SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on Dec. 20, 2016, is named 105464_000030_SL.txt and is 578,263 bytes in size.

BACKGROUND

Succinate (butanedioic acid) is a four carbon dicarboxylic acid that plays a key role in the citric acid cycle. Succinate was recently listed by the US Department of Energy at the top of its list of value added chemicals from biomass. Succinate is a precursor for a number of compounds, including tetrahydrofuran, 1,4-butanediol, and γ-butyrolactone. Succinate has a wide variety of potential applications including use in liquid antigels, heat transfer fluids, the solvents gamma butyrolactone (GBL) and dimethyl isosorbide, pigments, the polyesters poly-butylene succinate (PBS) and PEIT, synthesis intermediates and plasticizers.

Succinate has traditionally been derived from maleic anhydride, which is produced by oxidation of butane. In recent years, there have been several attempts to move away from these traditional production methods to biological production methods. Biological production provides several advantages over derivation from petrochemical sources, including increased efficiency and cost effectiveness and decreased environmental impact.

Previously developed biological succinate production methods have primarily utilized bacterial fermentation hosts. Although several bacterial species have been used successfully to produce succinate, bacteria present certain drawbacks for large-scale organic acid production. As organic acids are produced, the fermentation medium becomes increasingly acidic. These lower pH conditions result in lower costs for organic acid production, because the resultant product is partially or wholly in the acid form. However, most bacteria do not perform well in strongly acidic environments, and therefore either die or begin producing so slowly that they become economically unviable. To prevent this, it becomes necessary to buffer the medium to maintain a higher pH. However, this makes recovery of the organic acid product more difficult and expensive.

There has been increasing interest in recent years around the use of yeast to ferment sugars to organic acids. Yeast are used as biocatalysts in a number of industrial fermentations, and present several advantages over bacteria. While many bacteria are unable to synthesize certain amino acids or proteins that they need to grow and metabolize sugars efficiently, most yeast species can synthesize their necessary amino acids or proteins from inorganic nitrogen compounds. Yeast are also not susceptible to bacteriophage infection, which can lead to loss of productivity or of whole fermentation runs in bacteria.

Although yeast are attractive candidates for organic acid production, they present several difficulties. First, pathway engineering in yeast is typically more difficult than in bacteria. Enzymes in yeast are compartmentalized in the cytoplasm, mitochondria, or peroxisomes, whereas in bacteria they are pooled in the cytoplasm. This means that targeting signals may need to be removed in yeast to ensure that all the enzymes of the biosynthetic pathway co-exist in the same compartment within a single cell. Control of transport of pathway intermediates between the compartments may also be necessary to maximize carbon flow to the desired product. Second, not all yeast species meet the necessary criteria for economic fermentation on a large scale. In fact, only a small percentage of yeast possess the combination of sufficiently high volumetric and specific sugar utilization rates with the ability to grow robustly under low pH conditions. The Department of Energy has estimated that production rates of approximately 2.5 g/L/hour are necessary, using a minimal media, for economic fermentations of organic acid.

The yeast strains that have been developed thus far for succinate production have not exhibited high enough yields for economic production on an industrial scale. Therefore, there is a need for improved yeast strains that generate succinate on a larges scale in a more cost-effective manner.

SUMMARY

Provided herein in certain embodiments are genetically modified yeast cells comprising an active succinate fermentation pathway from phosphoenolpyruvate or pyruvate to succinate. In certain embodiments, the yeast cells provided herein are succinate resistant. In certain embodiments, the active succinate fermentation pathway includes at least the following reactions 1) conversion of pyruvate and/or phosphoenolpyruvate to oxaloacetate, 2) conversion of oxaloacetate to malate, 3) conversion of malate to fumarate, and 4) conversion of fumarate. In certain embodiments, the pathway also includes export of succinate from inside the cell to the extracellular environment. Each of the reactions in the active succinate fermentation pathway is catalyzed by one or more enzymes, which in turn are encoded by one or more exogenous or endogenous succinate fermentation pathway genes. In certain embodiments, all of the enzymes catalyzing reactions in the active succinate fermentation pathway are encoded by endogenous genes. In other embodiments, all of the enzymes catalyzing reactions in the active succinate fermentation pathway are encoded by exogenous genes. In still other embodiments, the enzymes catalyzing reactions in the active succinate fermentation pathway are encoded by a mix of endogenous and exogenous genes.

In certain embodiments, the genetically modified yeast cells provided herein comprise one or more endogenous genes encoding enzymes that catalyze various reactions in the active succinate fermentation pathway, and in certain of these embodiments the cells comprise one or more copies of endogenous pyruvate carboxylase, phosphoenolpyruvate carboxylase, malate dehydrogenase, fumarase, fumarate reductase, and/or succinate exporter genes. In certain of these embodiments, the endogenous genes are operatively linked to endogenous regulatory elements only. In other embodiments, one or more of the endogenous genes are operatively linked to one or more exogenous regulatory elements.

In certain embodiments, the genetically modified yeast cells provided herein comprise one or more exogenous genes encoding enzymes that catalyze various reactions in the active succinate fermentation pathway, and in certain of these embodiments the cells comprise one or more copies of exogenous pyruvate carboxylase, phosphoenolpyruvate carboxylase, malate dehydrogenase, fumarase, fumarate reductase, and/or succinate exporter genes. In certain of these embodiments, the exogenous genes are operatively linked to exogenous regulatory elements only. In other embodiments, one or more of the exogenous genes are operatively linked to one or more endogenous regulatory elements.

In certain embodiments, the genetically modified yeast cells provided herein comprise an endogenous and/or exogenous pyruvate carboxylase gene. In certain of those embodiments where the cells comprise an exogenous pyruvate carboxylase gene, the exogenous pyruvate carboxylase gene is derived from a yeast source gene such as anIssatchenkia orientalis, Saccharomyces cerevisiae, orKluyveromyces marxianussource gene, and in certain of these embodiments the exogenous pyruvate carboxylase gene encodes a poly peptide with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to a polypeptide encoded by the yeast source gene. In certain embodiments, the exogenous pyruvate carboxylase gene encodes a polypeptide that comprises the amino acid sequence of SEQ ID NOs:8, 10, or 12, or an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs:8, 10, or 12. In certain embodiments, the exogenous pyruvate carboxylase gene comprises the nucleotide sequence of SEQ ID NOs:7, 9, or 11, or a nucleotide sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NOs:7, 9, or 11. In certain other embodiments where the cells comprise an exogenous pyruvate carboxylase gene, the exogenous pyruvate carboxylase gene is derived from a fungal source gene other than aRhizopus oryzaesource gene.

In certain embodiments, the genetically modified yeast cells provided herein comprise an endogenous and/or exogenous phosphoenolpyruvate carboxylase gene. In certain of these embodiments where the cells comprise an exogenous phosphoenolpyruvate carboxylase gene, the exogenous phosphoenolpyruvate carboxylase gene is derived from a bacterial source gene such as anEscherichia coliorMannheimia succiniciproducenssource gene, and in certain of these embodiments the exogenous phosphoenolpyruvate carboxylase gene encodes a polypeptide with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to a polypeptide encoded by the bacterial source gene. In certain embodiments, the exogenous phosphoenolpyruvate carboxylase gene encodes a polypeptide that comprises the amino acid sequence of SEQ ID NOs:4 or 6, or an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs:4 or 6. In certain embodiments, the exogenous phosphoenolpyruvate carboxy lase gene comprises the nucleotide sequence of SEQ ID NOs:3 or 5, or a nucleotide sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NOs:3 or 5.

In certain embodiments, the genetically modified yeast cells provided herein comprise an endogenous and/or exogenous malate dehydrogenase gene. In certain of these embodiments where the cells comprise an exogenous malate dehydrogenase gene, the exogenous malate dehydrogenase gene is derived from a yeast source gene such as anI. orientalis, Zygosaccharomyces rouxii, orK. marxianussource gene, and in certain of these embodiments the exogenous malate dehydrogenase gene encodes a polypeptide with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to a polypeptide encoded by the yeast source gene. In certain embodiments, the exogenous malate dehydrogenase gene encodes a polypeptide that comprises the amino acid sequence of SEQ ID NOs:14, 16, 18, 168, 20, 22, or 24, or an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs:14, 16, 18, 168, 20, 22, or 24. In certain embodiments, the exogenous malate dehydrogenase gene comprises the nucleotide sequence of SEQ ID NOs:13, 15, 17, 167, 19, 21, or 23, or a nucleotide sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NOs:13, 15, 17, 167, 19, 21, or 23. In certain other embodiments where the cells comprise an exogenous malate dehydrogenase gene, the exogenous malate dehydrogenase gene is derived from a bacterial source gene such as anE. colisource gene, and in certain of these embodiments the exogenous malate dehydrogenase gene encodes a polypeptide with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to a polypeptide encoded by the bacterial source gene. In certain embodiments, the exogenous malate dehydrogenase gene encodes a polypeptide that comprises the amino acid sequence of SEQ ID NO:170, or an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:170. In certain embodiments, the exogenous malate dehydrogenase gene comprises the nucleotide sequence of SEQ ID NO:169, or a nucleotide sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO:169. In certain other embodiments where the cells comprise an exogenous malate dehydrogenase gene, the exogenous malate dehydrogenase gene is derived from a fungal source gene such as aR. oryzaesource gene, and in certain of these embodiments the exogenous malate dehydrogenase gene encodes a polypeptide with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to a polypeptide encoded by the fungal source gene. In certain embodiments, the exogenous malate dehydrogenase gene encodes a poly peptide that comprises the amino acid sequence of SEQ ID NO:172, or an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:172. In certain embodiments, the exogenous malate dehydrogenase gene comprises the nucleotide sequence of SEQ ID NO:171, or a nucleotide sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO:171.

In certain embodiments, the genetically modified yeast cells provided herein comprise an endogenous and/or exogenous fumarase gene. In certain of these embodiments where the cells comprise an exogenous fumarase gene, the exogenous fumarase gene is derived from a yeast source gene such as anI. orientalissource gene, and in certain of these embodiments the exogenous fumarase gene encodes a polypeptide with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to a polypeptide encoded by the yeast source gene. In certain embodiments, the exogenous fumarase gene encodes a polypeptide that comprises the amino acid sequence of SEQ ID NO:2, or an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:2. In certain embodiments, the exogenous fumarase gene comprises the nucleotide sequence of SEQ ID NO:1, or a nucleotide sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO:1.

In certain embodiments, the genetically modified yeast cells provided herein comprise an endogenous and/or exogenous fumarate reductase gene. In certain of these embodiments where the cells comprise an exogenous fumarate reductase gene, the exogenous fumarase gene is derived from a yeast source gene such as anS. cerevisiae, Saccharomyces mikatae, Kluyveromyces polyspora, orK. marxianussource gene, and in certain of these embodiments the exogenous fumarate reductase gene encodes a polypeptide with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to a polypeptide encoded by the yeast source gene. In certain embodiments, the exogenous fumarate reductase gene encodes a polypeptide that comprises the amino acid sequence of SEQ ID NOs:26, 28, 30, or 32, or an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs:26, 28, 30, or 32. In certain embodiments, the exogenous fumarate reductase gene comprises the nucleotide sequence of SEQ ID NOs:25, 27, 29, or 31, or a nucleotide sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NOs:25, 27, 29, or 31. In certain other embodiments where the cells comprise an exogenous fumarate reductase gene, the exogenous fumarate reductase gene is derived from a protozoan source gene such as aTrypanosoma brucei, Trypanosoma cruzi, Leishmania braziliensis, orLeishmania mexicanasource gene, and in certain of these embodiments the exogenous fumarate reductase gene encodes a polypeptide with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to a polypeptide encoded by the protozoan source gene. In certain embodiments, the exogenous fumarate reductase gene encodes a polypeptide that comprises the amino acid sequence of SEQ ID NOs:174, 176, 178, or 180, or an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs:174, 176, 178, or 180. In certain embodiments, the exogenous fumarate reductase gene comprises the nucleotide sequence of SEQ ID NOs:173, 175, 177, or 179, or a nucleotide sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NOs:173, 175, 177, or 179.

In certain embodiments, the genetically modified yeast cells provided herein comprise an endogenous and/or exogenous succinate exporter gene. In certain of these embodiments where the cells comprise an exogenous succinate exporter gene, the exogenous succinate exporter gene is derived from a fungal source gene such as anSchizosaccharomyces pombeorAspergillus oryzaesource gene, and in certain of these embodiments the exogenous succinate exporter gene encodes a polypeptide with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to a polypeptide encoded by the fungal source gene. In certain embodiments, the exogenous succinate exporter gene encodes a polypeptide that comprises the amino acid sequence of SEQ ID NOs:182 or 184, or an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NOs:182 or 184. In certain embodiments, the exogenous succinate exporter gene comprises the nucleotide sequence of SEQ ID NOs:181 or 183, or a nucleotide sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NOs:181 or 183.

In certain embodiments, the genetically modified yeast cells provided herein comprise, in addition to an active succinate fermentation pathway, an active reduction pathway from glucose-6-phosphate to ribulose-5-phosphate. In certain embodiments, this pathway includes at least the following reactions 1) conversion of glucose 6-phosphate to 6-phosphogluconaolactone, 2) conversion of 6-phosphogluconaolactone to 6-phosphogluconate, and 3) conversion of 6-phosphogluconate to ribulose 5-phosphate. Each of the reactions in the active reduction pathway is catalyzed by one or more enzymes, which in turn are encoded by one or more exogenous or endogenous reduction pathway genes. In certain embodiments, all of the enzymes catalyzing reactions in the active reduction pathway are encoded by endogenous genes. In other embodiments, all of the enzymes catalyzing reactions in the active reduction pathway are encoded by exogenous genes. In still other embodiments, the enzymes catalyzing reactions in the active reduction pathway are encoded by a mix of endogenous and exogenous genes.

In certain embodiments, the genetically modified yeast cells provided herein comprise one or more endogenous genes encoding enzymes that catalyze various reactions in the active reduction pathway, and in certain of these embodiments the cells comprise one or more copies of endogenous glucose 6-phosphate dehydrogenase, gluconolactonase, and/or 6-phosphogluconate dehydrogenase genes. In certain of these embodiments, the endogenous genes are operatively linked to endogenous regulatory elements only. In other embodiments, one or more of the endogenous genes are operatively linked to one or more exogenous regulatory elements.

In certain embodiments, the genetically modified yeast cells provided herein comprise one or more exogenous genes encoding enzymes that catalyze various reactions in the active reduction pathway, and in certain of these embodiments the cells comprise one or more copies of exogenous glucose 6-phosphate dehydrogenase, gluconolactonase, and/or 6-phosphogluconate dehydrogenase genes. In certain of these embodiments, the exogenous genes are operatively linked to exogenous regulatory elements only. In other embodiments, one or more of the exogenous genes are operatively linked to one or more endogenous regulatory elements.

In certain embodiments, the genetically modified yeast cells provided herein comprise an endogenous and/or exogenous glucose 6-phosphate dehydrogenase gene. In certain of these embodiments where the cells comprise an exogenous glucose 6-phosphate dehydrogenase gene, the exogenous glucose 6-phosphate dehydrogenase gene is derived from a yeast source gene such as anI. orientalissource gene, and in certain of these embodiments the exogenous glucose 6-phosphate dehydrogenase gene encodes a polypeptide with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to a poly peptide encoded by the yeast source gene. In certain embodiments, the exogenous glucose 6-phosphate dehydrogenase gene encodes a polypeptide that comprises the amino acid sequence of SEQ ID NO:34, or an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:34. In certain embodiments, the exogenous glucose 6-phosphate dehydrogenase gene comprises the nucleotide sequence of SEQ ID NO:33, or a nucleotide sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO:33.

In certain embodiments, the genetically modified yeast cells provided herein comprise an endogenous and/or exogenous gluconolactonase gene. In certain of these embodiments where the cells comprise an exogenous gluconolactonase gene, the exogenous gluconolactonase gene is derived from a yeast source gene such as anI. orientalissource gene, and in certain of these embodiments the exogenous gluconolactonase gene encodes a polypeptide with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to a polypeptide encoded by the yeast source gene. In certain embodiments, the exogenous gluconolactonase gene encodes a polypeptide that comprises the amino acid sequence of SEQ ID NO:36, or an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:36. In certain embodiments, the exogenous gluconolactonase gene comprises the nucleotide sequence of SEQ ID NO:35, or a nucleotide sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO:35.

In certain embodiments, the genetically modified yeast cells provided herein comprise an endogenous and/or exogenous 6-phosphogluconate dehydrogenase gene. In certain of these embodiments where the cells comprise an exogenous 6-phosphogluconate dehydrogenase gene, the exogenous 6-phosphogluconate dehydrogenase gene is derived from a yeast source gene such as anI. orientalissource gene, and in certain of these embodiments the exogenous 6-phosphogluconate dehydrogenase gene encodes a polypeptide with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to a polypeptide encoded by the yeast source gene. In certain embodiments, the exogenous 6-phosphogluconate dehydrogenase gene encodes a polypeptide that comprises the amino acid sequence of SEQ ID NO:38, or an amino acid sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:38. In certain embodiments, the exogenous 6-phosphogluconate dehydrogenase gene comprises the nucleotide sequence of SEQ ID NO:37, or a nucleotide sequence with at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99% sequence identity to the nucleotide sequence of SEQ ID NO:37.

In certain embodiments, the genetically modified yeast cells provided herein comprise a deletion or disruption of one or more endogenous genes. In certain of these embodiments, the cells comprise a deletion or disruption of an endogenous pyruvate carboxykinase, malic enzyme, pyruvate decarboxylase, and/or succinate importer gene.

In certain embodiments, the genetically modified yeast cells are derived from host yeast cells that exhibit a relatively high degree of succinate resistance. In certain embodiments, the cells provided herein belong to the genusIssatchenkia, Candida, Pichia, Zygosaccharomyces, Kluyveromyces, Saccharomyces, Debaryomyces, orSaccharomycopsis, and in certain of these embodiments the cells belong to thePichia fermentans/I. orientalisclade. In certain embodiments, the cells belong to the speciesI. orientalis, Candida lambica, Candida sorboxylosa, Candida zemplinina, Candida geochares, Pichia membranifaciens, Zygosaccharomyces kombuchaensis, Candida sorbosivorans, K. marxianus, Candida vanderwaltii, Candida sorbophila, Zygosaccharomyces bisporus, Zygosaccharomyces lentus, Saccharomyces bayanus, Saccharomyces bulderi, Debaryomyces castellii, Candida boidinii, Candida etchellsii, Kluyveromyces lactis, Pichia jadinii, Pichia anomala, orSaccharomycopsis crataegensis.

Provided herein in certain embodiments are methods of producing succinate by culturing the genetically modified yeast cells provided herein in the presence of at least one carbon source, then isolating the succinate from the culture. In certain embodiments, the carbon source is one or more of glucose, xylose, arabinose, sucrose, fructose, cellulose, glucose oligomers, and glycerol. Also provided herein are processes for converting succinate produced by the methods provided herein to a product such as poly-butylene succinate (PBS), other polymers, cosmetics, foodstuff, feed, or pharmaceuticals.

