of glucose is present in aerobic culture medium. Suitable crabtree- positive yeast are viable in culture and include, but are not limited to, Saccharomyces,

Schizosaccharomyces, and Issatchenkia. Suitable species include, but are not limited to,

Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces thermotolerans, Candida glabrata, Issatchenkia orientalis.

[0215] Crabtree-positive yeast cells may be grown with high aeration and in low glucose concentration to maximize respiration and cell mass production, as known in the art, rather than butanol production. Typically the glucose concentration is kept to less than about 0.2 g/L. The aerated culture can grow to a high cell density and then be used as the present production culture. Alternatively, yeast cells that are capable of producing butanol may be grown and concentrated to produce a high cell density culture.

[0216] In some embodiments, the yeast is Crabtree-negative. Crabtree-negative yeast cells do not demonstrate the crabtree effect when a high concentration of glucose is added to aerobic culture medium, and therefore, in crabtree-negative yeast cells, alcoholic fermentation is absent after an excess of glucose is added. Suitable Crabtree-negative yeast genera are viable in culture and include, but are not limited to, Hansenula, Debaryomyces, Yarrowia, Rhodotorula, and Pichia. Suitable species include, but are not limited to, Candida utilis, Hansenula nonfermentans, Kluyveromyces marxianus, Kluyveromyces lactis, Pichia stipitis, and Pichia pastoris.

[0217] Suitable microbial hosts include, but are not limited to, members of the genera

Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Vibrio, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Issatchenkia, Hansenula, Kluyveromyces, and Saccharomyces. Suitable hosts include: Escherichia coli, Alcaligenes eutrophus, Bacillus licheniformis, Paenibacillus macerans, Rhodococcus erythropolis, Pseudomonas putida,

Lactobacillus plantarum, Enterococcus faecium, Enterococcus gallinarium, Enterococcus faecalis, Bacillus subtilis and Saccharomyces cerevisiae. In some embodiments, the host cell is Saccharomyces cerevisiae. S. cerevisiae yeast are known in the art and are available from a variety of sources, including, but not limited to, American Type Culture Collection (Rockville, MD), Centraalbureau voor Schimmelcultures (CBS) Fungal Biodiversity Centre, LeSaffre, Gert Strand AB, Ferm Solutions, North American Bioproducts, Martrex, and Lallemand. S. cerevisiae include, but are not limited to, BY4741, CEN.PK 113-7D, Ethanol Red® yeast, Ferm Pro™ yeast, Bio-Ferm® XR yeast, Gert Strand Prestige Batch Turbo alcohol yeast, Gert Strand Pot Distillers yeast, Gert Strand Distillers Turbo yeast, FerMax™ Green yeast, FerMax™ Gold yeast, Thermosacc® yeast, BG-1, PE-2, CAT-1, CBS7959, CBS7960, and CBS7961.

[0218] Recombinant microorganisms containing the necessary genes that will encode the enzymatic pathway for the conversion of a fermentable carbon substrate to a desired product (eg. butanol) can be constructed using techniques well known in the art. For example, genes encoding the enzymes of one of the isobutanol biosynthetic pathways of the invention, for example, acetolactate synthase, acetohydroxy acid isomeroreductase, acetohydroxy acid dehydratase, branched-chain a-keto acid decarboxylase, and branched-chain alcohol

dehydrogenase, can be obtained from various sources, as described above.

[0219] Methods of obtaining desired genes from a genome are common and well known in the art of molecular biology. For example, if the sequence of the gene is known, suitable genomic libraries can be created by restriction endonuclease digestion and can be screened with probes complementary to the desired gene sequence. Once the sequence is isolated, the DNA can be amplified using standard primer-directed amplification methods such as polymerase chain reaction (U.S. 4,683,202) to obtain amounts of DNA suitable for transformation using

appropriate vectors. Tools for codon optimization for expression in a heterologous host are readily available (described elsewhere herein).

[0220] Once the relevant pathway genes are identified and isolated they can be transformed into suitable expression hosts by means well known in the art. Vectors or cassettes useful for the transformation of a variety of host cells are common and commercially available from companies such as EPICENTRE ^® (Madison, WI), Invitrogen Corp. (Carlsbad, CA), Stratagene (La Jolla, CA), and New England Biolabs, Inc. (Beverly, MA). Typically the vector or cassette contains sequences directing transcription and translation of the relevant gene, a selectable marker, and sequences allowing autonomous replication or chromosomal integration. Suitable vectors comprise a region 5' of the gene which harbors transcriptional initiation controls and a region 3' of the DNA fragment which controls transcriptional termination. Both control regions can be derived from genes homologous to the transformed host cell, although it is to be understood that such control regions can also be derived from genes that are not native to the specific species chosen as a production host.

[0221] Initiation control regions or promoters, which are useful to drive expression of the relevant pathway coding regions in the desired host cell are numerous and familiar to those skilled in the art. Virtually any promoter capable of driving these genetic elements, including those used in the Examples, is suitable for the present invention including, but not limited to, CYC1, HIS3, GAL1, GAL 10, ADH1, PGK, PH05, GAPDH, ADC1, TRP1, URA3, LEU2, ENO, TPI (useful for expression in Saccharomyces); AOX1 (useful for expression in Pichia); and lac, ara, tet, trp, IPL, IPR, T7, tac, and trc (useful for expression in Escherichia coli,

Alcaligenes, and Pseudomonas) as well as the amy, apr, npr promoters and various phage promoters useful for expression in Bacillus subtilis, Bacillus licheniformis, and Paenibacillus macerans. For yeast recombinant host cells, a number of promoters can be used in constructing expression cassettes for genes, including, but not limited to, the following constitutive promoters suitable for use in yeast: FBA1, TDH3 (GPD), ADH1, ILV5, and GPM1; and the following inducible promoters suitable for use in yeast: GAL1, GAL 10, OLE1, and CUP1. Other yeast promoters include hybrid promoters UAS(PGKl)-FBAlp, UAS(PGKl)-EN02p, UAS(FBAl)- PDClp, UAS(PGKl)-PDClp, and UAS(PGK)-OLElp.

[0222] Promoters, transcriptional terminators, and coding regions can be cloned into a yeast 2 micron plasmid and transformed into yeast cells (Ludwig, et al. Gene, 132: 33-40, 1993; US Appl. Pub. No. 20080261861A1).

[0223] Adjusting the amount of gene expression in a given host may be achieved by varying the level of transcription, such as through selection of native or artificial promoters. In addition, techniques such as the use of promoter libraries to achieve desired levels of gene transcription are well known in the art. Such libraries can be generated using techniques known in the art, for example, by cloning of random cDNA fragments in front of gene cassettes (Goh et al. (2002) AEM 99, 17025), by modulating regulatory sequences present within promoters (Ligr et al. (2006) Genetics 172, 2113),or by mutagenesis of known promoter sequences (Alper et al. (2005) PNAS, 12678; Nevoigt et al. (2006) AEM 72, 5266).

[0224] Termination control regions can also be derived from various genes native to the hosts. Optionally, a termination site can be unnecessary or can be included.

[0225] Certain vectors are capable of replicating in a broad range of host bacteria and can be transferred by conjugation. The complete and annotated sequence of pRK404 and three related vectors-pR 437, pR 442, and pRK442(H) are available. These derivatives have proven to be valuable tools for genetic manipulation in Gram-negative bacteria (Scott et al., Plasmid, 50: 74-79, 2003). Several plasmid derivatives of broad-host-range Inc P4 plasmid RSF1010 are also available with promoters that can function in a range of Gram-negative bacteria. Plasmid pAYC36 and pAYC37, have active promoters along with multiple cloning sites to allow for the heterologous gene expression in Gram-negative bacteria.

[0226] Chromosomal gene replacement tools are also widely available. For example, a thermosensitive variant of the broad-host-range replicon pWVlOl has been modified to construct a plasmid pVE6002 which can be used to effect gene replacement in a range of Gram-positive bacteria (Maguin, et al., J. Bacteriol, 174: 5633-5638 (1992)). Additionally, in vitro

transposomes are available to create random mutations in a variety of genomes from commercial sources such as EPICENTRE®.

[0227] The expression of a biosynthetic pathway in various microbial hosts is described in more detail in the Examples herein and in the art.U.S. Patent 7,851,188 and PCT App. No. WO2012/129555, both incorporated by reference, which disclose the engineering of recombinant microorganisms for production of isobutanol. U.S. Appl. Pub. No. 2008/0182308A1, incorporated by reference, discloses the engineering of recombinant microorganisms for production of 1-butanol. U.S. Appl. Pub. Nos. 2007/0259410A1 and 2007/0292927A1, both incorporated by reference, disclose the engineering of recombinant microorganisms for production of 2-butanol. Multiple pathways are described for biosynthesis of isobutanol and 2- butanol. The methods disclosed in these publications can be used to engineer the recombinant host cells of the present invention. The information presented in these publications is hereby incorporated by reference in its entirety.

Modifications

[0228] In some embodiments, the host cells comprising a biosynthetic pathway as provided herein may further comprise one or more additional modifications. U.S. Appl. Pub. No. 2009/0305363, incorporated herein by reference, discloses increased conversion of pyruvate to acetolactate by engineering yeast for expression of a cytosol-localized acetolactate synthase and substantial elimination of pyruvate decarboxylase activity. Modifications to reduce glycerol- 3-phosphate dehydrogenase activity and/or disruption in at least one gene encoding a polypeptide having pyruvate decarboxylase activity or a disruption in at least one gene encoding a regulatory element controlling pyruvate decarboxylase gene expression as described in U.S. Appl. Pub. No. 2009/0305363, incorporated herein by reference, modifications to a host cell that provide for increased carbon flux through an Entner-Doudoroff Pathway or reducing equivalents balance as described in U.S. Appl. Pub. No. 2010/0120105, incorporated herein by reference. Other modifications include integration of at least one polynucleotide encoding a polypeptide that catalyzes a step in a pyruvate -utilizing biosynthetic pathway. Other modifications are described in PCT. Pub. No. WO2012/129555, incorporated herein by reference. Modifications include at least one deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having acetolactate reductase activity. In embodiments, the polypeptide having acetolactate reductase activity is YMR226C of Saccharomyces cerevisae or a homolog thereof. Additional modifications include a deletion, mutation, and/or substitution in an endogenous polynucleotide encoding a polypeptide having aldehyde dehydrogenase and/or aldehyde oxidase activity. In embodiments, the polypeptide having aldehyde dehydrogenase activity is ALD6 from Saccharomyces cerevisiae or a homolog thereof. A genetic modification which has the effect of reducing glucose repression wherein the yeast production host cell is pdc- is described in U.S. Appl. Pub. No. 2011/0124060, incorporated herein by reference. In some embodiments, the pyruvate decarboxylase that is deleted or downregulated is selected from the group consisting of: PDC1, PDC5, PDC6, or combinations thereof. In some embodiments, host cells contain a deletion or downregulation of a polynucleotide encoding a polypeptide that catalyzes the conversion of glyceraldehyde-3 -phosphate to glycerate 1,3, bisphosphate. In some embodiments, the enzyme that catalyzes this reaction is glyceraldehyde-3 -phosphate dehydrogenase.

[0229] Recombinant host cells may further comprise (a) at least one heterologous polynucleotide encoding a polypeptide having dihydroxy-acid dehydratase activity; and (b)(i) at least one deletion, mutation, and/or substitution in an endogenous gene encoding a polypeptide affecting Fe-S cluster biosynthesis; and/or (ii) at least one heterologous polynucleotide encoding a polypeptide affecting Fe-S cluster biosynthesis, described in PCT Publication No.

WO2011/103300, incorporated herein by reference. In embodiments, the polypeptide affecting Fe-S cluster biosynthesis is encoded by AFTl, AFT2, FRA2, GRX3, or CCCl . In embodiments, the polypeptide affecting Fe-S cluster biosynthesis is constitutive mutant AFTl L99A, AFTl L102A, AFTl C291F, or AFTl C293F.

Differential Expression

[0230] As demonstrated in the Examples, a recombinant host cell comprising promoter nucleic acid sequences may be subjected to different conditions, such as conditions

corresponding to those in a propagation vs. a production phase, and differential expression of a suppressor tRNA and/or anti-sense RNA will result in differential expression of a target polynucleotide or its encoded polypeptide, which may may be confirmed using methods known in the art and/or provided herein. Differential expression of a polynucleotide encoding a biocatalyst polypeptide can be confirmed by comparing transcript levels under different conditions using reverse transcriptase polymerase chain reaction (RT-PCR) or real-time PCR using methods known in the art and/or exemplified herein. In some embodiments, a reporter, such as green fluorescent protein (GFP) can be used in combination with flow cytometry to confirm the capability of a promoter nucleic acid sequence to affect expression under different conditions. Furthermore, as demonstrated in the Examples, the activity of a biocatalyst polypeptide may be determined under different conditions to confirm the differential expression of the polypeptide. For example, where ALS is the biocatalyst polypeptide, the activity of ALS present in host cells subjected to different conditions may be determined (using, for example, methods described in Westerfeld, W.W., J. Biol. Chem. 161:495-502 (1945), modified as described in the Examples herein). A difference in ALS activity can be used to confirm differential expression of the ALS. It is also envisioned that differential expression of a biocatalyst polypeptide can be confirmed indirectly by measurement of downstream products or byproducts. For example, a decrease in production of isobutyraldehyde may be indicative of differential ALS expression.

[0231] It will be appreciated that other useful methods to confirm differential expression include measurement of biomass and/or measurement of biosynthetic pathway products under different conditions. For example, spectrophotometric measurement of optical density (O.D.) can be used as an indicator of biomass. Measurement of pathway products or by-products, including, but not limited to butanol concentration, DHMB concentration, or isobutyric acid can be carried out using methods known in the art and/or provided herein such as high pressure liquid chromatography (HPLC; for example, see PCT. Pub. No. WO2012/129555, incorporated herein by reference) Likewise, the rate of biomass increase, the rate of glucose consumption, or the rate of butanol production can be determined, for example by using the indicated methods. Biomass yield and product (eg. butanol) yield can likewise be determined using methods disclosed in the art and/or herein.

Methods for Producing Fermentation Products

[0232] Another embodiment of the present invention is directed to methods for producing various fermentation products including, but not limited to, lower alkyl alcohols. These methods employ the recombinant host cells of the invention. In one embodiment, the method of the present invention comprises providing a recombinant host cell as discussed above, contacting the recombinant host cell with a fermentable carbon substrate in a fermentation medium under conditions whereby the fermentation product is produced and, optionally, recovering the fermentation product.

[0233] It will be appreciated that a process for producing fermentation products may comprise multiple phases. For example, process may comprise a first biomass production phase, a second biomass production phase, a fermentation production phase, and an optional recovery phase. In embodiments, processes provided herein comprise more than one, more than two, or more than three phases. It will be appreciated that process conditions may vary from phase to phase. For example, one phase of a process may be substantially aerobic, while the next phase may be substantially anaerobic. Other differences between phases may include, but are not limited to, source of carbon substrate (e.g., feedstock from which the fermentable carbon is derived), carbon substrate (e.g.,. glucose) concentration, dissolved oxygen, pH, temperature, or concentration of fermentation product (e.g., butanol). Promoter nucleic acid sequences and nucleic acid sequences encoding biocatalyst polypeptides and recombinant host cells comprising such promoter nucleic acid sequences may be employed in such processes. In embodiments, a biocatalyst polypeptide is preferentially expressed in at least one phase.

[0234] The propagation phase generally comprises at least one process by which biomass is increased. In embodiments, the temperature of the propagation phase may be at least about 20, at least about 30, at least about 35, or at least about 40°C. In embodiments, the pH in the propagation phase may be at least about 4, at least about 5, at least about 5.5, at least about 6, or at least about 6.5. In embodiments, the propagation phase continues until the biomass concentration reaches at least about 5, at least about 10, at least about 15, at least about 20, at least about 30, at least about 50, at least about 70, or at least about 100 g/L. In embodiments, the average glucose or sugar concentration is about or less than about 2 g/L, about or less than about 1 g/L, about or less than about 0.5 g/L or about or less than about 0.1 g/L. In embodiments, the dissolved oxygen concetration may average as undetectable, or as at least about 10%, at least about 20%, at least about 30%, or at least about 40%.

[0235] In one non-limiting example, a stage of the propagation phase comprises contacting a recombinant yeast host cell with at least one carbon substrate at a temperature of about 30 to about 35°C and a pH of about 4 to about 5.5, until the biomass concentration is in the range of about 20 to about 100 g/L. The dissolved oxygen level over the course of the contact may average from about 20 to 40% (0.8 - 3.2 ppm). The source of the carbon substrate may be molasses or corn mash, or pure glucose or other sugar, such that the glucose or sugar concentration is from about 0 to about 1 g/L over the course of the contacting or from about 0 to about 0.1 g/L. In a subsequence or alternate stage of the propagation phase, a recombinant yeast host cell may be subjected to a further process whereby recombinant yeast at a concentration of about 0.1 g/L to about 1 g/L is contacted with at least one carbon substrate at a temperature of about 25 to about 35°C and a pH of about about 4 to about 5.5 until the biomass concentration is in the range of about 5 to about 15 g/L. The dissolved oxygen level over the course of the contact may average from undetectable to about 30% (0-2.4 ppm). The source of the carbon substrate may be corn mash such that the glucose concentration averages about 2 to about 30 g/L over the course of contacting.

[0236] It will be understood that the propagation phase may comprise one, two, three, four, or more stages, and that the above non-limiting example stages may be practiced in any order or combination.

[0237] The production phase typically comprises at least one process by which a product is produced. In embodiments, the average glucose concentration during the production phase is at least about 0.1, at least about 1, at least about 5, at least about 10 g/L, at least about 30 g/L, at least about 50 g/L, or at least about 100 g/L. In embodiments, the temperature of the production phase may be at least about 20, at least about 30, at least about 35, or at least about 40°C. In embodiments, the pH in the production phase may be at least about 4, at least about 5, or at least about 5.5. In embodiments, the production phase continues until the product titer reaches at least about 10 g/L, at least about 15 g/L, at least about 20 g/L, at least about 25 g/L, at least about 30 g/L, at least about 35 g/L or at least about 40 g/L. In embodiments, the dissolved oxygen concetration may average as less than about 5%, less than about 1%, or as negligible such that the conditions are substantially anaerobic.

[0238] In one non-limiting example production phase, recombinant yeast cells at a concentration of about 0.1 to about 6 g/L are contacted with at least one carbon substrate at a concentration of about 5 to about 100 g/L, temperature of about 25 to about 30°C, pH of about 4 to about 5.5. The dissolved oxygen level over the course of the contact may be negligible on average, such that the contact occurs under substantially anaerobic conditions. The source of the carbon substrate may mash such as corn mash, such that the glucose concentration averages about 10 to about 100 g/L over the course of the contacting, until it is substantially completely consumed. [0239] In embodiments, the glucose concentration is about 100-fold to about 1000-fold higher in the production phase than in the propagation phase. In embodiments, the glucose concentration in production is at least about 5X, at least about 10X, at least about 5 OX, at least about 100X, or at least about 500X higher than that in propagation. In embodiments, the temperature in the propagation phase is about 5 to about 10 degrees lower in the production phase than in the propagation phase. In embodiments, the average dissolved oxygen

concentration is anaerobic in the production phase and microaerobic to aerobic in the propagation phase.

[0240] One of skill in the art will appreciate that the conditions for propagating a host cell and/or producing a fermentation product utilizing a host cell may vary according to the host cell being used. In one embodiment, the method for producing a fermentation product is performed under anaerobic conditions. In one embodiment, the method for producing a fermentation product is performed under microaerobic conditions.

[0241] Further, it is envisioned that once a recombinant host cell comprising a suitable genetic switch has been identified, the process may be further refined to take advantage of the differential expression afforded thereby. For example, if the genetic switch provides preferential expression in high glucose conditions, one of skill in the art will be able to readily determine the glucose levels necessary to maintain minimal expression. As such, the glucose concentration in the phase of the process under which minimal expression is desired can be controlled so as to maintain minimal expression. In one non-limiting example, polymer-based slow-release feed beads (available, for example, from Kuhner Shaker, Basel, Switzerland) may be used to maintain a low glucose condition. A similar strategy can be employed to refine the propagation or production phase conditions relevant to the differential expression using the compositions and methods provided herein.

[0242] Carbon substrates may include, but are not limited to, monosaccharides (such as fructose, glucose, mannose, rhamnose, xylose or galactose), oligosaccharides (such as lactose, maltose, or sucrose), polysaccharides such as starch, maltodextrin, or cellulose, fatty acids, or mixtures thereof and unpurified mixtures from renewable feedstocks such as corn mash, cheese whey permeate, cornsteep liquor, sugar beet molasses, and barley malt. Other carbon substrates may include ethanol, lactate, succinate, or glycerol.

[0243] Additionally, the carbon substrate may also be a one carbon substrate such as carbon dioxide, or methanol for which metabolic conversion into key biochemical intermediates has been demonstrated. In addition to one and two carbon substrates, methylotrophic organisms are also known to utilize a number of other carbon containing compounds such as methylamine, glucosamine and a variety of amino acids for metabolic activity. For example, methylotrophic yeasts are known to utilize the carbon from methylamine to form trehalose or glycerol (Bellion, et al, Microb. Growth CI Compd., [Int. Symp.], 7th (1993), 415 32, Editor(s): Murrell, J.

Collin; Kelly, Don P. Publisher: Intercept, Andover, UK). Similarly, various species of Candida will metabolize alanine or oleic acid (Suiter, et al., Arch. Microbiol. 755:485-489 (1990)).

Hence, it is contemplated that the source of carbon utilized in the present invention may encompass a wide variety of carbon containing substrates and will only be limited by the choice of organism.

[0244] Although it is contemplated that all of the above mentioned carbon substrates and mixtures thereof may be suitable suitable in the present invention, preferred carbon substrates are glucose, fructose, and sucrose, or mixtures of these with C5 sugars such as xylose and/or arabinose for yeasts cells modified to use C5 sugars. Sucrose may be derived from renewable sugar sources such as sugar cane, sugar beets, cassava, sweet sorghum, and mixtures thereof. Glucose and dextrose may be derived from renewable grain sources through saccharification of starch based feedstocks including grains such as corn, wheat, rye, barley, oats, and mixtures thereof. In addition, fermentable sugars may be derived from renewable cellulosic or lignocellulosic biomass through processes of pretreatment and saccharification, as described, for example, in U.S. Appl. Pub. No. 2007/0031918 Al, which is herein incorporated by reference. Biomass in reference to a carbon source refers to any cellulosic or lignocellulosic material and includes materials comprising cellulose, and optionally further comprising hemicellulose, lignin, starch, oligosaccharides and/or monosaccharides. Biomass may also comprise additional components, such as protein and/or lipid. Biomass may be derived from a single source, or biomass can comprise a mixture derived from more than one source; for example, biomass may comprise a mixture of corn cobs and corn stover, or a mixture of grass and leaves. Biomass includes, but is not limited to, bioenergy crops, agricultural residues, municipal solid waste, industrial solid waste, sludge from paper manufacture, yard waste, wood and forestry waste. Examples of biomass include, but are not limited to, corn grain, corn cobs, crop residues such as corn husks, corn stover, grasses, wheat, wheat straw, barley, barley straw, hay, rice straw, switchgrass, waste paper, sugar cane bagasse, sorghum, soy, components obtained from milling of grains, trees, branches, roots, leaves, wood chips, sawdust, shrubs and bushes, vegetables, fruits, flowers, animal manure, and mixtures thereof.

[0245] The carbon substrates may be provided in any media that is suitable for host cell growth and reproduction. Non- limiting examples of media that can be used include M122C, MOPS, SOB, TSY, YMG, YPD, 2XYT, LB, Ml 7, or M9 minimal media. Other examples of media that can be used include solutions containing potassium phosphate and/or sodium phosphate. Suitable media can be supplemented with NADH or NADPH.

[0246] In one embodiment, the method for producing a fermentation product results in a titer of at least about 20 g/L of a fermentation product. In another embodiment, the method for producing a fermentation product results in a titer of at least about 30 g/L of a fermentation product. In another embodiment, the method for producing a fermentation product results in a titer of at least about 10 g/L, 15 g/L, 20 g/L, 25 g/L, 30 g/L, 35 g/L or 40 g/L of fermentation product.

[0247] In embodiments, the rate of production of a fermentation product is increased. In embodiments, the rate of biomass production is increased. In embodiments, the yield of fermentation product is increased. In embodiments, the yield of biomass is increased. Such improvements may be observed by comparison to that obtained using the control recombinant host cell without a genetic switch.

[0248] Non- limiting examples of lower alkyl alcohols which may be produced by the methods of the invention include butanol (for example, isobutanol), propanol, isopropanol, and ethanol. In one embodiment, isobutanol is produced.

[0249] In one embodiment, the recombinant host cell of the invention produces a fermentation product at a yield of greater than about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, or 75% of theoretical. In one embodiment, the recombinant host cell of the invention produces a fermentation product at a yield of greater than about 25% of theoretical. In another embodiment, the recombinant host cell of the invention produces a fermentation product at a yield of greater than about 40% of theoretical. In another embodiment, the recombinant host cell of the invention produces a fermentation product at a yield of greater than about 50% of theoretical. In another embodiment, the recombinant host cell of the invention produces a fermentation product at a yield of greater than about 75% of theoretical.

[0250] Non- limiting examples of lower alkyl alcohols produced by the recombinant host cells of the invention include butanol, isobutanol, propanol, isopropanol, and ethanol. In one embodiment, the recombinant host cells of the invention produce isobutanol. In another embodiment, the recombinant host cells of the invention do not produce ethanol.

Methods for Isobutanol Isolation from the Fermentation Medium

[0251] Bioproduced butanol may be isolated from the fermentation medium using methods known in the art for ABE fermentations (see, e.g., Durre, Appl. Microbiol. Biotechnol. 4P:639-648 (1998), Groot, et al, Process. Biochem. 27:61-75 (1992), and references therein). For example, solids may be removed from the fermentation medium by centrifugation, filtration, decantation, or the like. Then, the butanol may be isolated from the fermentation medium using methods such as distillation, azeotropic distillation, liquid-liquid extraction, adsorption, gas stripping, membrane evaporation, or pervaporation.

[0252] Because butanol forms a low boiling point, azeotropic mixture with water, distillation can be used to separate the mixture up to its azeotropic composition. Distillation may be used in combination with another separation method to obtain separation around the azeotrope. Methods that may be used in combination with distillation to isolate and purify butanol include, but are not limited to, decantation, liquid-liquid extraction, adsorption, and membrane-based techniques. Additionally, butanol may be isolated using azeotropic distillation using an entrainer (see, e.g., Doherty and Malone, Conceptual Design of Distillation Systems, McGraw Hill, New York, 2001).

[0253] The butanol-water mixture forms a heterogeneous azeotrope so that distillation may be used in combination with decantation to isolate and purify the butanol. In this method, the butanol containing fermentation broth is distilled to near the azeotropic composition. Then, the azeotropic mixture is condensed, and the butanol is separated from the fermentation medium by decantation. The decanted aqueous phase may be returned to the first distillation column as reflux. The butanol-rich decanted organic phase may be further purified by distillation in a second distillation column.

[0254] The butanol can also be isolated from the fermentation medium using liquid-liquid extraction in combination with distillation. In this method, the butanol is extracted from the fermentation broth using liquid-liquid extraction with a suitable solvent. The butanol-containing organic phase is then distilled to separate the butanol from the solvent.

[0255] Distillation in combination with adsorption can also be used to isolate butanol from the fermentation medium. In this method, the fermentation broth containing the butanol is distilled to near the azeotropic composition and then the remaining water is removed by use of an adsorbent, such as molecular sieves (Aden et al., Lignocellulosic Biomass to Ethanol Process Design and Economics Utilizing Co-Current Dilute Acid Prehydrolysis and Enzymatic

Hydrolysis for Corn Stover, Report NREL/TP-510-32438, National Renewable Energy

Laboratory, June 2002).

[0256] Additionally, distillation in combination with pervaporation may be used to isolate and purify the butanol from the fermentation medium. In this method, the fermentation broth containing the butanol is distilled to near the azeotropic composition, and then the remaining water is removed by pervaporation through a hydrophilic membrane (Guo, et al., J. Membr. Sci. 245: 199-210 (2004)).

[0257] In situ product removal (ISPR) (also referred to as extractive fermentation) can be used to remove butanol (or other fermentative alcohol) from the fermentation vessel as it is produced, thereby allowing the microorganism to produce butanol at high yields. One method for ISPR for removing fermentative alcohol that has been described in the art is liquid-liquid extraction. In general, with regard to butanol fermentation, for example, the fermentation medium, which includes the microorganism, is contacted with an organic extractant at a time before the butanol concentration reaches a toxic level. The organic extractant and the

fermentation medium form a biphasic mixture. The butanol partitions into the organic extractant phase, decreasing the concentration in the aqueous phase containing the microorganism, thereby limiting the exposure of the microorganism to the inhibitory butanol.

[0258] Liquid-liquid extraction can be performed, for example, according to the processes described in U.S. Patent Appl. Pub. No. 2009/0305370, the disclosure of which is hereby incorporated in its entirety. U.S. Patent Appl. Pub. No. 2009/0305370 describes methods for producing and recovering butanol from a fermentation broth using liquid-liquid extraction, the methods comprising the step of contacting the fermentation broth with a water immiscible extractant to form a two-phase mixture comprising an aqueous phase and an organic phase. Typically, the extractant can be an organic extractant selected from the group consisting of saturated, mono-unsaturated, poly-unsaturated (and mixtures thereof) C ₁₂ to C ₂₂ fatty alcohols, C ₁₂ to C ₂₂ fatty acids, esters of C ₁₂ to C ₂₂ fatty acids, C ₁₂ to C ₂₂ fatty aldehydes, and mixtures thereof. The extractant(s) for ISPR can be non-alcohol extractants. The ISPR extractant can be an exogenous organic extractant such as oleyl alcohol, behenyl alcohol, cetyl alcohol, lauryl alcohol, myristyl alcohol, stearyl alcohol, 1-undecanol, oleic acid, lauric acid, myristic acid, stearic acid, methyl myristate, methyl oleate, undecanal, lauric aldehyde, 20-methylundecanal, and mixtures thereof.

[0259] In some embodiments, the alcohol can be formed by contacting the alcohol in a fermentation medium with an organic acid (e.g., fatty acids) and a catalyst capable of esterfiying the alcohol with the organic acid. In such embodiments, the organic acid can serve as an ISPR extractant into which the alcohol esters partition. The organic acid can be supplied to the fermentation vessel and/or derived from the biomass supplying fermentable carbon fed to the fermentation vessel. Lipids present in the feedstock can be catalytically hydrolyzed to organic acid, and the same catalyst (e.g., enzymes) can esterify the organic acid with the alcohol. The catalyst can be supplied to the feedstock prior to fermentation, or can be supplied to the fermentation vessel before or contemporaneously with the supplying of the feedstock. When the catalyst is supplied to the fermentation vessel, alcohol esters can be obtained by hydrolysis of the lipids into organic acid and substantially simultaneous esterification of the organic acid with butanol present in the fermentation vessel. Organic acid and/or native oil not derived from the feedstock can also be fed to the fermentation vessel, with the native oil being hydrolyzed into organic acid. Any organic acid not esterified with the alcohol can serve as part of the ISPR extractant. The extractant containing alcohol esters can be separated from the fermentation medium, and the alcohol can be recovered from the extractant. The extractant can be recycled to the fermentation vessel. Thus, in the case of butanol production, for example, the conversion of the butanol to an ester reduces the free butanol concentration in the fermentation medium, shielding the microorganism from the toxic effect of increasing butanol concentration. In addition, unfractionated grain can be used as feedstock without separation of lipids therein, since the lipids can be catalytically hydrolyzed to organic acid, thereby decreasing the rate of build-up of lipids in the ISPR extractant.

[0260] In situ product removal can be carried out in a batch mode or a continuous mode.

In a continuous mode of in situ product removal, product is continually removed from the reactor. In a batchwise mode of in situ product removal, a volume of organic extractant is added to the fermentation vessel and the extractant is not removed during the process. For in situ product removal, the organic extractant can contact the fermentation medium at the start of the fermentation forming a biphasic fermentation medium. Alternatively, the organic extractant can contact the fermentation medium after the microorganism has achieved a desired amount of growth, which can be determined by measuring the optical density of the culture. Further, the organic extractant can contact the fermentation medium at a time at which the product alcohol level in the fermentation medium reaches a preselected level. In the case of butanol production according to some embodiments of the present invention, the organic acid extractant can contact the fermentation medium at a time before the butanol concentration reaches a toxic level, so as to esterify the butanol with the organic acid to produce butanol esters and consequently reduce the concentration of butanol in the fermentation vessel. The ester-containing organic phase can then be removed from the fermentation vessel (and separated from the fermentation broth which constitutes the aqueous phase) after a desired effective titer of the butanol esters is achieved. In some embodiments, the ester-containing organic phase is separated from the aqueous phase after fermentation of the available fermentable sugar in the fermentation vessel is substantially complete.

[0261] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application including the definitions will control. Also, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. All publications, patents and other references mentioned herein are incorporated by reference in their entireties for all purposes.

Examples

[0262] The present invention is further defined in the following Examples. It should be understood that these Examples, while indicating embodiments of the invention, are given by way of illustration only. From the above discussion and these Examples, one skilled in the art can ascertain the essential characteristics of this invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various uses and conditions.

General Methods

[0263] Standard recombinant DNA, molecular cloning techniques and transformation protocols used in the Examples are well known in the art and are described by Sambrook et al. (Sambrook, J., Fritsch, E. F. and Maniatis, T. (Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, 1989, here in referred to as Maniatis), by Ausubel et al. (Ausubel et al, Current Protocols in Molecular Biology, pub. by Greene

Publishing Assoc. and Wiley-Interscience, 1987) and by Amberg et al (Amberg, D. C, Burke, D. J. and Strathern, J. N. (Methods in Yeast Genetics: A Cold Spring Harbor Laboratory Course Manual, Cold Spring Harbor Press, 2005). Materials and methods suitable for the maintenance and growth of bacterial cultures are well known in the art. Techniques suitable for use in the following examples may be found as set out in Manual of Methods for General Bacteriology (Phillipp et al, eds., American Society for Microbiology, Washington, DC, 1994) or by Thomas D. Brock in (Brock, Biotechnology: A Textbook of Industrial Microbiology, Second Edition, Sinauer Associates, Inc., Sunderland, MA (1989). All reagents, restriction enzymes and materials used for the growth and maintenance of bacterial cells were obtained from Sigma- Aldrich Chemicals (St. Louis, MO), BD Diagnostic Systems (Sparks, MD), Invitrogen (Carlsbad, CA), HiMedia (Mumbai, India), SD Fine chemicals (India), or Takara Bio Inc. (Shiga,, Japan), unless otherwise specified.

[0264] The meaning of abbreviations is as follows: "sec" means second(s), "min" means minute(s), "h" means hour(s), "nm" means nanometers, "uL" means microliter(s), "mL" means milliliter(s), "mg/mL" means milligram per milliliter, "L" means liter(s), "nm" means

nanometers, "mM" means millimolar, "M" means molar, "mmol" means millimole(s), "μιηοΐε" means micromole(s), "kg" means kilogram, "g" means gram(s), '^g" means microgram(s) and "ng" means nanogram(s), "PCR" means polymerase chain reaction, "OD" means optical density, "OD600" means the optical density measured at a wavelength of 600 nm, "kDa" means kilodaltons, "g" can also mean the gravitation constant, "bp" means base pair(s), "kbp" means kilobase pair(s), "kb" means kilobase, "%" means percent, "% w/v" means weight/volume percent, "% v/v" means volume/volume percent, "HPLC" means high performance liquid chromatography, "g/L" means gram per liter, '^g/L" means microgram per liter, "ng^L" means nanogram per microliter, "pmol^L" means picomol per microliter, "RPM" means rotation per minute, '^mol/min/mg" means micromole per minute per milligram, "w/v" means weight per volume, "v/v" means volume per volume.

Example 1 : Overview of Promoter Prospecting

[0265] A "promoter prospecting" experiment was carried out as set forth below to examine the pattern of gene expression in an isobutanologen resulting from the transition from propagation to isobutanol production. RNA was extracted at the end of the propagation culture, and at 3 points during the production culture. Microarray analysis identified a number of genes that were up-regulated (up to 200-fold) and highly expressed in one or more timepoints during production, but not in the propagation sample. Twelve of these were selected for further study; their promoters were fused to the green fluorescent protein (GFP) as a reporter for expression, and their transcriptional activity was monitored during fermentation (including scaled-down models of simultaneous saccharification and fermentation). The twelve genes are tabulated in Table 1. They include IMAl, encoding isomaltase (involved in fermentation of residual sugars produced by a-amylase-catalyzed starch hydrolysis), genes induced by cell wall damage, genes involved in thermotolerance and halotolerance, in pseudohyphal growth (known to be induced by isobutanol), and genes encoding proteasomal subunits (the proteasome degrades misfolded proteins, which increase in abundance under certain kinds of stress).

[0266] The promoters that were identified through promoter prospecting would not necessarily have been selected based on a rational, a priori, approach. It may be necessary to periodically repeat promoter prospecting experiments, as isobutanologen strains and processes evolve. Also, promoters of genes that displayed dynamic expression profiles during this promoter prospecting experiment may also be screened for utility in a second round of testing.

[0267] The induction of the IMAl gene, observed in the promoter prospecting experiment, may be in response to maltrins present in the corn mash. Yeast has an active transcriptional response to corn oil fatty acid fractions, particularly oleic acid, resulting in the activation of genes involved in peroxisomal biogenesis and function. This response is overridden by glucose repression.

[0268] In studies of transcriptional responses to isobutanol challenge, a number of genes were observed to be induced, including GRE2 (encoding 3-methylbutanal reductase), PDR5 (encoding a drug-efflux pump), and heat shock genes. Upon fusing the GRE2 promoter to a gene encoding GFP, it was determined that GFP expression is indeed activated by isobutanol challenge.

Promoter Prospecting Experiment

[0269] The purpose of this experiment was to simulate an isobutanol fermentation of corn mash so that responsive promoters could be identified for subsequent exploitation. An aerobic propagation tank with excess glucose was followed by an anoxic production tank of limiting glucose fed by simultaneous saccharification and fermentation of a corn mash. During the production phase changes in gene expression are modest in number. A set of candidate promoters were identified and a means to test these candidates was developed. The "transcript off changes" were also tabulated with the most dramatic differences being off by at least 10 fold. The shut off of Fe and Zn genes may suggest that the medium has excess divalent metal ions while the induction of MALI genes indicates both glucose limitation and maltose availability.

Fermentation

[0270] Biological triplicate cultures were performed at all steps. A CEN.PK gpd2- pdc- yeast strain (PNY1504, described in US Appn. Pub. No. 20120237988, incorporated herein by reference) was grown in 3 g/L glucose + 3 g/L ethanol salt medium for ~24 hrs. 13-15 mL of each 250 mL culture was transferred to -270 mL medium in a 2 L, baffled, vented flask at an OD 2.0-2.5. Four 2L flasks were started for each propagation tank. 24 hrs after inoculating the 2 L flasks. Subsequently, 30 mL of YEP stock solution (200 g/L peptone, 100 g/L yeast extract) was added to each flask, then 300 mL of sterile, virgin, 90-95% Cognis oleyl alcohol was added and the flasks were returned to the shaker for ~20 hrs. The aqueous phase and oleyl alcohol phase were allowed to settle for ~5 minutes. The aqueous phase was pooled together from all 12 flasks and ~1.2 L was distributed to three pressure cans that were used to inoculate each propagation tank. The glucose concentration in the media was allowed to drop from ~l-2 g/L at inoculation to < 0.5 g/L and a 50% w/w glucose feed was started with a rate of ~m=0.17 until OD > 25. Thereafter, the concentration of dissolved oxygen was maintained at 30%. Approximately 1.1 L of propagation tank culture was then transferred to its corresponding production tank.

Simultaneous Saccharification and Fermentation (SSF) were carried out using a glucoamylase (β- amylase), maintaining an excess of glucose and a low concentration of dissolved oxygen (3%) for the entire production stage. The triplicate cultures were designated as follows:

GLNOR714PROP -> GLNOR715FERM

GLNOR716PROP -> GLNOR717FERM

GLNOR718PROP -> GLNOR719FERM

Molecular Biology

[0271] RNA was isolated from propagation and production tanks by standard methods.

RNA species were quantified using Agilent arrays by standard methods. Data was averaged and statistical analysis was performed. The abundance of RNA transcripts were interpreted with regard to physiology. Hence, relevant physiological data is summarized for the propagation tank first. Physiology

[0272] In fermentation tanks, it was observed that at 26 and 37 hrs, isobutanol is being synthesized. However, by 50 hrs isobutanol accumulation ceases. See Figure 10. Note however, that cumulative rate data (volumetric isobutanol produced/EFT) is misleading in that the catalyst is not performing optimally until about 40 hrs of culture. See Figure 11. This was verified by studying the carbon dioxide evolution and oxygen consumption rates, which both decline precipitously after 30 hrs and are close to zero at 50 hrs. See Figures 12 and 13. Thus, the 26, 37, and 50 hr transcript measures were chosen are most relevant for the purposes of this promoter prospecting experiment.

[0273] All promoter elements that are significantly up or down regulated in the production tank are summarized in the following table:

Table 6

[0274] Thus, although relatively few transcripts appreciably changed between the propagation and production stages, there are numerous candidatepromoters.

[0275] In these experiments, the aim was to identify RNAs that are regulated by the anoxic conditions of the fermentation tank. The promoters of such RNAs could be useful switches (e.g., OFF -> ON) or amplifier modules (e.g., LOW -> HIGH) that elevate expression from a significant basal level. Both "switch" and "amplifier" promoter elements are desirable.

[0276] Two ORFs (YJL 171 C and YGR287C) were transcribed at 26 hrs and retained elevated expression levels while DIA1 was transcribed at 37 hrs and was still highly expressed at 50 hrs. There are also choices to throw a switch at 26 hrs (IMD2), 37 hrs (CHA1 and YJL195 and 50 hrs (PRM6).

[0277] Initially, the top 45 transcripts were categorized under each of the four conditions in the following table: Table 7

From the propagation tank, 37 of the top 45 transcripts fall into the listed categories. In the production tank, the fraction is 40/45, 38/45, and 35/45 at 26, 37 and 50 hr EFT, respectively. As expected, translation is critical during exponential growth (26 hrs/production) but not to the other three conditions (15 hr, 37 hr, and 50 hr) which are more akin to stationary phase.

Example 2 (Prophetic): Controlling Expression of alsS (Encoding Acetolactate Synthase) in Isobutanologen Yeast Using a Genetic Switch

[0278] The biocatalyst pathway polypeptide alsS is chosen to measure differential expression using a genetic switch. An alsS polynucleotide is designed that contains a nonsense mutation that results in no translation of the full-length, functional biocatalyst protein and is under the control of a constitutive promoter. A second polynucleotide is designed containing a promoter identified in this application as differentially activated in response to a specific environmental cue and a suppressor tRNA specific for the nonsense mutation of the biocatalyst polypeptide. In the absence of the cue, the suppressor tRNA is not expressed, and the biocatalyst polypeptide is not translated. In the presense of the cue, the suppressor tRNA is expressed, and the biocatalyst polypeptide is translated.

Example 3 (Prophetic): Controlling Expression of alsS (Encoding Acetolactate Synthase) in Isobutanologen Yeast Using a Genetic Switch

[0279] The biocatalyst pathway polypeptide alsS is chosen to measure differential expression using a genetic switch. An alsS polynucleotide is designed that is under the control of a constitutive promoter. A second polynucleotide is designed containing a promoter identified in this application as differentially activated in response to a specific environmental cue and an anti- sense RNA specific for the alsS mRNA. In the absence of the cue, the anti-sense RNA is not expressed, and the biocatalyst polypeptide is translated. In the presense of the cue, the anti-sense RNA is expressed, and the biocatalyst polypeptide is not translated.