METHODS AND COMPOSITIONS FOR GROWTH OF YEAST ON ALGINATE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 61/594,306, filed February 2, 2012, which is hereby incorporated by reference, in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0002] This invention was made with government support under contract DE-FOA- 0000065-25 A4374 awarded by the Department of Energy. The government has certain rights in the invention.

SUBMISSION OF SEQUENCE LISTING AS ASCII TEXT FILE

[0003] The content of the following submission on ASCII text file is incorporated herein by reference in its entirety: a computer readable form (CRF) of the Sequence Listing (file name: 690212001240SEQLIST.TXT, date recorded: January 8, 2013, size: 580 KB).

FIELD

[0004] The present disclosure relates to the degradation of alginate by yeast. In particular, the present disclosure relates to methods of growing yeast on oligoalginate and recombinant yeast cells capable of growing on oligoalginate by expressing one or more proteins in an alginate metabolic pathway.

BACKGROUND

[0005] The demand for energy sources other than petroleum-based fuels and chemicals is ever growing. Consumption of industrial petroleum-based fuels and chemicals is a major source of greenhouse-gas emissions into the atmosphere, a central factor contributing to global climate change. From an economic and environmental standpoint, use of traditional fuels to power the globe is not sustainable, given that these fuels are non-renewable sources of energy. This presents the threat of "peak oil," the point at which the consumption of crude oil exceeds the supply. Consequences for the continued use of traditional petroleum fuels include environmental damage and increased cost for crude oil, both of which are highly undesirable. Thus, there exists a need to produce alternative sources of energy that are cost-effective, environmentally-friendly, and curb carbon emissions. Early efforts at producing alternative sources of biofuels and renewable chemicals included the production of bioethanol, or ethanol produced from biomass. One method of producing bioethanol for use as a commodity fuel involved the breakdown of lignocellulosic materials from plant biomass. This was once viewed as an extremely promising method of producing alternative energy. Indeed, in the United States alone, the government has provided generous subsidies for research and development on the use of plants for the production of bioethanol, particularly in crop plants such as maize and switchgrass.

[0006] Despite generation of a clean fuel, the production of bioethanol from plant biomass has many negative attributes. One consequence includes a required shift in land-use from traditional food crop space to biomass for biofuels, which requires either a reduction in food production or an increase in land cultivated specifically for economic plant growth. One study has indicated that the use of plant biomass to produce biofuels, as a result of increased land-use, would actually double greenhouse gas emissions over 30 years (Searchinger et ah, Science, 319: 1238-1240, 2008) resulting from altering carbon source/sink relationships and deforestation. From an economic standpoint, some studies have suggested that production of plant-based biofuels will lead to a decrease in livestock production and increased food prices (Tokgoz et ah, Review of Agricultural Economics, 30:4, 1-19, 2008). Therefore, lignocellulosic biofuel production in plants, although a useful model, will likely not be able to significantly address the alternative energy needs of a growing global population.

[0007] The use of microorganisms in biofuel production is on the rise. Current uses of microorganisms to produce biofuels involve their use in converting sugars into commodity chemicals that can serve as biofuels (Sung et ah, Current Opinion in Biotechnology, 19: 556- 563, 2010). One disadvantage to this method, related again to plants, is that often plant starting material such as cellulose requires industrial enzymatic processing to liberate the sugars, which is a complex process to accomplish in an industrial setting. The only alternative to enzymatic liberation includes a combination of heat, pressure, and use of strong acids. Microorganism- based processing of sugars also traditionally involves the use of plant materials, which carries its own disadvantages discussed previously. Alternatives to plant starting material for use in microorganism biofuel production include algae, which are space-efficient and possess suitable sugars for biofuel production. One such sugar is alginate, an abundant carbohydrate in some brown algae (Larsen et al., Carbohydrate Research, 17:2: 287-296, 1971). Unfortunately, and similar to the scenario in plants, these sugars require further processing before they can be converted into biofuels.

[0008] Despite advantages present in the use of algae rather than non-aquatic plants for biofuel production, there are currently no known industrial microbes that can natively digest and metabolize alginate to produce biofuel and renewable chemicals from the carbohydrates present in brown seaweed. Accordingly, there is a need for methods and compositions for producing biofuels and renewable chemicals, including suitable starting materials.

BRIEF SUMMARY

[0009] In order to meet the above needs, the present disclosure relates to methods and compositions for producing biofuels and renewable chemicals, including suitable starting materials, by engineering microorganisms, such as yeat, that metabolize major carbohydrate components, such as oligoalginate, in brown seaweed. In particular, the present disclosure provides methods and compositions for growing yeast on a substrate containing oligoalginate {e.g., DEHU) as a primary carbon source.

[0010] Brown seaweed contains approximately 50% of carbohydrates including alginate, mannitol, and glucan {e.g., cellulose, and polymers of glucose). For the most economical way of producing renewable fuels and chemicals from brown seaweed, it is essential to have a microbial strain that can metabolize the majority of carbohydrates, alginate, mannitol, and glucan contained in the seaweed. Several groups have demonstrated the production of commodity chemicals, such as ethanol, from mannitol and glucan compositions in brown seaweed using various microbial species (S.J. Horn et al., J. Ind. Microbiol. Biotechnol., 2000, 25, 249-254; S.J. Horn et al., J. Ind. Microbiol. Biotechnol., 2000, 24, 51-57; Adams et al., J. Appl. Phycol, 2009, 21, 569-574; and US2011/0020881). However, production of commodity chemicals from alginate has been carried out only using bacterial species that are not commonly used in industry (Takeda et al., Energy & Environmental Science, 2011, 4, 2575-2581; and Moen et al., Journal of Applied Phycology, 1997, 9, 157-166). Therefore, in order to utilize brown seaweed as a source of biomass, it is important to engineer more industrially relevant microbial strains to metabolize alginate.

[0011] We have previously engineered a strain of E. coli to enable alginate metabolism and demonstrated successful production of ethanol from all three sugars in brown seaweed

(Wargacki et al., Science, 2012, 335, 6066, 308-313; List of BAL patent applications). However, E. coli is natively more susceptible to microbial contamination, phage infection, pH and temperature shifts, and accumulation of toxic metabolites. Thus, engineering industrially more robust microbial strains, such as yeast strains, that are not subject to the problems of E. coli is a more attractive alternative for producing commodity chemicals from seaweed at a commercial scale. Although we have found that many intracellular enzymes responsible for alginate metabolism in bacteria can also be functionally over-expressed in yeast, membrane transporters responsible for the uptake of oligoalginate may not be functional due to differences in membrane composition between bacteria and eukaryotes. Therefore, we screened for alginate transporters from the marine alginolytic fungi Asteromyces cruciatus and Dendryphiella salina that can be used to engineer yeast to grow on alginate, and thus convert brown seaweed into commodity chemicals.

[0012] Thus, the present disclosure is based, at least in part, on the surprising discovery that a yeast cell can be engineered to transport oligoalginate, such as DEHU, into the yeast cell by expressing at least one transporter protein from an a marine alginolytic fungi, such as

Asteromyces cruciatus or Dendryphiella salina. Advantageously, a yeast cell with the ability to transport oligoalginate into the yeast cell can be further engineered to metabolize oligoalginate to a commodity chemical, such as a biofuel. Additionally, yeast is a versatile organism that can utilize the biofuel feedstock produced by alginate metabolism to directly produce commodity chemicals such as ethanol.

[0013] Accordingly, one aspect of the present disclosure includes a method of growing yeast on a substrate containing oligoalginate as a primary carbon source, by providing a yeast cell, where the yeast cell contains at least one recombinant polynucleotide encoding an oligoalginate transporter protein or permease; and culturing the yeast cell with the substrate containing oligoalginate under conditions whereby the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in transport of oligoalginate into the yeast cell, and where the yeast cell contains the proteins necessary for the yeast cell to metabolize oligoalginate.

[0014] Yet another aspect of the present disclosure includes a method of producing a commodity chemical, by providing a yeast cell, where the yeast cell contains at least one recombinant polynucleotide encoding an oligoalginate transporter protein or permease; culturing the yeast cell with a fermentable substrate containing oligoalginate under conditions whereby the recombinant polynucleotide is expressed and a commodity chemical is produced; and collecting the commodity chemical, where expression of the recombinant polynucleotide results in transport of oligoalginate into the yeast cell, and where the yeast cell contains the proteins necessary for the yeast cell to metabolize oligoalginate.

[0015] Still another aspect of the present disclosure includes a recombinant yeast cell, containing at least one recombinant polynucleotide encoding an oligoalginate transporter protein or permease, where expression of the recombinant polynucleotide results in transport of oligoalginate into the recombinant yeast cell, and the recombinant yeast cell contains the proteins necessary for the recombinant yeast cell to metabolize oligoalginate.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 depicts a schematic of alginate metabolism to pyruvate.

[0017] Figure 2 depicts amino acid sequence alignments of regions of yeast hexose transporter protein sequences containing conserved motifs.

[0018] Figure 3 depicts amino acid sequence alignments of a region of permease protein sequences containing a conserved sequence motif.

[0019] Figure 4 depicts a schematic of plant, fungi, and bacterial mannitol metabolism to fructose-6-phospahte.

[0020] Figure 5 depicts a schematic of the enzymatic conversion of mannitol to mannose by the mannitol dehydrogenase (MTD) that is in plants. [0021] Figure 6 depicts a schematic of the reactions carried out by mannitol dehydrogenase (MDH) and hexokinase to convert mannitol into fructose for its entry into glycolysis.

[0022] Figure 7 graphically depicts growth and ethanol production of engineered yeasts on mannitol. Figure 7A depicts the growth of engineered yeast strains that express a mannitol transporter (matl from celery), and one or two mannitol dehydrogenases (mtd from celery and yel070w from S. cerevisiae) on 2% mannitol. Figure 7B depicts the growth of an engineered yeast strain that expresses a mannitol dehydrogenase (M3.2 and M3.3: YNR073C, e56: HXT13 (YEL069C), e59: HXT17 (YNR072W), and EV: empty vector) on 5% mannitol compared with their growth on no substrate and 5% glucose. Figure 7C depicts ethanol production using engineered yeast strains on 5% mannitol compared with their ethanol production on 5% glucose in an aerobic condition.

[0023] Figure 8 depicts a phylogram derived from ClustalW of a query list of transporter gene, including putative mannitol, polyol, hexose, and other sugar transporters and permeases.

[0024] Figure 9 depicts a schematic of three vectors used for Al-I alginate lyase expression in yeast. Each plasmid harbors an antibiotic marker (Zeocin or Blasticidin marker) under the control of TEF promoter such that the yeast cells harboring these plasmids can be selected by growth in the presence of inhibitory concentration of these antibiotics. Figure 9A depicts the PGKp-ENOlt promoter/terminator cassette used to express the Al-I gene. Figure 9B depicts the GPMp-TPIt promoter/terminator cassette used to express the Al-I gene. Figure 9C depicts the PDCp-EN02t promoter/terminator cassette used to express the Al-I gene.

[0025] Figure 10 graphically depicts alginate lyase activity detection in yeast cells transformed with three plasmids (YML26, LML48 and LML52). The left hand graphs show the activity in the cell lysates, and the right hand graph shows the activity in the culture supernatant (secreted enzyme). Figure 10A graphically depicts expression from the YML26 plasmid. Figure 10B graphically depicts expression from the LML48 plasmid. Figure IOC graphically depicts expression from the LML52 plasmid.

[0026] Figure 11 depicts an immunoblot analysis indicating the presence of bands corresponding to the expected sizes of Al-I lyase in yeast culture supernatants and cell lysates from yeast cells transformed with the LML48 or LML51 plasmid. [0027] Figure 12 depicts an HPLC profile of an alginate sample after incubation with the supernatant sample of a yeast culture expressing the Al-I lyase enzyme. Figure 12A depicts the UV absorbance profile. Figure 12B depicts the mass spectrometry profile (selected ion monitoring using negative mode).

[0028] Figure 13 depicts Al-I expressing colonies and vector control colonies grown on two selective medium (SD minus Uracil) or YPD plus Blasticidin antibiotic medium. After about 4 days of growth, both the plates were flooded with 10% cetyl-pyridinium chloride and incubated for an hour. The dye was decanted and the plates were used to take pictures. For the YPD medium plates, the clearing zones are only visible for the colonies expressing Al-I lyase.

[0029] Figure 14 depicts increased Al-I lyase activity in yeast cells co-transformed with two Al-I expression plasmids. The alginate lyase assay was performed on several co- transformed colonies.

[0030] Figure 15 depicts amino acid sequence alignment of two alginate lyases. Figure 15A depicts the A. Vibrio QY101 Aly VI lyase protein sequence (SEQ ID NO: 55). Figure 15B depicts the Sphingomonas sp. Al Al-I lyase protein sequence (SEQ ID NO: 56).

[0031] Figure 16 depicts a vector system that is used to over-express alginate transporters.

[0032] Figure 17 graphically depicts growth of yeast on DEHU. The yeast strains,

BAL2193 background, express a DEHU reductase from Sphingomonas sp. Al, KdgK from Saccharophagus degradans 2-40, kdgpA from Escherichia coli, and a transporter isolated from one of Aspergillus niger (e20), Dendryphiella salina (el 15), and Asteromyces cruciatus (el74). EV denotes an empty vector.

[0033] Figure 18 depicts growth of yeast and DEHU consumed on varied concentration of DEHU (0-lOOmM). The yeast strains, BAL2269 background, express a DEHU reductase from Sphingomonas sp. Al, KdgK from Saccharophagus degradans 2-40, kdgpA from Escherichia coli, and transporter isolated from one Dendryphiella salina (el 15).

[0034] Figure 19 depicts the reaction scheme for the assay of OAL activity.

[0035] Figure 20 depicts the reaction scheme for the assay of DEHU reductase. [0036] Figure 21 depicts the reaction scheme for the assay of KDG kinase.

[0037] Figure 22 depicts the reaction scheme for the assay of KDGP aldolase.

[0038] Figure 23 graphically depicts the alginate metabolism pathway dependency of the growth of engineered yeast strain on DEHU. Variants of BAL2193 were individually created lacking each of the pathway genes and each transporter was introduced. The growth is depicted.

[0039] Figure 24 depicts the alignment of the identified transporters.

[0040] Figure 25 depicts the phylogram of e20, el 15, el74, and their homologues.

[0041] Figure 26A depicts consumption of mannitol and DEHU, growth curve, and ethanol production for the recombinant yeast strain BAL2575. Figure 26B depicts consumption of mannitol and DEHU, growth curve, and ethanol production for the recombinant yeast strain BAL2576. Optical density (A600) is read from the scale on the left and is represented by filled circles. DEHU concentration in mM is read from the scale on the left and is represented by open squares. Mannitol concentration in mM per liter is read from the scale on the right and is represented by open circles. Ethanol concentration in mM per liters read from the scale on the right and is represented by filled squares.

[0042] Figure 27 depicts growth of the recombinant yeast stain BAL2293 with different DEHU transporters on DEHU as a carbon source.

DETAILED DESCRIPTION

Overview

[0043] The present disclosure is based on the surprising discovery that yeast, which is otherwise incapable of growing on alginate, can be engineered to grow on alginate as a primary source of carbon. However, by expressing at least one oligoalginate transporter protein or permease in a yeast cell that contains the proteins necessary for metabolizing oligoalginate, it was unexpectedly found that the yeast cell was able to utilize alginate as a primary carbon source. [0044] Alginate is a linear copolymer with homopolymeric blocks of (l-4)-linked β-D- mannuronate (M) and its C-5 epimer a-L-guluronate (G) residues, respectively, covalently linked together in different sequences or blocks. Alginate monomers may appear in

homopolymeric blocks of consecutive G-residues (G-blocks), consecutive M-residues (M- blocks), alternating M and G-residues (MG-blocks), or randomly organized blocks. Suitable sources of alginate may include, without limitation, kelp, giant kelp, sargasso, seaweed, algae, brown algae, brown seaweed, marine microflora, microalgae, and sea grass.

[0045] As used herein, the term "oligoalginate" refers to any non-full length alginate polymer. Non-full length alginate polymers include, without limitation, short alginate polymers, alginate 20-mers, alginate 15-mers, alginate 10-mers, alginate 9-mers, alginate 8-mers, alginate 7-mers, alginate hexamers, alginate pentamers, alginate tetramers, alginate trimers, alginate dimers, and alginate monomers.

[0046] As used herein, the term "oligoalginate transporter protein(s)" refers to an

endogenous or heterologous protein that when expressed in a yeast cell transports oligoalginate into the yeast cell. As used herein, an "oligoalginate transporter protein" encompasses a wild- type protein and a mutant protein that has been modified to alter the expression level of the protein or to alter the substrate specificity of the protein in order to allow transport of oligoalginate into a yeast cell when expressed in yeast.

[0047] Yeast does not readily utilize oligoalginate, such as DEHU, as a carbon source, as its primary means of metabolism is glycolysis. The sugars derived from oligoalginate polymers (i.e., mannuronate and guluronate) cannot be utilized in glycolysis. Oligoalginate-derived sugars can be metabolized via the Entner-Duodoroff (ED) pathway. The entry point for the ED pathway is 6-phosphogluconate. Oligoalginate polymers are broken down by endo- and exo- type alginate lyase enzymes (i.e., alginate lyases) to short alginate polymers (e.g., alginate 10- mers, alginate hexamers, alginate pentamers, alginate tetramers, etc.) and to alginate monomers by oligoalginate lyases and then transported. Subsequent to digestion by alginate lyases, it is believed that short polymeric and monomeric alginate is transported into the cell. Alginate monomers are generated via a beta-elimination type reaction which creates 4-deoxy-L-5-erythro- hexoseulose uronic acid (DEHU), which can then be reduced to 2-keto-3-deoxy-D-gluconate (Kdg) by a DEHU reductase and enter the ED pathway, eventually leading to production of pyruvate (Fig. 1). As used herein, DEHU is a type of alginate monomer (i.e., an oligoalginate).

[0048] However, there is no demonstrated example of a eukaryotic organism that uses the ED pathway for metabolism. Without wishing to be bound by theory, it is believed that yeast can be engineered to metabolize oligoalginate-derived sugars via the Entner-Duodoroff (ED) pathway. Alternatively, yeast can be engineered to utilize an endogenous metabolic pathway to metabolize oligoalginate-derived sugars.

[0049] One advantage of engineering yeast rather than bacteria to grow on substrates containing oligoalginate is that yeast is a more versatile organism. For example, yeast is more robust (e.g. , has higher tolerance to harsh conditions), grows at a lower pH that is not favorable to bacterial growth, is not susceptible to phages, requires less nutrients for growth, and has been well studied because of its more frequent use in industry. Additionally, yeast can utilize the products of oligoalginate metabolism to produce commodity chemicals such as ethanol.

Consequently, engineering yeast to grow on substrates containing oligoalginate allows for an all- inclusive system that is capable of both saccharification and fermentation of oligoalginate to commodity chemicals such as biofuels. Additionally, using substrates containing oligoalginate provides advantages over other biomass sources in the production of commodity chemicals, such as biofuels. For example, large-scale aquatic-farming can generate a significant amount of oligoalginate-containing biomass. Moreover, oligoalginate-containing biomass typically lacks lignin, making it is easy to degrade. For example, aquatic biomass such as brown seaweed, may be easily converted to monosaccharides using recombinantly expressed enzymes, as aquatic biomass such as brown seaweed has significantly simpler major sugar components (e.g., oligoalginate: 25%, mannitol: 20%, and glucan: 5%).

[0050] Accordingly, certain aspects of the present disclosure relate to methods of growing yeast on a substrate containing oligoalginate, such as DEHU, as a primary carbon source, by providing cells containing at least one recombinant polynucleotide encoding an oligoalginate transporter protein or permease, and culturing the yeast cell with the substrate containing oligoalginate under conditions whereby the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in transport of oligoalginate into the yeast cell, and where the yeast cell contains the proteins necessary for the yeast cell to metabolize oligoalginate.

[0051] Still other aspects of the present disclosure relate to methods of producing a commodity chemical, by providing a yeast cell, where the yeast cell contains at least one recombinant polynucleotide encoding an oligoalginate transporter protein or permease, culturing the yeast cell with a fermentable substrate containing oligoalginate under conditions whereby the recombinant polynucleotide is expressed and a commodity chemical is produced, and collecting the commodity chemical, where expression of the recombinant polynucleotide results in transport of oligoalginate into the yeast cell, and wherein the yeast cell includes the proteins necessary for the yeast cell to metabolize oligoalginate.

[0052] Further aspects of the present disclosure relate to recombinant yeast cells containing at least one recombinant polynucleotide encoding an oligoalginate transporter protein or permease, where expression of the recombinant polynucleotide results in transport of oligoalginate into the recombinant yeast cell, and the yeast cell contains the proteins necessary for the yeast cell to metabolize oligoalginate.

Methods of Growing Yeast on Oligoalginate

[0053] Certain aspects of the present disclosure relate to methods of growing yeast on a substrate containing oligoalginate as a primary carbon source. Other aspects of the present disclosure relate to methods of growing yeast on a substrate containing oligoalginate and mannitol.

[0054] Advantageously, yeast cells that grow on oligoalginate, mannitol, and/or glucan can more completely metabolize the sugar content of aquatic biomass, such as seaweed and brown seaweed. Additionally, mannitol and oligoalginate metabolism generate opposite redox reaction imbalances, and so metabolizing mannitol and oligoalginate in a 2: 1 ratio may be helpful in balancing their respective redox reactions.

Oligoalginate Metabolism Components

[0055] Certain aspects of the present disclosure relate to methods of growing yeast on a substrate containing oligoalginate as the primary carbon source by expressing at least one recombinant polynucleotide encoding an oligoalginate transporter protein or permease and containing the proteins necessary for the yeast cell to metabolize oligoalginate.

[0056] In some embodiments, the proteins necessary for the yeast cell to metabolize oligoalginate contain at least one alginate lyase, at least one 4-deoxy-L-erythro-5-hexoseulose uronate (DEHU) reductase (DEHUH or DEHUR), at least one 2-keto-3-deoxy-D-gluconate (KDG) kinase (KdgK), or at least one 2-keto-3-deoxy D-gluconate-6-phosphate (KDGP) aldolase (KdgpA). In other embodiments, the yeast cell may further contain at least two of these proteins. In yet other embodiments, the yeast cell may further contain at least three of these proteins. In some preferred embodiments, the yeast cell further contains all four proteins (i.e. , alginate lyase, DEHU reductase, KDG kinase, and KDGP aldolase).

Suitable Oligoalginate Transporter Proteins

[0057] As disclosed herein, oligoalginate transporter proteins are plasma membrane sugar transporter proteins that allow the uptake of oligoalginate. However, yeast cells do not naturally express oligoalginate transporter proteins. Without wishing to be bound by theory, it is believed that a major limitation in engineering yeast that can grow on a substrate containing oligoalginate is the transport of oligoalginate into yeast. It is believed that transport of oligoalginate into the yeast cell is a limitation, because the transporter protein's functionality is highly dependent on extracellular conditions (e.g. , pH, salt, viscosity, sugar concentration, etc.) that may not be present in the extracellular environment of yeast, the yeast may not be able to target an exogenous or modified oligoalginate transporter protein to the cell membrane, thus preventing the protein from functioning. Additionally, wild-type (i.e., unmodified) exogenous oligoalginate transporter proteins may not be functional when expressed in yeast. For example, gram negative bacteria have two membranes as part of the cell envelope, and their oligoalginate transport systems typically use a multiprotein complex spanning both cellular membranes in order to uptake or excrete oligoalginate (Jain and Ohman 2005; and Moma et ah, 2000). However, yeast does not have two membranes.

[0058] Suitable oligoalginate transporter proteins may include, without limitation, proteins encoded by oligoalginate transporter genes derived from organisms known to grow on oligoalginate. Additionally, hexose/hexuronate transporter proteins (derived either from yeast or from other prokaryotic or eukaryotic organisms), ABC transporter proteins, and permeases may also be isolated or modified to facilitate the uptake of oligoalginate. Transporter proteins including, but not limited to, alginate transporters, oligoalginate transporters, alginate monomer transporters, hexose/hexuronate transporter proteins, ABC transporter proteins, and permeases may be modified to alter the expression level of the protein, or alter the substrate specificity of the protein in order to allow yeast to uptake oligoalginate. Methods for modifying these proteins are well known in the art. Moreover, optimal transport of oligoalginate into yeast may require the expression of multiple oligoalginate transporter proteins or the expression of a combination of oligoalginate transporter proteins and modified hexose/hexuronate transporter proteins.

[0059] Accordingly in certain embodiments, the oligoalginate transporter protein is selected from a GPH transporter, a pectin/cellodextrin transporter, a hexose transporter, an ABC transporter and a transporter derived from marine fungi.

GPH Transporters

[0060] One suitable oligoalginate transporter protein is the Vibrio splendidus Syml symporter protein. Additionally, its homologs (Sym2 and Sym3) may also be suitable for transporting oligoalginate into yeast. Many of the characterized oligoalginate transport systems include examples from gram negative bacteria. In these examples, multiple proteins make a pore in the outer membrane that allows import of oligomers, which are further transported across the inner membrane of the gram negative cell envelope via a function of another multimeric protein complex. Therefore, additional suitable oligoalginate transporter proteins may include multiple transporter proteins that achieve such a dual-membrane transport system in a yeast cell. However, single protein transport systems may also be used. Examples of such single protein systems may be found in pectin degrading prokaryotic as well as in eukaryotic systems. Pectin degrading prokaryotic systems have been described where transport of monomers {e.g., galacturonic acid) and dimers has been reported to occur via single protein transport systems. The ExuT-like proteins allow the transport of the monomeric substrate in these systems. The transport of the oligosaccharides occurs via transporters that belong to the Glycoside-Pentoside- Hexuronide (GPH) superfamily of proteins (Hugouvieux-Cotte-Pattat and Reverchon, 2001). Additionally, certain organisms also have KdgT-like proteins which allow the transport of KDG. Accordingly, suitable oligoalginate transporter proteins include, without limitation, the GPH transporter proteins listed in Table 1 below.

TABLE 1

[0061] In certain embodiments, the oligoalginate transporter protein is a GPH transporter selected from KdgT, ExuT, Syml, Sym2, Sym3, TogT, CAV18395.1, AAB84443.1, algS, algMl, algM2, KdgM, KdgN, GntT, GntU, GntP, and KgtP.

Pectin/Cellodextrin Transporters

[0062] Suitable oligoalginate transporter proteins also include those from other fungi that are known to utilize oligoalginate. Eukaryotic genes usually express well in yeast and may contain functional plasma membrane targeting sequences (PMTSs) for plasma membrane localization. Recent reports have identified and characterized pectin degradation pathways in fungi, such as Trichoderma, and Aspergillus species have revealed transport system candidates as well.

Another recent report has described the expression of a cellodextrin transporter from

Neurospora into yeast and the transporter system may be promiscuous in allowing the transport of oligoalginate substrates (Galazka et ah, 2010). Additionally, further suitable candidates may be identified using bioinforaiatic strategies known in the art, including without limitation, using BLAST to analyze available eukaryotic genomes. Without wishing to be bound by theory, it is believed that the galacturonate (monomers/oligomers) transport systems from these and similar species may be functional in the transport of oligoalginate substrates.

[0063] As disclosed herein, suitable oligoalginate transporter proteins also include, without limitation, the pectin/cellodextrin transporters listed in Table 2 below.

TABLE 2

[0064] In certain embodiments, the oligoalginate transporter protein is a pectin/cellodextrin transporter selected from XP_001391170.1, XP_001390064.1, XP_001401052.1,

XP_001401444.1, XP_963873.1, XP_963801.1, YP_001307595.1, YP_003843758.1,

BAE55061.1, XP_003017198.1, CAB16264.1, and XP_003021005.1. Hexose Transporters

[0065] Suitable oligoalginate transporter proteins may also include yeast hexose

transporters. Yeast contains about 6,000 putative genes, and transporter proteins may account for up to 5% of the proteome. Indeed, at least 20 genes have been identified that relate to hexose uptake. These genes encode for 18 transporters (HXT1 to HXT17 and GAL2) and 2 sensors (SNF3, RGT2). These Hxt transporters belong to the major facilitator superfamily (MFS) of transporters and function by passive, energy-independent facilitate diffusion in which hexose molecules (such as glucose) move down a concentration gradient. Without wishing to be bound by theory, it is believed that these hexose transporters may also be capable of uptake of

DEHU/monouronic acid molecules, since they are also 6-carbon sugars. A recent study (Youk, Nature 2009) describes the intricate relationship between glucose sensing, uptake, and growth and shows that proper sensing of sugar source might be essential for growth. Accordingly and without wishing to be bound by theory, it is believed that the glucose sensors SNF3 and RGT2 may be used as novel targets for regulation and mutagenesis in order to achieve oligoalginate uptake and metabolism. For example, SNF3 and RGT2 may be constitutively expressed such that the required HXT transported genes are actively transcribed. In an alternative example, the hexose transporter genes (HXT1 to HXT7, or a combination of high- and low-affinity transporters) can be constitutively expressed. Accordingly, suitable oligoalginate transporter proteins may also include, without limitation, the yeast hexose transporters listed in Table 3 below.

TABLE 3

(ίοηο Name Source Know n sugars

HXT13 Saccharomyces cerevis iae Hexose

HXT14 Saccharomyces cerevis iae Hexose

HXT15 Saccharomyces cerevis iae Hexose

HXT16 Saccharomyces cerevis iae Hexose

HXT17 Saccharomyces cerevis iae Hexose

GAL2 Saccharomyces cerevis iae Galactose

[0066] In certain embodiments, the oligoalginate transporter protein is a hexose transporter selected from HXT1, HXT2, HXT3, HXT4, HXT5, HXT6, HXT7, HXT8, HXT9, HXT10, HXT11, HXT12, HXT13, HXT14, HXT15, HXT16, HXT17, and GAL2.

[0067] Without wishing to be bound by theory, it is also believed that the redundant nature of hexose transporters in yeast gives rise to an evolutionary advantage in the sugar uptake system. Accordingly, suitable transporters for the disclosed methods and compositions for transporting oligoalginate into yeast also include oligoalginate transporter systems that have been evolved from hexose transporter proteins using methods and strategies known in the art including but not limited to, utilization of a cytostat. Additionally, any of the disclosed transporter proteins may be further optimized for expression in yeast. Methods of optimizing proteins are well known in the art and include, without limitation, the use of a chemostat/cytostat system.

ABC Transporters

[0068] Further suitable oligoalginate transporter proteins include, without limitation, "superchannel" proteins, by which oligoalginate may be directly incorporated into the cytosol and degraded inside a yeast cell. For example, a group of bacteria characterized as

Sphingomonads have a wide range in capability of degrading environmentally hazardous compounds such as polychlorinated polycyclic aromatics (dioxin). These bacteria contain characteristic large pleat- like molecules on their cell surfaces. In this regard, certain

Sphingomonas have structures characterized as "superchannel" that enable the bacteria to directly take up macromolecules.

[0069] In one non-limiting example of a microorganism containing a superchannel,

Sphingomonas sp. strain Al directly incorporates polysaccharides such as oligoalginate through a superchannel. Such superchannels may include a pit on the outer membrane (e. g., AlgR), oligoalginate-binding proteins in the periplasm (e.g., AlgQl and AlgQ2), and an ATP-binding cassette (ABC) transporter (e.g., AlgMl, AlgM2, and AlgS). Incorporated polysaccharides such as oligoalginate may subsequently be depolymerized by alginate lyases produced in the cytosol. Thus, certain embodiments may incorporate genes encoding a superchannel (e.g., algS, algMl, algM2, algQl, algQ2) to introduce this ability to a yeast cell. Accordingly, suitable

oligoalginate transporter proteins include, without limitation, the ABC transporters listed in Table 4 below.

TABLE 4

[0070] In certain embodiments, the oligoalginate transporter protein is an ABC transporter selected from Atu3021, Atu3022, Atu3023, Atu3024, algMl, algM2, AlgQl, AlgQ2, AlgS, OG2516-05558, OG2516-05563, OG2516-05568, OG2516-05573, TogM, TogN, TogA, and TogB.

Additional Oligoalginate Transporters from Marine Fungi

[0071] Several organisms, such as the marine fungi Asteromyces cruciatus and

Dendryphiella salina, can grow as prototroph in media with oligoalginate polymer as its sole carbon source. Without wishing to be bound by theory, it is believed that A. cruciatus, D. salina and other similar organisms contain the requisite genes for the breakdown and metabolism of oligoalginate. Accordingly, additional suitable oligoalginate transporter proteins that may be used with the disclosed methods and compositions for transporting oligoalginate into yeast may be identified and isolated from oligoalginate consuming organisms, such as A. cruciatus and D. salina. Methods for identifying and isolating proteins are well known in the art. In one non- limiting example, an oligoalginate transporter may be isolated from a complementation or gain- of-function type screen. In another non-limiting example, computationally mining of genomic DNA, such as that from A. cruciatus and D. salina, may be used to identify further oligoalginate transporter proteins. The sequencing may be done using, for example, the Illumina GS system. Assembly of the genome data may be done using any sequence analysis software known in the art. Additionally, oligoalginate transporter proteins may be identified from oligoalginate consuming organisms, such as A. cruciatus and D. salina, by mass spectrometry. In yet another non-limiting example, high throughput mRNA sequencing can be performed to identify candidates specifically expressed in oligoalginate consuming organisms upon growth on media utilizing oligoalginate as the sole carbon source to identify pathways and transporter genes for oligoalginate metabolism.

[0072] Further examples of proteins for transporting oligoalginate into a yeast cell include, without limitation, those encoded by the nucleotide sequences set forth in SEQ ID NOs: 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, and 198. In certain embodiments, a protein of the present disclosure for transporting oligoalginate into a yeast cell is encoded by a nucleotide sequence that is at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 65, 67, 69, 71, 73, 75, 77, 79, 81, 83, 85, 87, 89, 91, 93, 95, 97, 99, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157, 159, 160, 162, 164, 166, 168, 170, 172, 174, 176, 178, 180, 182, 184, 186, 188, 190, 192, 194, 196, and 198. In certain preferred embodiments, the protein for transporting oligoalginate into a yeast cell is encoded by a nucleotide sequence that is at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% identical to any one of the nucleotide sequences set forth in SEQ ID NOs: 133, 196, and 198.

[0073] In other embodiments, a protein of the present disclosure for transporting

oligoalginate into a yeast cell has an amino acid sequence with at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% amino acid identity to any one of SEQ ID NOs: 66, 68, 70, 72, 74, 76, 78, 80, 82, 84, 86, 88, 90, 92, 94, 96, 98, 100, 102, 104, 106, 108, 110, 112, 114, 116, 118, 120, 122, 124, 126, 128, 130, 132, 134, 136, 138, 140, 142, 144, 146, 148, 150, 152, 154, 156, 158, 161, 163, 165, 167, 169, 171, 173, 175, 177, 179, 181, 183, 185, 187, 189, 191, 193, 195, 197, and 199. In certain preferred embodiments, the protein for transporting oligoalginate into a yeast cell has an amino acid sequence with at least about 20%, at least about 25%, at least about 30%, at least about 40%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% amino acid identity to any one of SEQ ID NOs: 134, 197, and 199. Suitable Permeases

[0074] Further suitable proteins for transporting oligoalginate into a yeast cell include permeases. A genomic analysis of open reading frames from S. cerevisiae has revealed nearly 200 potential permeases with several transmembrane domains (Nielssen et al. GEMS Microbiol. Rev. 97). Accordingly, eukaryotic transporters that are known to be energy- independent, such as permeases, may also be used in the disclosed methods and compositions for transporting oligoalginate into yeast. Suitable permeases may include, without limitation, those listed in Table 5 below.

TABLE 5

[0075] In certain embodiments, the permease is selected from YBR241c, YBR298c, YCR098c, YDL199c, YDL247w, YDR387c, YDR497c, YDR536w, YFL040w, YGL104c, YGR289c, YJR160c, YML123c, and YOL103w.

[0076] It should be noted that one of skill in the art will understand that the transporter protein and permease genes listed in Tables 1-5 above may be found in genomic databases including, but not limited to, the National Center for Biotechnology Information (NCBI) database. One of skill in the art will further recognize that genomic databases may contain multiple nucleotide and/or amino acid sequences corresponding to each transporter protein and permease gene listed in Tables 1-5 above.

Oligoalginate Transporter Sequence Motifs and Core Domains

[0077] Amino acid sequence alignment of yeast hexose transporter proteins listed in Table 3 revealed that these proteins have three conserved motifs (Fig. 2). The first conserved sequence is: (I/V)A(F/V)GGFX(P/F)G(W/Y)D(S/T)GX(T/I) (SEQ ID NO: 1). The second conserved sequence is: (A/S)GF(I/V)(N/A)XXFX(M/R)(N/R)FG SEQ ID NO: 2). The third conserved sequence is: LSXVR (SEQ ID NO: 3).

[0078] Amino acid sequence alignment of the permease proteins listed in Table 5 revealed that these proteins have a conserved motif (Fig. 3). The conserved motif is:

XXXG(Q/R)X(L/I)XG(M/I)XXG (SEQ ID NO: 4).

[0079] Amino acid sequence alignment of the transporter proteins e20, el 15, and el74, identified from marine fungi, revealed that these proteins also have conserved motifs. The conserved motifs are listed in Table 6.

TABLE 6

[0080] Accordingly, certain aspects of the present disclosure relate to oligoalginate transporter proteins and permeases having a conserved motif. In certain embodiments, the oligoalginate transporter protein or permease contains a domain having an amino acid sequence selected from ( )A(F/V)GGFX(P/F)G(W/Y)D(S/T)GX(T/I) (SEQ ID NO: 1),

(A/S)GF(I/V)(N/A)XXFX(M/R)(N/R)FG(SEQ ID NO: 2), LSXVR (SEQ ID NO: 3),

XXXG(Q/R)X(L/I)XG(M/I)XXG (SEQ ID NO: 4), NI(V/A)SXXX(A/G)GXFX(G/A) (SEQ ID NO: 232), GR(R/K) (SEQ ID NO: 233),

GRXXXGXGXGXX(S/T)XXXPXXXXEXXPXX(I/V)RG (SEQ ID NO: 234), ESPR (SEQ ID NO: 235), (Y/F)XPXIFXXXG (SEQ ID NO: 236), LXXTGXYGXXK (SEQ ID NO: 237), and GR(R/K)XXL (SEQ ID NO: 238). In certain preferred embodiments of the methods and/or recombinant yeast cells of the present disclosure, the oligoalginate transporter protein or permease contains a domain having an amino acid sequence selected from

NI(V/A)SXXX(A/G)GXFX(G/A) (SEQ ID NO: 232), GR(R/K) (SEQ ID NO: 233),

GRXXXGXGXGXX(S/T)XXXPXXXXEXXPXX(I/V)RG (SEQ ID NO: 234), ESPR (SEQ ID NO: 235), (Y/F)XPXIFXXXG (SEQ ID NO: 236), LXXTGXYGXXK (SEQ ID NO: 237), and GR(R/K)XXL (SEQ ID NO: 238).

Suitable Alginate Lyases

[0081] Certain aspects of the disclosed methods of growing yeast on a substrate containing oligoalginate as a primary carbon source relate to a yeast cell further containing at least one alginate lyase. Alginate lyases are a family of enzymes that are capable of breaking down alginate and/or oligoalginate into smaller polysaccharides (e.g. , alginate 10-mers, alginate pentamers, alginate tetramers, alginate trimers), and/or monosaccharides. Alginate lyases are mainly classified into two distinctive subfamilies: endo-acting (EC 4.2.2.3) and exo-acting (EC 4.2.2.-) alginate lyases. Accordingly, in certain embodiments the alginate lyase is selected from an endo-acting alginate lyase and an exo-acting alginate lyase. Endo-acting alginate lyases are further classified based on their catalytic specificity; M specific and G specific alginate lyases. The endo-acting alginate lyases randomly cleave alginate via a β-elimination mechanism and mainly de-polymerize alginate and/or oligoalginate to di-, tri- and tetrasaccharides. The uronate at the non-reducing terminus of each oligosaccharide is converted to unsaturated sugar uronate, 4-deoxy-a-L-erythro-hex-4-ene pyranosyl uronates. The exo-acting alginate lyases catalyze further de-polymerization of these oligosaccharides and release unsaturated monosaccharides, which may be non-enzymatically converted to monosaccharides, including a-keto acid, 4-deoxy- L-erythro-hexoseulose uronate (DEHU; also called ((4S,5S)-4,5-dihydroxy-2,6- dioxohexanoate). Accordingly, as disclosed herein suitable alginate lyases include, without limitation, a fusion protein that contains endo M-, endo G-, and exo-acting alginate lyases, to degrade or depolymerize alginate and/or oligoalginate to a monosaccharide such as DEHU. As disclosed herein, alginate lyases also include, without limitation, alginate lyases, oligoalginate lyases, polymannuronate lyases, and polyguluronate lyases. Accordingly, in certain

embodiments, the alginate lyase is an oligoalginate lyase. Additionally, alginate lyases of the present disclosure may be secreted by the yeast cell or expressed on the surface of the yeast cell.

[0082] Moreover, as disclosed herein, heterologous alginate lyase proteins may further include a signal recognition peptide (SRP) and promoter combination that allows for protein secretion that retains an observable amount of enzymatic activity. Without wishing to be bound by theory, it is believed that there is no canonical SRP that ensures functional protein secretion in a yeast cell such as S. cerevisiae. However, there are a number of SRPs that have been demonstrated to work in a variety of conditions. As disclosed herein, proteins that are required to be secreted from the yeast cell may be cloned into a promoter/terminator (P/T) library using, for example, yeast gap-repair ligation and the SRP of Saccharomyces cerevisiae native and non- native secreted genes such as MATa, SUC2, etc. As disclosed herein, suitable SRPs include, without limitation, the sequences disclosed in Table 7 below.

TABLE 7

[0083] Additional suitable SRPs may be tested for their ability to secrete a protein of interest, such as an alginate lyase, from a yeast cell. Methods of testing signal peptide function are well known in the art. For example, functionality of an SRP may be assessed by comparing cell extracts to culture supernatants by SDS-PAGE/Western blotting for the presence of properly- sized proteins highly enriched in the supernatants. Degraded protein or lack of signal in the supernatant will be indicative of an insufficient SRP. [0084] As disclosed herein, alginate lyase sequences may also be synthesized and/or codon optimized for their use in yeast. Methods of sequence synthesis and codon optimization are well known in the art.

[0085] Alginate lyases that may be utilized in the methods and compositions disclosed herein include, without limitation, alginate lyases isolated from marine algae, mollusks, and from a wide variety of microbes, such as the genus Pseudomonas, Vibrio, Agwbacterium, and Sphingomonas. Many alginate lyases are endo-acting M specific, several are G specific, and few are exo-acting. For example, alginate lyases isolated from the Sphingomonas sp. strain AI include five endo-acting alginate lyases (AI-I, AI-II, ΑΙ-ΙΓ, AI-III, and AI-IV), and one exo- acting alginate lyase (AI-IV), while an alginate lyase isolated from the Agwbacterium

tumefaciens includes only a single exo-acting alginate lyase (Atu3025).

[0086] As disclosed herein, suitable alginate lyases may include, without limitation, those listed in Table 8 below.

TABLE 8

Protein I I) Source Mode ol action

XP_001839870.2 Coprinopsis cinerea okayama7#l 30 Unknown

XP_001878109.1 Laccaria bicolor S238N-H8 Exo-lytic

CAK40226.1 Aspergillus niger Endo-lytic

EAW16796.1 Neosartorya fischeri NRRL 181 Endo-lytic

XP_748403.1 Aspergillus fumigatus Af293 Endo-lytic

PDB: 2ZZJA Trichoderma Reesei Endo-lytic

XP_566624.1 Cryptococcus neoformans var. neoforn tans JEC21 Exo-lytic

BAE63841.1 Aspergillus oryzae Unknown

XP_002383604.1 Aspergillus flavus NRRL3357 Unknown

XP_002149780.1 Penicillium mameffei ATCC 18224 Unknown

EDP53576.1 Aspergillus fumigatus A1163 Endo-lytic

EDP49916.1 Aspergillus fumigatus A1163 Endo-lytic

XP_002340011.1 Talaromyces stipitatus ATCC 10500 Unknown

XP_001552887.1 Botryotinia fucke liana B05.10 Unknown

XP_001597195.1 Sclerotinia sclerotiorum 1980 Unknown

XP_778147.1 Cryptococcus neoformans var. neoforn tans B-3501A Endo-lytic

XP_001729882.1 Malassezia globosa CBS 7966 Endo-lytic

EEU36914.1 Nectria haematococca mpVI 77-13-4 Unknown

AF082561 Pseudoalteromonas sp. Unknown Protein I I) Source Mode ol action

ABO 18795 Halomonas marina Unknown

X70036 Photobacterium sp. Endo-lytic

CAA58650.1 Pseudomonas alginovora Unknown

AF114039 Vibrio halioticoli Endo-lytic

AF114037 Vibrio halioticoli Endo-lytic

AF114040 Vibrio halioticoli Endo-lytic

AF114038 Vibrio halioticoli Endo-lytic

XP_002383604.1 Aspergillus flavus NRRL3357 Unknown

EDP53576.1 Aspergillus fumigatus A1163 Endo-lytic

EDP49916.1 Aspergillus fumigatus A1163 Endo-lytic

XP_748403.1 Aspergillus fumigatus Af293 Endo-lytic

CAK40226.1 Aspergillus niger Endo-lytic

BAE63841.1 Aspergillus oryzae Unknown

XP_566624.1 Cryptococcus neoformans var. neoforman. _f JEC21 Exo-lytic

XP_001729882.1 Malassezia globosa CBS 7966 Unknown

XP_002149780.1 Penicillium mameffei ATCC 18224 Unknown

XP_001552887.1 Botryotinia fucke liana B05.10 Unknown

XP_001839870.2 Coprinopsis cinerea okayama 7 #130 Unknown

XP_001878109.1 Laccaria bicolor S238N-H8 Unknown

EEU36914.1 Nectria haematococca mpVI 77-13-4 Unknown

EAW16796.1 Neosartorya fischeri NRRL 181 Unknown

XP_001597195.1 Sclerotinia sclerotiorum 1980 Unknown

XP_002340011.1 Talaromyces stipitatus ATCC 10500 Unknown

AB026618.1 Haliotis discus discus Exo-lytic

XP_778147.1 Cryptococcus neoformans var. neoforman. _f B-3501A Exo-lytic

BAD90006.1 Sphingomonas Endo-lytic

BAD16656.1 Sphingomonas Endo-lytic

BAB03319.1 Sphingomonas Exo-lytic

BAB03312.1 Sphingomonas Endo-lytic

ADE10038.1 Tremella fuciformis Exo-lytic

ZP_00991979.1 Vibrio splendidus Exo-lytic

CAA11481.1 Azotobacter chroococcum ATCC 4412 Endo-lytic

AAC04567.1 Azotobacter vinelandii Endo-lytic

AAC32313.1 Azotobacter vinelandii Endo-lytic

BAA33966.1 Cobetia marina N-l Endo-lytic

AAA71990.1 Pseudomonas aeruginosa 8830 Endo-lytic

AAA91127.1 Pseudomonas aeruginosa FRD1 Endo-lytic

AAG06935.1 Pseudomonas aeruginosa PAOl Endo-lytic

NP_252237.1 Pseudomonas aeruginosa PAOl Endo-lytic Protein I I) Source Mode ol action

AAR23929.1 Pseudomonas sp. QD03 Endo-lytic

AAN63147.1 Pseudomonas sp. QDA Endo-lytic

AAF32371.1 Pseudomonas syringae pv. syringae FF5 Endo-lytic

2009330A Sphingomonas sp. AI Endo-lytic

BAA01182.1 Pseudomonas sp. OS-ALG-9 Endo-lytic

BAA83339.1 Corynebacterium sp. ALY-1 Endo-lytic

AAA25049.1 Klebsiella pneumoniae subsp. aerogenes Unknown

CAA49630.1 Photobacterium sp. ATCC 43367 Unknown

AAG04556.1 Pseudomonas aeruginosa PAOl Unknown

NP_249858.1 Pseudomonas aeruginosa PAOl Unknown

BAD16656.1 Sphingomonas sp. AI Unknown

2009330A Sphingomonas sp. AI Unknown

AAF22512.1 Vibrio halioticoli IAM14596T Unknown

ABB36771.1 Vibrio sp. 02 Unknown

ABB36772.1 Vibrio sp. 02 Unknown

AAP45155.1 Vibrio sp. QY101 Unknown

BAE81787.1 Haliotis discus hannai Exo-lytic

BAC87758.1 Haliotis discus hannai Exo-lytic

BAB 19127.1 Chlorella virus CVK2 Unknown

BAA19848.1 Pseudomonas sp. OS-ALG-9 Unknown

AAD 16034.1 Pseudoalteromonas sp. IAM14594 Endo-lytic

AAK90358.1 Agrobacterium tumefaciens str. C58 Exo-lytic

EJBO 1229.1 Rhizobium leguminosarum bv. viciae USl DA 2370 Exo-lytic

EAR52548.1 Oceanicola granulosus HTCC2516 Exo-lytic

EFU40869.1 Paenibacillus vortex V453 Exo-lytic

EHB63819.1 Paenibacillus lactis 154 Exo-lytic

ZP_09073475.1 Paenibacillus elgii B69 Exo-lytic

ABX40875.1 Clostridium phytofermentans ISDg Exo-lytic

EDL55721.1 Vibrio shilonii AK1 Exo-lytic

EFA99217.1 Victivallis vadensis ATCC BAA-548 Exo-lytic

[0087] In certain embodiments, the alginate lyase is selected from XP_001839870.2,

XP_001878109.1, CAK40226.1, EAW16796.1, XP_748403.1, PDB: 2ZZJA, XP_566624.1, BAE63841.1, XP_002383604.1, XP_002149780.1, EDP53576.1, EDP49916.1,

XP_002340011.1, XP_001552887.1, XP_001597195.1, XP_778147.1, XP_001729882.1, EEU36914.1, AF082561c, AB018795, X70036,CAA58650.1, AF114039f, AF114037f,

AF114040f, AF114038f, XP_002383604.1, EDP53576.1, EDP49916.1, XP_748403.1, CAK40226.1, BAE63841.1, XP .566624.1, XP_001729882.1, XP_002149780.1, XP_001552887.1, XP_001839870.2, XP_001878109.1, EEU36914.1, EAW16796.1,

XP_001597195.1, XP_002340011.1, AB026618.1, XP_778147.1, BAD90006.1, BAD16656.1, ADE10038.1, ZP_00991979.1, CAA11481.1, AAC04567.1, AAC32313.1, BAA33966.1, AAA71990.1, AAA91127.1, AAG06935.1, NP_252237.1, AAR23929.1, AAN63147.1, AAF32371.1, 2009330A, BAB03312.1, BAA01182.1, BAA83339.1, AAA25049.1,

CAA49630.1, AAG04556.1, NP_249858.1, BAD16656.1, 2009330A, BAB03312.1,

AAF22512.1, ABB36771.1, ABB36772.1, AAP45155.1, BAE81787.1, BAC87758.1,

BAB19127.1, BAA19848.1, AAD16034.1, AAK90358.1, EJB01229.1, EAR52548.1,

EFU40869.1, EHB63819.1, ZP_09073475.1, ABX40875.1, EDL55721.1, and EFA99217.1.

[0088] It should be noted that one of skill in the art will understand that the lyase genes listed in Table 8 above may be found in genomic databases including, but not limited to, the National Center for Biotechnology Information (NCBI) database. One of skill in the art will further recognize that genomic databases may contain multiple nucleotide and/or amino acid sequences corresponding to each of the lyase genes listed in Table 8 above.

[0089] Additional suitable alginate lyases may be identified and isolated from organisms, such as fungi, that can grow as prototroph in media with an alginate or oligoalginate polymer as its sole carbon source. Methods for identifying and isolating proteins are well known in the art. In one non-limiting example, cell extracts from a fungal species may be fractionated, for example by FPLC, to identify alginate lyases and other alginate and/or oligoalginate

metabolizing enzymes. The fractions can then be tested for alginate lyase activity by performing an alginate lyase activity assay. The protein responsible for lyase activity may then be identified by, for example, liquid chromatography/tandem mass spectrometry (LC-MS/MS). Alternatively, cell-free extracts may be directly tested for alginate lyase activity by measuring the rate of alginate and/or oligoalginate degradation using a capillary viscometer (Gimmestad et ah, 2003).

[0090] Another non-limiting example for identifying and isolating a suitable alginate lyase includes performing a complementation or gain-of-function type screen. In a further non- limiting example, computationally mining of genomic DNA may be used to identify further alginate lyases. The sequencing may be done using, for example, the Illumina GS system.

Assembly of the genome data may be done using any sequence analysis software known in the art. Additionally, suitable alginate lyases may be identified from alginate and/or oligoalginate consuming organisms by mass spectrometry. In yet another non-limiting example, high throughput mRNA sequencing can be performed to identify candidates specifically expressed in alginate and/or oligoalginate consuming organisms upon growth on media utilizing oligoalginate as a sole carbon source to identify pathways and lyase genes for alginate and/or oligoalginate metabolism.

Alginate Lyase Sequence Motifs and Core Domains

[0091] Without wishing to be bound by theory, it is believed that most alginate lyases fall into 3 classes based on molecular mass: 20-35 kDa, 40 kDa, and 60 kDa. The 40 kDa lyase proteins contain conserved sequences within the class. Two types of C-terminal conserved sequences, which are 9-amino acids in length, have been found within the 40 kDa lyase proteins. The first is a WLEPaC+LY (SEQ ID NO: 5). The second is a YFKhG+Y-Q (SEQ ID NO: 6) sequence. Without wishing to be bound by theory, it is believed that the 40 kDa lyase proteins contain 5 conserved alginate and/or oligoalginate binding motifs. The 5 alginate and/or oligoalginate binding motifs are:

1) NNHSYW (SEQ ID NO: 7)

2) NN-+Y-N (SEQ ID NO: 8)

3) -NN-SYp+ (SEQ ID NO: 57)

4) INNcop-+ (SEQ ID NO: 58)

5) hNNcSY-+ (SEQ ID NO: 59)

[0092] For the above motifs, a "+" represents a neutral residue, a "-" represents no consensus, a "c" represents a positively charged residue, an "h" represents a hydrophobic residue, an "o" represents an -OH side group, a "p" represents a planar side chain, and "a" represents an aromatic residue.

[0093] Accordingly, in certain embodiments the alginate lyase contains a conserved motif having an amino acid sequence selected from WLEPaC+LY (SEQ ID NO: 5), YFKhG+Y-Q (SEQ ID NO: 6), NNHSYW (SEQ ID NO: 7), NN-+Y-N (SEQ ID NO: 8), -NN-SYp+ (SEQ ID NO: 57), INNcop-+ (SEQ ID NO: 58), and hNNcSY-+ (SEQ ID NO: 59). [0094] Additionally, disclosed alginate lyases may contain the conserved catalytic amino acid residues from the atu_3025 alginate lyase (H311 and W467), Chlorella virus vAL-1 alginate lyase (K197 and S219), and from the P. aeruginosa PAl 167 alginate lyase (Y193 and Y 199).

[0095] Accordingly, in certain embodiments, the alginate lyase contains at least one catalytic residue selected from a residue corresponding to H311 of atu_3025 alginate lyase, a residue corresponding to W647 of atu_3025 alginate lyase, a residue corresponding to K197 of Chlorella virus vAL-1 alginate lyase, a residue corresponding to S219 of Chlorella virus vAL-1 alginate lyase, a residue corresponding to Y 193 of P. aeruginosa PAl 167 alginate lyase, and a residue corresponding to Y199 of P. aeruginosa PAl 167 alginate lyase.

Suitable DEHU Reductases, KDG Kinases, and KDGP Aldolases

[0096] Certain aspects of the disclosed methods of growing yeast on a substrate containing oligoalginate as a primary carbon source relate to a yeast cell further containing at least one 4- deoxy-L-erythro-5-hexoseulose uronate (DEHU) reductase, at least one 2-keto-3-deoxy-D- gluconate (KDG) kinase, and/or at least one 2-keto-3-deoxy D-gluconate-6-phosphate (KDGP) aldolase.

DEHU Reductases

[0097] As disclosed herein, suitable DEHU reductases may include, without limitation, those disclosed in Table 9 below.

TABLE 9

Protein I I) Source

YP 004087745.1 Asticcacaulis excentricus CB 48

[0098] It should be noted that one of skill in the art will understand that the DEHU reductase genes listed in Table 9 above may be found in genomic databases including, but not limited to, the National Center for Biotechnology Information (NCBI) database. One of skill in the art will further recognize that genomic databases may contain multiple nucleotide and/or amino acid sequences corresponding to each of the DEHU reductase genes listed in Table 9 above.

[0099] In certain embodiments, the DEHU reductase is selected from ZP_00990016.1, NP_357572.1, ABI71926.1, CAI86822.1, AAV95682.1, ZP_00990007.1, BAJ09322.1, YP_299653.1, ZP_06175136.1, YP_573809.1, and YP_004087745.1.

[0100] Additional suitable DEHU reductases may be identified and isolated from other organisms, such as fungi or bacteria, that utilize DEHU. Methods for identifying and isolating proteins are well known in the art. Methods for identifying and isolating proteins are well known in the art, and various non-limiting examples are disclosed herein.

[0101] The DEHU reductase reaction has been shown to require the addition of NADH or NADPH, thus certain embodiments of the methods and compositions disclosed herein may require redox balancing for full functionality.

KDG Kinases and KDGP Aldolases

[0102] As disclosed herein, suitable KDG kinases may include, without limitation, those disclosed in Table 10 below.

TABLE 10

(k'lic I I) Source

AAU42643.1 Bacillus licheniformis ATCC 14580

CAQ33844.1 Escherichia coli

NP 279296.1 Halobacterium sp. NRC-1

ZP 02184941.1 Carnobacterium sp. AT7

CAE11231.1 Bacillus amyloliquefaciens FZB42

AAK88038.2 Agrobacterium tumefaciens

ZP 00991972.1 Vibrio splendidus dene I I) Source

NP_172158.1 Arabidopsis thaliana

PDB: 1V1B_D Thermus thermophiles

CAA52959.1 Erwinia chrysanthemi

NP_347035.1 Clostridium acetobutylicum ATCC 824

YP_528747.1 Saccharophagus degradans 2-40

ABI72944.1 Shewanella frigidimarina NCIMB 400

[0103] In certain embodiments, the KDG kinase is selected from AAU42643.1,

CAQ33844.1, NP .279296.1, ZP_02184941.1, CAE11231.1, AAK88038.2, ZP_00991972.1 NP_172158.1, PDB: 1V1B_D, CAA52959.1, NP_347035.1, YP_528747.1, and ABI72944.1.

[0104] As disclosed herein, suitable KDGP aldolases may include, without limitation, those disclosed in Table 11 below.

TABLE 11

[0105] In certain embodiments, the KDGP aldolase is selected from ABS74160.1,

AAU42642.1, AAK78374.1, ACG62636.1, CAL81844.1, NP .416364.1, ZP_00991971.1, NP_288287.1, and NP_356163.2.

[0106] It should be noted that one of skill in the art will understand that the KDG kinases and KDGP aldolases genes listed in Tables 10 and 11 above may be found in genomic databases including, but not limited to, the National Center for Biotechnology Information (NCBI) database. One of skill in the art will further recognize that genomic databases may contain multiple nucleotide and/or amino acid sequences corresponding to each of the KDG kinases and KDGP aldolases genes listed in Tables 10 and 11.

[0107] Additional suitable KDG kinases and KDGP aldolases may be identified and isolated from organisms, such as fungi and bacteria, that utilize KDG kinase and KDGP aldolase.

Methods for identifying and isolating proteins are well known in the art, and various non- limiting examples are disclosed herein.

DEHU Reductase, KDG Kinase, and KDGP Aldolase Sequence Motifs and Core Domains

[0108] In certain embodiments, the DEHU reductase contains a conserved NADH/NADPH binding motif having an amino acid sequence of (T/V)GXXXG(I/L)G (SEQ ID NO: 60) (see Takase et al., 2010). In other embodiments, the KDG kinase contains a consensus domain having an amino acid sequence of DTTAAGDSFSAGYL (SEQ ID NO: 61) or

GGDTLNTAVYISROVKPDALDVHYV (SEQ ID NO: 62) (see Hugouvieux-Cotte-Pattat et al, 1994). In yet other embodiments, he KDGP aldolase contains a consensus domain having an amino acid sequence of ExTxRT (SEQ ID NO: 63).

Embodiments Relating to Oligoalginate Metabolism

[0109] Certain aspects of the disclosure relate to a method of growing yeast on a substrate containing oligoalginate as a primary carbon source by providing a yeast cell, where the yeast cell contains at least one recombinant polynucleotide encoding an oligoalginate transporter protein or permease; and culturing the yeast cell with the substrate containing oligoalginate under conditions whereby the recombinant polynucleotide is expressed, where expression of the recombinant polynucleotide results in transport of oligoalginate into the yeast cell, and where the yeast cell contains the proteins necessary for the yeast cell to metabolize oligoalginate.

[0110] In certain embodiments, the oligoalginate transporter protein is selected from a GPH transporter, a pectin/cellodextrin transporter, a hexose transporter, an ABC transporter and a transporter from marine fungi. In other embodiments, the oligoalginate transporter protein is a GPH transporter. Exemplary GPH transporters are as described in previous sections. In still other embodiments, the oligoalginate transporter protein is a pectin/cellodextrin transporter. Exemplary pectin/cellodextrin transporters are as described in previous sections.

[0111] In other embodiments, the oligoalginate transporter protein is a hexose transporter. Exemplary hexose transporters are as described in previous sections. In other embodiments, the oligoalginate transporter protein is an ABC transporter. Exemplary ABC transporters are as described in previous sections. In other embodiments, the oligoalginate transporter protein is a transporter from marine fungi. Exemplary transporters from marine fungi are as described in previous sections. In certain embodiments, the yeast cell contains at least 2, at least 3, at least 4, at least 5, at least 6, or more recombinant polynucleotides encoding an oligoalginate transporter proteins or permeases.

[0112] In certain embodiments, the proteins necessary for the yeast cell to metabolize oligoalginate contain at least one DEHU reductase. Exemplary DEHU reductases are as described in previous sections. In yet other embodiments, the proteins necessary for the yeast cell to metabolize oligoalginate contain at least one KDG kinase. Exemplary KDG kinases are as described in previous sections. In further embodiments, the proteins necessary for the yeast cell to metabolize oligoalginate contain at least one KDGP aldolase. Exemplary KDGP aldolases are as described in previous sections. In certain embodiments, the proteins necessary for the yeast cell to metabolize oligoalginate contain, at least one DEHU reductase, at least one KDG kinase, and at least one KDGP aldolase. In other embodiments, the yeast cell produces pyruvate from oligoalginate.

[0113] In other embodiments, the proteins necessary for the yeast cell to metabolize oligoalginate further contain at least one alginate lyase. In other embodiments, the proteins necessary for the yeast cell to metabolize oligoalginate may contain at least 1, at least 2, at least 3, at least 4, at least 5, or more alginate lyases. Exemplary alginate lyases are as described in previous sections. In certain embodiments, the proteins necessary for the yeast cell to metabolize oligoalginate contain, at least one DEHU reductase, at least one KDG kinase, and at least one KDGP aldolase. In other embodiments, the yeast cell produces pyruvate from oligoalginate.

[0114] In certain embodiments the yeast cell contains at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or more proteins necessary for the yeast cell to metabolize oligoalginate. In further embodiments, the yeast cell is selected from Saccharomyces sp., Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Schizosaccharomyces pombe, or

Saccharomyces oviformis Kluyveromyces lactis, Kluyveromyces marxianus, Pichia stipitis, Pichia pastoris, Candida shehatae, Yarrowia lipolytica, Brettanomyces custersii,

Zygosaccharomyces roux, Sporotrichum thermophile, Candida shehatae, and Neurospora crassa.

Mannitol Metabolism Components

[0115] Certain aspects of the present disclosure relate to methods of growing yeast on a substrate that further contains mannitol by expressing at least one recombinant polynucleotide encoding a mannitol transporter or a mannitol dehydrogenase, and containing the proteins necessary for the yeast cell to metabolize mannitol.

[0116] Mannitol is the most abundant sugar alcohol produced by bacteria, lichens, brown algae, brown seaweed, and several species of plants. Mannitol metabolism had been previously reported in bacteria, fungi, and plant species. The overall mannitol metabolic process can occur via three major pathways (Fig. 4). Each mannitol metabolic pathway contains distinct intermediates, which are the result of different enzymatic activities.

[0117] The first pathway, which appears to be predominant in plants, utilizes an enzyme called mannitol dehydrogenase (MTD) which converts intracellular mannitol into mannose while reducing NAD(P) ⁺ (Fig. 5). The mannose is converted to mannose 6-phosphate by hexokinase (HK). The mannose phosphate isomerase (MPI) converts the mannose 6-p into fructose 6-phosphate and the latter enters into glycolysis. The prerequisite for functionality of this pathway depends on transport of mannitol into the cells, which in plants is carried out by mannitol transporter (MaT). Only a handful of MaTs have been reported or characterized in plants. The yeast genome contains homologs of both HK and MPI. However, the MaT and MTD do not seem to be encoded by yeast.

[0118] The second pathway, which appears to be predominantly a fungal pathway, mostly operates in a reverse direction (resulting in synthesis of mannitol and industrial process for the same). The conversion of mannitol to fructose is carried out by an enzyme also known as mannitol dehydrogenase (but abbreviated as MDH to differentiate from MTD) (Fig. 6). Similar to the predominantly plant dehydrogenase (MTD), MDH utilizes NAD(P) ⁺ during the reaction. Most of these enzymes have higher Km for mannitol than fructose, favoring biosynthesis of mannitol. However, there are some reports of this enzyme catalyzing fructose biosynthesis and metabolism of mannitol. Once fructose is made, the hexokinase (HK) catalysis the

phosphorylation of fructose to fructose 6-p which enters glycolysis. The yeast genome harbors a gene called YEL070W that encodes a putative MDH. One prerequisite for utilizing this mode of mannitol metabolism appears to be the need for transport of mannitol into the cell.

[0119] The third pathway appears to be predominant in bacterial and fungal species. In bacterial species, the mannitol uptake occurs via a PTS system which results in transport of mannitol to the cytoplasm and its simultaneous conversion to mannitol 1 -phosphate. The Mannitol 1-P can be converted fructose 6-phosphate via a reversible reaction which utilizes reduction of NAD(P) while oxidizing mannitol-lp to fructose 6-p. This reaction is catalyzed by an enzyme known as mannitol 1-phophate dehydrogenase (MPD), which is present in several bacterial species and fungal species. This enzyme has been enzymatically characterized in some species from both origins. One prerequisite for utilizing this mode of mannitol metabolism appears to be the need for transport of mannitol into the cell.

[0120] The proteins necessary for a yeast cell to metabolize mannitol may make up any of the three disclosed mannitol metabolic pathways. The proteins may be endogenous yeast proteins, or they may be recombinantly expressed proteins. Additionally, the proteins necessary for a yeast cell to metabolize mannitol may be endogenous yeast proteins that make up an alternative metabolic pathway that is able to utilize mannitol. Suitable Mannitol Transporters

[0121] Suitable mannitol transporters include, without limitation, those listed in Table 12 below.

TABLE 12

[0122] In certain embodiments, the mannitol transporter is selected from MAT, AgvMaTl, AgvMaT2, ATPLT5, PLTl, PLT2, PLT4, PLT5, PLT6, AtMaTl, CeMaTl, VePoT5, MFS, NtMaTl, YDR497C(Itrl), YOL103W(Itr2), YGL104c(Vps73), YBR241c, KlMaTl,

AAL85876.2, AAG43998.1, NP_188513.1, NP_179671.2, AAB68028.1, NP_179438.1, NP_195385.1, AAL58131.1, XP_002468337.1, YP_001422011.1, CBN73775.1, AAY88181.2, AA039267.1, AAM44082.1, CAP94360.1, CAK37724.1., GL85070363, GL 12004316, GI: 821416, GL75338646, GL75338645, GL 117940083, GL 118573108, GL 118573109,

GL42569195, GL 182676628, GL302417138, GL 19114232, GL 19885, GL6320705,

GL37362691, GL6321334, GL6319718, GL50305573, NP_010845.1, and NP_014470.1.

Suitable Mannitol Dehydrogenases

[0123] Suitable mannitol dehydrogenases {i.e., MTD, MDH, and MPD) include, without limitation, those listed in Table 13 below.

TABLE 13

Protein/

M DH # (Jene l l) Orj ζηη is in (specilicitv - il know n ) Source

MDH1 NP_195510.1 plar it NAD (P) Arabidopsis thaliana

MDH2 Q0UEB6.1 fun, gi (M1P -DH) Phaeosphaeria nodorum

Phaeosphaeria nodorum

MDH3 EAT76858.1 fun, gi (M1P -DH) SN15

MDH4 AAC 15467.1 plar it (Celery) Apium graveolens

MDH5 AAB97617.1 plar it (Celery) Apium graveolens

MDH6 Q6UQ76.1 fun, gi (M1P -DH) Alternaria alternata

MDH7 S72477 plar it (tomato) Solanum lycopersicum Protein/

M l)l l # ( k no l l) ( )i ^• gaiiisni (siimncit> - ir know n ) Source

MDH8 P42754.1 pk tnt (Parsley) (NAD) Petroselinum crispum

MDH9 082515.1 pk tnt (alfa) Medicago sativa

MDH10 ACJ11737.1 pk tnt (upland cotton) Gossypium hirsutum

MDH11 AAK67169.1 fui lgi (NADP) Passalorafulva

MDH12 Q1DP56.2 fui lgi (M1P-DH) Coccidioides immiti

Ajellomyces capsulatus

MDH13 A6RGF3.2 fui lgi (M1P-DH) NAml

MDH14 P58708.1 ba rteria (Gm-) Ralstonia solanacearum

Bacillus amyloliquefaciens

MDH15 A7Z1E8.1 ba eria (M1P-DH) FZB42

MtdAG GI: 12643507 pk tnt (Celery) Apium graveolens

GI: 16087921 Clostridium

MtdCP 3 ba rteria phytofermentans

MtdBJ GL27381947 ba rteria Bradyrhizobium japonicum

GL 11661771

MtdLM 9 ba rteria Leuconostoc mesenteroides

MtdAF GL70993080 fui igus Aspergillus fumigatus

MtdTM GI: 15642843 ba rteria Thermotoga maritima

GL22750952

MtdLB 2 ba rteria Lactobacillus brevis

MtdPF GL2293418 ba rteria Pseudomonas fluorescens

MtdRS GL462654 ba rteria Rhodobacter sphaeroides

YEL070

W NP_010844.1 Fu ngi Saccharomyces cerevisiae

YNR073C NP_014471.3 Fu ngi Saccharomyces cerevisiae

[0124] In certain embodiments, the mannitol dehydrogenase is selected from MTD, MDH, MPD, MtdAG, MtdCP, MtdBJ, MtdLM, MtdAF, MtdTM, MtdLB, MtdPF, MtdRS,

NP_195510.1, Q0UEB6.1, EAT76858.1, AAC15467.1, AAB97617.1, Q6UQ76.1, S72477, P42754.1, 082515.1, ACJ11737.1, AAK67169.1, Q1DP56.2, A6RGF3.2, P58708.1, A7Z1E8.1, GL 12643507, GL 160879213, GL27381947, GL 116617719, GL70993080, GL 15642843, GL227509522, GL2293418, GL462654, NP_010844.1, and NP_014471.3.

[0125] It should be noted that one of skill in the art will understand that the mannitol transporter and mannitol dehydrogenase genes listed in Tables 12 and 13 above may be found in genomic databases including, but not limited to, the National Center for Biotechnology

Information (NCBI) database. One of skill in the art will further recognize that genomic databases may contain multiple nucleotide and/or amino acid sequences corresponding to each of the mannitol transporter and mannitol dehydrogenase genes listed in Tables 12 and 13 above.

Embodiments Relating to Mannitol Metabolism

[0126] Certain aspects of the disclosed methods relate to growing yeast on a substrate that further contains mannitol. Accordingly, in certain embodiments, the substrate containing oligoalginate further contains mannitol, the yeast cell further includes at least one recombinant polynucleotide encoding a mannitol transporter, the yeast cell is cultured under conditions whereby the recombinant polynucleotides encoding an oligoalginate transporter protein or permease and a mannitol transporter are expressed, where expression of the polynucleotide encoding a mannitol transporter results in transport of mannitol into the yeast cell, and the yeast cell further contains the proteins necessary for the yeast cell to metabolize mannitol. Exemplary mannitol transporters are as described in previous sections. In other embodiments, the proteins necessary for the yeast cell to metabolize mannitol include at least one mannitol dehydrogenase. Exemplary mannitol dehydrogenases are as described in previous sections.

[0127] In certain other embodiments, the substrate containing oligoalginate further contains mannitol, the yeast cell further includes at least one recombinant polynucleotide encoding a mannitol dehydrogenase, the yeast cell is cultured under conditions whereby the recombinant polynucleotides encoding an oligoalginate transporter protein or permease and a mannitol dehydrogenase are expressed, where expression of the polynucleotide encoding a mannitol dehydrogenase results in mannitol reduction, and the yeast cell further contains the proteins necessary for the yeast cell to metabolize mannitol. Exemplary mannitol dehydrogenases are as described in previous sections. In other embodiments, the proteins necessary for the yeast cell to metabolize mannitol include at least one mannitol transporter. Exemplary mannitol transporters are as described in previous sections.

[0128] In still other embodiments, the proteins necessary for the yeast cell to metabolize oligoalginate and mannitol include at least one DEHU reductase, at least one KDG kinase, at least one KDGP aldolase, and at least one mannitol dehydrogenase. Exemplary DEHU reductases, KDG kinases, and KDGP aldolases are as described in previous sections. In further embodiments, the yeast cell produces pyruvate from oligoalginate, and fructose 6-phosphate from mannitol.

Producing and Culturing Yeast Cells

[0129] Methods of producing and culturing yeast cells of the disclosure may include the introduction or transfer of expression vectors containing the recombinant polynucleotides of the disclosure into the yeast cell. Such methods for transferring expression vectors into yeast cells are well known to those of ordinary skill in the art.

[0130] The vectors preferably contain one or more selectable markers which permit easy selection of transformed hosts. A selectable marker is a gene encoding a product which provides, for example, biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Selection of recombinant cells may be based upon antimicrobial resistance that has been conferred by genes such as the zeo, blast, amp, gpt, neo, and hyg resistance genes.

[0131] Suitable markers for yeast hosts are, for example, ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.

[0132] Native yeast promoters and terminators may also be utilized with any suitable expression vector. Further a promoter/terminator sequence library may be constructed. The promoter/terminator sequences may be selected based on the known genes for their constitutive and/or high expression. Promoters and terminators may be cloned into a yeast-E. coli shuttle vector with an auxotrophic marker, and an antibiotic marker. A restriction enzyme site may also be designed between the promoter and the terminator to facilitate cloning of a gene of interest between the two. A conventional ligase based or a yeast gap-repair cloning based technique may be used to generate the library.

[0133] Table 14 below provides a list of yeast promoter and terminator sequences that may be utilized with the methods and compositions described herein. TABLE 14

Promoters Terminators

FBAlt GPMlt

PDClt ADH2t

TDH3t HXT2t

PGIlt TEFlt

TPIlt GNDlt

PMAlt TALlt

EN02t ACT It

HXT7t ENOlt

ADHlt CYClt

RPS lOBt CPRlt

VMA6t RPL16At

[0134] For integration into the host genome, the vector may rely on the sequence of a gene of interest or any other element of the vector for integration of the vector into the genome by homologous or nonhomologous recombination. Alternatively, the vector may contain additional nucleotide sequences for directing integration by homologous recombination into the genome of the host (e.g. , delta sequence). The additional nucleotide sequences enable the vector to be integrated into the host genome at a precise location(s) in the chromosome(s). To increase the likelihood of integration at a precise location, the integrational elements should preferably contain a sufficient number of nucleic acids, such as 40 to 100 base pairs, 40 to 10,000 base pairs, preferably more than about 500, 1,000, 1,500 or 2,000 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may be any sequence that is homologous with the target sequence in the genome of the host. Furthermore, the integrational elements may be non- coding or coding nucleotide sequences. On the other hand, the vector may be integrated into the genome of the host by non-homologous recombination.

[0135] For autonomous replication, the vector may further contain an origin of replication, enabling the vector to replicate autonomously in the host in question. The origin of replication may be any plasmid replicator mediating autonomous replication in a cell of interest. The term "origin of replication" or "plasmid replicator" is defined herein as a sequence that enables a plasmid or vector to replicate in vivo. Examples of origins of replication for use in a yeast cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6.

[0136] More than one copy of a gene may be inserted into the host to increase production of the gene product. An increase in the copy number of the gene can be obtained by integrating at least one additional copy of the gene into the host genome or by including an amplifiable selectable marker gene with the nucleotide sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the gene, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

[0137] Once the yeast cell has been transformed with the expression vector, the yeast cell is allowed to grow. Methods of the disclosure may include culturing the yeast cell such that recombinant nucleic acids in the cell are expressed. Typically cells are grown at 30°C in appropriate media. Preferred growth media in the present disclosure include, for example, common commercially prepared media such as Luria Bertani (LB) broth, Sabouraud Dextrose (SD) broth, Yeast Nitrogen Base (YNB) media, or Yeast medium (YM) broth. Other defined or synthetic growth media may also be used and the appropriate medium for growth of the particular yeast cell will be known by someone skilled in the art of microbiology or fermentation science.

[0138] According to some aspects of the disclosure, the culture media or substrate contains a carbon source for the yeast cell. Suitable carbon sources can be in various forms, including, but not limited to, polymers, carbohydrates, acids, alcohols, aldehydes, ketones, amino acids, peptides, etc. These include, but are not limited to, alginate, oligoalginate, unsaturated monouronic acids, 4-deoxy-L-erythro-hexoseulose uronic acid (DEHU), and mannitol.

[0139] In certain embodiments, the yeast cell can utilize alginate, oligoalginate, unsaturated monouronic acids, or 4-deoxy-L-erythro-hexoseulose uronic acid (DEHU) as the primary carbon source. In other embodiments, the yeast cell can further utilize mannitol as a secondary carbon source. In yet other embodiments, the yeast cell can further utilize glucose as a secondary carbon source.

[0140] In addition to an appropriate carbon source, culture media or substrate may contain suitable minerals, salts, cofactors, buffers and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathways necessary for the fermentation of various sugars and the production of hydrocarbons and hydrocarbon derivatives. Reactions may be performed under aerobic or anaerobic conditions, where aerobic, anoxic, or anaerobic conditions are preferred based on the requirements of the microorganism. As the yeast cell grows and/or multiplies, it expresses enzymes of the substrate utilization pathway necessary for growth on the substrate.

Suitable Substrates Containing Oligoalginate

[0141] Certain aspects of the present disclosure relate to growing yeast on a substrate that contains oligoalginate. In certain embodiments of the disclosed methods the oligoalginate is selected from short alginate polymers, alginate 10-mers, alginate 9-mers, alginate 8-mers, alginate 7-mers, alginate hexamers, alginate pentamers, alginate tetramers, alginate trimers, alginate dimers, and alginate monomers.

[0142] Additionally, suitable substrates may contain alginate that is enzymatically or chemically broken down to oligoalginate. In one non-limiting example, the yeast cells contain alginate lyases, which are either secreted or localized on the cell surface, that enzymatically degrade the alginate. Suitable substrates containing alginate include, but are not limited to, kelp, giant kelp, sargasso, seaweed, algae, brown algae, brown seaweed, marine microflora, microalgae, sea grass, and combinations thereof. Accordingly, in certain embodiments of the disclosed methods, the substrate containing alginate and/or oligoalginate is selected from kelp, giant kelp, sargasso, seaweed, algae, brown algae, brown seaweed, marine microflora, microalgae, sea grass, and combinations thereof.

Methods of Producing Commodity Chemicals

[0143] Certain aspects of the present disclosure relate to methods of producing commodity chemicals. Commodity chemicals include, without limitation, any saleable or marketable chemical that can be produced either directly or as a by-product of the disclosed methods and compositions, including biofuels, such biodiesels and ethanol. Biodiesel may refer generally to plant oil- or animal fat-based diesel fuel composed mainly of long-chain alkyl, methyl, propyl, or ethyl esters (i.e., fatty acid esters), though it can include other fatty acids, and terpenoids. [0144] General examples of commodity chemicals include, without limitation, biofuels, minerals, polymer precursors, carbohydrates, alcohols, fatty alcohols, surfactants, plasticizers, and solvents. Additionally, biofuels include, without limitation, solid, liquid, or gas fuels derived, at least in part, from a biological source, such as yeast. Additionally, commodity chemicals also include, without limitation, ethanol, biodiesel, methane, methanol, ethane, ethene, n-propane, 1-propene, 1-propanol, propanal, acetone, propionate, n-butane, 1-butene, 1- butanol, butanal, butanoate, isobutanal, isobutanol, 2-methylbutanal, 2-methylbutanol, 3- methylbutanal, 3-methylbutanol, 2-butene, 2-butanol, 2-butanone, 2,3-butanediol, 3-hydroxy-2- butanone, 2,3-butanedione, ethylbenzene, ethenylbenzene, 2-phenylethanol, phenylacetaldehyde, 1-phenylbutane, 4-phenyl- 1-butene, 4-phenyl-2-butene, l-phenyl-2-butene, l-phenyl-2-butanol, 4-phenyl-2-butanol, l-phenyl-2-butanone, 4-phenyl-2-butanone, l-phenyl-2,3-butandiol, 1- phenyl-3-hydroxy-2-butanone, 4-phenyl-3-hydroxy-2-butanone, l-phenyl-2,3-butanedione, n- pentane, ethylphenol, ethenylphenol, 2-(4-hydroxyphenyl) ethanol, , 4- hydroxyphenylacetaldehyde, l-(4-hydroxyphenyl) butane, 4-(4-hydroxyphenyl)-l-butene, 4-(4- hydroxyphenyl) -2-butene, l-(4-hydroxyphenyl)- 1-butene, l-(4-hydroxyphenyl)-2-butanol, 4-(4- hydroxyphenyl)-2-butanol, l-(4-hydroxyphenyl)-2-butanone, 4-(4-hydroxyphenyl)-2-butanone, l-(4-hydroxyphenyl)-2,3-butandiol, l-(4-hydroxyphenyl)-3-hydroxy-2-butanone, 4-(4- hydroxyphenyl)-3-hydroxy-2-butanone, 1 -(4-hydroxyphenyl)-2,3-butanonedione, indolylethane, indolylethene, 2-(indole-3-)ethanol, n-pentane,l -pentene, 1-pentanol, pentanal, pentanoate, 2- pentene, 2-pentanol, 3-pentanol, 2-pentanone, 3-pentanone, 4-methylpentanal, 4-methylpentanol, 2,3-pentanediol, 2-hydroxy-3-pentanone, 3-hydroxy-2-pentanone, 2,3-pentanedione, 2- methylpentane, 4-methyl-l -pentene, 4-methyl-2-pentene, 4-methyl-3 -pentene, 4-methyl-2- pentanol, 2-methyl-3-pentanol, 4-methyl-2-pentanone, 2-methyl-3-pentanone, 4-methyl-2,3- pentanediol, 4-methyl-2-hydroxy-3-pentanone, 4-methyl-3-hydroxy-2-pentanone, 4-methyl-2,3- pentanedione, 1-phenylpentane, 1 -phenyl- 1 -pentene, l-phenyl-2-pentene, l-phenyl-3-pentene, 1- phenyl-2-pentanol, l-phenyl-3-pentanol, l-phenyl-2-pentanone, l-phenyl-3-pentanone, 1- phenyl-2,3-pentanediol, l-phenyl-2-hydroxy-3-pentanone, l-phenyl-3-hydroxy-2-pentanone, 1- phenyl-2,3-pentanedione, 4-methyl- 1-phenylpentane, 4-methyl-l -phenyl- 1 -pentene, 4-methyl- 1- phenyl-2-pentene, 4-methyl- l-phenyl-3-pentene, 4-methyl- l-phenyl-3-pentanol, 4-methyl- 1- phenyl-2-pentanol, 4-methyl- l-phenyl-3-pentanone, 4-methyl- l-phenyl-2-pentanone, 4-methyl-

1- phenyl-2,3-pentanediol, 4-methyl- l-phenyl-2,3-pentanedione , 4-methyl- 1 -phenyl- 3 -hydroxy-

2- pentanone, 4-methyl- l-phenyl-2-hydroxy-3-pentanone, l-(4-hydroxyphenyl) pentane, l-(4- hydroxyphenyl)-l -pentene, l-(4-hydroxyphenyl)-2-pentene, l-(4-hydroxyphenyl)-3-pentene, 1- (4-hydroxyphenyl)-2-pentanol, l-(4-hydroxyphenyl)-3-pentanol, l-(4-hydroxyphenyl)-2- pentanone, l-(4-hydroxyphenyl)-3-pentanone, l-(4-hydroxyphenyl)-2,3-pentanediol, l-(4- hydroxyphenyl)-2-hydroxy-3-pentanone, l-(4-hydroxyphenyl)-3-hydroxy-2-pentanone, l-(4- hydroxyphenyl)-2,3-pentanedione, 4-methyl-l-(4-hydroxyphenyl) pentane, 4-methyl- 1- (4- hydroxyphenyl)-2-pentene, 4-methyl- l-(4-hydroxyphenyl)-3-pentene, 4-methyl- 1- (4- hydroxyphenyl) - 1 -pentene, 4-methyl- 1 - (4-hydroxyphenyl) - 3 -pentanol, 4-methyl- 1 - (4- hydroxyphenyl)-2-pentanol, 4-methyl- l-(4-hydroxyphenyl)-3-pentanone, 4-methyl- 1- (4- hydroxyphenyl)-2-pentanone, 4-methyl- l-(4-hydroxyphenyl)-2,3-pentanediol, 4-methyl- 1- (4- hydroxyphenyl)-2,3-pentanedione , 4-methyl- l-(4-hydroxyphenyl)-3-hydroxy-2-pentanone, 4- methyl-l-(4-hydroxyphenyl)-2-hydroxy-3-pentanone, l-indole-3-pentane, l-(indole-3)-l- pentene, l-(indole-3)-2-pentene, l-(indole-3)-3-pentene, l-(indole-3)-2-pentanol, l-(indole-3)-3- pentanol, l-(indole-3)-2-pentanone, l-(indole-3)-3-pentanone, l-(indole-3)-2,3-pentanediol, 1- (indole-3)-2-hydroxy-3-pentanone, l-(indole-3)-3-hydroxy-2-pentanone, l-(indole-3)-2,3- pentanedione, 4-methyl- l-(indole-3-)pentane, 4-methyl-l-(indole-3)-2-pentene, 4-methyl-l- (indole-3)-3-pentene, 4-methyl-l-(indole-3)-l-pentene, 4-methyl-2-(indole-3)-3-pentanol, 4- methyl- l-(indole-3)-2-pentanol, 4-methyl- l-(indole-3)-3-pentanone, 4-methyl- 1- (indole- 3) -2- pentanone, 4-methyl- l-(indole-3)-2,3-pentanediol, 4-methyl- l-(indole-3)-2,3-pentanedione, 4- methyl-l-(indole-3)-3-hydroxy-2-pentanone, 4-methyl- l-(indole-3)-2-hydroxy-3-pentanone, n- hexane, 1-hexene, 1-hexanol, hexanal, hexanoate, 2-hexene, 3-hexene, 2-hexanol, 3-hexanol, 2- hexanone, 3-hexanone, 2,3-hexanediol, 2,3-hexanedione, 3,4-hexanediol, 3,4-hexanedione, 2- hydroxy-3-hexanone, 3-hydroxy-2-hexanone, 3-hydroxy-4-hexanone, 4-hydroxy-3-hexanone, 2- methylhexane, 3-methylhexane, 2-methyl-2-hexene, 2-methyl-3-hexene, 5 -methyl- 1-hexene, 5- methyl-2-hexene, 4-methyl- 1-hexene, 4-methyl-2-hexene, 3-methyl-3-hexene, 3-methyl-2- hexene, 3-methyl- 1-hexene, 2-methyl-3-hexanol, 5-methyl-2-hexanol, 5-methyl-3-hexanol, 2- methyl-3-hexanone, 5-methyl-2-hexanone, 5-methyl-3-hexanone, 2-methyl-3,4-hexanediol, 2- methyl-3,4-hexanedione , 5-methyl-2,3-hexanediol, 5-methyl-2,3-hexanedione, 4-methyl-2,3- hexanediol, 4-methyl-2,3-hexanedione, 2-methyl-3-hydroxy-4-hexanone, 2-methyl-4-hydroxy-3- hexanone, 5-methyl-2-hydroxy-3-hexanone, 5-methyl-3-hydroxy-2-hexanone, 4-methyl-2- hydroxy-3-hexanone, 4-methyl-3-hydroxy-2-hexanone, 2,5-dimethylhexane, 2,5-dimethyl-2- hexene, 2,5-dimethyl-3-hexene, 2,5-dimethyl-3-hexanol, 2,5-dimethyl-3-hexanone, 2,5- dimethyl-3,4-hexanediol, 2,5-dimethyl-3,4-hexanedione, 2,5-dimethyl-3-hydroxy-4-hexanone, 5-methyl- 1-phenylhexane, 4-methyl- 1-phenylhexane, 5-methyl- 1 -phenyl- 1-hexene, 5-methyl- 1- phenyl-2-hexene, 5-methyl- l-phenyl-3-hexene, 4-methyl- 1 -phenyl- 1-hexene, 4-methyl- 1- phenyl-2-hexene, 4-methyl- l-phenyl-3-hexene, 5-methyl- l-phenyl-2-hexanol, 5-methyl- 1- phenyl-3-hexanol, 4-methyl- l-phenyl-2-hexanol, 4-methyl- l-phenyl-3-hexanol, 5-methyl- 1- phenyl-2-hexanone, 5-methyl- l-phenyl-3-hexanone, 4-methyl- l-phenyl-2-hexanone, 4-methyl - l-phenyl-3-hexanone, 5-methyl- l-phenyl-2,3-hexanediol, 4-methyl- l-phenyl-2,3-hexanediol, 5- methyl- l-phenyl-3-hydroxy-2-hexanone, 5-methyl- l-phenyl-2-hydroxy-3-hexanone, 4-methyl -

1- phenyl-3-hydroxy-2-hexanone, 4-methyl- l-phenyl-2-hydroxy-3-hexanone, 5-methyl- 1- phenyl-2,3-hexanedione, 4-methyl- l-phenyl-2,3-hexanedione, 4-methyl- 1- (4- hydroxyphenyl)hexane, 5-methyl- 1 -(4-hydroxyphenyl)- 1 -hexene, 5-methyl- 1- (4- hydroxyphenyl) -2-hexene, 5 -methyl- 1 - (4-hydroxyphenyl) - 3 -hexene, 4-methyl- 1 - (4- hydroxyphenyl)- 1 -hexene, 4-methyl- 1 -(4-hydroxyphenyl)-2-hexene, 4-methyl- 1 -(4- hydroxyphenyl)-3-hexene, 5-methyl- l-(4-hydroxyphenyl)-2-hexanol, 5-methyl- 1- (4- hydroxyphenyl)-3-hexanol, 4-methyl- 1 -(4-hydroxyphenyl)-2-hexanol, 4-methyl- 1 -(4- hydroxyphenyl)-3-hexanol, 5-methyl- l-(4-hydroxyphenyl)-2-hexanone, 5-methyl- 1- (4- hydroxyphenyl)-3-hexanone, 4-methyl- l-(4-hydroxyphenyl)-2-hexanone, 4-methyl- 1- (4- hydroxyphenyl)-3-hexanone, 5-methyl- l-(4-hydroxyphenyl)-2,3-hexanediol, 4-methyl- 1- (4- hydroxyphenyl)-2,3-hexanediol, 5-methyl- l-(4-hydroxyphenyl)-3-hydroxy-2-hexanone, 5- methyl-l-(4-hydroxyphenyl)-2-hydroxy-3-hexanone, 4-methyl- l-(4-hydroxyphenyl)-3-hydroxy-

2- hexanone, 4-methyl- l-(4-hydroxyphenyl)-2-hydroxy-3-hexanone, 5-methyl- 1- (4- hydroxyphenyl)-2,3-hexanedione, 4-methyl- l-(4-hydroxyphenyl)-2,3-hexanedione, 4-methyl- 1- (indole-3-)hexane, 5-methyl-l-(indole-3)-l-hexene, 5-methyl- l-(indole-3)-2-hexene, 5-methyl- l-(indole-3)-3-hexene, 4-methyl-l-(indole-3)-l-hexene, 4-methyl-l-(indole-3)-2-hexene, 4- methyl-l-(indole-3)-3-hexene, 5-methyl- l-(indole-3)-2-hexanol, 5-methyl- l-(indole-3)-3- hexanol, 4-methyl-l-(indole-3)-2-hexanol, 4-methyl-l-(indole-3)-3-hexanol, 5 -methyl- l-(indole- 3)-2-hexanone, 5-methyl-l-(indole-3)-3-hexanone, 4-methyl- l-(indole-3)-2-hexanone, 4-methyl- l-(indole-3)-3-hexanone, 5-methyl- l-(indole-3)-2,3-hexanediol, 4-methyl- l-(indole-3)-2,3- hexanediol, 5-methyl- l-(indole-3)-3-hydroxy-2-hexanone, 5-methyl- l-(indole-3)-2-hydroxy-3- hexanone, 4-methyl- l-(indole-3)-3-hydroxy-2-hexanone, 4-methyl- l-(indole-3)-2-hydroxy-3- hexanone, 5-methyl-l-(indole-3)-2,3-hexanedione, 4-methyl- l-(indole-3)-2,3-hexanedione, n- heptane, 1-heptene, 1-heptanol, heptanal, heptanoate, 2-heptene, 3-heptene, 2-heptanol, 3- heptanol, 4-heptanol, 2-heptanone, 3-heptanone, 4-heptanone, 2,3-heptanediol, 2,3- heptanedione, 3,4-heptanediol, 3,4-heptanedione, 2-hydroxy-3-heptanone, 3-hydroxy-2- heptanone, 3-hydroxy-4-heptanone, 4-hydroxy-3-heptanone, 2-methylheptane, 3-methylheptane,

6- methyl-2-heptene, 6-methyl-3-heptene, 2-methyl-3-heptene, 2-methyl-2-heptene, 5-methyl-2- heptene, 5-methyl-3-heptene, 3-methyl-3-heptene, 2-methyl-3-heptanol, 2-methyl-4-heptanol, 6- methyl-3-heptanol, 5-methyl-3-heptanol, 3-methyl-4-heptanol, 2-methyl-3-heptanone, 2-methyl- 4-heptanone, 6-methyl-3-heptanone, 5-methyl-3-heptanone, 3-methyl-4-heptanone, 2-methyl-

3.4- heptanediol, 2-methyl-3,4-heptanedione, 6-methyl-3,4-heptanediol, 6-methyl-3,4- heptanedione, 5-methyl-3,4-heptanediol, 5-methyl-3,4-heptanedione, 2-methyl-3-hydroxy-4- heptanone, 2-methyl-4-hydroxy-3-heptanone, 6-methyl-3-hydroxy-4-heptanone, 6-methyl-4- hydroxy-3-heptanone, 5-methyl-3-hydroxy-4-heptanone, 5-methyl-4-hydroxy-3-heptanone, 2,6- dimethylheptane, 2,5-dimethylheptane, 2,6-dimethyl-2-heptene, 2,6-dimethyl-3-heptene, 2,5- dimethyl-2-heptene, 2,5-dimethyl-3-heptene, 3,6-dimethyl-3-heptene, 2,6-dimethyl-3-heptanol, 2,6-dimethyl-4-heptanol, 2,5-dimethyl-3-heptanol, 2,5-dimethyl-4-heptanol, 2,6-dimethyl-3,4- heptanediol, 2,6-dimethyl-3,4-heptanedione, 2,5-dimethyl-3,4-heptanediol, 2,5-dimethyl-3,4- heptanedione, 2,6-dimethyl-3-hydroxy-4-heptanone, 2,6-dimethyl-4-hydroxy-3-heptanone, 2,5- dimethyl-3-hydroxy-4-heptanone, 2,5-dimethyl-4-hydroxy-3-heptanone, n-octane, 1 -octene, 2- octene, 1-octanol, octanal, octanoate, 3-octene, 4-octene, 4-octanol, 4-octanone, 4,5-octanediol,

4.5- octanedione, 4-hydroxy-5-octanone, 2-methyloctane, 2-methyl-3 -octene, 2-methyl-4-octene,

7- methyl-3-octene, 3-methyl-3-octene, 3-methyl-4-octene, 6-methyl-3-octene, 2-methyl-4- octanol, 7-methyl-4-octanol, 3-methyl-4-octanol, 6-methyl-4-octanol, 2-methyl-4-octanone, 7- methyl-4-octanone, 3-methyl-4-octanone, 6-methyl-4-octanone, 2-methyl-4,5-octanediol, 2- methyl-4,5-octanedione, 3-methyl-4,5-octanediol, 3-methyl-4,5-octanedione, 2-methyl-4- hydroxy-5-octanone, 2-methyl-5-hydroxy-4-octanone, 3-methyl-4-hydroxy-5-octanone, 3- methyl-5-hydroxy-4-octanone, 2,7-dimethyloctane, 2,7-dimethyl-3-octene, 2,7-dimethyl-4- octene, 2,7-dimethyl-4-octanol, 2,7-dimethyl-4-octanone, 2,7-dimethyl-4,5-octanediol, 2,7- dimethyl-4,5-octanedione, 2,7-dimethyl-4-hydroxy-5-octanone, 2,6-dimethyloctane, 2,6- dimethyl-3-octene, 2,6-dimethyl-4-octene, 3,7-dimethyl-3-octene, 2,6-dimethyl-4-octanol, 3,7- dimethyl-4-octanol, 2,6-dimethyl-4-octanone, 3,7-dimethyl-4-octanone, 2,6-dimethyl-4,5- octanediol, 2,6-dimethyl-4,5-octanedione, 2,6-dimethyl-4-hydroxy-5-octanone, 2,6-dimethyl-5- hydroxy-4-octanone, 3,6-dimethyloctane, 3,6-dimethyl-3-octene, 3,6-dimethyl-4-octene, 3,6- dimethyl-4-octanol, 3,6-dimethyl-4-octanone, 3,6-dimethyl-4,5-octanediol, 3,6-dimethyl-4,5- octanedione, 3,6-dimethyl-4-hydroxy-5-octanone, n-nonane, 1-nonene, 1-nonanol, nonanal, nonanoate, 2-methylnonane, 2-methyl-4-nonene, 2-methyl-5-nonene, 8-methyl-4-nonene, 2- methyl-5-nonanol, 8-methyl-4-nonanol, 2-methyl-5-nonanone, 8-methyl-4-nonanone, 8-methyl- 4,5-nonanediol, 8-methyl-4,5-nonanedione, 8-methyl-4-hydroxy-5-nonanone, 8-methyl-5- hydroxy-4-nonanone, 2,8-dimethylnonane, 2,8-dimethyl-3-nonene, 2,8-dimethyl-4-nonene, 2,8- dimethyl-5-nonene, 2,8-dimethyl-4-nonanol, 2,8-dimethyl-5-nonanol, 2,8-dimethyl-4-nonanone, 2,8-dimethyl-5-nonanone, 2,8-dimethyl-4,5-nonanediol, 2,8-dimethyl-4,5-nonanedione, 2,8- dimethyl-4-hydroxy-5-nonanone, 2,8-dimethyl-5-hydroxy-4-nonanone, 2,7-dimethylnonane, 3,8-dimethyl-3-nonene, 3,8-dimethyl-4-nonene, 3,8-dimethyl-5-nonene, 3,8-dimethyl-4- nonanol, 3,8-dimethyl-5-nonanol, 3,8-dimethyl-4-nonanone, 3,8-dimethyl-5-nonanone, 3,8- dimethyl-4,5-nonanediol, 3,8-dimethyl-4,5-nonanedione, 3,8-dimethyl-4-hydroxy-5-nonanone, 3,8-dimethyl-5-hydroxy-4-nonanone, n-decane, 1-decene, 1-decanol, decanoate, 2,9- dimethyldecane, 2,9-dimethyl-3-decene, 2,9-dimethyl-4-decene, 2,9-dimethyl-5-decanol, 2,9- dimethyl-5-decanone, 2,9-dimethyl-5,6-decanediol, 2,9-dimethyl-6-hydroxy-5-decanone, 2,9- dimethyl-5,6-decanedionen-undecane, 1-undecene, 1-undecanol, undecanal. undecanoate, n- dodecane, 1-dodecene, 1-dodecanol, dodecanal, dodecanoate, n-dodecane, 1-decadecene, 1- dodecanol, ddodecanal, dodecanoate, n-tridecane, 1-tridecene, 1-tridecanol, tridecanal, tridecanoate, n-tetradecane, 1-tetradecene, 1-tetradecanol, tetradecanal, tetradecanoate, n- pentadecane, 1-pentadecene, 1-pentadecanol, pentadecanal, pentadecanoate, n-hexadecane, 1- hexadecene, 1-hexadecanol, hexadecanal, hexadecanoate, n-heptadecane, 1-heptadecene, 1- heptadecanol, heptadecanal, heptadecanoate, n-octadecane, 1-octadecene, 1-octadecanol, octadecanal, octadecanoate, n-nonadecane, 1-nonadecene, 1-nonadecanol, nonadecanal, nonadecanoate, eicosane, 1-eicosene, 1-eicosanol, eicosanal, eicosanoate, 3-hydroxy propanal, 1, 3-propanediol, 4-hydroxybutanal, 1, 4-butanediol, 3-hydrxy-2-butanone, 2, 3-butandiol, 1,5- pentane diol, homocitrate, homoisocitorate, b-hydroxy adipate, glutarate, glutarsemialdehyde, glutaraldehyde, 2-hydroxy-l-cyclopentanone, 1,2-cyclopentanediol, cyclopentanone,

cyclopentanol, (S)-2-acetolactate, (R)-2,3-Dihydroxy-isovalerate, 2-oxoisovalerate, isobutyryl- CoA, isobutyrate, isobutyraldehyde, 5 -amino pentaldehyde, 1,10-diaminodecane, 1,10-diamino- 5-decene, l,10-diamino-5-hydroxydecane, l,10-diamino-5-decanone, l,10-diamino-5,6- decanediol, l,10-diamino-6-hydroxy-5-decanone, phenylacetoaldehyde, 1,4-diphenylbutane, 1,4-diphenyl-l-butene, l,4-diphenyl-2-butene, l,4-diphenyl-2-butanol, l,4-diphenyl-2-butanone, l,4-diphenyl-2,3-butanediol, l,4-diphenyl-3-hydroxy-2-butanone, l-(4-hydeoxyphenyl)-4- phenylbutane, l-(4-hydeoxyphenyl)-4-phenyl- 1-butene, l-(4-hydeoxyphenyl)-4-phenyl-2- butene, l-(4-hydeoxyphenyl)-4-phenyl-2-butanol, l-(4-hydeoxyphenyl)-4-phenyl-2-butanone, 1- (4-hydeoxyphenyl)-4-phenyl-2,3-butanediol, l-(4-hydeoxyphenyl)-4-phenyl-3-hydroxy-2- butanone, l-(indole-3)-4-phenylbutane, 1- (indole- 3 )-4-phenyl-l -butene, l-(indole-3)-4-phenyl-

2- butene, l-(indole-3)-4-phenyl-2-butanol, l-(indole-3)-4-phenyl-2-butanone, 1- (indole- 3) -4- phenyl-2,3-butanediol, l-(indole-3)-4-phenyl-3-hydroxy-2-butanone, 4- hydroxyphenylacetoaldehyde, 1 ,4-di(4-hydroxyphenyl)butane, 1 ,4-di(4-hydroxyphenyl)- 1 - butene, l,4-di(4-hydroxyphenyl)-2-butene, l,4-di(4-hydroxyphenyl)-2-butanol, l,4-di(4- hydroxyphenyl)-2-butanone, 1 ,4-di(4-hydroxyphenyl)-2,3-butanediol, 1 ,4-di(4-hydroxyphenyl)-

3- hydroxy-2-butanone, l-(4-hydroxyphenyl)-4-(indole-3-)butane, l-(4-hydroxyphenyl)-4- (indole-3)- 1 -butene, l-di(4-hydroxyphenyl)-4-(indole-3)-2-butene, l-(4-hydroxyphenyl)-4- (indole-3)-2-butanol, l-(4-hydroxyphenyl)-4-(indole-3)-2-butanone, l-(4-hydroxyphenyl)-4- (indole-3)-2,3-butanediol, l-(4-hydroxyphenyl-4-(indole-3)-3-hydroxy-2-butanone, indole-3- acetoaldehyde, l,4-di(indole-3-)butane, l,4-di(indole-3)-l -butene, l,4-di(indole-3)-2-butene, l,4-di(indole-3)-2-butanol, l,4-di(indole-3)-2-butanone, l,4-di(indole-3)-2,3-butanediol, 1,4- di(indole-3)-3-hydroxy-2-butanone, succinate semialdehyde, hexane-l,8-dicarboxylic acid, 3- hexene-l,8-dicarboxylic acid, 3-hydroxy-hexane-l,8-dicarboxylic acid, 3-hexanone-l,8- dicarboxylic acid, 3,4-hexanediol-l,8-dicarboxylic acid, 4-hydroxy-3-hexanone-l,8-dicarboxylic acid, alkylene diacid, succinic acid, fumaric acid, adipic acid, 6-aminohexanoic acid, glycerol, 1,3-propanediol, 1,4-butanediol, acrylic acid, amino acid, alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, Isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine, nucleic acid, adenosine, thymidine, cytidine, guanosine, adenine, thymine, cytosine, guanine, fatty acid, formic acid, acetic acid, propionic acid, butanoic acid, hexanoic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid linoleic acid, linoelaidic acid, linolenic acid, arachidonic acid, eicosapentanoic acid, erucic acid, docosahexaenoic acid, isoprenoids, lycopene, astaxanthine, squalene, farnesene, isoprene, lactic acid, citric acid, fucoidan, iodine, chlorophyll, carotenoid, calcium, magnesium, iron, sodium, potassium, phosphate, and the like.

[0145] Accordingly, certain embodiments provide a method of producing a commodity chemical, by providing a yeast cell, where the yeast cell contains at least one recombinant polynucleotide encoding an oligoalginate transporter protein or permease; culturing the yeast cell with a fermentable substrate containing oligoalginate under conditions whereby the recombinant polynucleotide is expressed and a commodity chemical is produced; and collecting the commodity chemical, where expression of the recombinant polynucleotide results in transport of oligoalginate into the yeast cell, and wherein the yeast cell contains the proteins necessary for the yeast cell to metabolize oligoalginate. Exemplary oligoalginate transporter proteins or permeases are as described in previous sections. In other embodiments, the proteins necessary for the yeast cell to metabolize oligoalginate contain at least one protein selected from a DEHU reductase, a KDG kinase, and a KDGP aldolase. Exemplary DEHU reductases, KDG kinases, and KDGP aldolases are as described in previous sections.

[0146] In other embodiments, the yeast cell further contains at least one recombinant polynucleotide encoding an alginate lyase. Exemplary alginate lyases are as described in previous sections. In other embodiments, the proteins necessary for the yeast cell to metabolize oligoalginate contain at least one protein selected from a DEHU reductase, a KDG kinase, and a KDGP aldolase. Exemplary DEHU reductases, KDG kinases, and KDGP aldolases are as described in previous sections.

[0147] In certain other embodiments, the fermentable substrate containing oligoalginate further contains mannitol, the yeast cell further contains at least one recombinant polynucleotide encoding a mannitol transporter, the yeast cell is cultured under conditions whereby the recombinant polynucleotides encoding an oligoalginate transporter protein or permease and a mannitol transporter are expressed, expression of the recombinant polynucleotide encoding a mannitol transporter results in transport of mannitol into the yeast cell, and the yeast cell contains the proteins necessary for the yeast cell to metabolize mannitol. Exemplary mannitol transporters are as described in previous sections. In yet other embodiments, the proteins necessary for the yeast cell to metabolize mannitol contain at least one mannitol dehydrogenase. Exemplary mannitol dehydrogenases are as described in previous sections.

[0148] In still other embodiments, the fermentable substrate containing oligoalginate further contains mannitol, the yeast cell further contains at least one recombinant polynucleotide encoding a mannitol dehydrogenase, the yeast cell is cultured under conditions whereby the recombinant polynucleotides encoding an oligoalginate transporter protein or permease and a mannitol dehydrogenase are expressed, expression of the recombinant polynucleotide encoding a mannitol dehydrogenase results in mannitol reduction, and the yeast cell contains the proteins necessary for the yeast cell to metabolize mannitol. Exemplary mannitol dehydrogenases are as described in previous sections. In yet other embodiments, the proteins necessary for the yeast cell to metabolize mannitol contain at least one mannitol transporter. Exemplary mannitol transporters are as described in previous sections.

[0149] In yet other embodiments, the commodity chemical is selected from biofuels, polymer precursors, carbohydrates, fatty acids, fatty alcohols, amino acids, nucleic acids, and alcohols. In still other embodiments, the commodity chemical is selected from ethanol, butanol, isobutanol, n-butanol, 2-butanol, and biodiesel. Preferably, the biodiesel is selected from a fatty acid, a fatty acid ester, and a terpenoid.

[0150] In other embodiments, the commodity chemical is selected from alkylene diacid, succinic acid, fumaric acid, adipic acid, 6-aminohexanoic acid, glycerol, 1,3-propanediol, 1,4- butanediol, acrylic acid, amino acid, alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, tyrosine, nucleic acid, adenosine, thymidine, cytidine, guanosine, adenine, thymine, cytosine, guanine, fatty acid, formic acid, acetic acid, propionic acid, butanoic acid, hexanoic acid, caprylic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid, myristoleic acid, palmitoleic acid, sapienic acid, oleic acid, elaidic acid, vaccenic acid linoleic acid, linoelaidic acid, linolenic acid, arachidonic acid, eicosapentanoic acid, erucic acid,

docosahexaenoic acid, isoprenoids, lycopene, astaxanthine, squalene, farnesene, isoprene, lactic acid, and citric acid.

[0151] In certain preferred embodiments, the commodity chemical is selected from succinic acid, fumaric acid, adipic acid, 6-aminohexanoic acid, glycerol, 1,3-propanediol, 1,4-butanediol, and acrylic acid. In other preferred embodiments, the commodity chemical is selected from alanine, cysteine, aspartate, glutamate, phenylalanine, glycine, histidine, Isoleucine, lysine, leucine, methionine, asparagine, proline, glutamine, arginine, serine, threonine, valine, tryptophan, and tyrosine. In further preferred embodiments, the commodity chemical is selected from adenosine, thymidine, cytidine, guanosine, adenine, thymine, cytosine, and guanine. [0152] In other embodiments, the fermentable substrate containing oligoalginate is selected from kelp, giant kelp, sargasso, seaweed, algae, brown algae, brown seaweed, marine microflora, microalgae, sea grass, and combinations thereof.

[0153] In certain embodiments, a bacterial organism such as E. coli may be used to metabolize alginate and/or oligoalginate and the products of alginate and/or oligoalginate metabolism may then be isolated and combined with engineered yeast to produce commodity chemicals such as ethanol and butanol. For example, see U.S. Patent Application Publication Nos. US 2009-0139134, US 2009/0155873, and US 2010/0185017.

Recombinant Yeast Cells

[0154] Certain aspects of the present disclosure relate to a recombinant yeast cell containing at least one recombinant polynucleotide encoding an oligoalginate transporter protein or permease, where expression of the recombinant polynucleotide results in transport of oligoalginate into the recombinant yeast cell, and contains the proteins necessary for the recombinant yeast cell to metabolize oligoalginate.

[0155] "Recombinant polynucleotide" or "recombinant nucleic acid" as used herein refers to a polymer of nucleic acids wherein at least one of the following is true: (a) the sequence of nucleic acids is foreign to {i.e., not naturally found in a given yeast cell); (b) the sequence may be naturally found in a given yeast cell, but in an unnatural {e.g., greater than expected) amount; or (c) the sequence of nucleic acids contains two or more subsequences that are not found in the same relationship to each other in nature. For example, regarding instance (c), a recombinant nucleic acid sequence will have two or more sequences from unrelated genes arranged to make a new functional nucleic acid. Specifically, the present disclosure is related to the introduction of an expression vector into a yeast cell, wherein the expression vector contains a nucleic acid sequence coding for a protein that is not normally found in a yeast cell or contains a nucleic acid coding for a protein that is normally found in a yeast cell but is under the control of different regulatory sequences. With reference to the yeast cell's genome, then, the nucleic acid sequence that codes for the protein is recombinant.

[0156] The term "recombinant yeast cell" refers to a cell into which a recombinant vector has been introduced or recombinant DNA has been integrated into its chromosome. It should be understood that such terms are intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term "recombinant yeast cell" as used herein.

[0157] The recombinant yeast cells of the present disclosure express proteins necessary for metabolizing oligoalginate and/or mannitol. The coding regions of the desired proteins may be heterologous to the yeast cell or endogenous to the yeast cell, but are operatively linked to heterologous promoters and/or terminators resulting in a different expression level of the coding region in the yeast cell. As used herein with reference to a nucleic acid or protein and a particular yeast cell, endogenous may refer to a nucleic acid or protein that is present in the cell and was not introduced into the cell using recombinant techniques (e.g. , a gene found in the cell when it was originally isolated from nature). In contrast, exogenous may refer to a nucleic acid or protein that is not present in the cell (e.g. , foreign nucleic acid or protein) and was introduced into the cell using recombinant techniques.

[0158] Heterologous, as used in reference to a coding region of a protein of interest and flanking sequences, such as a 5' promoter and/or UTR and a 3' terminator, may indicate that the flanking sequences are non-native to the coding region. For instance, a PGK1 promoter and an ENOlt terminator are heterologous to an oligoalginate transporter coding region.

[0159] Genetically engineered or genetically modified may refer to any recombinant DNA or RNA method used to create a yeast cell that expresses a protein at elevated levels, at lowered levels, or in a mutated form. In other words, the yeast cell has been transfected, transformed, or transduced with a recombinant polynucleotide molecule, and thereby has been altered so as to cause the cell to alter expression of a desired protein. Methods and vectors for genetically engineering yeast cells are well known in the art; for example various techniques are illustrated in Current Protocols in Molecular Biology, Ausubel et ah, eds. (Wiley & Sons, New York, 1988, and quarterly updates).

[0160] Genetic modifications that result in an increase in gene expression or function can be referred to as amplification, overproduction, overexpression, activation, enhancement, addition, or up-regulation of a gene. More specifically, reference to increasing the action (or activity) of enzymes or other proteins discussed herein generally refers to any genetic modification of the host cell in question which results in increased expression and/or functionality (biological activity) of the enzymes or proteins and includes higher activity or action of the proteins (e.g. , specific activity or in vivo enzymatic activity), reduced inhibition or degradation of the proteins, and overexpression of the proteins. For example, gene copy number can be increased, expression levels can be increased by use of a promoter that gives higher levels of expression than that of the native promoter, or a gene can be altered by genetic engineering or classical mutagenesis to increase the biological activity of an enzyme or action of a protein.

Combinations of some of these modifications are also possible.

[0161] In general, according to the present disclosure, an increase or decrease in a characteristic of a genetically modified yeast cell (e.g. , enzyme expression) is made with reference to the same characteristic of a reference yeast cell (e.g., wild- type yeast cell of the same species, preferably the same strain or a recombinant yeast cell of the same species, preferably the same strain, which has been transformed with an expression vector of a multi- enzyme pathway), under the same or equivalent conditions. Such conditions include the assay or culture conditions (e.g. , medium components, temperature, pH, etc.) under which the activity of the protein or other characteristic of the host cell is measured, as well as the type of assay used. Equivalent conditions are conditions (e.g. , culture conditions) which are similar, but not necessarily identical (e.g. , some conservative changes in conditions can be tolerated), and which do not substantially change the effect on cell growth or enzyme expression or biological activity as compared to a comparison made under the same conditions.

[0162] Additionally, yeast strains that grow on oligoalginate and mannitol may be constructed by engineering haploid yeast strains that only utilize one of the two carbon sources, and then mating the haploid strains to generate a diploid yeast strain that can utilize both carbon sources. Methods of generating haploid and diploid yeast strains are well known in the art. In one non-limiting example, a MAT A yeast strain containing the genes necessary for growth on oligoalginate and a MAT a strain containing all the genes necessary for growth on mannitol can be constructed, and then the MAT A and MAT a strains can be mated to generate a diploid strain that has all the genes necessary to grow on both oligoalginate and mannitol. Homologous proteins

[0163] It should be noted that the oligoalginate transporter proteins, permeases, alginate lyases, DEHU reductases, KDG kinases, KDGP aldolases, mannitol transporters, and mannitol dehydrogenases disclosed herein can be readily replaced using a homologous protein thereof. A homologous protein is a protein that has a polypeptide sequence that is at least 70 %, 75 %, 80 %, 85 %, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical or 100% identical to any one of the proteins described in the present disclosure or in an incorporated reference.

[0164] As used herein, the terms percent "identical," "percent identity," and "percent sequence identity" are defined as amount of identity between a reference nucleic acid or amino acid sequence and at least one other nucleic acid or amino acid sequence. Percent sequence identity can be determined by comparing two optimally aligned sequences, wherein the portion of the sequence being compared may contain additions or deletions (i.e. , gaps) as compared to the reference sequence (e.g., a nucleic acid or amino acid sequence of the disclosure), which does not contain additions or deletions, for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions being compared and multiplying the result by 100 to yield the percentage of sequence identity. Two sequences have percent identity if two sequences have a specified percentage of nucleic acids or amino acid residues that are the same (i.e., 75% identical over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.

[0165] One example of an algorithm that is suitable for determining percent sequence identity is the BLAST algorithm, which is described in Altschul et al. (1977) Nuc. Acids Res. 25:3389-3402. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. The BLASTN program (used for nucleic acid sequences) uses as defaults a wordlength (W) of 11, an expectation (E) or 10, M=5, N=-4 and a comparison of both strands. For amino acid sequences, the BLASTP program is used with default settings of a wordlength of 3, and expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Natl. Acad. Sci. USA 89: 10915) alignments (B) of 50, expectation (E) of 10, M=5, N=-4, and a comparison of both strands.

[0166] Homologous proteins retain amino acids residues that are recognized as conserved for the protein. The homologous protein may have non-conserved amino acid residues replaced or found to be of a different amino acid, or amino acid(s) inserted or deleted, but which do not affect or has insignificant effect on the activity of the homologous protein. The homologous protein has an activity that is essentially the same as the activity of any one of the protein described herein or in an incorporated reference in that it will catalyze the same reaction. The specific activity of the protein may be increased or decreased. The homologous protein may be found in nature, be an engineered mutant thereof, or be a synthetic protein thereof. The proteins described herein can also be replaced by an isozyme, a protein that may differ in amino acid sequence but that catalyzes the same chemical reaction.

Suitable Yeast Cells

[0167] Any yeast cell may be used in the present disclosure so long as it remains viable after being transformed with a sequence of nucleic acids. Preferably, the yeast cell is not adversely affected by the transduction of the necessary nucleic acid sequences, the subsequent expression of the proteins (e.g., enzymes), or the resulting intermediates. Suitable yeast cells include, without limitation, industrial yeast strains, laboratory yeast strains, and wild- type yeast strains. Suitable yeast strains include, without limitation, those that are tolerant to salt stress, thermal stress, and osmotic stress. Additionally, suitable yeast strains may have high tolerance to ethanol. Suitable yeast strains may further be capable of high ethanol production, and have a high ethanol yield.

[0168] In certain embodiments, the yeast cell is Candida, Hansenula, Kluyveromyces, Pichia, Saccharomyces, Schizosaccharomyces, or Yarrowia. In other embodiments, the yeast cell is a Saccharomyces carlsbergensis, Saccharomyces cerevisiae, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces monacensis, Saccharomyces bayanus, Saccharomyces pastorianus, Schizosaccharomyces pombe, or Saccharomyces oviformis. In yet other embodiments, the yeast cell is Kluyveromyces lactis, Kluyveromyces marxiamus, Pichia stipitis, Pichia pastoris, or Candida shehatae. In still other embodiments, the yeast cell is Yarrowia lipolytica, Brettanomyces custersii, or

Zygosaccharomyces roux. In preferred embodiments, the yeast cell is a Sacchawmyces sp.

[0169] In certain embodiments, the Sacchawmyces sp. is an industrial Sacchawmyces strain, such as those used in bioethanol production, as well as specific gene polymorphisms that are important for bioethanol production (Argueso et ah, Genome Research, 19: 2258-2270, 2009). Industrial yeast strains include, without limitation, Thermosacc, Stress tolerant yeast, N96, Lalvin/Bourgovin, Ethanol Red, Pasteur Red, Acid tolerant Brazilian yeast, S. cerevisiae D5A, S. cerevisiae Y1582, S. cerevisiae Y12687, and S. pastorianus. The yeast cells of the present disclosure is genetically modified in that recombinant nucleic acids have been introduced into the yeast cells, and as such the genetically modified yeast cells do not occur in nature.

[0170] Accordingly, in certain embodiments the recombinant yeast cell is selected from

Sacchawmyces sp., Sacchawmyces carlsbergensis, Sacchawmyces cerevisiae, Sacchawmyces diastaticus, Sacchawmyces douglasii, Sacchawmyces kluyveri, Sacchawmyces norbensis, Sacchawmyces monacensis, Sacchawmyces bayanus, Sacchawmyces pastorianus,

Schizosaccharomyces pombe, or Sacchawmyces oviformis Kluyveromyces lactis,

Kluyveromyces marxianus, Pichia stipitis, Pichia pastoris, Candida shehatae, Yarrowia lipolytica, Brettanomyces custersii, Zygosaccharomyces roux, Sporotrichum thermophile, Candida shehatae, and Neurospora crassa.

Embodiments Relating to Recombinant Yeast Cells

[0171] Certain embodiments relate to a recombinant yeast cell containing at least one recombinant polynucleotide encoding an oligoalginate transporter protein or permease, where expression of the recombinant polynucleotide results in transport of oligoalginate into the recombinant yeast cell, and the proteins necessary for the recombinant yeast cell to metabolize oligoalginate. In other embodiments, the oligoalginate transporter protein is selected from the group of a GPH transporter, a pectin/cellodextrin transporter, a hexose transporter, an ABC transporter, and a transporter from marine fungi. Exemplary GPH transporters,

pectin/cellodextrin transporters, hexose transporters, ABC transporters, transporters from marine fungi, and permeases are as described in previous sections. In certain embodiments, the recombinant yeast cell contains at least 2, at least 3, at least 4, at least 5, at least 6, or more recombinant polynucleotides encoding an oligoalginate transporter protein or permease.

[0172] In certain embodiments, the proteins necessary for the recombinant yeast cell to metabolize oligoalginate contain at least one DEHU reductase. Exemplary DEHU reductases are as described in previous sections. In yet other embodiments, the proteins necessary for the recombinant yeast cell to metabolize oligoalginate contain at least one KDG kinase. Exemplary KDG kinases are as described in previous sections. In further embodiments, the proteins necessary for the recombinant yeast cell to metabolize oligoalginate contain at least one KDGP aldolase. Exemplary KDGP aldolases are as described in previous sections. In certain embodiments, the proteins necessary for the recombinant yeast cell to metabolize oligoalginate contain at least one DEHU reductase, at least one KDG kinase, and at least one KDGP aldolase. In other embodiments, the recombinant yeast cell produces pyruvate from oligoalginate.

[0173] In other embodiments, the recombinant yeast cell further contains at least one alginate lyase. In other embodiments, the proteins necessary for the recombinant yeast cell to metabolize oligoalginate contain at least 2, at least 3, at least 4, at least 5, or more alginate lyases. Exemplary alginate lyases are as described in previous sections. In other embodiments, the proteins necessary for the recombinant yeast cell to metabolize oligoalginate contain at least one alginate lyase.

[0174] In certain embodiments the recombinant yeast cell contains at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or more proteins necessary for the yeast cell to metabolize oligoalginate. In other embodiments, the oligoalginate is selected from short alginate polymers, alginate pentamers, alginate tetramers, alginate trimers, alginate dimers, and alginate monomers. In yet other embodiments, the substrate containing oligoalginate is selected from kelp, giant kelp, sargasso, seaweed, algae, brown algae, brown seaweed, marine microflora, microalgae, sea grass, and combinations thereof.

[0175] Certain embodiments relate to the recombinant yeast cell further containing at least one recombinant polynucleotide encoding a mannitol transporter, where expression of the recombinant polynucleotide results in transport of mannitol into the recombinant yeast cell; and the proteins necessary for the recombinant yeast cell to metabolize mannitol. Exemplary mannitol transporters are as described in previous sections. In other embodiments, the proteins necessary for the recombinant yeast cell to metabolize mannitol contains at least one mannitol dehydrogenase. Exemplary mannitol dehydrogenases are as described in previous sections.

[0176] In other embodiments, the recombinant yeast cell further contains at least one recombinant polynucleotide encoding a mannitol dehydrogenase, where expression of the recombinant polynucleotide results in mannitol oxidation; and the proteins necessary for the recombinant yeast cell to metabolize mannitol. Exemplary mannitol dehydrogenases are as described in previous sections. In other embodiments, the proteins necessary for the

recombinant yeast cell to metabolize mannitol contains at least one mannitol transporter.

Exemplary mannitol transporters are as described in previous sections.

[0177] In yet other embodiments, the proteins necessary for the recombinant yeast cell to metabolize oligoalginate and mannitol contain at least one DEHU reductase, at least one KDG kinase, at least one KDGP aldolase, and at least one mannitol dehydrogenase. Exemplary DEHU reductases, KDG kinases, KDGP aldolases, and mannitol dehydrogenases are as described in previous sections. In further embodiments, the recombinant yeast cell produces pyruvate from oligoalginate, and fructose 6-phosphate from mannitol.

[0178] In further embodiments, the proteins necessary for the recombinant yeast cell to metabolize oligoalginate and mannitol contain at least one alginate lyase, at least one DEHU reductase, at least one KDG kinase, at least one KDGP aldolase, and at least one mannitol dehydrogenase. Exemplary alginate lyases, DEHU reductases, KDG kinases, KDGP aldolases, and mannitol dehydrogenases are as described in previous sections. In further embodiments, the recombinant yeast cell produces pyruvate from oligoalginate, and fructose 6-phosphate from mannitol.

[0179] It is to be understood that, while the methods and compositions disclosed herein have been described in conjunction with the preferred embodiments thereof, the foregoing description is intended to illustrate and not limit the scope thereof as defined in the appended claims. Other aspects, advantages, and modifications within the scope thereof as defined in the appended claims will be apparent to those skilled in the art to which the present disclosure pertains. [0180] All patents, patent applications, and publications mentioned herein are hereby incorporated by reference in their entireties.

[0181] The following Examples are merely illustrative and are not meant to limit any aspects of the present disclosure in any way.

EXAMPLES

Example 1: Analysis of a cDNA Library to Identify Putative Alginate Transporters

[0182] The following example describes the construction of a transporter cDNA library from genomic analysis of Asteromyces cruciatus and Dendryphiella salina to identify putative alginate transporters.

Transporter sources

[0183] Naturally occurring organisms that are able to consume alginate and/or oligoalginate were identified. For example, Asteromyces cruciatus and Dendryphiella salina have been reported to metabolize alginate. Growth on alginate or its alginate lyase degradation products {e.g., oligoalginate) was confirmed. Samples of mRNA from A. cruciatus and D. salina were isolated and high-throughput sequencing was performed to computationally mine A. cruciatus and D. salina for additional putative alginate transporters. The sequencing was performed using the niumina GS system. Assembly of the cDNA or contigs was performed using sequence analysis software, and generated a database of contigs that serve as templates for BLAST queries to find candidate transporter genes. A set of cDNAs encoding putative transporters expressed and/or up regulated during growth on alginate (compared to glucose) as carbon source was then identified. The identified cDNAs for putative transporters were directly amplified from an mRNA prep or were synthesized to be cloned into the expression vector p425CYC (Fig.16).

Construction of random cDNA library from Asteromyces cruciatus and Dendryphiella salina

[0184] Alternatively, total RNA was isolated from 1 L-scale cultures of A. cruciatus (BAL791) and D. salina (BAL788) grown on oligoalginate as the sole carbon source in a controlled environment (fixed temperature, pH, d0 ₂ ) using the QIAgen RNeasy Plant Mini Kit. Samples of mRNA were then purified with Invitrogen's Dynabead mRNA purification kit and used as the input for cDNA library construction with Invitrogen 's Superscript Full Length cDNA Library Construction Kit. cDNA inserts were transferred into a Gateway cloning-compatible yeast expression vector prior to transformation into host strains for screening.

Screening and identification of oligoalginate transporters

Host strains:

[0185] Host yeast strains contained the pathway genes for metabolism of DEHU to pyruvate integrated into the genome. The strains included one or more of the genes shown in Table 15.

TABLE 15

[0186] The genes were integrated into one or more of the following genomic loci: LEU2, LYS2, ADE2, HIS3, URA3, or CAN1. Strains were either diploid or haploid, and contained insertions at one or both loci. Some strains also included an oligoalginate lyase (OAL) integrated into the genome. Table 16 shows the sources of OAL.

TABLE 16

[0187] Alginate degradation pathway genes were expressed under different promoters. Host strains used the TEF2, TDH3, FBAl, or ACTl promoter and 5'UTR for expression of the gene.

[0188] Host yeast strains were derived from a number of laboratory or industrial

backgrounds including, but not limited to, S288C and NCYC3076.

Specific activity assays:

[0189] The specific activity of enzymes, such as oligoalginate lyase, DEHU reductase, KDG kinase, and KDGP aldolase, was determined using the in vitro described in Example 5 below.

Strains:

[0190] The genotypes of engineered yeast strains that were used for screening and identification of functional oligoalginate transporters are listed in the Table 17.

TABLE 17

Gene definition:

kdgK(Sd): KDG kinase from Sac char ophagus degradans 2-40

kdgpA(Vs): KDGP aldolase from Vibrio splendidus 12B01 DEHUH(Sph): DEHU reductase from Sphingomonas sp. Al OAL(At): exo-alginate lyase from Agrobacterium tumefaciens C58 Results

[0191] The specific activity of the oligoalginate lyase, DEHU reductase, KDG kinase, and KDGP aldolase of the S. cerevisiae strains BAL2139 and BAL2438 was determined from crude lysates for each enzymatic step. These results are shown in Table 18.

TABLE 18

[0192] The screen was carried out in deep well plates containing minimal media (YNB, lx CSM, lOOmM DEHU). Each strain was independently transformed with a plasmid encoding a putative transporter. To minimize background growth, a yeast minimal medium consisting of Yeast Nitrogen Base (YNB) without ammonium sulfate, supplemented with Complete

Supplement Mixture -uracil (from Sunrise Science Products) was used. 100 mM DEHU

(concentration quantified by DEHU reductase assays) was added to the experimental wells as the carbon source, and water was added to the negative controls (2-4 replicates of each, dependent on the screen). Assays were done in 96 well deep well plates, and the growth was done at 29°C, with 90% humidity, and shaking at 950 rpm.

[0193] The results of the screen are shown in Table 19. Using this method, the transporters "e20" and "el 15" were identified from the library of putative transporters. A third transporter ("el74") was identified during outgrowth after transformation of the random cDNA library of A. cruciatus into BAL2193. All three transporters were identified as DEHU transporters.

TABLE 19

[0194] The nucleic acid of the genes encoding the identified transporter proteins, as well as the amino acid sequences of the proteins are listed below.

1. Nucleic acid sequence encoding e20 (SEQ ID NO: 196)

ATGTCCTTGTTGAAAAACTATCGTGTCTACCTTTTAACTGCAGTGGCCTATTCAGGA

TCTCTTCTCTTTGGTTATGATACTGGAGTTATGGGTAGTGTGTTGTCCTTGACTTCA T

TCAAGGAAGATTTTGGTATTCCTACAGGAAGTTCTGGGTTTGCTTCATCCAAATCAT

CTGAAATTAGTTCCAATGTCGTATCACTGTTAACTGCAGGCTGTTTCTTTGGCGCCA T

TTTTGCAGCACCATTGAATGAACGTATTGGTAGAAGATACGCACTCATGATCTTTAC

AGTGATATTTCTGATAGGAGCTGCTGTTCAAGTTGCATCAAAACATCACATAGGTCA

GATCTACGGAGGAAGAGTCATAGCAGGATTAGGTATCGGTGGTATGAGTTCCATCA

CTCCAGTGTTTGTTTCAGAAAATTGTCCACCAAGTATCCGTGGCAGAGTAGCTGGAA

TGTTCCAAGAGTTTTTAGTTATTGGCAGTACCTTTGCCTACTGGTTAGATTACGGTG T

ATCATTGCATATTCCATCCTCAACAAAACAATGGAGAGTACCTGTTGCGGTTCAGTT

GATACCTGGTGGACTTATGTTATTAGGTTTGTTTTTCCTCAAGGAATCACCAAGATG

GCTAGCTGGGAAGGGTCGACATGAGGAAGCTTTACAATCTCTGGCGTACATTAGGA

ACGAATCTCCTGACTCCGAAGAGATCCAAAAAGAGTTCGCTGAGATCAGAGCAGCT

ATAGACGAAGAGGTAGCCGCCACAGAAGGCCTCACATATAAAGAGTTTATCCAACC

TTCTAATCTAAAAAGATTTGGTTTCGCCTTTACGCTTATGTTGAGCCAACAATTCAC T

GGGACTAATTCTATAGGGTACTATGCTCCTGAAATCTTTCAAACTATTGGACTATCT

GCGACAAACTCTTCTCTGTTTGCCACTGGCGTGTACGGTACAGTAAAAGTCGTGGCT

ACAGCAATCTTCCTATTCGTAGGTATTGACAGATGGGGTAGAAAGTTATCTTTGGTT

GGAGGCTCTATCTGGATGGCTTCTATGATGTTTATCATTGGTGCTGTGTTGGCAACC

CACCCACCAGATACGTCTGCAAGCGGTGTTAGTCAAGCTTCTATCGCTATGGTTGTC

ATGATCTACCTGTACGTTATCGGCTACTCAGCCTCTTGGGGTCCTACCCCATGGGTT T

ATGTCTCAGAAATTTTCCCAACAAGGCTTAGATCATACGGCGTAGGGTTGGCCGCA

ACCTCTCAATGGCTATGGTCTTTTGTCGTTACAGAAATTACTCCAAAGGCGGTTCAT

AACATCGGGTGGAGAACATTCCTAATGTTTGGTATTTTCTGCGTTGCAATGTGTGTA

TTCGTGATTGTTTTCGCCAAGGAAACAAAAGGTAGGAGCCTTGAAGATATGGACAT

CTTATTTGGAGCAGTTAATGAAGCTGATAGAAGAGCTGCTGTAGAACACACCATGC

ACAAAAGAGGCTCATCTCATATAGAAGATGTCGATGAGGAAACAGAGAGAGTTAG

ACATGAACAGGACAAGGTGTAA

2. Amino acid sequence of e20 (SEP ID NO: 197)

MSLLKNYRVYLLTAVAYSGSLLFGYDTGVMGSVLSLTSFKEDFGIPTGSSGFASSKS SEI

SSNVVSLLTAGCFFGAIFAAPLNERIGRRYALMIFTVIFLIGAAVQVASKHHIGQIY GGRV

IAGLGIGGMSSrrPVFVSENCPPSIRGRVAGMFQEFLVIGSTFAYWLDYGVSLHIPS STKQ

WRVPVAVQLIPGGLMLLGLFFLKESPRWLAGKGRHEEALQSLAYIRNESPDSEEIQK EF

AEIRAAIDEEVAATEGLTYKEFIQPSNLKRFGFAFTLMLSQQFTGTNSIGYYAPEIF QTIGL

SATNSSLFATGVYGTVKVVATAIFLFVGIDRWGRKLSLVGGSr MASMMFIIGAVLATH

PPDTSASGVSQASIAMVVMIYLYVIGYSASWGPTPWVYVSEIFPTRLRSYGVGLAAT SQ

WLWSFVVTEITPKAVHNIGWRTFLMFGIFCVAMCVFVIVFAKETKGRSLEDMDILFG AV

NEADRRAAVEHTMHKRGSSHIEDVDEETERVRHEQDKV- 3. Nucleic acid sequence encoding el 15 (SEQ ID NO: 133)

ATGGGGATCCTCGACAAACTGATTAAGAATGAATCCATGAAAAGTGACCCGAAAGA

GATATACGGATGGCGCATTTGGGCTCTGGCTTCATCCGCCTGCTTCGGCGGTATGCT

GTTTGGCTGGGACATAGGTGCTATTGGTGGAATTCTGGTGATGCCAAGTTTCCAAGA

AAAGTTCGGTCTCGCAGACAAGACGGACAGCGAAGTCGCTGATGTGACGAGCAACA

TTGTATCCGTGCTCCAGGCGGGATGCTTTGCAGGATCTCTGATTGCCTATTGGATTG

CAGACCGCTGGGGTCGAAAGCCGTCTCTACTTATCTCCGCTTCTATCGCCATTGTTG

GCGTGGTGATACAAACAGCGTCTCTGGGACATCTGGCCGCTCTCTTCGTGGGCCGTT

TCCTAGCCGGTCTCGGTGTTGGATCAGCCTCTATGTTAACTCCGCTCTACGTCTCTG A

GAACGCACCTCGATCCATTCGGGGAGCGCTGACAGGCCTTTACCAGCTGAATATCA

CGATTGGTATCATGTTGTCTTTCTGGGTCAATTTTGGGTCCCTCAAGCATAGCAAAG

GCGATAGTCAGTGGGAGGTACCTCTCGCTACTCAGATGCTTGCGGCAGTCTTCATGT

TTGCTGGTATCAGCCTCTGTGGAGAGTCTCCCCGCTATCTCGCTAAACAAGATAACT

GGGAATCAGCCACCACCGTCCTGTCCCAACTTCGAAATCTTCCGGCCGATCACCCCT

ATGTTGTCCAGGAGCTTCAGGATATGGCTGAACAGCTCGAGAAAGAACAAATTCAC

TCGGACAACTCTTTTATGGGCCTTCACCGCGATATGTGGACTATTCCAACCAATCGC

AATCGCGCACTTATATCCATTGGACTTATGGTATGCCAACAGATGACTGGTGTTAAT

GCCATCAATTACTATGCCCCAACGATCTTCAACGGCCTAGGTATCCAGGGCCCTTCC

AACGGTCTCTTTGCCACTGGTGTTTATGGAATCGTTAAAGTTTTTGGCGTCATCCTC T

TCGTTTTATTTGTCGCGGACTCTGTTGGGCGAAGGCGCTGCCTCCTCTGGACGGCTA

TTGGGCAGGGCATCTTCATGTTCATCATTGGTTGTTACGTTCTTACCAGTCCTCCAG T

GGAAGGGGAGCCTATTCCTGCATTTGGATATGTCGCCCTAGTTTCTATTTTCCTCTT T

GTGCTTTGCTTTGAGGTGGGCTGGGGACCTGCATGCTGGATTCTCGTTTCAGAGATT

CCAGCAGCACGTCTTCGCGCCTTGAATGTAGCCCTCGCCGCCGCTACTCAATGGCTC

TTCAACTTTGTTGTTGCTCAAGCCGTTCCACGCATGCTGATAACAACTGGCGAGGGT

GGGTACGGAACTTATTTTATCTTTGGCTCATTTTCTTTTTCCATGTTTTTCTTTGTC TG

GTTCCTCATCCCGGAGACGAAAGGTGTATCGCTTGAGAAAATGGACGAATTGTTCG

GTGTTAAGCCATCGTTCACGGGGGGTGAGGAAGGAAATGTAGGATTCCTTGGGAAA

TCTGGACCTCACACGGACACGGAAGAAGCAGTGTCAGGAAAATCTGGCGAGGCTAC

TCATATTGAACGCAAATAA

4. Amino acid sequence of el 15 (SEP ID NO: 134)

MGILDKLIKNESMKSDPKEIYGWRIWALASSACFGGMLFGWDIGAIGGILVMPSFQE KF

GLADKTDSEVADVTSNIVSVLQAGCFAGSLIAYWIADRWGRKPSLLISASIAIVGVV IQT

ASLGHLAALFVGRFLAGLGVGSASMLTPLYVSENAPRSIRGALTGLYQLNITIGIML SFW

VNFGSLKHSKGDSQWEVPLATQMLAAVFMFAGISLCGESPRYLAKQDNWESATTVLS Q

LRNLPADHPYVVQELQDMAEQLEKEQIHSDNSFMGLHRDMWTIPTNRNRALISIGLM V

CQQMTGVNAINYYAPTIFNGLGIQGPSNGLFATGVYGIVKVFGVILFVLFVADSVGR RR

CLLWTAIGQGIFMFIIGCYVLTSPPVEGEPIPAFGYVALVSIFLFVLCFEVGWGPAC WILV

SEIPAARLRALNVALAAATQWLFNFVVAQAVPRMLITTGEGGYGTYFIFGSFSFSMF FFV

WFLIPETKGVSLEKMDELFGVKPSFTGGEEGNVGFLGKSGPHTDTEEAVSGKSGEAT HI

ERK- 5. Nucleic acid sequence encoding e!74 (SEQ ID NO: 198)

ATGGGGATCCTCGACAAGCTGATCAAAAATGAATCCATGAAAAGCGACCCGAAGG

AAATATACGGATGGCGCATTTGGGCTTTGGCTTCATCCGCCTGCTTCGGCGGTATGT

TGTTTGGTTGGGACATAGGTGCTATTGGTGGAATTCTGGTGATGCCAAGTTTCCAAG

AAAAGTTCGGTCTTGCAGAAAAGTCGGAAAGCGAACTCGCTGATGTGGAGAGCAAC

ATTGTATCCGTGCTCCAGGCGGGATGCTTTGCAGGATCTCTGATCGCCTATTGGATT

GCAGACCGCTGGGGTCGCAAGCCATCTCTACTGGCCTCCGCTGCTATGTCCACCATT

GGCGTGGTGATACAAACAGCGTCTTCGGGACATCTGGCCGCTCTCTTCGTGGGCCGT

TTCTTAGCCGGTCTCGGTGTTGGAGCAGCCTCTATGTTGACTCCGCTCTACGTCTCT G

AGAATGCACCTCGCTCCATTCGAGGAGCGCTAACAGGCCTTTACCAGCTGAATATC

ACGATTGGCATCATGCTGTCTTTCTGGGTCAATTTTGGGTCCCTCAGGCATAGCGAA

GGCGATATTCAGTGGGAGGTACCTCTCGCTACTCAGATGCTTGCGGCTGTCTTTATG

TTTGTTGGTATCAGCCTCTGTGGAGAGTCTCCCCGCTTTCTCGCTAAACAAGATAAC

TGGGAAGCTGCCTCCGCCGTTCTGTCCAAACTTCGAAATCTTCCGGCCGACCACACT

TATATTGCCCAGGAGCTTCAGGATATGGCTGATCAGCTCGAGAAAGAACGAGGTCA

CTCCGACAACAACTCTTTTTGGGGCCTTCACCGCGATATGTGGACTGTTCCAACCAA

TCGCAAGCGCGCGCTTATATCCATCGGACTCATGATATGCCAACAGATGACTGGTGT

CAATGCTATCAATTACTATGCCCCAAAAATCTTCAACGGCCTAGGTATTCAGGGCCC

TTCCAACGGTCTCTTTGCCACTGGTGTTTACGGAATCGTCAAAGTTGTTGGCTGCGC

CCTTTTCGTTTTATTCGCCGCGGACTCTGTTGGGAGAAGGCTTTCCCTCCTCTGGAC G

GCTATTGCCCAGGGCATCTTCATGTTCATCATTGGTTGCTACGTTCTTACCAATCCT C

CGGTCGAAGGGGCGCCTATACCAGCATTTGGATATGTTGCCCTAGTCTCTATTTTTC

TGTTTGTGCTTTGCTTTGAGGTGGGCTGGGGGCCGGCATGCTGGATTCTCGTCTCAG

AGATTCCACAAGCACGTCTTCGCGCATTGAATGTAGCCCTCGCCGCCGCTACACAAT

GGCTCTTCAACTTTGTTGTTGCTCAAGCCGTCCCTCACATGTTAATCACGACTGGCG

AGGGTGGGTACGGAACTTATTTCATCTTTGGCTCGTTTTCTTTTTGCATGTTTTTCT TT

ACCTGGTTCCTCATCCCCGAGACTAAAGGTGTGTCGCTTGAGAAAATGGACGCATTG

TTCGGTGTCAAGGCACCTTTGGGGGGTGAGGAAGGGAATCCAGAATTCCTCGAGAA

GTCTGGGCCTCACACGGACGCGGATGCAGGGTCAGGAAAATTTGCCGAGACTACTC

ATATTGAACGCAAATGA

6. Amino acid sequence of e!74 (SEP ID NO: 199)

MGILDKLIKNESMKSDPKEIYGWRIWALASSACFGGMLFGWDIGAIGGILVMPSFQE KF

GLAEKSESELADVESNIVSVLQAGCFAGSLIAYWIADRWGRKPSLLASAAMSTIGVV IQT

ASSGHLAALFVGRFLAGLGVGAASMLTPLYVSENAPRSIRGALTGLYQLNITIGIML SFW

VNFGSLRHSEGDIQWEVPLATQMLAAVFMFVGISLCGESPRFLAKQDNWEAASAVLS K

LRNLPADHTYIAQELQDMADQLEKERGHSDNNSFWGLHRDMWTVPTNRKRALISIGL

MICQQMTGVNAINYYAPKIFNGLGIQGPSNGLFATGVYGIVKVVGCALFVLFAADSV GR

RLS LLWT AIAQGIFMFIIGC Y VLTNPP VEG APIP AFG Y V ALVS IFLF VLCFE VGWGP AC WI

LVSEIPQARLRALNVALAAATQWLFNFVVAQAVPHMLITTGEGGYGTYFIFGSFSFC MF

FFTWFLIPETKGVSLEKMDALFGVKAPLGGEEGNPEFLEKSGPHTDADAGSGKFAET TH

IERK- [0195] Figure 24 shows a sequence alignment of the transporter proteins e20, el 15, and el74. The sequence alignment shows regions of identity and homology between the three proteins. Additionally, the alignment was used to identify sequence motifs.

[0196] Figure 25 shows a phylogram of the e20, el 15, and el74 transporter proteins. The genes were selected from a wide range of species including, but not limited to, plants, fungi, and yeasts.

[0197] The growth on DEHU of engineered yeast strains each harboring a plasmid containing one of the three transporter proteins is shown in Figure 17. Growth was measured by the increase in OD6oo _nm over time. OD in this format is 3 times lower than OD measured in a spectrophotometer.

[0198] The same transporter proteins were also introduced into derivatives of the strain BAL2193, where each strain lacked one of the 4 essential enzymes in the alginate metabolism pathway (i.e. , OAL, kdgK, DEHUH, or kdgPA). Each strain was transformed with the plasmid encoding the transporter. Growth in deep well plates was assessed after incoluation into a well of a deep well plate containing a yeast minimal media consisting of Yeast Nitrogen Base (YNB) without ammonium sulfate, supplemented with Complete Supplement Mixture -uracil (from Sunrise Science Products). 100 mM DEHU (concentration quantified by DEHU reductase assays) was added to the experimental wells and water to the negative controls (2-4 replicates of each). The growth was assessed at 29°C, with 90% humidity, and shaking at 950 rpm. Growth was determined by ΟΌ οο reads every 1-3 days for 7-10 days. Figure 23 demonstrates that growth, as detected in OD ₆ oo increase over the "no substrate" control, is detected only in strains that contain the alginate metabolism pathway (i.e. , kdgK, DEHUH, and kdgPA). The results show that the transporter-containing strains did not grow when any one of the essential enzymes was knocked-out, indicating a direct dependence on the alginate degradation pathway (Fig. 23). Thus, the results demonstrate that growth of the transporter-expressing strains was dependent on the alginate metabolic pathway.

[0199] The strain lacking the OAL gene showed no effect on growth (Fig. 23). This result further supports the conclusion that the transporter proteins transport a DEHU substrate, as the absence of the OAL enzyme would not allow the yeast strain to process a larger alginate substrate (e.g., a dimer or trimer) (Fig. 23).

[0200] We confirmed the ability of the e20, el 15, and el74 transporter proteins to transport the oligoalginate DEHU in the BAL2193 host strain.

[0201] We also compared the relative performance of the transporter proteins to each other via their growth in YNB media containing 2x CSM, and 50mM DEHU as the carbon source. The results are shown in Figure 17. All three strains (e20, el 15, and el74) reached optical densities significantly greater than the control lacking the DEHU substrate or the control strain containing an empty vector (EV) (Fig. 17). The EV control is a strain that included all the metabolic components except the transporter gene. In particular, the strains containing e20, el 15, or el74 grown with the DEHU substrate reached optical densities of approximately, 0.6, 0.9, and 1.0, respectively (Fig. 17). However, the controls only reached optical densities that were less than approximately 0.35 (Fig. 17).

[0202] For the strain containing the el 15 construct, we also grew the cells in YNB + 2 x CSM with a range of DEHU concentrations (0, 10, 25, 50, and 100 mM) and confirmed that DEHU is consumed during growth with the oligoalginate monomer DEHU as the carbon source (Fig. 18).

[0203] Additionally, using e20, el 15, and el74 as queries, we have identified several homologues (Fig. 25). We have confirmed some of the transporter homologs listed in Figure 25 can be functionally expressed in a recombinant yeast strain (Fig. 27).

Example 2: Selection/ Forced Evolution of Transporter Proteins with Chemostat and Cytostat

[0204] Accelerated evolution is also applied to improve alginate uptake in two ways. Once a functional heterologous transporter is identified, the strain/protein is further improved by forced evolution. Alternatively, native yeast hexose sensors and transporters are forced to evolve to facilitate alginate uptake.

[0205] Forced evolution is performed by chemostat/cytostat evolution. A yeast strain carrying an alginate lyase and the other alginate metabolism pathway components (oligoalginate lyase, DEHU reductase, KDG kinase, and KDGP aldolase) is used to perform the chemostat/cytostat evolution. To facilitate the evolution of the heterologous transporter, in vitro mutagenesis of the transporter gene is performed using error-prone PCR or other physical and chemical mutagens (e.g., UV, methyl methane sulfonate, N-methyl-N'-nitro-N-nitrosoguanidine, etc.). To facilitate the evolution of the native yeast components, in vivo mutagenesis, such as UV exposure and chemical mutagens, is performed.

Example 3: Expression of Functional Alginate Transporters in S. cerevisiae

[0206] The following example describes the expression of the Syml symporter from Vibrio splendidus in yeast. The methods described below can be applied to any of the presently disclosed alginate transporter protein candidates.

[0207] Protein and nucleotide sequences used:

1. V12B01 24194 (Syml) protein sequence (SEP ID NO: 13)

MTIDTFVVLAYFFFLIAIGWMFRKFTTSTSDYFRGGGKMLWWMVGATAFMTQFSAWT

FTGAAGRAFNDGFVIVILFLANAFGYFMNYMYFAPKFRQLRVVTAIEAIRQRFGKTS EQ

FFTWAGMPDSLISAGr LNGLAIFVAAVFNIPMEATIVVTGMVLVLMAVTGGSWAVVA

SDFMQMLVIMAVTITCAVAAYFHGGGLTNIVANFDGDFMLGNNLNYMSIFVLWVVFI F

VKQFGVMNNSINAYRYLCAKDSENARKAAGLACILMVVGPLr FLPPWYVSAFMPDF

ALEYASMGDKAGDAAYLAFVQNVMPAGMVGLLMSAMFAATMSSMDSGLNRNAGIF

VMNFYSPILRQNATQKELVIVSKLTTIMMGIIIIAIGLFINSLRHLSLFDIVMNVGA LIGFP

MLIPVLLGMWIRKTPDWAGWSTLIVGGFVSYIFGISLQAEDIEHLFGMETALTGREW SD

LKVGLSLAAHVVFTGGYFILTSRFYKGLSPEREKEVDQLFTNWNTPLVAEGEEQQNL DT

KQRSMLGKLISTAGFGILAMALIPNEPTGRLLFLLCGSMVLTVGILLVNASKAPAKM NN

ESVAK

2. V12B01 24194 (Syml) nucleotide sequence codon- optimized for yeast (SEP ID NO: 14)

ATGACCATTGACACCTTCGTTGTATTAGCTTATTTCTTCTTCTTGATAGCCATAGGT T

GGATGTTCAGAAAGTTTACCACCAGTACCTCTGATTACTTTAGAGGTGGTGGTAAAA

TGTTGTGGTGGATGGTTGGTGCCACCGCTTTTATGACTCAATTCTCAGCATGGACCT

TTACTGGTGCTGCTGGTAGAGCCTTTAATGATGGTTTCGTTATAGTCATCTTGTTCT T

AGCAAACGCCTTCGGTTACTTCATGAACTACATGTACTTCGCTCCAAAATTCAGACA

ATTGAGAGTTGTCACTGCCATAGAAGCTATCAGACAAAGATTCGGTAAAACCTCCG

AACAATTTTTCACTTGGGCTGGTATGCCAGATTCCTTAATAAGTGCTGGTATCTGGT

TGAATGGTTTAGCAATTTTTGTTGCCGCTGTCTTCAACATACCTATGGAAGCCACAA

TCGTAGTTACCGGTATGGTATTGGTTTTAATGGCTGTTACTGGTGGTTCCTGGGCAG

TCGTAGCCAGTGATTTTATGCAAATGTTGGTAATCATGGCCGTTACAATTACCTGTG

CTGTCGCAGCCTACTTTCATGGTGGTGGTTTGACAAACATAGTTGCTAACTTCGATG GTGACTTCATGTTGGGTAACAACTTAAACTACATGTCTATCTTCGTTTTGTGGGTTGT

CTTTATATTCGTTAAACAATTCGGTGTCATGAACAACTCTATTAATGCATATAGATA

CTTATGTGCCAAAGATTCAGAAAACGCTAGAAAGGCTGCTGGTTTGGCATGCATCTT

AATGGTAGTTGGTCCATTGATTTGGTTTTTACCACCTTGGTATGTTTCCGCTTTTAT G

CCTGACTTCGCTTTGGAATACGCAAGTATGGGTGACAAGGCAGGTGACGCCGCTTA

TTTGGCCTTTGTACAAAATGTTATGCCAGCCGGTATGGTTGGTTTGTTAATGTCTGC T

ATGTTCGCAGCCACTATGTCTTCAATGGATTCAGGTTTGAACAGAAACGCAGGTATC

TTCGTTATGAACTTCTACTCCCCTATCTTAAGACAAAACGCTACACAAAAGGAATTG

GTCATAGTTAGTAAGTTAACTACAATCATGATGGGTATCATCATCATCGCAATAGGT

TTGTTTATAAATTCTTTGAGACACTTGTCATTATTCGATATAGTCATGAACGTAGGT G

CTTTGATTGGTTTTCCAATGTTAATACCTGTTTTGTTAGGCATGTGGATTAGAAAAA C

TCCAGACTGGGCTGGTTGGTCAACATTGATAGTCGGTGGTTTCGTATCTTACATCTT

CGGTATTTCATTACAAGCTGAAGATATCGAACATTTGTTCGGTATGGAAACTGCATT

AACAGGTAGAGAATGGTCCGACTTAAAGGTTGGTTTGAGTTTAGCTGCACACGTCG

TTTTTACTGGTGGTTACTTCATCTTGACCTCTAGATTCTACAAGGGTTTGTCACCAG A

AAGAGAAAAGGAAGTAGATCAATTGTTCACTAACTGGAACACACCTTTAGTTGCAG

AAGGTGAAGAACAACAAAATTTGGACACCAAACAAAGATCCATGTTGGGTAAATTG

ATTAGTACTGCTGGTTTTGGTATCTTGGCTATGGCATTAATTCCAAACGAACCTACA

GGTAGATTGTTATTCTTGTTATGTGGTTCTATGGTTTTAACCGTCGGTATTTTATTA G

TAAACGCTTCCAAGGCACCAGCAAAAATGAACAACGAATCCGTCGCCAAATGA

[0208] Plasma membrane localization, visualization, and improvement: The cellular localization of Syml was determined. The plasma membrane localization of Syml relies on the fact that this protein has multiple transmembrane domains and no signal/motif for its retention to ER/Nuclear/Mitochondrial membrane. The localization of this native form of the protein in yeast was evaluated by fusing GFP to its C-terminus. The GFP fusion also allowed

quantification the amount of transporter protein.

[0209] To improve plasma membrane localization, the Syml gene was fused to Plasma Membrane Targeting Sequences (PMTS) that are known to be sufficient for targeting proteins to the plasma membrane in yeast based on two post-translational protein modification systems (Farnesylation/Geranylgeranylation and Palmitoylation). The PMTS that were used are listed in Table 20. Confocal light microscopy was used to confirm and quantify protein localization.

TABLE 20

[0210] Cloning strategy: The above synthesized Syml sequence was PCR amplified and subcloned (flanked by Ascl/Pacl) into a 2-micron based high-copy yeast expression vector (YML9) for constitutive expression of Syml (pTLT24) or into a CEN-ARS low copy yeast expression vector (p425CYC). GFP was fused to the C-terminus of Syml to create Syml-GFP for constitutive expression (pTLT2). Yeast native PMTS's (Table 20) were fused to either the 5' or 3' end of the Syml/Syml-GFP by homologous recombination. In addition, an 8xHis tag was fused to the C-terminus of Syml to confirm proper protein expression. The maps of the three key vectors are shown in Figure 9.

[0211] Table 21 summarizes all constructs made for the evaluation of Syml in S. cerevisiae. The same cloning strategy is applied to further transporter candidates disclosed herein.

TABLE 21

[0212] *A[3] plasmid in Table 21 contains the following genes: Syml, KdgpA, KdgK, OAL, and DEHUH. To create pTLT3, the Syml gene was removed and replaced by a

Puromycin marker.

[0213] Alginate transport assay:

[0214] Monitoring uptake of alginate uptake by growing yeast: Alginate components (mannuronate and guluronate monomers, dimers, trimers, tetramers, and/or DEHU) were fed to growing yeast cells harboring the recombinantly expressed alginate transporter system. The uptake of these components was assessed by exhaustive HPLC, TLC, and MS analysis. The profiles of relevant species in both the culture broth and isolated cells was then determined. Uptake of individual molecules by yeast was monitored by quantifying the appearance (in isolated cells) or reduction (in culture broth) of peaks corresponding to oligoalginate products. To look at the profile of intracellular metabolites in isolated cells, the cells were washed and frozen with liquid nitrogen, followed by grinding with a mortar and pastel and methanol extraction.

[0215] Growth-based assay: To assay for alginate uptake, a yeast strain harboring the plasmid with oligoalginate lyase (OAL), DEHU reductase (DEHUH), KDG kinase (KdgK), and KDGP aldolase (KdgpA) (pTLT3) was used to screen for functional alginate transporters. This plasmid contains a Puromycin antibiotic marker for growth selection. Co-transformation of pTLT3 with a functional alginate transporter allowed growth on the oligoalginate DEHU.

Alternatively, the BAL2193 yeast strain, which contains stable integration of oligoalginate lyase (OAL), DEHU reductase (DEHUH), KDG kinase (KdgK), and KDGP aldolase (KdgpA), was transformed with the el 87 vector. The vector can be selected for utilizing its resistance to blasticidin, which is encoded on the plasmid.

[0216] Radiolabeled alginate derivatives based assay: To directly assay transporter function, the uptake of [ ³ H] or [ ¹⁴ C] labeled alginate monomers (DEHU, mannuronate, guluronate) is determined. Preparation of radiolabeled substrate and measurement of sugar uptake is performed according to Zheng et ah, J. Ind. Microb., 1994.

[0217] Identification of additional putative eukaryotic alginate transporters:

Eukaryotic/fungal libraries are mined for additional alginate transporter genes. Eukaryotic genes usually express well in yeast and may contain functional PMTSs for plasma membrane localization. Recent reports of identification and characterization of pectin degradation pathways in fungi such as Trichoderma and Aspergillus species have revealed transport system candidates in these fungi as well. It is possible that the galacturonate (monomers/oligomers) transport systems from these species may function in the transport of alginate substrate(s).

Accordingly, candidate genes from these systems are expressed in yeast. Expression of a cellodextrin transporter from Neurospora in yeast can also be used to test for substrate promiscuity that allows the transport of alginate substrates. Bioinformatic strategies, such as BLAST, are also performed on available eukaryotic genomes to identify suitable candidates.

Example 4: Expression and Secretion of Functional Sphingomonas Al-I Alginate Lyase in S. cerevisiae

Materials and methods

[0218] Protein and nucleotide sequences used:

1. Protein sequence used (Δ2-53 AA) (SEP ID NO: 17):

MHPFDQAVVKDPTASYVDVKARRTFLQSGQLDDRLKAALPKEYDCTTEATPNPQQGE MVIPRRYLSGNHGPVNPDYEPVVTLYRDFEKISATLGNLYVATGKPVYATCLLNMLDK WAKADALLNYDPKSQSWYQVEWSAATAAFALSTMMAEPNVDTAQRERVVKWLNRV

ARHQTSFPGGDTSCCNNHSYWRGQEATIIGVISKDDELFRWGLGRYVQAMGLINEDG SF

VHEMTRHEQSLHYQNYAMLPLTMIAETASRQGIDLYAYKENGRDIHSARKFVFAAVK N

PDLIKKYASEPQDTRAFKPGRGDLNWIEYQRARFGFADELGFMTVPIFDPRTGGSGT LL

AYKPQGAAAQAPVSAPAAAHSSIDLSKWKLQIPVDPIDVATRDLLKGYQDKYFYVDK D

GSLAFWCPASGFKTTANTKYPRSELREMLDPDNHAVNWGWQGTHEMNLRGAVMHVS

PSGKTrVMQIHAVMPDGSNAPPLVKGQFYKNTLDFLVKNSAAGGKDTHYVFEGIELG K

PYDAQIKVVDGVLSMTVNGQTKTVDFVAKDAGWKDLKFYFKAGNYLQDRQADGSDT

SALVKLYKLDVKHSS

2. DNA sequence used (which was codon optimized for E. coli) (SEQ ID NO: 18):

ATGCACCCGTTCGACCAAGCAGTTGTGAAAGATCCGACTGCGTCCTATGTTGACGTT

AAAGCGCGTCGTACTTTCCTGCAAAGCGGTCAACTGGATGATCGCCTGAAAGCAGC

GCTGCCGAAGGAATATGACTGTACCACCGAAGCGACGCCGAACCCACAGCAGGGTG

AAATGGTGATCCCACGCCGCTATCTGTCCGGTAACCACGGCCCGGTGAATCCGGATT

ACGAGCCGGTTGTCACTCTGTATCGCGACTTCGAAAAAATCAGCGCGACCCTGGGT

AACCTGTACGTTGCGACTGGTAAACCAGTGTACGCAACTTGTCTGCTGAACATGCTG

GACAAATGGGCTAAAGCAGACGCGCTGCTGAACTATGACCCGAAATCTCAGAGCTG

GTATCAAGTAGAATGGTCCGCAGCCACGGCGGCCTTTGCCCTGAGCACTATGATGG

CAGAGCCGAACGTGGACACCGCGCAGCGTGAGCGTGTTGTGAAATGGCTGAACCGT

GTAGCACGTCACCAGACTTCTTTTCCGGGTGGCGACACTAGCTGCTGTAACAATCAT

TCTTACTGGCGTGGTCAGGAGGCTACCATCATCGGCGTTATTTCCAAGGATGATGAA

CTGTTCCGTTGGGGTCTGGGTCGTTATGTACAGGCGATGGGTCTGATCAACGAAGAT

GGTTCCTTCGTTCACGAAATGACTCGTCACGAACAGAGCCTGCATTATCAGAACTAT

GCGATGCTGCCGCTGACCATGATCGCTGAGACTGCCTCTCGTCAGGGTATCGATCTG

TATGCTTACAAGGAAAACGGTCGTGATATCCATTCTGCTCGTAAATTCGTATTCGCG

GCCGTAAAGAATCCGGATCTGATCAAGAAATACGCGAGCGAACCGCAGGACACGC

GCGCTTTTAAACCGGGTCGCGGCGATCTGAACTGGATCGAATATCAGCGTGCGCGTT

TCGGCTTTGCAGATGAGCTGGGCTTTATGACCGTGCCAATCTTCGATCCGCGCACCG

GCGGCTCTGGCACTCTGCTGGCGTATAAGCCACAGGGTGCGGCTGCTCAGGCGCCG

GTTTCCGCTCCGGCGGCAGCACACTCTTCCATCGATCTGTCCAAATGGAAACTGCAG

ATCCCTGTTGACCCGATCGATGTTGCTACCCGCGATCTGCTGAAGGGTTATCAGGAC

AAGTATTTCTACGTGGATAAAGATGGTTCTCTGGCCTTCTGGTGCCCAGCATCCGGT

TTCAAAACCACGGCGAATACTAAGTATCCGCGTAGCGAGCTGCGTGAAATGCTGGA

CCCGGATAATCATGCTGTTAATTGGGGCTGGCAGGGCACCCACGAAATGAACCTGC

GCGGTGCAGTTATGCACGTTTCCCCGTCCGGTAAAACCATCGTCATGCAGATCCACG

CAGTTATGCCGGACGGTTCCAATGCGCCACCACTGGTTAAAGGCCAGTTCTACAAA

AACACGCTGGACTTCCTGGTGAAAAATTCTGCGGCTGGTGGTAAAGATACTCACTA

CGTGTTCGAAGGCATCGAACTGGGTAAACCATACGACGCTCAGATCAAAGTTGTAG

ATGGTGTCCTGTCTATGACCGTTAATGGTCAGACTAAAACTGTTGACTTCGTGGCTA

AAGATGCGGGCTGGAAGGATCTGAAATTCTATTTCAAGGCAGGTAACTATCTGCAG

GACCGCCAGGCCGACGGCTCCGATACCTCTGCCCTGGTAAAGCTGTACAAACTGGA

CGTTAAACATTCCAGCTAA 3. SRP nucleotide sequence (SEP ID NO: 19):

ATGCTTTTGCAAGCTTTCCTTTTCCTTTTGGCTGGTTTTGCAGCCAAAATATCTGCA

4. SRP protein sequence (SEP ID NO: 20):

M L L Q A F L F L L A G F A A K I S A

[0219] Cloning strategy: The above synthesized alginate lyase Al-I DNA sequence was PCR amplified and cloned into three vectors such that the expression of this gene could be driven by three different promoters. Each of these vectors is 2-micron based high-copy vectors. In each case, a yeast native Signal Recognition Particle (SRP) from SUC2 (Invertase) protein encoding gene was fused to the 5' end of the above nucleotide sequence (minus the ATG start codon). The SRP sequence is shown above. In all of the three vectors, an 8xHis tag was fused to the C-terminus of the protein. The maps of each of the three vectors are shown below (Fig. 9).

[0220] Alginate lyase assay: The following protocol describes the method used for preparing the cell lysates, supematants, and the 96-well plate for the alginate lyase assay which was done using a plate reader.

[0221] For preparing cell lysate: 1) measure the absorbance at CD ₆₀₀ of a 1: 10 dilution of yeast culture (16-24hrs old); 2) take about 2.5-3.0 OD ₆₀₀ of cells in a fresh falcon tube; 3) pellet yeast cells at 4300rpm for 5 minutes; 4) resuspend yeast cells in 1XM9 buffer; 5) wash 2x in M9 buffer. Each time pellet it down at 4300rpm for 5 min; 6) discard wash supernatant; 7) to each sample of pelleted cells add lOOul of Cellytic reagent to which DTT (final concentration of lOmM), Zymolase (final concentration 15units/ml), and Protease inhibitor (Roche tablet) - 1 tablet for a 5-10 ml lysis buffer have already been added; 8) shaking in the incubator at 30degC for 1.5 - 2 hours; and 9) add 25ul of cell lysate to alginate lyase assay plate.

[0222] For supematants: take about 2.5-3 ml of supernatant from a 16-24 hr old yeast culture, sterile filter through a 0.22 μιη filter into a fresh falcon tube; and add 20 μΐ of supernatant per well of the assay plate.

[0223] Setting up the plate: 1) a master mix of alginate (substrate), buffer (M9) and water was prepared as follows: for each reaction well: 1% Alginate stock - 20ul (final concentration of substrate = 0.1%), 5X M9 buffer - 40 μΐ, sterile filtered water - 120 μΐ, and volume of assay components (leaving out enzyme) - 180 μΐ; 2) 180 μΐ of the master mix was dispensed into each assay well, then 20 μΐ of enzyme source (cell supernatant or cell lysate) was added to each well; 3) for standard curve, 20ul of each dilution of the standard (Sigma Alginate Lyase) was pipetted into the wells; 4) for enzyme blank -the substrate and substitute was left out of 20 μΐ of water; 5) for substrate blank - the enzyme and substitute were left out of 20 μΐ of water; and 6) the increase of absorbance at 232 nm was read immediately after enzymes were added to the reaction wells.

[0224] Method for analyzing products of alginate lyase degradation on LC/LC-MS: The following parameters were used for the analysis:

[0225] LC method parameters: 1) column: 100 mm hypercarb column; 2) mobile phase: B HPLC grade methanol, A 0.2% TFA in HPLC grade water; 3) gradient: 0 min-30%B, 15.00 min-90%B, 15.50-90%B, 15.75-30%B, 21min-stop; 4) flow rate: 0.65 mL/min; 5) run time: 21 min; 6) UV detector wavelengths: 210 nm, 235 nm; 6) column oven: 65°C; and 7) injection volume: 5-40 μΐ per sample (if lower signal is observed with smaller volumes, higher volume injections are done).

[0226] Mass parameters: 1) m/z: 175, 193, 351, 369, 527, 545, 703, 879; 2) event time: 0.1 sec; 3) negative scanning; 4) DUIS interface; and 5) DL temperature 250°C, Heat block temperature 400°C.

Results

[0227] Detection of alginate lyase activity in culture lysates and supernatants: The alginate lyase expression vectors (YML26, LML48 and LML51) were transformed into a laboratory diploid yeast strain (ATCC201392) and the multiple isolates from each

transformation were selected. These isolates were grown into YPD in the presence of the respective antibiotics. The alginate lyase activity was tested for these isolates by an assay which involves incubation of the sample (culture lysate/supernatant) with a solution prepared using commercial alginate. The enzyme causes degradation of the alginate into products with unsaturated double bonds. This causes increase in optical density (OD) at 232 nm. Therefore, by measuring increase in OD _{232 nm} over time, the alginate lyase activity can be detected and measured. The results for alginate lyase assay for the isolates from each of the three plasmid transformants is shown in Figure 10.

[0228] Detection of the presence of a His-tagged protein corresponding to the size of Al-I in yeast cells harboring the Al-I expression plasmid: Cultures of different isolates from LML48 and LML51 transformants of yeast were used to prepare cell free extracts and culture supernatants samples for anti-His immunoblot analysis. As shown in Figure 11, two isolates of each of the transformants were used and bands corresponding to the expected Al-I size were present in the samples expressing Al-I but not in the vector control samples. The presence of two bands indicate processing at the N-terminus of the Al-I protein (His tag is at the C- terminus). Al-I enzyme is known to exhibit auto-catalysis to give rise to three alginate lyase enzymes (Al-I -60 kDa, Al-II -25 kDa and Al-III -40 kDa) in the Sphingomonas cytoplasm.

[0229] Analysis of the alginate degradation profile of yeast expressed Al-I enzyme:

The sample from alginate degradation using enzyme Al-I expressed and secreted in yeast culture supernatant was analyzed using LC/LC-MS. The degradation profile revealed that the yeast made Al-I enzyme is able to generate dimers from alginate degradation along with higher molecular weight oligomers such as trimers, tetramers. The presence of monomers was negligible. The profile is consistent with the endo-type activity of Al-I that has been reported. The results show that the LCMS profile for an alginate sample incubated with the yeast made Al-I enzyme was present in the culture supernatant (Fig. 12).

[0230] Detection of alginate lyase activity on solid growth medium: Since the recombinant yeast secreted the Al-I enzyme into the extracellular milieu, the degradation of alginate (0.5%) from commercial sources (sigma chemicals) was tested in the culture plate by making YPD media plates in the presence of alginate. After the alginate expressing colonies are grown on the plate, a dye called Cetyl-pyridinium chloride was used to stain the plates. This dye causes the medium to become opaque against which a zone of clearing can be seen if the alginate present in the medium has been degraded around the colonies expressing Al-I. The results indicated that the zone of clearing is present around the colonies harboring LML48 (Al-I expressing cells) (Fig. 13). However, the control colonies did not exhibit a surrounding zone of clearing due to alginate degradation. [0231] Increasing the expression of Al-I: In order to test if the levels of secreted Al-1 can be increased by increasing the gene dosage in the yeast cells, YML26 and LML48 were co- transformed and screened for activity. The results show that by co-transforming the two plasmids and selecting on two antibiotics (Zeo and Blasticidin) yeast strains were isolated with increased (up to 2 fold) expression of Al-I compared to the strain harboring single plasmids (Fig. 14).

[0232] Primary sequence and domain comparison of the Sphingomonas Al-1 alginate lyase and the Vibrio sp. QY101 ALy VI alginate lyase: Based on the review by Wong et al. (Wong et αΙ., Αηηιι. Rev. Microbio., 2000) and amino acid sequence data, most alginate lyases fall into 3 classes -based on molecular mass: 20-35 kDa, 40 kDa, and 60 kDa. The Vibrio Aly VI lyase falls into the 40 kDa class and the Sphingomonas Al-1 lyase falls into the 60 kDa class. Additionally, conserved sequences were found in the 40 kDa class of lyases.

[0233] Two types of 9-amino acid C-terminal conserved sequences are found within the 40 kDa class of lyases. The first is a WLEPaC+LY (SEQ ID NO: 5) sequence, which is absent in both the Vibrio Aly VI and the Sphingomonas Al-1 lyases. The second conserved sequence is a YFKhG+Y-Q (SEQ ID NO: 6) sequence, which is present in both the Vibrio Aly VI and

Sphingomonas Al-1 lyases (Fig. 15).

[0234] The 40 kDa class of lyases also contains conserved amino acid sequences that are thought to be alginate binding motifs. These include a -NNHSYW (SEQ ID NO: 7) consensus sequence, which is absent in the Vibrio Aly VI lyase but present in the Sphingomonas Al-1 lyase (Fig. 15). There is also an -NN-+Y-N (SEQ ID NO: 8) consensus sequence, which is absent in both the Vibrio Aly VI and Sphingomonas Al-1 lyases.

Example 5: Assays for functional Oligoalginate Metabolism Pathway in Yeast

Introduction

[0235] The pathway for degrading oligoalginate includes several enzymes. Endo and exo alginate lyases break down alginate polymers and/or oligoalginate polymers into smaller products, and the ultimate product of alginate digestion is the monosaccharide DEHU. This monosaccharide is further metabolized intracellularly by a series of enzymes (DEHU reductases, KdgK, and KdgpA) that utilize one molecule of NADH or NADPH and one molecule of ATP to split the monomer into pyruvate and glyceraldehyde-3-phosphate. Glyceraldehyde- 3 -phosphate enters the glycolysis pathway (EM pathway) and produces NADH (or NADPH) and ATP along with the co-production of pyruvate (Fig. 1).

In vitro assay for exo-alginate lyase

[0236] An in vitro assay was developed to assess functionality of the exo alginate lyase. This assay involves detection of NADPH consumption via absorbance at 340 nm when crude lysate of the engineered yeast is supplied with endo-alginate lyase-oligoalginate as a substrate.

[0237] A spectrophotometric-coupled enzyme assay for exo-alginate lyase (OAL) activity was developed. Figure 19 shows the assay. This assay measures the oxidation of NADPH, which is dependent on the formation of DEHU. Dimers and larger multimers are prepared by incubation of 0.10 mg/mL Sigma alginate lyase-1 with 50 g/L sodium alginate in the presence of 2 mM DTT for > 10 hr at 37°C, and this preparation is used directly in the assay. Excess partially purified DEHU reductase (enough to give > 0.2 OD/min rate with 10 mM DEHU and 2 mM NADPH) is present, as is 2 mM NADPH. Table 22 gives the assay conditions for reaction mixtures of 0.050 mL with the addition of 0.005 mL of yeast lysate initiating the reaction.

TABLE 22

In vitro assay for DEHU reductase

[0238] An in vitro assay was developed to assess functionality of the DEHU reductase. This assay involves detection of NADPH consumption via absorbance at 340 nm when crude lysate of the engineered yeast is supplied with DEHU as a substrate.

[0239] A spectrophotometric assay for the identification of DEHU reductase in lysates of yeast was developed. The assay directly measures the oxidation of NADH or NADPH while DEHU is reduced to KDG (Fig. 20). Table 23 gives the final reaction conditions of the assay that has a final volume of 0.050 mL, and the reactions are initiated by the addition of 0.005 mL of lysate of yeast. The assay readily detects DEHU reductase activity in lysates of yeast.

TABLE 23

In vitro assay for KDG kinase (KdgK)

[0240] An in vitro assay was developed to assess functionality of KdgK. This assay involves detection of NADH or NADPH consumption via absorbance at 340 nm when crude lysate of the engineered yeast is supplied with KDG as a substrate.

[0241] A spectrophotometric assay for the identification of Kdg kinase activity in lysates of yeast was developed. Figure 21 describes the assay whereby KDG, which is synthesized for the assay by the reaction of mannonic acid and mannonic acid dehydratase, is reacted with ATP in the presence of the kinase. The product phosphorylated KDG (KDG-P) is cleaved by added excess partially purified Kdgp aldolase (KdgpA) to give pyruvate as a product, which is then reduced by excess lactate dehydrogenase. This reduction reaction results in the loss of NADH, which is measured as a decrease in absorbance at 340 nm. Table 24 gives the reaction mixture composition for assays with a final volume of 0.050 mL in which the reaction is initiated by the addition of 0.005 mL of yeast lysate.

TABLE 24

In vitro assay for KDGP aldolase (KdgpA)

[0242] An in vitro assay was developed to assess functionality of the KdgpA. This assay involves detection of NADH or NADPH consumption via absorbance at 340 nm when crude lysate of the engineered yeast is supplied with KDGP as a substrate.

[0243] A spectrophotometric assay for the identification of Kdg-P aldolase in lysates of was developed. Figure 22 describes the assay whereby KDGP, which is synthesized by the reaction of KDG and ATP with Kdg kinase, is reacted with the aldolase. The reaction gives pyruvate as a product, which is then reduced by excess lactate dehydrogenase. This reduction reaction results in the loss of NADH, which is measured as a decrease in absorption at 340 nm. Table 25 gives the reaction mixture composition for assays with a final volume of 0.050 mL in which the reaction is initiated by the addition of 0.005 mL of yeast lysate.

TABLE 25

1 inal Slock W orking Added ml.

DTT (M) 0.0020 1 0.0022 0.002

Triton x 100 (% w/v) 0.0050 10 0.0056 0.0006

+/- KDGP (M) 0.0050 0.025 0.006 0.222

NADH (M) 0.0040 0.020 0.0044 0.222

LDH (mg/ml) 0.050 5.0 0.0556 0.011

dH ₂ 0 0.431

Example 6: Yeast Strains Capable of Growth on Mannitol

Yeast strain capable of utilizing mannitol as a sole carbon source

[0244] This experiment demonstrates the ability of an engineered yeast strain to grown on mannitol. The yeast strain was engineered to express a mannitol transporter and a mannitol dehydrogenase.

Materials and Methods

[0245] Strains: Yeast strain BAL1620 (M1.2 in FY1679) contains pTEF- AgMaT 1 -pTEF- AgMtd-pTEF-YEL070w (high copy URA3 plasmid yeplacl95). Yeast strain BAL1640 (Ml .O in FY 1679) contains pTEF- AgMaT 1 -pTEF- AgMtd (high copy URA3 plasmid yeplacl95). Yeast strain BAL1642) contains pTEF- pTEF-AgMtd (high copy URA3 plasmid yeplacl95). Yeast strain BAL1643 contains pTEF-AgMaTl (high copy URA3 plasmid yeplacl95). Yeast strain BAL1644 contains high copy URA3 plasmid yeplacl95 only. Yeast strain BAL2500 (BAL2129 with plasmid eEV) is a wild- type yeast strain (SEY6210/11) carrying an empty URA3/BlasticidinR vector. Yeast strain BAL2501 (BAL2129 with plasmid M3.2) is a wild-type yeast strain (SEY6210/1 1) carrying plasmid M3.2 (pTDH3-YNR073c, URA3/NeoR). Yeast strain BAL2502 (BAL2129 with plasmid M3.3) is a wild-type yeast strain (SEY6210/11) carrying plasmid M3.3 (pTDH3-YNR073c, URA3/NeoR). Yeast strain BAL2503 (BAL2129 with plasmid e56) is a wild-type yeast strain (SEY6210/11) carrying plasmid e56 (pTDFB- HXT13, URA3/BlasticidinR). Yeast strain BAL2504 (BAL2129 with plasmid e59) is a wild- type yeast strain (SEY6210/11) carrying plasmid e59 (pTDH3-HXT17, URA3/BlasticidinR). Yeast strain BAL2505 (BAL2129 with plasmids M3.2 and eEV) is a wild-type yeast strain (SEY6210/11) carrying plasmids M3.2 (pTDH3-YNR073c, URA3/NeoR) and an empty URA3/BlasticidinR vector. Yeast strain BAL2506 (BAL2129 with plasmids M3.3 and eEV) is a wild-type yeast strain (SEY6210/11) carrying plasmids M3.3 (pTDH3-YNR073c, URA3/NeoR) and an empty URA3/BlasticidinR vector. Yeast strain BAL2507 (BAL2129 with plasmids M3.2 and e56) is a wild-type yeast strain (SEY6210/11) carrying plasmids M3.2 (pTDH3-YNR073c, URA3/NeoR) and e56 (pTDH3-HXT13, URA3/BlasticidinR). Yeast strain BAL2508

(BAL2129 with plasmids M3.3 and e56) is a wild-type yeast strain (SEY6210/11) carrying plasmids M3.3 (pTDH3-YNR073c, URA3/NeoR) and e56 (pTDH3-HXT13,

URA3/BlasticidinR). Yeast strain BAL2509 (BAL2129 with plasmids M3.2 and e59) is a wild- type yeast strain (SEY6210/11) carrying plasmids M3.2 (pTDH3-YNR073c, URA3/NeoR) and e59 (pTDH3-HXT17, URA3/BlasticidinR). Yeast strain BAL2510 (BAL2129 with plasmids M3.3 and e59) is a wild-type yeast strain (SEY6210/11) carrying plasmids M3.3 (pTDFB- YNR073c, URA3/NeoR) and e59 (pTDH3-HXT17, URA3/BlasticidinR).

[0246] Gene definitions:

AgMaTl: mannitol transporter from celery (Apium graveolens) AgMtd: mannitol dehydrogenase from celery {Apium graveolens) YEL070w: putative mannitol dehydrogenase (S. cerevisiae) YNR073c: putative mannitol dehydrogenase (S. cerevisiae) HXT13 (YEL069C): putative mannitol transporter (S. cerevisiae) HXT17 (YNR072W): putative mannitol transporter (S. cerevisiae) YNR071C: putative mutorase (S. cerevisiae)

[0247] Yeast Cultures: For each genotype, single colonies were picked from two independent transformants, inoculated to YNB 2% glucose + amino acids minus uracil cultures, and grown overnight at 30°C. The colonies were then washed 3 times with water and absorbance at Οϋ ₆ οο nm was measured. Colonies were then inoculated to YNB 2% mannitol or 2% glucose + amino acids minus uracil to a starting OD6oo _nm of 0.1 in 30 mL, using 250 mL baffle flasks. For each time point, absorbance was measured at OD ₆ oo nm and samples were taken to measure sugar concentration using HPLC.

[0248] For the strains BAL2500-2510 the growth took place in 96-well deep well plates, each culture at 0.55 mL. 3 single colonies from each strain were inoculated in YNB + amino acids minus uracil + 2% glucose + drugs (Blasticidin for BAL2500, 2503, 2504; G418 for BAL2501, 2502; and Blasticidin+G418 for 2505, 2506, 2507, 2508, 2509, 2510) and grown overnight. The overnight cultures were then diluted back to an OD6oo _nm of 0.1 in YNB + amino acids minus uracil, and for each of the 3 isolates either + 2% glucose or + 5% Mannitol. 2 of the 3 isolates were also set up with water instead of added sugar ("No Substrate" control). The same drugs as in the overnight cultures were also added to the experimental cultures. Time points were taken at 44 hours and at 115 hours to measure Οϋ ₆ οο nm, and samples were taken for sugar and ethanol concentration using HPLC at the 44 hour time point.

Results

[0249] Yeast strain FY 1679 containing high copy versions of mannitol dehydrogenase and a mannitol transporter from Apium graveolens (celery) grew to an OD ₆ oo _nm of 4 in 140 hours in YNB 2% mannitol (Fig. 7A). Yeast strain FY1679 containing high copy versions of mannitol dehydrogenase and transporter from Apium graveolens (celery) and overexpressed native Mtd YEL070w grew to an OD ₆₀ o nm of 12 in 140 hours in YNB 2% mannitol. Yeast strain FY1679 containing high copy versions of only mannitol dehydrogenase or transporter from Apium graveolens (celery) had negligible growth (OD ₆ oo _nm < 1.0) in 140 hours in YNB 2% mannitol. Yeast strain FY1679 containing the empty plasmid did not grow (OD ₆ oo _nm < 0.5) in 140 hours in YNB 2% mannitol (Fig. 7A). Yeast strain SEY6210/11 containing CEN/ARS plasmids that overexpress the native yeast mannitol dehydrogenase (YNR073c expressed from the TDH3 promoter) and the native yeast transporters HXT13 and HXT17 (also expressed from the TDH3 promoter) grew to an OD ₆ oo nm-piate Reader of 1.4 in 115 hours in YNB+5 mannitol (this OD6oo _nm was measured in a plate reader and corresponds to an OD ₆ oo nm of 4.2 by a regular

spectrophotometer) .

[0250] After 44 hours, an ethanol concentration of around 1.5 g/liter was detected in these cultures. If either gene alone was expressed, no growth on mannitol or ethanol production was detected (Figs 7B and 7C).

Mannitol transporter proteins

[0251] A phylogram of putative mannitol, polyol, hexose, and other sugar transporters, as well as permeases was constructed (Fig. 8). The genes were selected from a wide range of species including, but not limited to, plants, fungi, and yeasts. The tree branch at the top of Figure 8, from AgMaTl (celery mannitol transporter) through MFS (Schizosaccharomyces pombe), depicts proteins that cluster based on proposed function, rather than by species. As such, this branch represents candidates for mannitol transporters and are synthesized and expressed in conjunction with synthesized and expressed mannitol dehydrogenases.

Mannitol consumption assay

[0252] During the course of a growth experiment, aliquots were removed, absorbance was measured at Οϋ ₆ οο nm, and HPLC sampling was performed. For HPLC sampling, samples were diluted 1: 10 in DI water so that maximum sugar concentration is ~2 g/L. Samples were then run on HPLC using an ethanol method. The carbohydrates present were quantitated by measuring the area underneath each appropriate peak. Standard curves existed for mannitol and glucose within this range of concentration. Sugar consumption was calculated by the change (decrease) in measured sugar as a function of total cell mass (dry cell weight or OD ₆ oo nm) and time (per hour).

Example 7: Assays for Ethanol Production from DEHU and Mannitol in Yeast

[0253] This experiment demonstrates the ability of two engineerd yeast strains to produce ethanol from DEHU and mannitol.

Materials and methods

Yeast strains

[0254] Two strains of recombinant yeast were used. Recombinant yeast strain BAL2575 was engineered to consume mannitol but not DEHU, while recombinant yeast strain BAL2576 was engineered to consume both mannitol and DEHU. The background strain used to generate both BAL2575 and BAL2576 was the strain BAL2533. The genotype of the BAL2533 strain is shown in Table 26. The BAL2533 strain contains the bacterial alginate degradation pathway. It has also been confirmed that the BAL2533 strain has the ability to grow on DEHU as the carbon source when a DEHU transporter is expressed. [0255] Additionally, both the BAL2575 and BAL2576 strains carry the plasmid M4.3. Plasmid M4.3 is described in Table 27. This plasmid contains three native yeast genes expressed under constitutive promoters. Moreover, this plasmid has been shown to support the growth of yeast on mannitol.

[0256] The BAL2575 strain also carries a second plasmid, eEV (plasmid type: "e", with no transporter gene: "Empty Vector"). Plasmid eEV.3 is described in Table 27. This plasmid confers blasticidin resistance to the strain.

[0257] The BAL2576 strain carries a second plasmid, e207. Plasmid e207 is described in Table 27. This plasmid contains a DEHU transporter, which allows the strain to utilize DEHU

TABLE 27

Bioreactors and culture conditions

[0258] Two Sartorius Biostat A+ bioreactors were used in this study. Reactors were automatically kept at 29°C and continuously mixed using two six-blade Rushton turbines at 300 RPM. The pH was continuously monitored with an Easyferm Plus K8 probe (Hamilton) and automatically kept at a pH of 5.5 by the addition of 5 N sodium hydroxide and/or 5 N

phosphoric acid as needed. A continuous flow of 0.4 L/min of air was introduced through the fermenter headspace. The level of dissolved oxygen in the bioreactor was continuously monitored with an Oxyferm 0 ₂ sensor (Hamilton).

[0259] The media was prepared to have the following final concentrations: DEHU 50 mM (prepared in house using alginate lyase purchased from SIGMA and A. tumefasiens oligoalginate lyase produced from engineered E. coli), mannitol 2% w/v, Yeast Nitrogen Base without amino acids or ammonium sulfate from Difco at the manufacturer's recommended concentration, Complete Supplement Mixture without uracil (CSM-Ura) from Sunrise Science Products at twice the manufacturer's recommended concentration, kanamycin 100 μg/mL, carbeniciUin 200 μg/mL, blasticidin 100 μg/mL, and G418 50 μg/mL. The media was filter sterilized.

Bioreactor preparation

[0260] The bioreactors were autoclaved with one liter of M9 minimal salts solution

(Amresco) to ensure the sterility of the setup and probes. Prior to inoculation, this solution was sterilely removed and approximately one liter of the sterile culture media was added in. Once the solution reached the final desired temperature the dissolved oxygen probe was calibrated using nitrogen and air.

Inoculum preparation

[0261] Overnight yeast cultures (100 mL) were set up at OD ₆ oo=0.1 from saturated liquid cultures in 2% glucose, Yeast Nitrogen Base without amino acids or ammonium sulfate from Difco at the manufacturer's recommended concentration, Complete Supplement Mixture without uracil (CSM-Ura) from Sunrise Science Products at twice the manufacturer' s recommended concentration, kanamycin 100 μg/mL, carbeniciUin 200 μg/mL, blasticidin 100 μg/mL, and G418 50 μg/mL.

[0262] The next day, cultures were measured at an OD ₆ oo=l-l for BAL2575 and 0.8 for BAL2576, at which time the full volumes were used for inoculation of the fermenters. Inoculation

[0263] Each reactor was sterilely inoculated with approximately 100 mL of inoculum, yielding an initial A ₆ oo in the reactor of around 0.1 units.

Bioreactor sampling

[0264] Reactors were sampled twice a day. One filtered aliquot was immediately frozen and stored at -80°C for DEHU analysis. A second filtered aliquot was stored at 4°C for HPLC analysis. An unfiltered aliquot was used for optical density determination using a Genesys 10S spectrophotometer (Thermo Scientific).

Mannitol and ethanol measurements

[0265] Mannitol and ethanol were quantified by HPLC (Shimadzu) equipped with an organic acid column (Phenomenex). HPLC was operated at 60°C using 5 mM H2S04 as a mobile phase at 1 mL/min flow rate (5 injection volume, 15 min isocratic method). Mannitol and ethanol were detected using a refractive index detector. For the DEHU, quantification was performed by reacting it with ammonia to form 5-hydroxypicolinic acid by quantification via HPLC using a Thermo Hypercarb column and detection of UV absorbance at 235 nm.

Results

[0266] Figures 26A and 26B show the optical density and metabolite profiles for both the BAL2575 and BAL2576 cultures. A minimal amount of growth (as measured by the increase in optical density) was seen in both strains. Both BAL2575 and BAL2576 were able to consume mannitol (Figs. 26A and 26B). However, only BAL2576 was able to consume both mannitol and DEHU (Fig. 26B).

[0267] The BAL2575 strain showed a slow reduction in DEHU concentration during the fermentation (Fig. 26 A). However, without wishing to be bound by theory, it is believed that this result is primarily due to spontaneous degradation of DEHU. The BAL2575 strain produced a peak amount of approximately 75 mM/L ethanol during the fermentation (Fig. 26A). [0268] In contrast to the BAL2575 strain, the BAL2576 strain demonstrated a 60% increase in ethanol titer under the same fermentation conditions (Fig. 26B). As can be seen in Figure 26, the BAL2576 strain had a peak ethanol production of 120 mM/L, as compared to the peak production of 75 mM/L ethanol of BAL2575.

[0269] These results clearly demonstrate that the BAL2576 strain has the ability to ferment mannitol and DEHU into ethanol with a high efficiency. The results indicate that both mannitol and DEHU are required to produce ethanol, as both are needed for redox balance. Accordingly, the results show that the engineered yeast strain is able to more efficiently produce ethanol when a mixture of mannitol and DEHU is used, as compared to using just mannitol.