# Patent

## Title
Methods for succinate production

## Abstract

Provided herein are genetically modified yeast cells for the production of succinate, methods of making these yeast cells, and methods of using these cells to produce succinate.

## Background
RELATED APPLICATIONS
The present application claims priority to U.S. Provisional Patent Application No. 61/590,795, filed Jan. 25, 2012, and to PCT/US2013/023300, filed Jan. 25, 2013, the disclosures of which are incorporated by reference herein in their entirety, including drawings.


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.
SUMMARY
Provided herein in certain embodiments are methods of producing succinate, for example succinic acid, 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.
In one aspect, provided is a method of producing a succinate-containing fermentation broth, the method comprising: culturing in a fermenter a genetically modified yeast cell in the presence of at least one carbon source to provide succinate; and introducing into the fermenter sufficient oxygen to provide an oxygen uptake rate of greater than about 5 mmol/L/h (for example, greater than about 6 mmol/L/h, greater than about 8 mmol/L/h, greater than about 10 mmol/L/h, greater than about 12 mmol/L/h, or greater than about 14 mmol/L/h), wherein a final concentration of succinate in the fermentation broth is greater than about 20 g/L (for example, greater than about 40 g/L, greater than about 60 g/L, or greater than about 80 g/L).
In some embodiments, a production rate of succinate is greater than about 0.25 g/L/h (for example, greater than about 1 g/L/h, greater than about 1.5 g/L/h, greater than about 2 g/L/h, or greater than about 3 g/L/h). In some embodiments, a specific production rate of succinate is greater than about 0.1 g/g/h (for example, greater than about 0.15 g/g/h, greater than about 0.5 g/g/h, or greater than about 1.0 g/g/h). In some embodiments, the product yield of succinate is greater than about 25%. In some embodiments, the cell dry weight at end of fermentation is less than about 10 g/L (for example, less than about 7 g/L, less than about 5 g/L, or less than about 3 g/L). In some embodiments, the genetically modified yeast cell dry weight at inoculation is less than about 5 g/L (for example, less than about 3 g/L).
In some embodiments, the genetically modified yeast cell has an active succinate fermentation pathway from phosphoenolpyruvate or pyruvate to succinate. In some embodiments, the active succinate fermentation pathway includes the reactions: (a) pyruvate to oxaloacetate; (b) oxaloacetate to malate; (c) malate to fumarate; and (d) fumarate to succinate. In some embodiments, the succinate fermentation pathway further includes the reaction (e) export of succinate from inside the yeast cell to the extracellular environment. In some embodiments, the genetically modified yeast cell comprises one or more copies of one or more endogenous genes that encode an enzyme that catalyzes any of reactions (a) though (e). In some embodiments, the genetically modified yeast cell comprises one or more copies of one or more endogenous genes selected from the group consisting of pyruvate carboxylase, malate dehydrogenase, fumarase, fumarate reductase, and succinate exporter genes. In some embodiments, one or more of the endogenous genes are operatively linked to an exogenous regulatory element selected from the group consisting of an exogenous promoter and an exogenous terminator. In some embodiments, the genetically modified yeast cell comprises one or more copies of one or more exogenous genes that encode an enzyme that catalyzes any of reactions (a) though (e). In some embodiments, the genetically modified yeast cell comprises one or more copies of one or more exogenous genes selected from the group consisting of pyruvate carboxylase, malate dehydrogenase, fumarase, fumarate reductase, and succinate exporter genes. In some embodiments, the exogenous malate dehydrogenase genes are Rhizopus delemar malate dehydrogenase genes encoding the amino acid sequence of SEQ ID NO:167, and in certain of these embodiments the Rhizopus delemar malate dehydrogenase genes comprise the nucleotide sequence of SEQ ID NO:166. In some embodiments, the genetically modified yeast cell comprises a genetic modification to enhance succinate export. In some embodiments, the genetically modified yeast cell belongs to a genus selected from the group consisting of Issatchenkia, Candida, Pichia, Zygosaccharomyces, Kluyveromyces, Saccharomyces, Debaryomyces, and Saccharomycopsis. In some embodiments, the genetically modified yeast cell is a species selected from the group consisting of Issatchenkia orientalis, Candida lambica, Candida sorboxylosa, Candida zemplinina, Candida geochares, Pichia membranifaciens, Zygosaccharomyces kombuchaensis, Candida sorbosivorans, Kluyveromyces 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, Saccharomycopsis crataegensis, and Pichia jadinii. In some embodiments, the genetically modified yeast cell is from the Pichia fermentans/Issatchenkia orientalis clade.
In some embodiments, the carbon source is selected from the group consisting of glucose, xylose, arabinose, sucrose, fructose, cellulose, glucose oligomers, and glycerol. In some embodiments, a final pH of the fermentation broth is less than about 6.0 (for example, less than about 5.5, less than about 5.0, less than about 4.0, or less than about 3.5). In some embodiments, a final concentration of succinate comprises greater than about 20 g/L of succinate (for example, greater than about 40 g/L, greater than about 80 g/L, or greater than about 90 g/L). In some embodiments, the active succinate fermentation pathway further includes step (e) a genetic modification to enhance succinate export. In some embodiments, the genetically modified yeast cell has a deletion or disruption in pyruvate decarboxylase (“PDC”) gene. In some embodiments, the genetically modified yeast cell is a succinate resistant yeast cell.
In another aspect, provided is a method of producing succinate, the method comprising: culturing in a fermenter a genetically modified yeast cell in the presence of at least one carbon source to produce succinate; and providing sufficient oxygen to the fermenter to obtain an oxygen uptake rate of from about 8 mmol/L/h to about 25 mmol/L/h, wherein the cell dry weight at end of fermentation is less than about 10 g/L, and wherein the method comprises at least one of the following properties: i) a final concentration of succinate in the fermentation broth is greater than about 20 g/L, ii) a production rate of succinate greater than about 0.25 g/L/h, iii) a product yield of succinate greater than 25%, and iv) a specific succinate production rate greater than about 0.1 g/g/h. In some embodiments, a final pH of the fermentation broth is less than about 6.0, suitably less than about 5.5, desirably less than about 5.0, preferably less than about 4.0, and more preferably less than about 3.5. In some embodiments, the genetically modified yeast cell has an oxygen uptake rate of from about 8 mmol/L/h to about 20 mmol/L/h (for example, from about 8 mmol/L/h to about 15 mmol/L/h). In some embodiments, the production rate of succinate is greater than about 0.25 g/L/h (for example, greater than about 1 g/L/h, greater than about 1.5 g/L/h, greater than about 2 g/L/h, or greater than about 3 g/L/h). In some embodiments, the product yield of succinate is greater than about 50% (for example, greater than about 60%, greater than about 70%, greater than about 80%, or greater than about 90%). In some embodiments, the genetically modified yeast is Crabtree negative. In some embodiments, the dissolved oxygen in the fermentation broth is maintained at less than about 10% of air saturation at one atmosphere for greater than about 10 hours during the batch time.



BRIEF DESCRIPTION OF DRAWINGS
FIG. 1 shows plasmid pMI449, CYB2A deletion construct.
FIG. 2 shows plasmid pMI454, CYB2A deletion construct.
FIG. 3 shows plasmid pHJJ17.
FIG. 4 shows plasmid pVB10, PDC:cre construct.
FIG. 5 shows plasmid pVB32.
FIG. 6 shows plasmid pKF031, PGK:MEL5 construct.
FIG. 7 shows plasmid pKF044, CYB2A construct.
FIG. 8 shows plasmid pKF043, I. orientalis PYC1 expression construct.
FIG. 9 shows plasmid pKF045, I. orientalis PYC1 expression construct.
FIG. 10 shows plasmid pHJJ23.
FIG. 11 shows plasmid pGPB11, ADHa deletion construct.
FIG. 12 shows plasmid pGPB14, ADHa deletion construct.
FIG. 13 shows plasmid pGPB28, ADHa deletion construct.
FIG. 14 shows plasmid pGPB34, ADHa deletion construct.
FIG. 15 shows plasmid pGPB20, 22, 25, and 26, FRD1 expression constructs.
FIG. 16 shows plasmid pGPB36, 37, 39, and 40, FRD1 expression constructs.
FIG. 17 shows plasmid pKWB18, ATO2 deletion construct.
FIG. 18 shows plasmid pKWB23, ATO2 deletion construct.
FIG. 19 shows plasmid pKWB28, ATO2 deletion construct.
FIG. 20 shows plasmid pGPB54, MDH expression construct.
FIG. 21 shows plasmid pGPB55, MDH expression construct.
FIG. 22 shows plasmid pGPB64, Z. rouxii MDH expression construct.
FIG. 23 shows plasmid pGPB66, Z. rouxii MDH expression construct.
FIG. 24 shows plasmid pGPB30, FUM1 expression constructs.
FIG. 25 shows plasmid pGPB44, FUM1 expression constructs.
FIG. 26 shows plasmid pGPB43, FUM1 expression constructs.
FIG. 27 shows plasmid pGPB47, FUM1 expression constructs.
FIG. 28 shows plasmid pVMB54, RIOR43690 deletion construct.
FIG. 29 shows plasmid pKWB95, SpMAE expression construct.
FIG. 30 shows plasmid pKWB96, SpMAE expression construct.
FIG. 31 shows plasmid pKWB97, SpMAE expression construct.
FIG. 32 shows plasmid pHJJ77.
FIG. 33 shows plasmid pGPB119, ADHb deletion construct.
FIG. 34 shows plasmid pGPB126 (Trypanosoma brucei), pGPB159 (Trypanosoma cruzi), pGPB160 (Leishmania braziliensis), and pGPB161 (Leishmania mexicana) FRD expression constructs.
FIG. 35 shows plasmid pGPB127, Trypanosoma brucei expression construct.
FIG. 36 shows plasmid pGPB168, ADHb deletion construct.
FIG. 37 shows plasmid pGPB148, ADHb deletion construct.
FIG. 38 shows a dissolved oxygen profile of fermentation; straight batch glucose, pH 3.0, 8.6% CO2, OUR 10.2 (mmol/L/h).
FIG. 39 shows succinate and free sucinate production (g/L) at 90 h: straight batch glucose, pH 3.0, 10% CO2, OUR (mmol/L/h) variable.
FIG. 40 shows succinate and free sucinate production (g/L) at 90 h: fed-batch glucose, pH 3.0, 10% CO2, OUR (mmol/L/h) variable.
FIG. 41 shows yield to succinate (g/g glucose) at 90 h fermentation time at pH 3.0, 10% CO2, OUR (mmol/L/h) variable, straight batch glucose.
FIG. 42 shows succinate production (g/L) at 90 h: straight batch glucose, pH 3.0, 10% CO2, OUR (mmol/L/h) variable. FIG. 42 also shows the product of: succinate (g/L) multiplied by the succinate production rate (g/L/h) multiplied by the yield (g succinate/g glucose) with OUR as the variable.



DETAILED DESCRIPTION
The following description of the invention is merely intended to illustrate various embodiments of the invention. As such, the specific modifications discussed are not to be construed as limitations on the scope of the invention. It will be apparent to one skilled in the art that various equivalents, changes, and modifications may be made without departing from the scope of the invention, and it is understood that such equivalent embodiments are to be included herein. All references cited herein are incorporated by reference in their entirety.
ABBREVIATIONS
α-KGDH, α-ketoglutarate dehydrogenase; CYB2, L-(+)-lactate:ferricytochrome c oxidoreductase; CYC, iso-2-cytochrome c; ENO1, enolase; FRD, fumarate reductase; FUM, fumarase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPD, glycerol 3-phosphate dehydrogenase; G6PD, glucose 6-phosphate dehydrogenase; IDH, isocitrate dehydrogenase; MDH, malate dehydrogenase; OAA, oxaloacetate; OUR, oxygen uptake rate; PCK, phosphoenolpyruvate carboxykinase; PDC, pyruvate decarboxylase; PEP, phosphoenolpyruvate; 6PGDH, 6-phosphogluconate dehydrogenase; PGK, phosphoglycerate kinase; PFL, pyruvate formate lyase; PPC, phosphoenolpyruvate carboxylase; PYC, pyruvate carboxylase; RKI, ribose 5-phosphate ketol-isomerase; TAL, transaldolase; TCA, tricarboxylic acid; TEF, translation elongate factor; TKL, transketolase; URA3, orotidine 5′-phosphate decarboxylase; XDH, xylitol dehydrogenase; XR, xylose reductase; 6PGDH, 6-phosphogluconate dehydrogenase; SpMAE, succinate exporter.
Definitions
As used herein, “inoculation” is defined as the point in time wherein a microorganism capable of producing succinate is introduced into the fermentation media. This is a term that is well known to those skilled in the art.
As used herein, “end of fermentation” is defined as point in time where the fermentation is stopped to harvest the succinate. This point is triggered by meeting any of the following criteria: exhaustion of the desired fraction of carbon source supplied, cessation of carbon source consumption, or cessation of succinate formation.
As used herein, “cell dry weight at inoculation” refers to the concentration of dry cell mass present in the fermentation medium at inoculation, as measured in a fermentation sample. For fed-batch fermentations, the initial cell dry weight is calculated based on the final volume of fermentation media. Measurement of dry cell weight is a method known to those skilled in the art. Cell dry weight at inoculation is commonly expressed in units of g/L.
As used herein, “cell dry weight at end of fermentation” refers to the concentration of dry cell mass present in the fermentation medium at end of fermentation, as measured in a fermentation sample. Cell dry weight at end of fermentation is commonly expressed in units of g/L.
As used herein, “final titer” shall be defined as the concentration of a substance in the fermentation broth at the end of fermentation. The final titer is commonly expressed in units of grams/liter (g/L).
As used herein, “initial titer” shall be defined as the concentration of a substance present at inoculation. The initial titer is commonly expressed in units of grams/liter (g/L).
As used herein, “batch time” shall be defined as the amount of time that has elapsed between the inoculation and the end of fermentation. The batch time is commonly expressed in units of hours (h).
As used herein, “production rate” shall be defined as the final titer of succinate at end of fermentation divided by the batch time. The production rate is commonly expressed in units of grams per liter per hour (g/L/h).
One skilled in the art will recognize that the unit designation “x/y/z” is equivalent to and represents a shorthand version of the units xy−1z−1 or x/(y*z). For example, g/L/h is equivalent to gL−1 h−1 or g/(L*h).
As used herein, the “specific production rate” refers to the production rate divided by the cell dry weight at the end of fermentation.
As used herein, “product yield” for succinate shall be defined as a ratio of two quantities: a) mass of product (succinate) produced in the course of the batch (numerator) b) the mass of carbon source added to the batch (denominator). The product yield as a percentage is commonly expressed in units of gram per gram (g/g) times 100. Particular note should be taken that product yield is calculated as a ratio of masses (g). The mass of succinate produced should account for the mass of succinate present in the fermentation medium at the end of the batch, as well as the mass of any succinate harvested during the course of the batch, less the mass of succinate present at the start of batch, and further less the mass of any succinate added during the course of the batch. The mass of carbon source added to the batch should include the mass of all carbon source(s) present in the fermenter at the start of the batch in addition to the mass of any carbon source(s) added during the course of the batch less the mass of carbon source(s) present in the fermenter at the end of fermentation.
As used herein, a yeast cell having a Crabtree-negative phenotype is any yeast cell that does not exhibit the Crabtree effect. The term “Crabtree-negative” refers to both naturally-occurring and genetically-modified organisms. Briefly, the Crabtree effect is defined as the inhibition of oxygen consumption by a microorganism when cultured under aerobic conditions due to the presence of glucose (e.g., 50 grams of glucose/L). That is, a yeast cell having a Crabtree-positive phenotype continues to ferment irrespective of oxygen availability due to the presence of glucose, while a yeast cell having a Crabtree-negative phenotype does not exhibit glucose mediated inhibition of oxygen consumption. Examples of yeast cells typically having a Crabtree-negative phenotype include, without limitation, yeast cells from the following genera: Kluyveromyces, Pichia, Hansenula, Trichosporon, and Yamadazyma. 
“Succinate” as used herein includes the anions of a succinate salt and acid forms of succinate such as succinic acid.
“Oxygen uptake rate” (“OUR”) as used herein refers to the volumetric rate at which oxygen is consumed during the fermentation. Inlet and outlet oxygen concentrations can be measured with exhaust gas analysis, for instance by mass spectrometers. OUR is calculated by one of ordinary skill in the relevant arts, using the Direct Method described in Bioreaction Engineering Principles 2nd Edition, 2003, Kluwer Academic/Plenum Publishers, p. 449, equation 1.
As used herein, “final pH” refers to fermentation medium pH at end of fermentation, as measured in a fermentation sample.
DESCRIPTION
Provided herein are genetically modified yeast cells for the production of succinate, methods of making these yeast cells, and methods of using these cells to produce succinate.
There are three primary fermentation pathways for producing succinate from a microorganism: reductive TCA, oxidative TCA, and glyoxylate shunt.
The reductive TCA pathway begins with carboxylation of the three carbon glycolytic intermediate phosphoenolpyruvate (PEP) or pyruvate to oxaloacetate (OAA) (by PEP carboxylase (PPC) and pyruvate carboxylase (PYC), respectively). OAA is converted to malate by malate dehydrogenase (MDH), malate is converted to fumarate by fumarase (FUM, also known as fumarate hydratase), and fumarate is converted to succinate by fumarate reductase (FRD). When written from the perspective of redox state, the net stoichiometry for this succinate production pathway is: 1 glucose (C6H12O6)+2 CO2+2 (NADH+H+)→2 succinic acid (C4H6O4)+2H2O+2 NAD+. The reductive TCA pathway provides the highest succinate yield of the three succinate fermentation pathways, but it results in a net deficit in reducing power (NADH). This means that in isolation the pathway results in a redox imbalance. In order to provide redox balance, the reductive TCA pathway can be combined with one or both of the oxidative TCA or glyoxylate shunt pathways, or with one or more unrelated pathways that produce NADH or NADPH.
The oxidative TCA pathway begins with the conversion of OAA and acetyl-CoA to citrate by citrate synthase. OAA can be generated from carboxylation of PEP or pyruvate, while acetyl-CoA is generated from the decarboxylation of pyruvate by PDH or pyruvate formate lyase (PFL). Citrate is converted to isocitrate by aconitase, isocitrate is converted to a-ketoglutarate by isocitrate dehydrogenase (IDH), α-ketoglutarate is converted to succinyl-CoA by α-ketoglutarate dehydrogenase (α-KGDH), and succinyl-CoA is converted to succinate by succinyl coenzyme A synthetase (succinate thiokinase). The net stoichiometry for this succinate production pathway is: 1 glucose (C6H12O6)+2H2O+5 NAD+→1 succinic acid (C4H6O4)+2 CO2+5 (NADH+H+). This pathway has a lowest carbon yield of the three succinate fermentation pathways, but the highest yield of reducing power.
Like the oxidative TCA pathway, the glyoxylate shunt pathway begins with the generation of citrate from OAA and acetyl-CoA and the conversion of citrate to isocitrate. Isocitrate is converted to glyoxylate and succinate by isocitrate lyase. Glyoxylate is condensed with acetyl-CoA to form malate by malate synthase, and the resultant malate is converted to succinate via a fumarate intermediate. The net stoichiometry for this succinate production pathway is: 1 glucose (C6H12O6)+% H2O+2% NAD+→11⁄3 succinic acid (C4H6O4)+2⁄3 CO2+22⁄3 (NADH+H+).
Previous attempts to produce succinate from microorganisms at commercially viable levels have utilized bacterial fermentation hosts. These bacterial hosts are either native succinate producers or non-native succinate producers that have been genetically engineered to produce succinate. Examples of native succinate producers are Actinobacillus succinogenes (see, e.g., U.S. Pat. No. 5,504,004) and Mannheimia succiniciproducens, each of which primarily utilizes a reductive TCA pathway. A. succinogenes and M. succiniciproducens both produce relatively high titers of succinate, but they also produce various organic acid by-products. The presence of these by-products decreases yield and complicates succinate recovery. An example of a non-native succinate producer is Escherichia coli. Although E. coli is capable of producing trace levels of succinate naturally, genetic modification is required to obtain useful titers. Significant efforts have been made previously to increase succinate yield in genetically modified E. coli by decreasing the formation of other organic acids and combining different succinate fermentation pathways. Although E. coli strains have been developed that produce fewer organic acid by-products, they still produce lower succinate titers than the native producers. In addition, E. coli requires aerobic conditions to grow, but produces succinic acid at high yields only under anaerobic conditions. This means that succinate production in E. coli requires a two-phase fermentation.
One drawback common to all of the bacterial hosts developed to date for succinate production is relatively poor performance in strongly acidic environments. However, allowing the low pH conditions to develop as organic acids are produced is preferred for commercial succinate production. The ideal host for commercial succinate production should produce high levels of succinate and relatively low levels of other organic acids, and should possess a high degree of pH resistance and the ability to both grow and ferment under anaerobic or substantially anaerobic conditions.
As disclosed herein, a set of yeast cells from various species were tested for succinate resistance. Cells exhibiting succinate resistance were further evaluated based on their growth rates and glucose consumption rates in media containing varying concentrations of succinate. Based on these experiments, a set of ideal host cells for succinate production were identified. These host cells were then genetically modified to contain an active succinate fermentation pathway, resulting in a set of genetically modified yeast cells that produce succinate under low pH conditions.
Provided herein in certain embodiments are genetically modified succinate-resistant yeast cells having at least one active succinate fermentation pathway from PEP or pyruvate to succinate. A yeast cell having an “active succinate fermentation pathway” as used herein produces active enzymes necessary to catalyze each reaction in a succinate fermentation pathway, and therefore is capable of producing succinate in measurable yields when cultured under fermentation conditions in the presence of at least one fermentable sugar. A yeast cell having an active succinate fermentation pathway comprises one or more succinate fermentation pathway genes. A “succinate fermentation pathway gene” as used herein refers to the coding region of a nucleotide sequence that encodes an enzyme involved in an active succinate fermentation pathway.
In certain embodiments, the yeast cells provided herein have a reductive TCA active succinate fermentation pathway that proceeds through PEP or pyruvate, OAA, malate, and fumarate intermediates. In these embodiments, the yeast cells comprise a set of succinate fermentation pathway genes comprising MDH, FUM, and FRD genes, one or both of PPC and PYC genes, and a succinate exporter.
In those embodiments where the yeast cells provided herein have a reductive TCA active succinate fermentation pathway, the cells may further have an active reduction pathway. An “active reduction pathway” as used herein produces NADH or NADPH from NAD or NADP, respectively, thereby helping to balance out redox imbalances generated by a reductive TCA pathway. A yeast cell having an active reduction pathway comprises one or more reduction pathway genes. A “reduction pathway gene” as used herein refers to the coding region of a nucleotide sequence that encodes an enzyme involved in an active reduction pathway.
In certain embodiments, the yeast cells provided herein have a pentose phosphate active reduction pathway that proceeds through glucose 6-phosphate, 6-phosphogluconaolactone, 6-phosphogluconate, and ribulose 5-phosphate intermediates. In these embodiments, the yeast cells comprise a set of reduction pathway genes comprising glucose 6-phosphate dehydrogenase (G6PD), gluconolactonase, and 6-phosphogluconate dehydrogenase (6PGDH) genes.
In certain embodiments, the yeast cells provided herein may have one or more active succinate fermentation pathways, or portions of such pathways, that are not reductive TCA active succinate fermentation pathways. In these embodiments, the other pathways or portions thereof may be present in addition to or in lieu of the reductive TCA pathway. For example, the cells may comprise a reductive TCA active succinate fermentation pathway and all or a part of an oxidative TCA or glyoxylate shunt active succinate fermentation pathway.
The succinate fermentation pathway and reduction pathway genes in the yeast cells provided herein may be endogenous or exogenous. “Endogenous” as used herein with regard to genetic components such as genes, promoters, and terminator sequences means that the genetic component is present at a particular location in the genome of a native form of a particular yeast cell. “Exogenous” as used herein with regard to genetic components means that the genetic component is not present at a particular location in the genome of a native form of a particular yeast cell. “Native” as used herein with regard to a yeast cell refers to a wild-type yeast cell of a particular yeast species. “Native” as used herein with regard to a metabolic pathway refers to a metabolic pathway that exists and is active in a native yeast cell.
An exogenous genetic component may have either a native or non-native sequence. An exogenous genetic component with a native sequence comprises a sequence identical to (apart from individual-to-individual mutations which do not affect function) a genetic component that is present in the genome of a native cell (i.e., the exogenous genetic component is identical to an endogenous genetic component). However, the exogenous component is present at a different location in the host cell genome than the endogenous component. For example, an exogenous MDH gene that is identical to an endogenous MDH gene may be inserted into a yeast cell, resulting in a modified cell with a non-native (increased) number of MDH gene copies. Similarly, an exogenous PDC promoter that is identical to an endogenous PDC promoter can be inserted into a yeast cell such that it is operatively linked to an endogenous gene such as an MDH gene, resulting in altered expression of the endogenous gene. An exogenous genetic component with a non-native sequence comprises a sequence that is not found in the genome of a native cell. For example, an exogenous MDH gene from a particular species may be inserted into a yeast cell of another species. Similarly, an exogenous PDC promoter from a particular species may be inserted into a yeast cell of another species.
An exogenous gene is preferably integrated into the host cell genome in a functional manner, meaning that it is capable of producing an active protein in the host cell. However, in certain embodiments the exogenous gene may be introduced into the cell as part of a vector that is stably maintained in the host cytoplasm.
In certain embodiments, the genetically modified yeast cells provided herein comprise one or more exogenous succinate fermentation and/or reduction pathway genes.
In certain embodiments, the yeast cells provided herein comprise one or more endogenous succinate fermentation and/or reduction pathway genes.
In certain embodiments, the yeast cells provided herein comprise one or more endogenous succinate fermentation and/or reduction pathway genes and one or more exogenous succinate fermentation and/or reduction pathway genes. In certain embodiments, the yeast cells may comprise both endogenous and exogenous copies of a single succinate fermentation pathway gene. For example, a yeast cell may comprise both endogenous and exogenous copies of an MDH gene.
Succinate fermentation and/or reduction pathway genes in the modified yeast cells provided herein may be operatively linked to one or more regulatory elements such as a promoter or terminator. As used herein, the term “promoter” refers to an untranslated sequence located upstream (i.e., 5′) to the translation start codon of a gene (generally within about 1 to 1000 base pairs (bp), preferably within about 1 to 500 bp) which controls the start of transcription of the gene. The term “terminator” as used herein refers to an untranslated sequence located downstream (i.e., 3′) to the translation finish codon of a gene (generally within about 1 to 1000 bp, preferably within about 1 to 500 bp, and especially within about 1 to 100 bp) which controls the end of transcription of the gene. A promoter or terminator is “operatively linked” to a gene if its position in the genome relative to that of the gene is such that the promoter or terminator, as the case may be, performs its transcriptional control function. Suitable promoters and terminators are described, for example, in WO99/14335, WO00/71738, WO02/42471, WO03/102201, WO03/102152 and WO03/049525 (all incorporated by reference herein in their entirety).
Regulatory elements linked to succinate fermentation and/or reduction pathway genes in the yeast cells provided herein may be endogenous or exogenous. In certain embodiments, exogenous regulatory elements may comprise a sequence with a relatively high degree of sequence identity to a native regulatory element. For example, an exogenous gene may be linked to an exogenous promoter or terminator having at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% sequence identity to a native promoter or terminator. Sequence identity percentages for nucleotide or amino acid sequences can be calculated by methods known in the art, such as for example using BLAST (National Center for Biological Information (NCBI) Basic Local Alignment Search Tool) version 2.2.1 software with default parameters. For example, a sequence having an identity score of at least 90% using the BLAST version 2.2.1 algorithm with default parameters is considered to have at least 90% sequence identity. The BLAST software is available from the NCBI, Bethesda, Md. In those embodiments wherein multiple exogenous genes are inserted into a host cell, each exogenous gene may be under the control of a different regulatory element, or two or more exogenous genes may be under the control of the same regulatory elements.
Examples of promoters that may be linked to one or more succinate fermentation and/or reduction pathway genes in the yeast cells provided herein include, but are not limited to, promoters for pyruvate decarboxylase (PDC1), phosphoglycerate kinase (PGK), xylose reductase (XR), xylitol dehydrogenase (XDH), L-(+)-lactate-cytochrome c oxidoreductase (CYB2), translation elongation factor-1 or -2 (TEF1, TEF2), enolase (ENO1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and orotidine 5′-phosphate decarboxylase (URA3) genes. In these examples, the succinate fermentation and/or reduction pathway genes may be linked to endogenous or exogenous promoters for PDC1, PGK, XR, XDH, CYB2, TEF1, TEF2, ENO1, GAPDH, or URA3 genes. Where the promoters are exogenous, they may be identical to or share a high degree of sequence identity (i.e., at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%) with native promoters for PDC1, PGK, XR, XDH, CYB2, TEF1, TEF2, ENO1, GAPDH, or URA3 genes.
Examples of terminators that may be linked to one or more succinate fermentation and/or reduction pathway genes in the yeast cells provided herein include, but are not limited to, terminators for PDC1, XR, XDH, transaldolase (TAL), transketolase (TKL), ribose 5-phosphate ketol-isomerase (RKI), CYB2, or iso-2-cytochrome c (CYC) genes or the galactose family of genes (especially the GAL10 terminator). In these examples, the succinate fermentation and/or reduction pathway genes may be linked to endogenous or exogenous terminators for PDC1, XR, XDH, TAL, TKL, RKI, CYB2, or CYC genes or galactose family genes. Where the terminators are exogenous, they may be identical to or share a high degree of sequence identity (i.e., at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%) with native terminators for PDC1, XR, XDH, TAL, TKL, RKI, CYB2, or CYC genes or galactose family genes. In certain embodiments, succinate fermentation and/or reduction pathway fermentation pathway genes are linked to a terminator that comprises a functional portion of a native GAL10 gene native to the host cell or a sequence that shares at least 80%, at least 85%, at least 90%, or at least 95% sequence identity with a native GAL10 terminator.
Exogenous genes may be inserted into a yeast host cell via any method known in the art. In preferred embodiments, the genes are integrated into the host cell genome. Exogenous genes may be integrated into the genome in a targeted or a random manner. In those embodiments where the gene is integrated in a targeted manner, it may be integrated into the loci for a particular gene, such that integration of the exogenous gene is coupled to deletion or disruption of a native gene. Alternatively, the exogenous gene may be integrated into a portion of the genome that does not correspond to a gene.
Targeted integration and/or deletion may utilize an integration construct. The term “construct” as used herein refers to a DNA sequence that is used to transform a cell. The construct may be, for example, a circular plasmid or vector, a portion of a circular plasmid or vector (such as a restriction enzyme digestion product), a linearized plasmid or vector, or a PCR product prepared using a plasmid or genomic DNA as a template. Methods for transforming a yeast cell with an exogenous construct are described in, for example, WO99/14335, WO00/71738, WO02/42471, WO03/102201, WO03/102152, and WO03/049525.
An integration or deletion construct may comprise one or more selection marker cassettes cloned into the construct between the two target DNA sequences. The selection marker cassette contains at least one selection marker gene that allows for selection of transformants. A “selection marker gene” is a gene that encodes a protein needed for the survival and/or growth of the transformed cell in a selective culture medium, and therefore can be used to apply selection pressure to the cell. Successful transformants will contain the selection marker gene, which imparts to the successfully transformed cell at least one characteristic that provides a basis for selection. Typical selection marker genes encode proteins that (a) confer resistance to antibiotics or other toxins (e.g., resistance to bleomycin or zeomycin (e.g., Streptoalloteichus hindustanus ble gene), aminoglycosides such as G418 or kanamycin (e.g., kanamycin resistance gene from transposon Tn903), or hygromycin (e.g., aminoglycoside antibiotic resistance gene from E. coli)), (b) complement auxotrophic deficiencies of the cell (e.g., deficiencies in leucine (e.g., Kluyveromyces marxianus LEU2 gene), uracil (e.g., K. marxianus, Saccharomyces cerevisiae, or Issatchenkia orientalis URA3 gene), or tryptophan (e.g., K. marxianus, S. cerevisiae, or I. orientalis TRP gene)), (c) enable the cell to synthesize critical nutrients not available from simple media, or (d) confer the ability for the cell to grow on a particular carbon source (e.g., MEL5 gene from S. cerevisiae, which encodes the alpha-galactosidase (melibiose) enzyme and confers the ability to grow on melibiose as the sole carbon source). Preferred selection markers include the URA3 gene, zeocin resistance gene, G418 resistance gene, MEL5 gene, and hygromycin resistance gene. Another preferred selection marker is a CYB2 gene cassette, provided that the host cell either natively lacks such a gene or that its native CYB2 gene(s) are first deleted or disrupted. A selection marker gene is operatively linked to one or more promoter and/or terminator sequences that are operable in the host cell. In certain embodiments, these promoter and/or terminator sequences are exogenous promoter and/or terminator sequences that are included in the selection marker cassette. Suitable promoters and terminators are as described above.
An integration or deletion construct is used to transform the host cell. Transformation may be accomplished using, for example, electroporation and/or chemical transformation (e.g., calcium chloride, lithium acetate-based, etc.) methods. Selection or screening based on the presence or absence of the selection marker may be performed to identify successful transformants. In successful transformants, a homologous recombination event at the locus of the target site results in the disruption or the deletion of the target site sequence. Where the construct targets a native gene for deletion or disruption, all or a portion of the native target gene, its promoter, and/or its terminator may be deleted during this recombination event. The expression cassette, selection marker cassette, and any other genetic material between the target sequences in the integration construct is inserted into the host genome at the locus corresponding to the target sequences. Analysis by PCR or Southern analysis can be performed to confirm that the desired insertion/deletion has taken place.
In some embodiments, cell transformation may be performed using DNA from two or more constructs, PCR products, or a combination thereof, rather than a single construct or PCR product. In these embodiments, the 3′ end of one integration fragment overlaps with the 5′ end of another integration fragment. In one example, one construct will contain the first sequence from the locus of the target sequence and a non-functional part of the marker gene cassette, while the other will contain the second sequence from the locus of the target sequence and a second non-functional part of the marker gene cassette. The parts of the marker gene cassette are selected such that they can be combined to form a complete cassette. The cell is transformed with these pieces simultaneously, resulting in the formation of a complete, functional marker or structural gene cassette. Successful transformants can be selected for on the basis of the characteristic imparted by the selection marker. In another example, the selection marker resides on one fragment but the target sequences are on separate fragments, so that the integration fragments have a high probability of integrating at the site of interest. In other embodiments, transformation from three linear DNAs can be used to integrate exogenous genetic material. In these embodiments, one fragment overlaps on the 5′ end with a second fragment and on the 3′ end with a third fragment.
An exogenous succinate fermentation or reduction pathway gene in the modified yeast cells provided herein may be derived from a source gene from any suitable source organism. For example, an exogenous gene may be derived from a yeast, fungal, bacterial, plant, insect, or mammalian source. As used herein, an exogenous gene that is “derived from” a source gene encodes a polypeptide that 1) is identical to a polypeptide encoded by the source gene, 2) shares 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 with a polypeptide encoded by the source gene, and/or 3) has the same function in a succinate fermentation or reduction pathway as the polypeptide encoded by the source gene. For example, a FUM gene that is derived from an I. orientalis FUM gene may encode a polypeptide comprising the amino acid sequence of SEQ ID NO:2, 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 the amino acid sequence of SEQ ID NO:2, and/or a polypeptide that has the ability to catalyze the conversion of malate to fumarate. A gene derived from a source gene may comprise 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 coding region of the source gene. In certain embodiments, a gene derived from a source gene may comprise a nucleotide sequence that is identical to the coding region of the source gene. For example, a FUM gene that is derived from an I. orientalis FUM gene may comprise 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 of the modified yeast cells provided herein, an exogenous succinate fermentation or reduction pathway gene may be derived from the host yeast species. For example, where the host cell is I. orientalis, an exogenous gene may be derived from a native I. orientalis gene. In these embodiments, the exogenous gene may comprise a nucleotide sequence identical to the coding region of the native gene, such that incorporation of the exogenous gene into the host cell increases the copy number of a native gene sequence and/or changes the regulation or expression level of the gene if under the control of a promoter that is different from the promoter that drives expression of the gene in a wild-type cell. In other embodiments, the exogenous gene may comprise a nucleotide sequence that differs from the coding region of a native gene, but nonetheless encodes a polypeptide that is identical to the polypeptide encoded by the native gene. In still other embodiments, the exogenous gene may comprise a nucleotide sequence that 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 one or more native genes. In certain of these embodiments, the exogenous gene comprises 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 coding region of one or more native genes. In still other embodiments, the exogenous gene may encode a polypeptide that has less than 50% sequence identity to a polypeptide encoded by a native gene, but which nonetheless has the same function as the native polypeptide in a succinate fermentation or reduction pathway (i.e., the ability to catalyze the same reaction between reaction intermediates).
In other embodiments, an exogenous succinate fermentation or reduction pathway gene may be derived from a species that is different than that of the host yeast cell. In certain of these embodiments, the exogenous gene may be derived from a different yeast species than the host cell. For example, where the host cell is I. orientalis, the exogenous gene may be derived from S. cerevisiae. In other embodiments, the exogenous gene may be derived from a fungal, bacterial, plant, insect, or mammalian source. For example, where the host cell is I. orientalis, the exogenous gene may be derived from a bacterial source such as E. coli. In those embodiments where the exogenous gene is derived from a non-yeast source, the exogenous gene sequence may be codon optimized for expression in a yeast host cell.
In those embodiments where the exogenous succinate fermentation or reduction pathway gene is derived from a species other than the host cell species, the exogenous gene may encode a polypeptide identical to a polypeptide encoded by a native gene from the source organism. In certain of these embodiments, the exogenous gene may be identical to a native gene from the source organism. In other embodiments, the exogenous gene may share 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 coding region of a native gene from the source organism. In other embodiments, the exogenous gene may encode a polypeptide that shares 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 with a polypeptide encoded by a native gene from the source organism. In certain of these embodiments, the exogenous gene may comprise 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 coding region of one or more native genes from the source organism. In still other embodiments, the exogenous gene may encode a polypeptide that has less than 50% sequence identity to a polypeptide encoded by a native gene from the source organism, but which nonetheless has the same function as the native polypeptide from the source organism in an active succinate fermentation or active reduction pathway. An exogenous source gene may be subjected to mutagenesis if necessary to provide a coding sequence starting with the usual eukaryotic starting codon (ATG), or for other purposes.
In certain embodiments, the genetically modified yeast cells provided herein have a reductive TCA active succinate fermentation pathway that proceeds via PEP or pyruvate, OAA, malate, and fumarate intermediates. In these embodiments, the cells comprise one or more succinate fermentation pathway genes encoding enzymes selected from the group consisting PPC, PYC, MDH, FUM, FRD, and succinate exporter genes. In certain embodiments, the cells also have one or more active reduction pathways. In these embodiments, the cells comprise one or more reduction pathway genes encoding enzymes selected from the group consisting of G6PD, gluconolactonase, and 6PGDH. In certain embodiments, the cells may comprise all or part of an active oxidative TCA or glyoxylate shunt succinate fermentation pathway. In these embodiments, the cells comprise one or more genes encoding enzymes selected from the group consisting of citrate synthase, PDH, PFL, aconitase, IDH, α-KGDH, succinate thiokinase, isocitrate lyase, and malate synthase. In certain embodiments, the cells have reduced activity for the endogenous succinate dehydrogenase (SDH) gene.
A “PEP carboxylase gene” or “PPC gene” as used herein refers to any gene that encodes a polypeptide with PEP carboxylase activity, meaning the ability to catalyze the conversion of PEP to OAA. In certain embodiments, a PPC gene may be derived from a bacterial source. For example, a PPC gene may be derived from an E. coli PPC gene encoding the amino acid sequence set forth in SEQ ID NO:4 or a Mannheimia succiniciproducens PPC gene encoding the amino acid sequence set forth in SEQ ID NO:6. In other embodiments, the gene may encode 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, a PPC gene may comprise the nucleotide sequence set forth in 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 set forth in SEQ ID NOs:3 or 5. In other embodiments, a PPC gene may be derived from a plant source.
A “pyruvate carboxylase gene” or “PYC gene” as used herein refers to any gene that encodes a polypeptide with pyruvate carboxylase activity, meaning the ability to catalyze the conversion of pyruvate to OAA. In certain embodiments, a PYC gene may be derived from a yeast source. For example, the PYC gene may be derived from an I. orientalis PYC gene encoding the amino acid sequence set forth in SEQ ID NO:8, an S. cerevisiae PYC1 gene encoding the amino acid sequence set forth in SEQ ID NO:10, or a K. marxianus PYC1 gene encoding the amino acid sequence set forth in SEQ ID NO:12. In other embodiments, the gene may encode 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, a PYC gene may comprise the nucleotide sequence set forth in 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 set forth in SEQ ID NOs:7, 9, or 11. In other embodiments, a PYC gene may be derived from a fungal source other than R. oryzae. 
A “malate dehydrogenase gene” or “MDH gene” as used herein refers to any gene that encodes a polypeptide with malate dehydrogenase activity, meaning the ability to catalyze the conversion of OAA to malate. In certain embodiments, an MDH gene may be derived from a yeast source. For example, the MDH gene may be derived from an I. orientalis MDH1, MDH2, or MDH3 gene encoding the amino acid sequence set forth in SEQ ID NOs:14, 16, or 18, respectively, a K. marxianus MDH1, MDH2, or MDH3 gene encoding the amino acid sequence set forth in SEQ ID NOs:20, 22, or 24, respectively, a Zygosaccharomyces rouxii MDH1 gene encoding the amino acid sequence set forth in SEQ ID NO:26, an E. coli MDH gene encoding the amino acid sequence set forth in SEQ ID NO:28, or a Rhizopus delemar MDH gene encoding the amino acid sequence set forth in SEQ ID NO:167. In other embodiments, the gene may encode 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, 20, 22, 24, 26, 28, or 167. In certain embodiments, an MDH gene may comprise the nucleotide sequence set forth in SEQ ID NOs:13, 15, 17, 19, 21, 23, 25, 27, or 166 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 set forth in SEQ ID NOs:13, 15, 17, 19, 21, 23, 25, 27, or 166.
A “fumarase gene” or “FUM gene” as used herein refers to any gene that encodes a polypeptide with fumarase activity, meaning the ability to catalyze the conversion of malate to fumarate. In certain embodiments, a FUM gene may be derived from a yeast source. For example, the FUM gene may be derived from an I. orientalis FUM gene encoding the amino acid sequence set forth in SEQ ID NO:2. In other embodiments, the gene may encode 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, a FUM gene may comprise the nucleotide sequence set forth in 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 set forth in SEQ ID NO:1. In other embodiments, a FUM gene may be derived from a bacterial source outside of the Mannheimia genus.
A “fumarate reductase gene” or “FRD gene” as used herein refers to any gene that encodes a polypeptide with fumarate reductase activity, meaning the ability to catalyze the conversion of fumarate to succinate. In certain embodiments, an FRD gene may be derived from a yeast source. For example, the FRD gene may be derived from an S. cerevisiae FRD1 gene encoding the amino acid sequence set forth in SEQ ID NO:30, a Saccharomyces mikatae FRD1 gene encoding the amino acid sequence set forth in SEQ ID NO:32, a Kluyveromyces polyspora FRD1 gene encoding the amino acid sequence set forth in SEQ ID NO:34, or a K. marxianus FRD1 gene encoding the amino acid sequence set forth in SEQ ID NO:36. In other embodiments, the gene may encode 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:30, 32, 34, or 36. In certain embodiments, a yeast-derived FRD gene may comprise the nucleotide sequence set forth in SEQ ID NOs:29, 31, 33, or 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 set forth in SEQ ID NOs: 29, 31, 33, or 35. In other embodiments, an FRD gene may be derived from a protozoan source. For example, the FRD gene may be derived from a Trypanosoma brucei FRD1 gene encoding the amino acid sequence set forth in SEQ ID NO:38, a Trypanosoma cruzi FRD1 gene encoding the amino acid sequence set forth in SEQ ID NO:40, a Leishmania braziliensis FRD1 gene encoding the amino acid sequence set forth in SEQ ID NO:42, or a Leishmania mexicana FRD1 gene encoding the amino acid sequence set forth in SEQ ID NO:44. In other embodiments, the gene may encode 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:38, 40, 42, or 44. In certain embodiments, a protozoan-derived FRD gene may comprise the nucleotide sequence set forth in SEQ ID NOs:37, 39, 41, or 43, 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 set forth in SEQ ID NOs:37, 39, 41, or 43.
A “succinate exporter gene” as used herein refers to any gene that encodes a polypeptide which gives cells the ability to transport succinate out of the cell into the extracellular environment. In certain embodiments, the succinate exporter gene may be derived from a fungal source. For example, the MAE (malic anion exporter) gene is a characterized succinate transporter and may be derived from an Schizosaccharomyces pombe gene encoding the amino acid sequence set forth in SEQ ID NO:46 or an Aspergillus oryzae malic anion transporter (MAE) gene encoding the amino acid sequence set forth in SEQ ID NO:48. In other embodiments, the gene may encode 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:46 or 48. In certain embodiments, a succinate exporter gene may comprise the nucleotide sequence set forth in SEQ ID NOs:45 or 47, 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 set forth in SEQ ID NOs:45 or 47.
In certain embodiments, the genetically modified yeast cells provided herein further comprise a deletion or disruption of one or more native genes. “Deletion or disruption” with regard to a native gene means that either the entire coding region of the gene is eliminated (deletion) or the coding region of the gene, its promoter, and/or its terminator region is modified (such as by deletion, insertion, or mutation) such that the gene no longer produces an active enzyme, produces a severely reduced quantity (at least 75% reduction, preferably at least 90% reduction) of an active enzyme, or produces an enzyme with severely reduced (at least 75% reduced, preferably at least 90% reduced) activity.
In certain embodiments, deletion or disruption of one or more native genes results in a deletion or disruption of one or more native metabolic pathways. “Deletion or disruption” with regard to a metabolic pathway means that the pathway is either inoperative or else exhibits activity that is reduced by at least 75%, at least 85%, or at least 95% relative to the native pathway. In certain embodiments, deletion or disruption of a native metabolic pathway is accomplished by incorporating one or more genetic modifications that result in decreased expression of one or more native genes that reduce succinate production.
In certain embodiments, deletion or disruption of native gene can be accomplished by forced evolution, mutagenesis, or genetic engineering methods, followed by appropriate selection or screening to identify the desired mutants. In certain embodiments, deletion or disruption of a native host cell gene may be coupled to the incorporation of one or more exogenous genes into the host cell, i.e., the exogenous genes may be incorporated using a gene expression integration construct that is also a deletion construct. In other embodiments, deletion or disruption may be accomplished using a deletion construct that does not contain an exogenous gene or by other methods known in the art.
In certain embodiments, the modified yeast cells provided herein comprise a deletion or disruption of one or more native genes encoding an enzyme involved in ethanol fermentation or consumption, including for example pyruvate decarboxylase (PDC, catalyzes the conversion of pyruvate to acetaldehyde) and/or alcohol dehydrogenase 1 (ADH1, catalyzes the conversion of acetaldehyde to ethanol) or 2 (ADH2, catalyzes the conversion of ethanol to acetaldehyde). Such modifications decrease the ability of the yeast cell to produce ethanol, thereby maximizing succinate production. In certain embodiments wherein the modified yeast cell is I. orientalis, the cells comprise a deletion of a PDC gene encoding the amino acid sequence of SEQ ID NO:50, an ADHa gene encoding the amino acid sequence of SEQ ID NO:52, and/or a gene encoding 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:50 or 52. In certain of these embodiments, the deleted gene may comprise the nucleotide sequence of SEQ ID NOs:49 or 51, 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:49 or 51.
In certain embodiments, the modified yeast cells provided herein comprise a deletion or disruption of one or more native genes encoding an enzyme involved in producing alternate fermentative products such as glycerol or other by-products such as acetate or diols, including for example glycerol 3-phosphate dehydrogenase (GPD, catalyzes the conversion of dihydroxyacetone phosphate to glycerol 3-phosphate). In certain embodiments wherein the modified yeast cell is I. orientalis, the cells comprise a deletion of a GPD gene encoding the amino acid sequence of SEQ ID NO:54 or a gene encoding 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:54. In certain of these embodiments, the deleted GPD gene may comprise the nucleotide sequence of SEQ ID NO:53 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:53.
In certain embodiments, the modified yeast cells provided herein comprise a deletion or disruption of one or more native genes encoding an enzyme that catalyzes a reverse reaction in a succinate fermentation pathway. For example, in certain embodiments the modified yeast cells provided herein comprise a deletion or disruption of a native PEP carboxykinase (PCK) gene, which encodes an enzyme that converts OAA to PEP. In certain embodiments wherein the modified yeast cell is I. orientalis, the cells comprise a deletion of a PCK gene encoding the amino acid sequence of SEQ ID NO:56 or a gene encoding 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:56. In certain of these embodiments, the deleted PCK gene may comprise the nucleotide sequence of SEQ ID NO:55 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:55. In another example, the modified yeast cells provided herein comprise a deletion or disruption of a native malic enzyme gene, which encodes an enzyme that converts malate to pyruvate. In certain embodiments wherein the modified yeast cell is I. orientalis, the cells comprise a deletion of a malic enzyme gene encoding the amino acid sequence of SEQ ID NO:58 or a gene encoding 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:58. In certain of these embodiments, the deleted malic enzyme gene may comprise the nucleotide sequence of SEQ ID NO:57 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:57. In another embodiment, the modified yeast cells provided herein comprise a deletion or disruption of an endogenous succinate importer gene (RIOR43690), which encodes an protein that allows for growth on and consumption of succinate. In certain embodiments, wherein the modified yeast cell is I. orientalis, the cells comprise a deletion of a RIOR43690 gene encoding the amino acid sequence of SEQ ID NO:60 or a gene encoding 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:60. In certain of these embodiments, the deleted RIOR43690 gene may comprise the nucleotide sequence of SEQ ID NO:59 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:59.
In certain embodiments, the genetically modified yeast cells provided herein comprise a deletion or disruption of one or more native genes encoding an enzyme involved in an undesirable reaction with a succinate fermentation pathway product or intermediate.
In certain embodiments, the genetically modified yeast cells provided herein comprise a deletion or disruption of one or more native genes encoding an enzyme that has a neutral effect on a succinate fermentation pathway, including for example native genes encoding an enzyme selected from the group consisting of ammonia transport outward (ATO), L-lactate cytochrome-c oxidoreductase (CYB2A or CYB2B, catalyzes the conversion of lactate to pyruvate), and alcohol dehydrogenase (ADHa, ADHb catalyzes conversion between acetaldehyde and ethanol). Deletion or disruption of neutral genes allows for insertion of one or more exogenous genes without affecting succinate fermentation pathways. In certain embodiments wherein the modified yeast cell is I. orientalis, the cells comprise a deletion of a CYB2A gene encoding the amino acid sequence of SEQ ID NO:62, a CYB2B gene encoding the amino acid sequence of SEQ ID NO:64, an ATO2 gene encoding the amino acid sequence of SEQ ID NO:66, an ADHb gene encoding the amino acid sequence of SEQ ID NO:68, and/or a gene encoding 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:62, 64, 66, or 68. In certain of these embodiments, the deleted gene may comprise the nucleotide sequence of SEQ ID NOs:61, 63, 65, or 67 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:61, 63, 65, or 67.
In certain embodiments, the genetically modified yeast cells provided herein are succinate resistant yeast cells. A “succinate-resistant yeast cell” (which may also be referred to as “succinate-resistant yeast”) as used herein refers to a yeast cell that exhibits a growth rate (as described in Example 1) in media containing 75 g/L or greater succinate at pH 2.8 that is at least 50% as high as its growth rate in the same media containing 0 g/L succinate. In certain of these embodiments, the yeast cells may exhibit succinate resistance in their native form. In other embodiments, the cells may have undergone mutation and/or selection before, during, or after introduction of genetic modifications related to an active succinate fermentation pathway, such that the mutated and/or selected cells possess a higher degree of resistance to succinate than wild-type cells of the same species. In certain embodiments, mutation and/or selection may be carried out on cells that exhibit succinate resistance in their native form. Cells that have undergone mutation and/or selection may be tested for sugar consumption and other characteristics in the presence of varying levels of succinate in order to determine their potential as industrial hosts for succinate production. In addition to succinate resistance, the yeast cells provided herein may have undergone mutation and/or selection for resistance to one or more additional organic acids or to other fermentation products, by-products, or media components.
Selection for resistance to succinate or other compounds may be accomplished using methods well known in the art. For example, selection may be carried out using a chemostat. A chemostat is a device that allows for a continuous culture of microorganisms (e.g., yeast) wherein the specific growth rate and cell number can be controlled independently. A continuous culture is essentially a flow system of constant volume to which medium is added continuously and from which continuous removal of any overflow can occur. Once such a system is in equilibrium, cell number and nutrient status remain constant, and the system is in a steady state. A chemostat allows control of both the population density and the specific growth rate of a culture through dilution rate and alteration of the concentration of a limiting nutrient, such as a carbon or nitrogen source. By altering the conditions as a culture is grown (e.g., decreasing the concentration of a secondary carbon source necessary to the growth of the inoculum strain, among others), microorganisms in the population that are capable of growing faster at the altered conditions will be selected and will outgrow microorganisms that do not function as well under the new conditions. Typically such selection requires the progressive increase or decrease of at least one culture component over the course of growth of the chemostat culture. The operation of chemostats and their use in the directed evolution of microorganisms is well known in the art (see, e.g., Novick Proc Natl Acad Sci USA 36:708-719 (1950), Harder J Appl Bacteriol 43:1-24 (1977).
As disclosed herein, yeast strains exhibiting succinate resistance were identified based on their growth rate and glucose consumption rates in succinate containing media. One such succinate resistant strain was I. orientalis strain CD1822. Strain CD1822 was generated by evolving I. orientalis ATCC PTA-6658 for 91 days in a glucose limited chemostat. The system was fed with 15 g/L glucose in a DM medium, and operated at a dilution rate of 0.06 h−1 at pH=3 with added lactic acid in the feed medium. The conditions were maintained with a low oxygen transfer rate of approximately 2 mmol L−1 h−1, and dissolved oxygen concentration remained constant at 0% of air saturation. Single colony isolates from the final time point were characterized in two shake flask assays. In the first assay, the strains were characterized for their ability to ferment glucose to ethanol in the presence of 25 g/L total lactic acid with no pH adjustment in the DM defined medium. In the second assay, the growth rate of the isolates were measured in the presence of 25, 32 and 45 g/L of total lactic, with no pH adjustment in DM defined medium. Strain CD1822 was a single isolate selected based on the measured fermentation rates and growth rates.
Yeast strains exhibiting the best combinations of growth and glucose consumption in succinate media as disclosed in the examples below are preferred host cells for various genetic modifications relating to succinate fermentation pathways. Yeast genera that possess the potential for a high degree of succinate resistance, as indicated by growth in the presence of 150 g/L succinate at a pH of 2.8, include for example Issatchenkia and Candida. Other yeast genera with the potential for a relatively high degree of succinate resistance, as indicated by growth in the presence of 100 g/L succinate, include for example Pichia, Zygosaccharomyces, Kluyveromyces, Saccharomyces, Debaryomyces, and Saccharomycopsis. Species exhibiting a high degree of succinate resistance included I. orientalis (also known as Candida krusei), Candida lambica (also known as Pichia fermentans), Candida sorboxylosa, Candida zemplinina, Candida geochares, Pichia membranifaciens, Zygosaccharomyces kombuchaensis, Candida sorbosivorans, Kluyveromyces 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, Saccharomycopsis crataegensis, and Pichia jadinii. I. orientalis and C. lambica belong to the I. orientalis/P. fermentans clade. Specific strains exhibiting succinate resistance included I. orientalis strains PTA-6658, 60585, and 24210, C. lambica strain 38617, and C. sorboxylosa strain 24120.
Other wild-type yeast or fungi may be tested in a similar manner and identified to have acceptable levels of growth and glucose utilization in the presence of high levels of succinate as described herein. For example, Gross and Robbins (Hydrobiologia 433(103):91-109) have compiled a list of 81 fungal species identified in low pH (<4) environments that could be relevant to test as potential production hosts.
In certain embodiments, the modified yeast cells provided herein are generated by incorporating one or more genetic modifications into a Crabtree-negative host yeast cell. In certain of these embodiments the host yeast cell belongs to the genus Issatchenkia or Candida, and in certain of these embodiments the host cell belongs to the I. orientalis/P. fermentans clade. In certain of embodiments, the host cell is I. orientalis or C. lambica. 
The I. orientalis/P. fermentans clade is the most terminal clade that contains at least the species I. orientalis, Pichia galeiformis, Pichia sp. YB-4149 (NRRL designation), Candida ethanolica, Pichia deserticola, Pichia membranifaciens, and P. fermentans. Members of the I. orientalis/P. fermentans clade are identified by analysis of the variable D1/D2 domain of the 26S ribosomal DNA of yeast species, using the method described by Kurtzman and Robnett in “Identification and Phylogeny of Ascomycetous Yeasts from Analysis of Nuclear Large Subunit (26S) Ribosomal DNA Partial Sequences,” Antonie van Leeuwenhoek 73:331-371, 1998, incorporated herein by reference (see especially p. 349). Analysis of the variable D1/D2 domain of the 26S ribosomal DNA from hundreds of ascomycetes has revealed that the I. orientalis/P. fermentans clade contains very closely related species. Members of the I. orientalis/P. fermentans clade exhibit greater similarity in the variable D1/D2 domain of the 26S ribosomal DNA to other members of the clade than to yeast species outside of the clade. Therefore, other members of the I. orientalis/P. fermentans clade can be identified by comparison of the D1/D2 domains of their respective ribosomal DNA and comparing to that of other members of the clade and closely related species outside of the clade, using Kurtzman and Robnett's methods.
In certain embodiments, the genetically modified yeast cells provided herein belong to the genus Issatchenkia, and in certain of these embodiments the yeast cells are I. orientalis. When first characterized, the species I orientalis was assigned the name Pichia kudriavzevii. The anamorph (asexual form) of I. orientalis is known as C. krusei. Numerous additional synonyms for the species I. orientalis have been listed elsewhere (Kurtzman and Fell, The Yeasts, a Taxonomic Study. Section 35. Issatchenkia Kudryavtsev, pp 222-223 (1998)).
The ideal yeast cell for succinate production is ideally capable of conducting fermentation at low pH levels. The ability to conduct fermentation at a low pH decreases downstream recovery costs, resulting in more economical production. Therefore, in certain embodiments the yeast host cell is capable of conducting fermentation at low pH levels.
A suitable host cell may possess one or more favorable characteristics in addition to succinate resistance and/or low pH growth capability. For example, potential host cells exhibiting succinate resistance may be further selected based on glycolytic rates, specific growth rates, thermotolerance, tolerance to biomass hydrolysate inhibitors, overall process robustness, and so on. These criteria may be evaluated prior to any genetic modification relating to a succinate fermentation or reduction pathway, or they may be evaluated after one or more such modifications have taken place.
The level of gene expression and/or the number of exogenous genes to be utilized in a given cell will vary depending upon the identity of the host cell. For fully genome-sequenced yeasts, whole-genome stoichiometric models may be used to determine which enzymes should be expressed to develop a desired pathway succinate fermentation pathway. Whole-genome stoichiometric models are described in, for example, Hjersted Biotechnol Bioeng 97:1190 (2007) and Famili Proc Natl Acad Sci USA 100:13134 (2003).
For yeasts without a known genome sequence, sequences for genes of interest (either as overexpression candidates or as insertion sites) can typically be obtained using techniques known in the art. Routine experimental design can be employed to test expression of various genes and activity of various enzymes, including genes and enzymes that function in a succinate fermentation or reduction pathway. Experiments may be conducted in which each enzyme is expressed in the yeast individually and in blocks of enzymes up to and including preferably all pathway enzymes, to establish which are needed (or desired) for improved succinate production. One illustrative experimental design tests expression of each individual enzyme as well as of each unique pair of enzymes, and further can test expression of all required enzymes, or each unique combination of enzymes. A number of approaches can be taken, as will be appreciated.
In certain embodiments, methods are provided for producing succinate from a genetically modified yeast cell as provided herein. In certain embodiments, these methods comprise providing a modified yeast cell as provided herein with at least one carbon source and culturing the yeast cell such that succinate is produced. The carbon source may be any carbon source that can be fermented by the yeast cell. Examples include, but are not limited to, twelve carbon sugars such as sucrose, hexose sugars such as glucose or fructose, glycan, starch or other polymer of glucose, glucose oligomers such as maltose, maltotriose and isomaltotriose, panose, and fructose oligomers, and pentose sugars such as xylose, xylan, other oligomers of xylose, or arabinose. In certain embodiments, more than one type of genetically modified yeast cell may be present in the culture. Likewise, in certain embodiments one or more native yeast cells of the same or a different species than the genetically modified yeast cell may be present in the culture.
In certain embodiments, culturing of the cells provided herein to produce succinate may be divided up into phases. For example, the cell culture process may be divided into a cultivation phase, a production phase, and a recovery phase. The following represent examples of specific conditions that may be used for each of these phases. One of ordinary skill in the art will recognize that these conditions may be varied based on factors such as the species of yeast being used, the desired yield, or other factors. The medium will typically contain nutrients as required by the particular cell, including a source of nitrogen (such as amino acids, proteins, inorganic nitrogen sources such as ammonia or ammonium salts, and the like), and various vitamins, minerals and the like. In some embodiments, the cells of the invention can be cultured in a chemically defined medium. In one example, the medium is a DM medium containing around 5 g/L ammonium sulfate, around 3 g/L potassium dihydrogen phosphate, around 0.5 g/L magnesium sulfate, trace elements, vitamins and around 150 g/L glucose. The pH is adjusted may be allowed to range freely during cultivation, or may be buffered if necessary to prevent the pH from falling below or rising above predetermined levels. For example, the medium may be buffered to prevent the pH of the solution from falling below around 2.0 or rising above around 8.0 during cultivation. In certain of these embodiments, the medium may be buffered to prevent the pH of the solution from falling below around 3.0 or rising above around 7.0, and in certain of these embodiments the medium may be buffered to prevent the pH of the solution from falling below around 4.0 or rising above around 6.0. In certain embodiments, the fermentation medium is inoculated with sufficient yeast cells that are the subject of the evaluation to produce an OD600 of 1.0. In some embodiments, the genetically modified yeast cell dry weight at inoculation is less than about 5 g/L and desirably less than about 3 g/L. Unless explicitly noted otherwise, OD600 as used herein refers to an optical density measured at a wavelength of 600 nm with a 1 cm pathlength using a model DU600 spectrophotometer (Beckman Coulter). The cultivation temperature may range from about 25 to about 50° C., and the cultivation time may be up to about 120 hours. During cultivation, aeration, and agitation, conditions are selected to produce a desired OUR such as, for example, from about 2 to about 25 mmol/L/hr, from about 5 to about 20 mmol/L/h, from about 8 mmol/L/h to about 25 mmol/L/h, from about 8 mmol/L/h to about 20 mmol/L/h, or from about 8 to about 15 mmol/L/h. In some embodiments, oxygen uptake rate may be greater than about 2 mmol/L/h, greater than about 5 mmol/L/h, or greater than about 8 mmol/L/h. In some embodiments, the oxygen uptake rate may be less than about 25 mmol/L/h, less than about 20 mmol/L/h, or less than about 15 mmol/L/h.
In one example, the cell dry weight at end of fermentation is typically in the range of about 0.1 to 20 g dry cells/liter, preferably from 0.1 to 10 g dry cells/liter, even more preferably from 2 to 5 g dry cells/liter of fermentation medium during the production phase. In some embodiments, the cell dry weight at end of fermentation is less than about 10 g/L, suitably less than about 7 g/L, desirably less than about 5 g/L, and preferably less than about 3 g/L. The fermentation may be conducted microaerobically, as described herein. If desired, oxygen uptake rate can be varied throughout fermentation as a process control (see, e.g., WO03/102200). In certain embodiments, the modified yeast cells provided herein may perform especially well when cultivated under microaerobic conditions characterized by an OUR of from about 2 to about 25 mmol/L/hr. The medium may be buffered during the production phase such that the pH typically is maintained in a range of about 2.0 to about 7.0, about 2.0 to 6.0, about 3.0 to about 6.0, or about 3.0 to about 5.0. In some embodiments, a final pH of the fermentation broth is less than about 6.0, suitably less than about 5.5, desirably less than about 5.0, preferably less than about 4.0, and more preferably less than about 3.5.
Suitable buffering agents are basic materials that neutralize the acid as it is formed, and include, for example, calcium hydroxide, calcium carbonate, sodium hydroxide, potassium hydroxide, potassium carbonate, sodium carbonate, ammonium carbonate, ammonia, ammonium hydroxide and the like. In general, those buffering agents that have been used in conventional fermentation processes are also suitable here.
In those embodiments where a buffered fermentation is utilized, acidic fermentation products may be neutralized to the corresponding salt as they are formed. In these embodiments, recovery of the acid involves regeneration of the free acid. This may be done by removing the cells and acidulating the fermentation broth with a strong acid such as sulfuric acid. This results in the formation of a salt by-product. For example, where a calcium salt is utilized as the neutralizing agent and sulfuric acid is utilized as the acidulating agent, gypsum is produced as a salt by-product. This by-product is separated from the broth, and the acid is recovered using techniques such as liquid-liquid extraction, distillation, absorption, and others (see, e.g., T. B. Vickroy, Vol. 3, Chapter 38 of Comprehensive Biotechnology, (ed. M. Moo-Young), Pergamon, Oxford, 1985; R. Datta, et al., FEMS Microbiol Rev, 1995, 16:221-231; U.S. Pat. Nos. 4,275,234, 4,771,001, 5,132,456, 5,420,304, 5,510,526, 5,641,406, and 5,831,122, and WO93/00440.
In other embodiments, the pH of the fermentation medium may be permitted to drop during cultivation from a starting pH that is at or above the lower pKa (4.207) of succinate, typically 4.5 or higher, to at or below the lower pKa of the acid fermentation product, such as in the range of about 2.0 to about 4.2, in the range of from about 3.0 to about 4.2, or in the range from about 3.8 to about 4.2.
In still other embodiments, fermentation may be carried out to produce a product acid by adjusting the pH of the fermentation broth to at or below the lower pKa of the product acid prior to or at the start of the fermentation process. The pH may thereafter be maintained at or below the lower pKa of the product acid throughout the cultivation. In certain embodiments, the pH may be maintained at a range of about 2.0 to about 4.2, in the range of from about 3.0 to about 4.2, or in the range from about 3.8 to about 4.2.
In certain embodiments of the methods provided herein, the genetically modified yeast cells produce relatively low levels of ethanol. In certain embodiments, ethanol may be produced in a yield of 10% or less, preferably in a yield of 2% or less. In certain of these embodiments, ethanol is not detectably produced. In other embodiments, however, succinate and ethanol may be co-produced. In these embodiments, ethanol may be produced at a yield of greater than 10%, greater than 25%, or greater than 50% of the theoretical mass yield.
In some embodiments, a production rate of succinate is greater than about 0.25 g/L/h, greater than about 1 g/L/h, greater than about 1.5 g/L/h, greater than about 2 g/L/h, or greater than about 3 g/L/h. In some embodiments, the production rate may be less than about 4 g/L/h or less than about 3 g/L/h. In some embodiments, the production rate may be about 0.25 g/L/h to about 4 g/L/h or about 1 g/L/h to about 3 g/L/h.
In some embodiments, a specific production rate of succinate is greater than about 0.1 g/g/h, greater than about 0.15 g/g/h, greater than about 0.5 g/g/h, and greater than about 1.0 g/g/h. In some embodiments, the specific production rate of succinate is less than about 2 g/g/h and less than about 1 g/g/h. In some embodiments, the specific production rate of succinate is about 0.1 g/g/h to about 2 g/g/h or about 0.15 g/g/h to about 1.0 g/g/h.
In certain embodiments of the methods provided herein, the product yield of succinate on the carbon source is at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, or greater than 50% of the theoretical yield. In some embodiments, the product yield of succinate is greater than about 50%, suitably greater than about 60%, desirably greater than about 70%, preferably greater than about 80%, and more preferably greater than about 90%. In some embodiments, the product yield of succinate is less than about 100%, less than about 90%, less than about 80%, or less than about 70%. In some embodiments, the product yield of succinate is about 10% to about 100%, about 20% to about 90%, about 30% to about 80%, or about 40% to about 70%.
In certain embodiments, the cells provided herein are capable of converting at least 80% or at least 90% by weight of a carbon source to succinate. The concentration, or titer, of succinate will be a function of the yield as well as the starting concentration of the carbon source. In certain embodiments, the titer may reach at least about 1 to about 3, at least about 5, at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, or greater than 50 g/L at some point during the fermentation, and preferably at the end of the fermentation. In some embodiments, a final concentration of succinate in the fermentation broth is greater than about 20 g/L, greater than about 40 g/L, greater than about 60 g/L, greater than about 70 g/L, greater than about 80 g/L, and greater than about 90 g/L. In some embodiments, the final concentration of succinate in the fermentation broth is less than about 90 g/L, less than about 80 g/L, or less than about 70 g/L. In some embodiments, the final concentration of succinate in the fermentation broth is about 20 g/L to about 90 g/L, about 40 g/L to about 80 g/L, or about 40 g/L to about 70 g/L. In certain embodiments, the final yield of succinate may be increased by decreasing the temperature of the fermentation medium, particularly during the production phase.
The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit the invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention. It will be understood that many variations can be made in the procedures herein described while still remaining within the bounds of the present invention. It is the intention of the inventors that such variations are included within the scope of the invention.
EXAMPLES
Example 1A: Selection of Host Yeast Cells Based on Succinate Tolerance
A set of wild-type yeast strains were tested for their ability to grow in the presence of succinate.
The range of succinate concentrations to utilize in primary screening procedures was determined by evaluating the ability of seven wild-type yeast strains (Candida sonorensis, Candida zemplinina, I. orientalis strain PTA-6658, Kluyveromyces lactis, K. marxianus, S. cerevisiae strain CENPK 113-7D, and I. orientalis strain CD1822) to grow on media containing varying levels of succinate. Cells were streaked onto YPD plates and grown overnight. A cell slurry with an OD600 of around 4 was made in YPD media, pH 3.0, and this slurry was used to inoculate microtiter wells containing various concentrations of succinate to an OD600 of 0.05. Plates were covered with a gas permeable membrane and incubated in a 30° C./300 rpm shaker overnight. The optical densities of each well were measured at a wavelength of 600 nm in a GENios model plate reader (Tecan), and plates were observed visually for growth. The highest succinate concentration that one or more of the strains grew in (150 g/L) was chosen as the upper range for primary screening procedure.
For the primary screening procedure, 91 yeast strains were screened for growth on microtiter plates at 0 g/L, 100 g/L, or 150 g/L succinate and pH 2.8 using the same protocol used for range finding. Solubility issues presented challenges when trying to test succinate concentrations greater than 150 g/L, so low pH rather than higher succinate concentration was used to test more stringent conditions. For these samples, strains were screened for growth at 150 g/L succinate and pH 2.5.
A fresh YPD plate was used for each strain, and a slurry with an OD600 of around 4 was made in YPD media, pH 3.0. The slurry was used to inoculate each well to an OD600 of 0.05. Plates were covered with a gas permeable membrane, and incubated in a 30° C./300 rpm shaker overnight. Optical densities of each well were measured at 600 nm in a GENios model plate reader, and plates were observed visually for growth.
A similar protocol was run to evaluate growth at lactic acid concentration of 0 g/L, 30 g/L, 45 g/L, and 60 g/L. Table 1 summarizes the highest concentrations of succinate or lactic acid at which growth was observed.







TABLE 1







Primary Screen for Growth on Succinate or Lactic Acid












Lactic





acid
Succinate


ATCC#
Genus/species
(g/L)
(g/L)













PTA-6658
Issatchenkia orientalis
60
150 (pH 2.5)


CD1822
Issatchenkia orientalis
60
150 (pH 2.5)


(Cargill





collection)





PYCC 04-501
Candida zemplinina
60
100


Cargill
Candida geochares
60
100


NCYC 2696
Pichia membranifaciens
60
100


NCYC 2897
Zygosaccharomyces
60
100



kombuchaensis




mya-402
Saccharomyces bulderi
60
0


38619
Candida sorbosivorans
60
100


NCYC 535
Schizosaccharomyces pombe
60
0


52486
Kluyveromyces marxianus
45
100


113-7D
Saccharomyces cerevisiae-
45
0



CENPK




MUCL
Candida vanderwaltii
45
100


300000





Cargill
Candida sorbophila
45
100


NCYC 3134
Zygosaccharomyces bisporus
45
100


NCYC 2928
Zygosaccharomyces lentus
45
100


NCYC 734
Saccharomyces ludwigii
45
0


60585
Issatchenkia orientalis
45
150 (pH 2.8)


46330
Yarrowia lipolytica
45
0


36946
Zygosaccharomyces bailii
45
0


60592
Candida milleri
45
0


38617
Candida lambica
45
150 (pH 2.8)


20306
Candida rugosa
45
0


28525
Candida valida
45
0


20347
Candida zeylanoides
45
0


24210
Issatchenkia orientalis
45
150 (pH 2.5)


20282
Kodamaea ohmeri
45
0


90739
Saccharomyces bayanus
45
100


MYA-404
Saccharomyces bulderi
45
0/100


MUCL 31237
Saccharomycopsis javensis
45
0


32109
Candida sonorensis
30
0


PYCC 70-1022
Debaryomyces castellii
30
100


PYCC 70-104
Candida boidinii
30
100


PYCC 60-8
Candida etchellsii
30
100


44691
Candida kefyr
30
0


34890
Zygosaccharomyces rouxii
30
0


60591
Candida milleri
30
0


24120
Candida sorboxylosa
30
150 (pH 2.5)


28526
Pichia fermentans
30
0


96784
Saccharomyces cerevisiae
0/30
0


52709
Kluyveromyces thermotolerans
0/30
0


NCYC 614
Pachysolen tannophilus
0/30
0


CBS 8452
Wickerhamiella occidentalis
0/30
0


18735
Candida blankii
0/30
0


8585
Kluyveromyces lactis
0
100


9950
Pichia jadinii
0
100


38623
Candida fluviatilis
0
0


20033
Saccharomyces capsularis
0
NG


20284
Candida famata
0
NG


20118
Candida guilliermondii
0
0


20178
Candida intermedia
0
0


20179
Candida parapsilosis
0
0


96309
Candida pseudolambica
0
0


20280
Debaryomyces polymorphus
0
0


20277
Dekkera anomala
0
0


10563
Dekkera lambica
0
0


20030
Hyphopichia burtonii
0
0


9889
Metschnikowia pulcherrima
0
0


2102
Pichia anomala
0
0/100


24116
Pichia nakasei
0
0


16768
Pichia silvicola
0
0


34024
Pichia strasburgensis
0
0


2261
Pichia tannicola
0
0


76514
Saccharomyces uvarum
0
0


52714
Torulaspora delbrueckii
0
0


90197
Yamadazyma guilliermondii
0
0


20321
Yamadazyma halophila
0
0


MUCL 44417
Saccharomycopsis
0
100



crataegensis




NRRL Y-7290
Saccharomycopsis vini
0
0


9950
Pichia jadinii
0
100


CBS 6054
Pichia stipitis
0
0


NCYC 2389
Candida shehatae
0
0


201225
Yamadazyma stipites
0
0


10660
Schizosaccharomyces
0
NG



japonicus




12659
Lipomyces starkeyi
0
0


42479
Torulaspora pretoriensis
0
0


90624
Debaryomyces hansenii
NG
NG


20117
Candida catenulata
NG
NG


96927
Candida lactiscondensi
NG
NG


36592
Candida pignaliae
NG
NG


34087
Citeromyces matritensis
NG
NG


36591
Kluyveromyces yarrowii
NG
NG


20292
Nematospora coryli
NG
NG


28778
Pichia fluxuum
NG
NG


58362
Pichia toletana
NG
NG


96272
Bulleromyces albus
NG
NG


MUCL 47216
Candida tenuis
NG
NG


or MUCL





31253B





20361
Candida methanosorbosa
NG
NG


NCYC 813
Brettanomyces naardenensis
NG
NG/0


76214
Myxozyma kluyveri
NG
NG


56306
Lipomyces tetrasporus
NG
NG


56465
Candida naeodendra
NG
NG









All six strains that exhibited growth at 150 g/L succinate were selected for secondary screening. For the first secondary screen, growth rates were measured in YPD media containing 0 g/L succinate at pH 3.0 or 75 g/L succinate at pH 2.85. Shaken flasks were inoculated with biomass harvested from seed flasks grown overnight to an OD600 of 6 to 10. 250 mL baffled growth rate flasks (50 mL working volume) were inoculated to an OD600 of 0.1 and grown at 250 rpm and 30° C. Samples were taken throughout the time course of the assay and analyzed for biomass growth via OD600. The resulting OD600 data was plotted and growth rates were established. Results are summarized in Table 2.







TABLE 2







Growth Rate in Succinate












0 g/L succinate
75 g/L succinate




(pH 3.0)
(pH 2.85)



Strain
(h−1)
(h−1)






Issatchenkia orientalis
0.71
0.50



ATCC PTA-6658





Issatchenkia orientalis
0.69
0.47



CD1822





Issatchenkia orientalis
0.73
0.46



ATCC 60585





Candida lambica
0.81
0.48



ATCC 38617





Candida sorboxylosa
0.66
0.36



ATCC 24120





Issatchenkia orientalis
0.74
0.45



ATCC 24210









For the second secondary screen, glucose consumption was measured in YPD media containing 0 g/L succinate at pH 3.0 or 75 g/L succinate at pH 2.85. Shake flasks were inoculated with biomass harvested from seed flasks grown overnight to an OD600 of 6 to 10. 250 mL baffled glycolytic assay flasks (50 mL working volume) were inoculated to an OD600 of 0.1 and grown at 250 RPM and 30° C. Samples were taken throughout the time course of the assay and analyzed for glucose consumption using a 2700 Biochemistry Analyzer (Yellow Springs Instruments, YSI). The resulting data was plotted and glucose consumption rates were established. Results are summarized in Table 3.







TABLE 3







Glucose Consumption Rate in Succinate












0 g/L succinate
75 g/L succinate




(pH 3.0)
(pH 2.85)



Strain
(g L−1 h−1)
(g L−1 h−1)






Issatchenkia orientalis
>4.2 g/L/h
>2.3 g/L/h



ATCC PTA-6658





Issatchenkia orientalis
>4.2 g/L/h
>2.3 g/L/h



CD1822





Issatchenkia orientalis
>4.2 g/L/h
2.3-2.8 g/L/h 



ATCC 60585





Candida lambica
>4.2 g/L/h
>2.3 g/L/h



ATCC 38617





Candida sorboxylosa
 3.1 g/L/h
 0.7 g/L/h



ATCC 24120





Issatchenkia orientalis
>4.2 g/L/h
>2.3 g/L/h



ATCC 24210









To identify the most attractive candidates for succinate production, strain performance was graded in three categories. Two of these categories were based on different aspects of growth rate: 1) growth rate at highest acid concentration and 2) slope of the growth rates plotted against acid concentration. The third category was the glycolytic rate at the highest acid concentration. Grading was done on a normalized scale using the highest and lowest value for each rating as the normalized boundaries. Each strain thus received a grade of 0 to 1 for each category, with 1 being the highest possible score. The overall rating of a strain was the sum of the normalized value for the three categories. A weighted score was made in which the growth rate and glycolytic rate were equally weighted. In this case the glycolytic rate at the highest acid concentration was weighted at 50%, while the two growth rate ratings were weighted at 25% each. In accordance with the description above, the final score for each strain was calculated as follows:




  
    Final
    ⁢
    
      
    
    ⁢
    score
    ⁢
    
      
    
    ⁢
    for
    ⁢
    
      
    
    ⁢
    strain
    ⁢
    
      
    
    ⁢
    X
  
  =
  
    
      
        (
        
          
            actual
            ⁢
            
              
            
            ⁢
            growth
            ⁢
            
              
            
            ⁢
            rate
            ⁢
            
              
            
            ⁢
            in
            ⁢
            
              
            
            ⁢
            highest
            ⁢
            
              
            
            ⁢
            acid
            ⁢
            
              
            
            ⁢
            concentration
          
          -
          
            slowest
            ⁢
            
              
            
            ⁢
            growth
            ⁢
            
              
            
            ⁢
            rate
          
        
        )
      
      *
      0.25
      ⁢
      
        
      
      ⁢
      
        (
        
          
            fastest
            ⁢
            
              
            
            ⁢
            growth
            ⁢
            
              
            
            ⁢
            rate
          
          -
          
            slowest
            ⁢
            
              
            
            ⁢
            growth
            ⁢
            
              
            
            ⁢
            rate
          
        
        )
      
    
    +
    
      
        (
        
          
            slope
            ⁢
            
              
            
            ⁢
            of
            ⁢
            
              
            
            ⁢
            growth
            ⁢
            
              
            
            ⁢
            rates
            ⁢
            
              
            
            ⁢
            of
            ⁢
            
              
            
            ⁢
            strain
            ⁢
            
              
                
              
              ⁢
              
                
              
            
            ⁢
            X
          
          -
          
            lowest
            ⁢
            
              
            
            ⁢
            growth
            ⁢
            
              
            
            ⁢
            rate
            ⁢
            
              
            
            ⁢
            slope
          
        
        )
      
      *
      0.25
      ⁢
      
        
      
      ⁢
      
        (
        
          
            largest
            ⁢
            
              
            
            ⁢
            growth
            ⁢
            
              
            
            ⁢
            rate
            ⁢
            
              
            
            ⁢
            slope
          
          -
          
            lowest
            ⁢
            
              
            
            ⁢
            growth
            ⁢
            
              
            
            ⁢
            rate
            ⁢
            
              
            
            ⁢
            slope
          
        
        )
      
    
    +
    
      
        (
        
          
            actual
            ⁢
            
              
            
            ⁢
            glycolytic
            ⁢
            
              
            
            ⁢
            rate
            ⁢
            
              
            
            ⁢
            in
            ⁢
            
              
            
            ⁢
            highest
            ⁢
            
              
            
            ⁢
            acid
            ⁢
            
              
            
            ⁢
            concentration
          
          -
          
            slowest
            ⁢
            
              
            
            ⁢
            glycolytic
            ⁢
            
              
            
            ⁢
            rate
          
        
        )
      
      *
      0.50
      ⁢
      
        
      
      ⁢
      
        (
        
          
            fastest
            ⁢
            
              
            
            ⁢
            glycolytic
            ⁢
            
              
                
              
              ⁢
              
                
              
            
            ⁢
            rate
          
          -
          
            slowest
            ⁢
            
              
            
            ⁢
            glycolytic
            ⁢
            
              
                
              
              ⁢
              
                
              
            
            ⁢
            rate
          
        
        )
      
    
  




Normalized values for each category and the final weighted score for each strain are summarized in Table 4.







TABLE 4







Normalized Strain Grades in Succinate












Growth






rate @






75 g/L
Growth
Glycolic
Weighted


Strain
succinate
rate slope
rate
score





Issatchenkia orientalis
1.00
1.00
0.76
0.88


ATCC PTA-6658






Issatchenkia orientalis
0.79
0.94
0.76
0.81


CD1822






Issatchenkia orientalis
0.71
0.50
1.00
0.80


ATCC 60585






Candida lambica
0.86
0.00
0.76
0.60


ATCC 38617






Candida sorboxylosa
0.00
0.25
0.00
0.06


ATCC 24120






Issatchenkia orientalis
0.64
0.31
0.79
0.63


ATCC 24210









The same procedures were utilized to screen, rate, and score the original 91 yeast strains from the primary screen with media containing 0, 30, 45, and 60 g/L lactic acid at pH 3.0 (about 80% free acid). Due to difficulties in properly scoring some very weak growth that occasionally occurred at 60 g/L, 21 strains were re-tested in the primary screen. Of these 21 strains, eight were eliminated due to very slow growth relative to the rest of the test group. The remaining 13 strains were advanced into secondary screening, and normalized values and weighted and summed scores were derived for each strain. These results are summarized in Table 5.







TABLE 5







Normalized Strain Grades in Lactic acid












Growth rate






50 g/L lactic
Growth
Glycolic
Weighted


Strain
acid
rate slope
rate
score














Candida lambica
0.92
1
1
0.98


ATCC 38617






Issatchenkia orientalis
0.94
0.95
1
0.97


ATCC PTA-6658






Issatchenkia orientalis
1.00
0.86
1
0.97


CD1822






Issatchenkia orientalis
0.89
0.73
1
0.91


ATCC 24210






Candida zemplinina
0.22
0.95
1
0.79


Saccharomyces
0.47
0.45
1
0.73


bulderi






ATCC MYA-404






Saccharomyces
0.08
0.91
0.96
0.73


bayanus






Saccharomyces
0.5
0.23
1
0.68


bulderi






ATCC MYA-402






Candida milleri
0
0.64
0.92
0.62


ATCC 60592






Candida
0.28
0.95
0.59
0.60


sorbosivorans






Kodamaea ohmeri
0.42
0
0.76
0.49


Candida geochares
0.17
0.27
0.69
0.46


Saccharomyces
0.11
0.68
0
0.20


javensis









Of the strains tested in lactic acid, only S. javensis did not achieve a 2.5 g/L/hr glucose utilization rate at pH 2.85 in media with 50 g/L lactic acid. While I. orientalis and C. lambica showed tolerance for both succinic and lactic acids, there were a number of species and strains that were tolerant for only one of the acids. Additionally, the rank order of the strains is different for each acid. This is even more clearly illustrated in the primary screen results (Table 1), where more strains were included. The most succinate tolerant strains are scattered among the top three tiers for lactic acid tolerance. Further, one of the strains that grew at the highest lactic acid concentration in the primary screen and scored highly in the secondary screen (S. bulderi) did not grow even at the lowest non-zero concentration of succinic acid tested. Thus, tolerance to lactic acid was shown to be a very poor predictor of tolerance to succinic acid, meaning that ideal strains for succinate production cannot be identified based on tolerance to lactic acid. This is further highlighted by comparing the strains that showed succinate resistance above with the list of eight strains identified as preferred hosts for organic acid production in WO03/049525. While two of those strains (C. diddensiae and C. entomophila) could not be obtained for testing, the other six (C. sonorensis, C. methanosorbosa, C. parapsilosis, C. naeodendra, C krusei, and C. blankii) were included in the primary screen described above. Of these six, only C. krusei (tested as I. orientalis) was able to grow in the presence of 150 g/L succinate at either pH 2.5 or pH 2.8.
Example 1B: Mutagenesis and Selection of Mutant Strains Having Succinate Resistance
Yeast cells selected in Example 1A are subjected to mutagenesis and exposed to selection pressure in order to identify mutants with high succinate tolerance.
For example, yeast cells from a fresh YP (yeast extract/peptone)+20 g/L glucose plate or liquid culture (OD6001-4) are resuspended in sterile water to an OD600 of around 10. 200 μL aliquots of this cell suspension are pipetted into individual tubes and exposed to 3 μL ethane methyl sulfonate (EMS) for approximately one hour, which kills around 65% of the cells. Higher EMS concentrations can also be used to increase the kill rate. After exposure, cells are neutralized with 5% sodium thiosulfate, washed in PBS buffer, recovered in rich media for approximately four hours, and cultured on selective media. Mock samples (no EMS) are also run to ensure that the conditions are selective. Alternatively, cell can be mutagenized using UV irradiation.
To select for succinate resistant mutant strains, aliquots of the EMS-treated cell suspension (approximately 2×108 of mutagenized cells) are plated onto a potato glucose agar (PDA) or another media containing succinate at a level at which the parental strain does not grow or grows very slowly. These plates are incubated for several days until colonies appear. Single colonies are purified, streaked on non-selective media to eliminate any adaptive effects of the selection, and re-tested on selective media to confirm increased resistance. Resistant strains are then tested in a shake flask format with periodic sampling for HPLC analysis of products and substrates. Alternatively, selection for succinate tolerance may be done by chemostat or serial shake flask evolution. Additional rounds of mutagenesis and selection can be performed. Mutagenesis can be used to increase the resistance of a host that does not natively meet succinate production requirements so that it has the necessary attributes for commercial succinate production.
Example 2: Deletion of Both Alleles of CYB2A from I. orientalis Strain CD1822
Both alleles of CYB2A are deleted from I. orientalis strain CD1822. As discussed above, CD1822 is an evolved lactic acid resistant strain isolated from a chemostat that also exhibited a high degree of succinate tolerance.
Plasmids pMI449 (FIG. 1) and pMI454 (FIG. 2) are used to delete both copies of the L-lactate:ferricytochrome c oxidoreductase (CYB2A) gene (SEQ ID NO:61) in I. orientalis strain CD1822, a lactic acid resistant strain of I. orientalis isolated from the environment. pMI449 and pMI454 were both described in WO07/106524. Each plasmid contains 5′ and 3′ flanking regions from I. orientalis CYB2A separated by a selection marker cassette comprising the S. cerevisiae MEL5 gene operatively linked to a PGK promoter. This selection marker cassette is flanked on either end by a sequence (“KtSEQ”) from K. thermotolerans. The 5′ and 3′ CYB2A flanking regions in pMI449 correspond to nucleotides from 913 to 487 bp upstream of the start of the predicted ORF and nucleotides from 90 to 676 bp downstream of the stop codon of the predicted ORF, respectively. The 5′ and 3′ CYB2A flanking regions in pMI454 correspond to nucleotides from 466 to 7 bp upstream of the predicted ORF and nucleotides from 402 bp upstream to 77 bp downstream of the predicted stop codon, respectively.
The first CYB2A allele is deleted by transforming strain CD1822 with pMI449 digested with SacI using lithium acetate transformation (Gietz Met Enzymol 350:87 (2002)). Transformants are selected on yeast nitrogen base (YNB) (Becton, Dickinson and Co, Sparks, Md.; REF#239210)+2% melibiose plate containing x-α-gal (5-bromo-4-chloro-3-indolyl-α-D-galactoside). Blue-colored transformants are visible after around 4 days of growth at 30° C. Transformants are picked and plated for single colonies on Yeast Extract/Peptone/20 g/L glucose plates (YPD) containing x-α-gal. A single blue colony for each transformant is picked and re-streaked to YPD plates. Genomic DNA is isolated from the purified transformants, and replacement of the CYB2A gene is confirmed by PCR. To obtain strains where the MEL5 marker has undergone spontaneous recombination to excise it from the chromosome, the transformant is grown for several rounds in liquid YPD (100 g/L glucose) at 250 rpm and 30° C. A dilution series is plated onto YPD plates overlaid with x-α-gal, and grown overnight at 30° C. A white colony (indicative of the loop-out of the MEL5 marker cassette) is selected and re-streaked to YPD+x-α-gal plates. A white colony is selected and genomic DNA is prepared. Disruption of one allele of the native CYB2A gene is verified by PCR using primers oMM173 (SEQ ID NO:69) and oTM123 (SEQ ID NO:70).
The second CYB2A allele is deleted from this transformant by transforming with pMI454 digested with SacI. Transformants are obtained and purified as described above and analyzed by PCR for the absence of a 1000 bp CYB2A-specific PCR product using primers oMM175 (SEQ ID NO:71) and oMM176 (SEQ ID NO:72). The MEL5 marker derived from plasmid pMI454 is looped out of a transformant having a deletion of both CYB2A alleles via recombination as before, and confirmed by PCR using primers oMM172 (SEQ ID NO:73) and oMM173 (SEQ ID NO:69). This transformant is designated strain 2610.
The CYB2A deletion strain generated in Example 2 are summarized in Table 6.







TABLE 6







I. orientalis CYB2A Deletion Strain









Strain name
Description
Parent strain





CD1822
Lactic acid-resistant parent
—



strain



2610
CYB2A deletion (2)
1822









Example 3: Construction of Cre Expression Plasmids pVB10 and pVB32
The cre recombinase gene is synthesized using the native cre protein sequence as a reference. This gene is PCR amplified from template DNA (Blue Heron Biotechnology, Bothell, Wash.) representing a codon-optimized version of the bacteriophage P1 CRE gene (SEQ ID NO:74, encoding polypeptide of SEQ ID NO:75) using PCR primers oVB5 (SEQ ID NO:76) and oVB6 (SEQ ID NO:77) and cloned into pCR2.1-TOPO (Invitrogen) to produce pVB15a. pVB15a is digested with BamHI and PacI to generate a 1 kb cre fragment, and this fragment is ligated into similarly digested pHJJ17 (FIG. 3). The resultant vector, pVB10 (FIG. 4), contains the cre gene operatively linked to a PDC promoter and terminator. The vector also contains a URA3 selection marker gene from I. orientalis. 
To replace the URA3 selection marker in pVB10 with the SUC2 selection marker, a SUC2 expression cassette was amplified from pTMC82. This SUC2 expression cassette contains the S. cerevisiae SUC2 gene operatively linked to the I. orientalis PGK1 promoter and the S. cerevisiae CYC1 terminator. Amplification was performed using primers oTM298 (SEQ ID NO:78) and oTM299 (SEQ ID NO:79), which add NsiI and SalI restriction sites to the product. The PCR product was digested with NsiI and SalI and ligated to pVB10 digested with SalI and PstI (NsiI and PstI have compatible cohesive ends) to produce pVB32 (FIG. 5).
Example 4: Insertion of PYC1 at the PDC1 Locus in I. orientalis Strain 2610
A PYC1 expression cassette is inserted at one or both PDC1 alleles in I. orientalis strain 2610 (Example 2).
Example 4A: Construction of I. orientalis PYC1 Expression Constructs pKF043 and pKF045
The PYC1 gene from I. orientalis (SEQ ID NO:7) is amplified from genomic DNA using Phusion polymerase and primers oKF245 (SEQ ID NO:80) and oKF246 (SEQ ID NO:81), which contain an MluI site and an SbfI site, respectively. After amplification, the product is gel purified, digested with MluI and SbfI, and ligated to similarly digested pKF031 and pKF044. pKF031 (FIG. 6) and pKF044 (FIG. 7) are constructed from pUC19 backbones, and both contain a multiple cloning site containing MluI, NotI, and SbfI sites operatively linked to the I. orientalis PENO promoter and the S. cerevisiae GAL10 terminator. pKF031 also contains a selection marker cassette comprising the S. cerevisiae MEL5 gene operatively linked to the I. orientalis PPGK promoter. This selection marker cassette is flanked by loxP sites. pKF044 contains an expression cassette comprising the I. orientalis CYB2A promoter, gene, and terminator. This expression cassette is flanked by loxP sites.
The plasmids are transformed into E. coli, and transformants are selected on LB plates containing 100 μg/ml carbenicillin and screened using primers flanking the NotI site of pKF031 and pKF044 (oKW93 (SEQ ID NO:82) and oKW95 (SEQ ID NO:83)). Quickchange PCR is performed using primers oKW96 (SEQ ID NO:84) and oKW97 (SEQ ID NO:85) to eliminate an internal NdeI site (T2847C). Correct plasmids are confirmed by sequencing, and the final constructs are designated pKF043 (MEL5 marker) (FIG. 8) and pKF045 (CYB2A marker) (FIG. 9).
Example 4B: Insertion of I. orientalis PYC1 at the First and Second PDC1 Loci in I. orientalis Strain 2610
pKF043 and pKF045 are both amplified from the loxP site on the 5′ end to the GAL10 terminator on the 3′ end. pKF043 is amplified using primers oKF243 (SEQ ID NO:86) and oKF244 (SEQ ID NO:87), and pKF045 is amplified using primers oKF255 (SEQ ID NO:88) and oKF244 (SEQ ID NO:87). Each of these primers contains on their 5′ end 65 bp of sequence specific to the 65 bp immediately upstream and downstream of the PDC1 locus in I. orientalis. This recombination sequence enables double recombination and integration at the PDC1 locus.
The PCR product amplified from pKF043 is used to transform I. orientalis strain 2610. Transformants are selected on YNB+2% melibiose+x-α-gal and, and integration of PYC1 at a first PDC1 allele is confirmed by PCR using primers oCM566 (SEQ ID NO:89), oKF151 (SEQ ID NO:90), oKF252 (SEQ ID NO:91), and oCM587 (SEQ ID NO:92). The correct heterozygous strain is designated ySBCK43.
Strain ySBCK43 is transformed with the PCR product from pKF045 amplification to generate a homozygous strain with PYC1 inserted at both PDC1 alleles. Integration is confirmed by PCR using the primers oCM566 (SEQ ID NO:89), oMM174 (SEQ ID NO:93), oCM587 (SEQ ID NO:92), and oCA397 (SEQ ID NO:94). The correct homozygous strain is designated 12629.
For marker recycling, I. orientalis 12629 was grown to around OD600 of 1.0 in YP+100 g/L glucose (50 ml media in a 250 ml flask; 30° C./250 rpm). Cells were transformed with pVB32 using lithium acetate transformation, and transformants were selected on YNB+2% sucrose plates overlaid with x-α-gal. After 4 to 5 days, white colonies were streaked to YP+20 g/L glucose plates overlaid with x-α-gal and grown at 37° C. for 2 days. Genomic DNA from white colonies was screened for retention of the expression cassette at the I. orientalis PDC1 locus and for loss of the selectable markers using PCR primers oGPB9 (SEQ ID NO:95), oGPB10 (SEQ ID NO:96), oGPB11 (SEQ ID NO:97), and oGPB12 (SEQ ID NO:98). Positive transformants were confirmed to have lost the marker by a phenotypic screen showing no growth on YNB+2% lactic (pH 5.5), 2% melabiose, or 2% sucrose. The homozygous strain with both markers removed was designated 12481.
The various PYC1 insertion/PDC1 deletion strains generated in Example 4 are summarized in Table 7.







TABLE 7







PYC1 Insertion Strains









Strain name
Description
Parent strain





ySBCK43
CYB2A deletion (2)
 2610



I. orientalis PYC1 insertion at PDC1 (1)


12629
CYB2A deletion (2)
ySBCK43



I. orientalis PYC1 insertion at PDC1 (2)


12481
CYB2A deletion (2)
12629



I. orientalis PYC1 insertion at PDC1 (2)









Example 5: Insertion of FRD1 at the ADHa Locus in I. orientalis Strains 12481
An FRD1 expression cassette is inserted at one or both ADHa alleles of I. orientalis strains 12481 (Example 4).
Example 5A: Construction of ADHa Deletion Constructs pGPB11, pGPB14, pGPB28, and pGPB34
pKF044 was used as a template for quickchange mutagenesis using oligonucleotides oKW64 (SEQ ID NO:99) and oKW65 (SEQ ID NO:100) to delete an EcoRI site at nucleotide 932 of the I. orientalis CYB2A gene. The resulting plasmid was designated pKW49. pKW49 was digested with EcoRI and BgIII and the resultant fragment ligated to EcoRI and BamHI digested pHJJ23 (FIG. 10). The resulting ADHa deletion construct, designated pGPB11 (FIG. 11), contains the I. orientalis PDC1 promoter (amplified using primers oJLJ3 (SEQ ID NO:101) and oJLJ19 (SEQ ID NO:102)) and terminator (amplified using primers oJLJ1 (SEQ ID NO:103) and oJLJ2 (SEQ ID NO:104)) and a CYB2A marker element between an 858 bp fragment corresponding to the region immediately 5′ of the I. orientalis AHD2a open reading frame (amplified using primers oHJJ71 (SEQ ID NO:105) and oHJJ72 (SEQ ID NO:106)) and a 996 bp fragment corresponding to the region immediately 3′ of the I. orientalis ADHa open reading frame (amplified using primer oHJJ73 (SEQ ID NO:107) and oHJJ74 (SEQ ID NO:108)).
pKF046, which contains an S. cerevisiae MEL5 marker gene operatively linked to a I. orientalis PGK promoter and an S. cerevisiae MEL5 terminator and flanked by LoxP sites, was used as a template for quickchange mutagenesis using oligonucleotides oKW74 (SEQ ID NO:109) and oKW75 (SEQ ID NO:110) to delete an EcoRI site at nucleotide 2392 of the plasmid. The resulting plasmid was designated pKW50. pKW50 was digested with EcoRI and BgIII and the resultant fragment ligated to EcoRI and BamHI digested pHJJ23. The resulting plasmid, designated pGPB14 (FIG. 12), contains the same elements as pGPB11, but with the CYB2A selectable marker element replaced by the S. cerevisiae MEL5 selectable marker element.
pGPB11 and pGPB14 were each digested with EcoRI and BamHI to remove those portions of the plasmids corresponding to the PDC promoter and terminator, and each plasmid backbone was blunted with Klenow fragment and ligated to recircularize the plasmid. The plasmids were then transformed into E. coli. Plasmid isolated from positive colonies was designated pGPB28 (FIG. 13, derived from pGPB11) and pGPB34 (FIG. 14, derived from pGPB14).
Example 5B: Construction of FRD1 Expression Constructs pGPB26, pGPB39
Expression cassettes for the FRD1 gene from S. cerevisiae (SEQ ID NO:29) is inserted into the ADHa deletion construct pGPB11.
Plasmids containing S. cerevisiae FRD1 gene was digested with XbaI and PacI, and the FRD1 fragments were ligated to similarly digested pGPB11. The resulting plasmid, which contained the FRD1 coding sequence flanked by the I. orientalis PDC1 promoter and terminator and also contained the I. orientalis CYB2A selectable marker, was designated pGPB26 (S. cerevisiae FRD1) (FIG. 15).
pGPB26 was digested with BamHI and NdeI and ligated to similarly digested pGPB14. The resulting plasmids, which contained the FRD1 coding sequence flanked by the I. orientalis PDC1 promoter and terminator and also contained the S. cerevisiae MEL5 selectable marker was designated pGPB39 (S. cerevisiae FRD1) (FIG. 16).
Example 5C: Insertion of FRD1 at First and Second ADHa Loci of I. orientalis Strain 12481
pGPB39, was digested with SacI and ApaI and transformed into I. orientalis strain 12481 by lithium acetate transformation. Transformants are selected on YNB+2% melibiose plates overlaid with x-α-gal. After around six days, blue transformants are picked and plated for single colonies on YP+20 g/L glucose plates containing x-α-gal. Blue colonies are picked, and genomic DNA is isolated and screened by PCR to confirm integration of the FRD1 expression cassette at the ADHa locus using primers oGPB47 (SEQ ID NO:111), oGPB56 (SEQ ID NO:112), oGPB54 (SEQ ID NO:113), and oGPB46 (SEQ ID NO:114). Strains with the correct integration of the FRD1 gene are designated ySBCG101.
pGPB26 (S. cerevisiae FRD1) are digested with SacI and ApaI and transformed into strain ySBCG101 by lithium acetate transformation. Transformants are screened by PCR to confirm correct integration of the FRD1 expression cassette at a first ADHa locus using primers oGPB47 (SEQ ID NO:111), oGPB53 (SEQ ID NO:115), oGPB52 (SEQ ID NO:116), oGPB54 (SEQ ID NO:113), and oGPB46 (SEQ ID NO:114). The resulting strains are designated ySBCG126.
Marker recycling is carried out with plasmid pVB32. The correct homozygous strain with both markers removed is designated 12752.
The various FRD1 insertion/ADHa deletion strains generated in Example 5 are summarized in Table 8.







TABLE 8







I. orientalis FRD1 Insertion Strains









Strain name
Description
Parent strain





ySBCG101
CYB2A deletion (2)
12481



I. orientalis PYC1 insertion at PDC1 (2)



S. cerevisiae FRD1 insertion at ADHa (1)


ySBCG126
CYB2A deletion (2)
ySBCG101



I. orientalis PYC1 insertion at PDC1 (2)



S. cerevisiae FRD1 insertion at ADHa (2)


12752
CYB2A deletion (2)
ySBCG126



I. orientalis PYC1 insertion at PDC1 (2)



S. cerevisiae FRD1 insertion at ADHa (2)









Example 6: Insertion of MDH at the ATO2 Locus in I. orientalis Strain 12752
An MDH expression cassette is inserted at one or both ATO2 alleles of I. orientalis strain 12752 (Example 5).
Example 6A: Construction of ATO2 Deletion Construct pKWB18, pKWB23, and pKWB28
Upstream and downstream regions of I. orientalis ATO2 (SEQ ID NO:65) were amplified in order to generate an ATO2 deletion construct. The upstream and downstream regions correspond to nucleotides from 419 bp upstream to the start codon of ATO2 and from the stop codon to 625 bp downstream, respectively. Amplification of the upstream region is performed using primers oKW66 (forward, SEQ ID NO:117) and oKW67 (reverse, SEQ ID NO:118), which adds a PmeI restriction site and NdeI, NotI, and SacI restriction sites, respectively, to the product. Amplification of the downstream region is performed using primers oKW68 (forward, SEQ ID NO:119) and oKW69 (reverse, SEQ ID NO:120), which adds NdeI, NotI, and SacI restriction sites and a PmeI restriction site, respectively, to the product. The two fragments are amplified independently, then assembled into a full-length product with a two stage PCR protocol. The first stage uses 10 cycles (98° C. 10 s, 55° C. 20 s, 72° C. 60 s) with no primers, and the second stage uses 20 cycles (98° C. 10 s, 55° C. 20 s, 72° C. 60 s) with upstream forward and downstream reverse primers. The full-length product is gel purified, cloned into pCR-BluntII (Invitrogen), and sequenced. The plasmid confirmed to have correct sequence is subjected to quickchange PCR using Phusion polymerase to eliminate the plasmid-borne SacI site. Correct plasmids are confirmed by digestion with SacI and sequencing. The final ATO2 deletion construct is designated pKWB18 (FIG. 17). pKWB18 was digested with NdeI and SacI restriction enzymes and ligated to like cut pKF031 (ScMEL5 selectable marker) and pKF044 (CYB2A selectable marker). The resulting ATO2 deletion constructs with ScMEL5 and CYB2A selectable markers are designated pKWB23 (ScMEL5) and pKWB28 (CYB2A) (FIG. 18 and FIG. 19 respectively).
Example 6B: Construction of MDH Expression Constructs pGPB64 and 66
The MDH gene from Z. rouxii (SEQ ID NO:25) is amplified from genomic DNA using primers designed to add a MluI restriction enzyme site 5′ end of the start codon, and a SbfI restriction enzyme site to the 3′ end of the stop codon. Z. rouxii MDH is amplified using primers oGPB67 (SEQ ID NO:121) and oGPB68 (SEQ ID NO:122) After amplification, the product is gel purified and cloned into pZeroBluntII and transformed into E. coli. Transformants are selected on LB plates containing 50 μg/ml kanamycin. The correct plasmid is confirmed by sequencing, and the final construct is designated pGPB58 (Z. rouxii MDH). pGPB58 is digested with MluI and SbfI to liberate the fragment containing the MDH gene. The fragment is gel purified and cloned into like cut pGPB54 and pGPB55 (FIG. 20 and FIG. 21). The resulting plasmids are designated pGPB64 (Z. rouxii MDH, MEL5 marker, FIG. 22) and pGPB66 (Z. rouxii MDH, CYB2A selectable marker, FIG. 23).
Example 6C: Insertion of MDH at First and Second ATO2 Loci in I. orientalis Strain 12752
pGPB64 is digested with PmeI and the appropriate fragments are used to transform I. orientalis strain 12752 (Example 5) by lithium acetate transformation. Transformants are selected by growth on YNB+2% melibiose and screened by PCR with primers flanking the ATO2 locus (oKW214 (SEQ ID NO:123) and oKWB155 (SEQ ID NO:124)) along with nested primers specific to the MEL5 integration cassette (oGPB55 (SEQ ID NO:125), and oGPB11 (SEQ ID NO:97). Colony with the correct insertion of MDH at a first ATO2 locus is designated ySBCG160.
pGPB66 is digested with PmeI and the appropriate fragments are used to transform I. orientalis strain ySBCG160. Transformants are selected by growth on YNB+2% lactic+x-α-gal and screened by PCR with primers flanking the ATO2 locus (oKW214 (SEQ ID NO:123) and oKWB155 (SEQ ID NO:124)) along with nested primers specific to the MEL5 integration cassette (oGPB55 (SEQ ID NO:125), and the CYB2A integration cassette oGPB52 (SEQ ID NO:116), oGPB53 (SEQ ID NO:115). A strain homozygous for MDH at the ATO2 loci is designated 12787.
Marker recycling is carried out using pVB32, and homozygous strain with both markers removed is designated 12792.
The various MDH insertion/ATO2 deletion strains generated in Example 6 are summarized in Table 9.







TABLE 9







I. orientalis MDH Insertion Strains









Strain name
Description
Parent strain





ySBCG160
CYB2A deletion (2)
12752



I. orientalis PYC1 insertion at



PDC1 (2)



S. cerevisiae FRD1 insertion at



ADHa (2)



Z. rouxii MDH insertion at ATO2 (1)


12787
CYB2A deletion (2)
ySBCG160



I. orientalis PYC1 insertion at



PDC1 (2)



S. cerevisiae FRD1 insertion at



ADHa (2)



Z. rouxii MDH insertion at ATO2 (2)


12792
CYB2A deletion (2)
12787



I. orientalis PYC1 insertion at



PDC1 (2)



S. cerevisiae FRD1 insertion at



ADHa (2)



Z. rouxii MDH insertion at ATO2 (2)









Example 7: Insertion of I. orientalis FUM1 Genes at the CYB2B Locus in I. orientalis Strains 12790-12792
I. orientalis FUM1 expression cassettes are inserted at both alleles of CYB2A of I. orientalis strain 12792 (Example 6).
Example 7A: Construction of I. orientalis FUM1 Expression Constructs pGPB30, pGPB42, pGPB44, and pGPB47
An expression cassette for the I. orientalis FUM1 gene (SEQ ID NO:1) is inserted into the ADHa deletion construct pGPB11. PCR primers oGPB38 (SEQ ID NO:127) and oGPB40 (SEQ ID NO:128) are used to amplify FUM1 using I. orientalis genomic DNA as the template. The 5′ primer adds an XbaI site at the start site of the coding sequence and the 3′ primer adds a PacI site 3′ of the stop codon. The resulting PCR product is digested with XbaI and PacI and ligated to similarly digested pGPB11. The resulting plasmid, which contains the FUM1 coding sequence flanked by the I. orientalis PDC1 promoter and terminator and the CYB2A selectable marker, is designated pGPB30 (FIG. 24).
pGPB30 is digested with BamHI and NdeI and ligated into similarly digested pGPB14. The resulting plasmid is designated pGPB44 (FIG. 25).
The expression cassette from pGPB30 is excised using NotI and ligated to the NotI cut pKW22. The resulting plasmid is designated pGPB43 (FIG. 26).
The expression cassette from pGPB44 is excised using NotI and ligated to the NotI cut pKW22. The resulting plasmid is designated pGPB47 (FIG. 27).
Example 7B: Insertion of I. orientalis FUM1 at One or Both I. orientalis CYB2B Loci
Integration of the first copy of the FUM1 expression cassette at the CYB2B locus is performed using plasmids containing the MEL5 selectable marker. pGPB47 is digested with SacI and ApaI and transformed into 12792 using lithium acetate transformation. Transformants are screened by PCR to confirm correct integration of the FUM1 expression cassette at the second CYB2B locus using primers oKW202 (SEQ ID NO:129), oGPB54 (SEQ ID NO:113), oKW195 (SEQ ID NO:130), and oGPB56 (SEQ ID NO:112). The resulting strains is designated 12826.
Integration of the second copy of the FUM1 expression cassette at the CYB2B locus is performed using plasmids containing the CYB2A selectable marker. pGPB43 is digested with SacI and ApaI and transformed into I. orientalis strain 12826 using lithium acetate transformation. Transformants are screened by PCR to confirm correct integration of the FUM1 expression cassette at the first CYB2B locus using primers oKW202 (SEQ ID NO:129), oGPB54 (SEQ ID NO:113), oGPB52 (SEQ ID NO:116), oKW195 (SEQ ID NO:130), and oGPB53 (SEQ ID NO:115). The resulting strain is designated 12833.
Marker recycling is carried out with plasmid pVB32. The correct homozygous strain with both markers removed is designated 12868.
The various FUM1 insertion/CYB2B deletion strains generated in Example 7 are summarized in Table 10.







TABLE 10







I. orientalis FUM1 Insertion Strains









Strain name
Description
Parent strain





12826
CYB2A deletion (2)
12792



I. orientalis PYC1 insertion at PDC1 (2)



S. cerevisiae FRD1 insertion at ADHa (2)



Z. rouxii MDH insertion at ATO2 (2)



I. orientalis FUM1 insertion at CYB2B (1)


12833
CYB2A deletion (2)
12826



I. orientalis PYC1 insertion at PDC1 (2)



S. cerevisiae FRD1 insertion at ADHa (2)



Z. rouxii MDH insertion at ATO2 (2)



I. orientalis FUM1 insertion at CYB2B (2)


12868
CYB2A deletion (2)
12833



I. orientalis PYC1 insertion at PDC1 (2)



S. cerevisiae FRD1 insertion at ADHa (2)



Z. rouxii MDH insertion at ATO2 (2)



I. orientalis FUM1 insertion at CYB2B (2)









Example 8: Insertion of SpMAE at the Putative RIOR43690 Locus in I. orientalis Strain 12868
A SpMAE expression cassette was inserted at one or both RIOR43690 alleles in I. orientalis strain 12868 (Example 7).
Example 8A: Synthesis of RIOR43690 Deletion Construct pVMB54
Deletion constructs for RIOR43690 (SEQ ID NO:59) were created. Oligonucleotide sequences identical to the upstream and downstream regions of the RIOR43690 locus (SEQ ID NOs:131 and 132, respectively) was synthesized to create the RIOR43690 deletion construct. The upstream and downstream regions correspond to nucleotides from 180 bp upstream to the start codon of RIOR43690 and from the stop codon to 180 bp downstream, respectively. PmeI restriction sites were added to the 5′ end of the upstream identity sequence and to the 3′ end of the downstream identity sequence. A multiple cloning site was added between the two sequences which include the following restriction sites, ordered from 5′ to 3′: NdeI, and SacI. The full length construct oligonucleotide fragment (SEQ ID NO:133) was cloned into the pIDTSMART Kan vector (Integrated DNA Technologies (“IDT”), Coralville, Iowa). The resulting plasmid was confirmed to have the correct sequence via Sanger sequencing. The final RIOR43690 deletion construct was designated pVMB54 (FIG. 28).
Example 8B: Construction of S. pombe SpMAE Bipartite Integration Constructs pKWB95, pKWB96 and pKWB97
The SpMAE gene from S. pombe (SEQ ID NO:45) was synthesized such that the coding sequence was segmented into five fragments with overlapping regions of homology. Fragments 2-5 (SEQ ID NO:134) were joined into one larger fragment via multi-fragment primerless PCR. The resulting fragment, which encoded the last 1154 bp of the S. pombe transporter, was topo cloned and sequenced for verification. Fragment 1 (SEQ ID NO:135) was topo cloned separately and encoded the first 384 bp of the S. pombe transporter. Each of the fragments was flanked by mluI and sbfI restriction sites. After restriction digest, the fragments were gel purified, and ligated to similarly digested pKWB86 and pKWB87. pKWB86 and pKWB87 are constructed from pVMB54 backbones, and both contain a multiple cloning site containing MluI, NotI, and SbfI sites operatively linked to the I. orientalis ENO promoter and the S. cerevisiae GAL10 terminator. pKWB86 also contains a selection marker cassette comprising the S. cerevisiae MEL5 gene operatively linked to the I. orientalis PGK promoter. This selection marker cassette is flanked by loxP sites. pKWB87 contains an expression cassette comprising the I. orientalis CYB2A promoter, gene, and terminator. This expression cassette is flanked by loxP sites.
The plasmids were transformed into E. coli, and transformants were selected on LB plates containing 50 μg/ml Kanamycin and screened using primers flanking the NotI site of pKWB87 and pKWB86 (oKW93 (SEQ ID NO:82) and oKWB95 (SEQ ID NO:126)). Correct plasmids were designated pKWB95 (fragment 1 with MEL5 marker) (FIG. 29 pKWB95), pKWB96 (fragment 1 with CYB2A marker) (FIG. 30 pKWB96) and pKWB97 (Fragment 2 with MEL5 marker) (FIG. 31 pKWB97).
Example 8C: Integration of SpMAE at CB1 RIOR43690 in 12868
Plasmids pKWB95 and pKWB96 were digested with pmeI and sbfI and gel purified. Each of these digests served to liberate a DNA fragment containing the upstream flanking region, the selective marker and fragment 1 from the vector backbone. pKWB97 was digested with pmeI and mluI. This digest served to liberate fragment 2 through the gal10 terminator and the downstream flanking region from the vector backbone. Strain 12868 (example 8) was transformed with the fragments purified from both pKWB95 and pKWB97 simultaneously. This transformation was designed such that integration of both fragments reconstitutes the intact coding sequence intracellularly to produce a functional transport protein. Crossover events occur between the flanking regions of the fragments and genomic DNA as well as the overlapping regions of the SpMAE gene. Transformants are selected on YNB+2% melibiose+x-α-gal and, and integration of SpMAE at a first RIOR43690 allele is confirmed by PCR. The correct heterozygous strain is designated strain 13050.
A second integration, targeting the second RIOR43690 allele is performed using the fragment purified from pKWB96 and pKWB97. Transformants are selected on YNB+2% lactic+x-α-gal and, and integration of SpMAE at the second RIOR43690 allele is confirmed by PCR. The correct heterozygous strain is designated strain 13051.
Marker recycling is carried out with plasmid pVB32. The correct homozygous strains with both markers removed are designated 13053.
The various SpMAE insertion/RIOR43690 deletion strains generated in Example 8 are summarized in Table 11.







TABLE 11







I. orientalis SpMAE Strains









Strain name
Description
Parent strain





13050
I. CYB2A deletion (2)
12868



I. orientalis PYC1 insertion at PDC1 (2)



S. cerevisiae FRD1 insertion at ADHa (2)



Z. rouxii MDH insertion at ATO2 (2)



I. orientalis FUM1 insertion at CYB2B (2)



S. pombe MAE insertion at RIOR43690 (1)


13051
I. CYB2A deletion (2)
13050



I. orientalis PYC1 insertion at PDC1 (2)



S. cerevisiae FRD1 insertion at ADHa (2)



Z. rouxii MDH insertion at ATO2 (2)



I. orientalis FUM1 insertion at CYB2B (2)



S. pombe MAE insertion at RIOR43690 (2)


13053
I. CYB2A deletion (2)
13051



I. orientalis PYC1 insertion at PDC1 (2)



S. cerevisiae FRD1 insertion at ADHa (2)



Z. rouxii MDH insertion at ATO2 (2)



I. orientalis FUM1 insertion at CYB2B (2)



S. pombe MAE insertion at RIOR43690 (2)









Example 9: Insertion of the FRD1 from Trypanosoma brucei, Trypanosoma cruzi, Leishmania braziliensis, or Leishmania mexicana at the ADHb Locus in I. orientalis Strain 13053
A FRD1 expression cassette is inserted at one or both ADHb alleles of I. orientalis strains 13053 (Example 8).
Example 9A: Construction of ADHb Deletion Construct pGPB119
pGPB26 was digested with NotI and the resultant fragment ligated to NotI digested pHJJ77 (FIG. 32). The resulting ADHb deletion construct, designated pGPB119 (FIG. 33), contains the I. orientalis PDC1 promoter (amplified using primers oJLJ3 (SEQ ID NO:101) and oJLJ19 (SEQ ID NO:102)) and terminator (amplified using primers oJLJ1 (SEQ ID NO:103) and oJLJ2 (SEQ ID NO:104)) and a CYB2A marker element between a 770 bp fragment corresponding to the region immediately 5′ of the I. orientalis AHD2b open reading frame (amplified using primers oHJJ124 (SEQ ID NO:137) and oHJJ125 (SEQ ID NO:138)) and a 615 bp fragment corresponding to the region immediately 3′ of the I. orientalis ADHb open reading frame (amplified using primer oHJJ126 (SEQ ID NO:139) and oHJJ127 (SEQ ID NO:140)).
Example 9B: Construction of FRD Expression Constructs pGPB126, pGPB127, pGPB159, pGPb160, pGPB161, and ADHb Null Constructs pGPB148 and pGPB168
Expression cassettes for the FRD1 genes were inserted into the ADHb deletion construct pGPB119 (Example 9A); the FRD1 genes from T. brucei (SEQ ID NO:37), T. cruzi (SEQ ID NO:39), L. braziliensis (SEQ ID NO:41), and L. mexicana (SEQ ID NO:43), were codon optimized to I. orientalis, and lacked the C-terminal glyoxysomal targeting sequence from the native gene.
Plasmids containing the codon optimized FRD1 genes were digested with XbaI and PacI, and the FRD1 fragments were ligated to similarly digested pGPB119. The resulting plasmids, containing the FRD1 coding sequence flanked by the I. orientalis PDC1 promoter and terminator and the I. orientalis CYB2A selectable marker, were designated pGPB126 (T. brucei), pGPB159 (T. cruzi), pGPB160 (L. braziliensis), and pGPB161 (L. mexicana) (FIG. 34).
pGPB126 was digested with NdeI and a partial NotI digest to remove the CYB2A selectable marker, and the resulting vector was ligated to the NdeI and NotI fragment from pGPB14 which contained the MEL5 selectable marker. The resulting plasmid, which contained the FRD1 coding sequence flanked by the I. orientalis PDC1 promoter and terminator and the S. cerevisiae MEL5 selectable marker was designated pGPB127 (FIG. 35).
pGPB126 (CYB2A) and pGPB127 (MEL5) were each digested with NdeI and BamHI to remove those portions of the plasmids corresponding to the PDC promoter, T. brucei FRD and PDC terminator, and each plasmid backbone was blunted with Klenow fragment and ligated to recircularize the plasmid. The plasmids were then transformed into E. coli. Plasmids isolated from positive colonies are designated pGPB168 (FIG. 36, derived from pGPB126) and pGPB148 (FIG. 37, derived from pGPB127).
Example 9C: Insertion of FRD at First and Second ADHb Loci of I. orientalis Strain 13053
pGPB126pGPB159, pGPB160, pGPB161 and pGPB148 were digested with SacI and ApaI and transformed into I. orientalis strain 13053 by lithium acetate transformation. Transformants are selected on YNB+2% lactic plates or YNB+2% melibiose plates (pGPB148). After around six days, transformants were picked and plated for single colonies on YP+20 g/L glucose plates. Colonies are picked, and genomic DNA was isolated and screened by PCR to confirm integration of the FRD1 expression cassette at the ADHb locus using primers oGPB106 (SEQ ID NO:141), oGPB56 (SEQ ID NO:112), oGPB52 (SEQ ID NO:116), oGPB54 (SEQ ID NO:113), oGPB55 (SEQ ID NO:125), and oGPB107 (SEQ ID NO:142). Strains with the correct integration of the FRD1 gene are designated 13171 (Tb FRD), 13256 (Tc FRD), 13257 (Lb FRD), and 13258 (Lm FRD).
The various FRD1 insertion/ADHb deletion strains generated in Example 9 are summarized in Table 12.







TABLE 12







I. orientalis FRD1 Insertion Strains









Strain name
Description
Parent strain





13171
CYB2A deletion (2)
13053



I. orientalis PYC1 insertion at PDC1 (2)



S. cerevisiae FRD1 insertion at ADHa (2)



Z. rouxii MDH insertion at ATO2 (2)



I. orientalis FUM1 insertion at CYB2B (2)



T. brucei FRD1 insertion at ADHb (1)


13256
CYB2A deletion (2)
13053



I. orientalis PYC1 insertion at PDC1 (2)



S. cerevisiae FRD1 insertion at ADHa (2)



Z. rouxii MDH insertion at ATO2 (2)



I. orientalis FUM1 insertion at CYB2B (2)



T. cruzi FRD1 insertion at ADHb (1)


13257
CYB2A deletion (2)
13053



I. orientalis PYC1 insertion at PDC1 (2)



S. cerevisiae FRD1 insertion at ADHa (2)



Z. rouxii MDH insertion at ATO2 (2)



I. orientalis FUM1 insertion at CYB2B (2)



L. braziliensis FRD1 insertion at ADHb (1)


13258
CYB2A deletion (2)
13053



I. orientalis PYC1 insertion at PDC1 (2)



S. cerevisiae FRD1 insertion at ADHa (2)



Z. rouxii MDH insertion at ATO2 (2)



I. orientalis FUM1 insertion at CYB2B (2)



L. mexicana FRD1 insertion at ADHb (1)









Example 10: Succinic Acid Production of Strain 13171
The yeast strain 13171 is run in fermenters to test succinic acid production. Fermenters are inoculated with biomass grown in defined medium (adapted from Verduyn, et. al, 1992, Yeast 8, 501-517, see Tables 13, 15, and 16). Seeds are run in 1 L baffled flasks (250 mL working volume) at 250 rpm and 30° C. The contents of the flasks are harvested at approximately 24 hours incubation time with 10% v/v inoculum used to start fermenters. Fermenter initial working volume is 1.2 L for fed-batch glucose and 1.5 L for straight batch glucose. The cell dry weight at inoculation is found in Table 17. Fermenter media is outlined in Table 14, 15, 16. Glucose is either provided by either a) the addition of 115 g/L at the start of the batch (straight batch). or b) providing a glucose feed addition to the fermenter controlled to maintain the glucose concentration at <10 g/L residual glucose (fed-batch). pH is controlled at 3.0 with 5 N KOH. The fermenter systems are sparged at 0.24 slpm with a blend of pure CO2 and air to target 10% CO2 in the inlet gas stream. Different oxygen uptake rates are applied to the vessels by changing vessel agitation rate. These fermentations are operated such that after the cells achieve a sufficient density, oxygen limitation is achieved and subsequently maintained throughout the rest of the fermentation (e.g., dissolved oxygen less than about 10%). A DO curve is demonstrated in FIG. 36. Dissolved oxygen is measured using Mettler Toledo INPRO® 6800 sensor (Mettler-Toledo GmbH, Urdorf, Switzerland), calibrated prior to inoculation. 0% is calibrated by sparging nitrogen at 0.24 slpm, 100% is calibrated using the run conditions in the vessel as detailed above (prior to inoculation).







TABLE 13







Defined Media for Seed Flask Cultures










Compound
Concentration (g/kg)














C6H12O6
20.0



(NH2)2CO
2.3



KH2PO4
3.0



MgSO4—7H2O
0.5



1000x Vitamin Solution
1



1000x Trace Solution
1

















TABLE 14







Defined Media for 2 L Fermenters










Compound
Concentration (g/kg)







C6H12O6
Fed-batch at <10 g/L or straight batch at




115 g/L initial glucose



(NH4)2SO4
2.3



KH2PO4
3.0



MgSO4—7H2O
0.5



1000x Vitamin
1



Solution



100x Trace
1



Solution

















TABLE 15







Trace Element 1000x Stock Solution










Chemical
g/1.0 L














C10H14N2Na2O8•2H2O
15.00



ZnSO4•7H2O
4.50



MnCl2•2H2O
0.84



CoCl2•6H2O
0.30



CuSO4•5H2O
0.30



Na2MoO4•2H2O
0.40



CaCl2•2H2O
4.50



FeSO4•7H2O
3.00



H3BO3
1.00



KI
0.10

















TABLE 16







Vitamin 1000x Stock Solution










Chemical
g/1.0 L














Biotin (D−)
0.05



Ca D(+) panthothenate
1.00



Nicotinic acid
1.00



Myo-inositol
25.00



Thiamine chloride
1.00



hydrochloride



Pyridoxol hydrochloride
1.00



p-aminobenzoic acid
0.20










Cell concentration is obtained from an optical density measurement using an established conversion factor between dry cell mass and optical density. Optical density is measured at wavelength of 600 nm with a 1 cm pathlength using a model Genesys20 spectrophotometer (Thermo Scientific). Unless explicitly noted otherwise, an experimentally derived conversion factor of 1.68 OD600 units per 1 g dry cell mass is used to estimate cell dry weight.
OUR is calculated using methods known to those in the art as described above. For this example, oxygen and CO2 values are measured by an EGAS L instrument (Sartorious). While a mass spectrometer was not used, the results obtained by the EGAS L are believed to be substantially the same. Nitrogen value was calculated as 100% less % measured CO2 minus, less % measured Oxygen.
Samples are taken at 90 h batch time and analyzed for biomass growth via OD600, succinate via gas chromatography with flame ionization detector and glucose by high performance liquid chromatography with refractive index detector.
Table 17 illustrates the concentration of cells in straight batch at end of fermentation is variable with oxygen concentration, ranging from 0.4 to 2.3 g/L.







TABLE 17







Final Cell Concentration (g/L) at 90 h Fermentation Time: Straight


Batch Glucose at pH 3.0, 10% CO2, OUR (mmol/L/h) Variable











Cell Dry Weight
Cell Dry Weight at



OUR
at Inoculation
End of
Mode of


(mmol/L/h)
(g/L)
Fermentation (g/L)
Operation













1.9
0.14
0.4
Straight Batch


4.2
0.16
1.1
Straight Batch


6.1
0.14
1.7
Straight Batch


10.2
0.17
2.3
Straight Batch









FIG. 37 and FIG. 38 illustrate succinate acid production for both straight batch and fed batch fermentations. A previous example (WO2010/003728, pg 9, line 15-18), tells us that “an OUR above 5 mmol/L/h resulted in lower succinic acid production”. Surprisingly, in both modes of operation, we find improving production above 5 mmol/L/h, with succinic acid titer increasing with increasing OUR, such that the no decrease in succinic production is observed even at the highest OUR tested, 20 mmol/L/h.
Furthermore, FIG. 39 indicates no significant reduction in succinate yield on carbon source associated with increasing OUR across the range tested. These data indicate OUR greater than about 10 mmol/L/h to be a preferred condition for succinate production.
Example 11: Construction of Preparatory Strains 11-1 to 11-6
An I. orientalis host strain is generated by evolving I. orientalis strain ATCC PTA-6658 for 91 days in a glucose-limited chemostat. The system is fed 15 g/L glucose in a defined medium and operated at a dilution rate of 0.06 h−1 at pH 3 with added lactic acid in the feed medium. The conditions are maintained with an oxygen transfer rate of approximately 2 mmol L−1 h−1, and dissolved oxygen concentration remains constant at 0% of air saturation. Single colony isolates from the final time point are characterized in two shake flask assays. In the first assay, the isolates are characterized for their ability to ferment glucose to ethanol in the presence of 25 g/L total lactic acid with no pH adjustment in the defined medium. In the second assay, the growth rate of the isolates is measured in the presence of 45 g/L of total lactic acid, with no pH adjustment in the defined medium. Strain 11-1 is a single isolate exhibiting the highest glucose consumption rate in the first assay and the highest growth rate in the second assay.
Strain 11-1 is transformed with linearized integration fragment P2 (having nucleotide sequence SEQ ID NO:143) designed to disrupt the URA3 gene, using the LiOAc transformation method as described by Gietz et al., in Met. Enzymol. 350:87 (2002). Integration fragment P2 includes a MEL5 selection marker gene. Transformants are selected on YNB+2% melibiose plates and screened by PCR to confirm the integration of the integration piece and deletion of a copy of the URA3 gene. A URA3-deletant strain is grown for several rounds until PCR screening identifies an isolate in which the MEL5 selection marker gene has looped out. The PCR screening is performed using the primers of SEQ ID NOs:144 and 145 to confirm the 5′-crossover and the primers of SEQ ID NOs:146 and 147 to confirm the 3′ crossover. That isolate is again grown for several rounds until PCR screening using the primers of SEQ ID NOs:144 and 147 identifies an isolate in which both URA3 alleles have been deleted. This isolate is named strain 11-2.
Strain 11-2 is transformed with integration fragment P3 (SEQ ID NO:148), which is designed to disrupt the PDC gene. Integration piece P2 contains the following elements, 5′ to 3′: a DNA fragment with homology for integration corresponding to the region immediately upstream of the I. orientalis PDC open reading frame, a PDC transcriptional terminator, the URA3 promoter, the I. orientalis URA3 gene, an additional URA3 promoter direct repeat for marker recycling, and a DNA fragment with homology for integration corresponding to the region directly downstream of the I. orientalis PDC open reading frame. A successful integrant (and single-copy PDC deletant) is identified on selection plates lacking uracil and confirmed by PCR using the primers of SEQ ID NOs:149 and 150 to confirm the 5′-crossover and the primers of SEQ ID NOs:151 and 152 to confirm the 3′-crossover. That integrant is grown for several rounds and plated on 5-fluoroorotic acid (FOA) plates to identify a strain in which the URA3 marker has looped out. Loopout of the URA3 marker is confirmed by PCR. The resultant strain is again transformed with integration fragment P3 to delete the second copy of the native PDC gene. A successful transformant is again identified by selection on selection plates lacking uracil, and further confirmed by culturing the strain over two days and measuring ethanol production. Lack of ethanol production further demonstrates a successful deletion of both copies of the PDC gene in a transformant. The resultant transformant is grown for several rounds and plated on FOA plates until PCR identifies a strain in which the URA3 marker has looped out. That strain is plated on selection plates lacking uracil to confirm the loss of the URA3 marker, and is designated strain 11-3.
Integration fragment P4-1 (SEQ ID NO:153) contains the following elements, 5′ to 3′: a DNA fragment with homology for integration corresponding to the region immediately upstream of the I. orientalis ADHa open reading frame, an I. orientalis PDC1 promoter, the I. orientalis PYC gene (SEQ ID NO:7), the I. orientalis TAL terminator, the I. orientalis URA3 promoter, and the first 530 bp of the I. orientalis URA3 open reading frame.
Integration fragment P4-2 (SEQ ID NO:155) contains the following elements, 5′ to 3′: a DNA fragment corresponding to the last 568 bp of the I. orientalis URA3 open reading frame, the I. orientalis URA3 terminator, the I. orientalis URA3 promoter, the I. orientalis TDH3 promoter, the S. pombe MAE gene (SEQ ID NO:45), the I. orientalis TKL terminator, and a DNA fragment with homology for integration corresponding to the region immediately downstream of the I. orientalis ADHa open reading frame.
Strain 11-3 is transformed simultaneously with integration fragments P4-1 and P4-2 using lithium acetate methods to insert the I. orientalis PYC gene and the S. pombe MAE gene at the ADHa locus. Integration occurs via three cross-over events: in the regions of the ADHa upstream homology, in the regions of the ADHa downstream homology, and in the region of URA3 homology between SEQ ID NO:153 and SEQ ID NO:155. Transformants are streaked to isolates and the correct integration of the cassette at the AHD9091 locus is confirmed in a strain by PCR. PCR screening is performed using the primers of SEQ ID NOs:111 and 112) to confirm the 5′-crossover and the primers of SEQ ID NOs:159 and 114 to confirm the 3′-crossover. That strain is grown and plated on FOA as before until the loopout of the URA3 marker from an isolate is confirmed by PCR.
The resultant isolate is then transformed simultaneously with integration fragments P4-3 and P4-4 using LiOAc transformation methods to insert a second copy of each of the I. orientalis PYC gene and the S. pombe MAE gene at the ADHa locus.
Integration fragment P4-3 (SEQ ID NO:161) contains the following elements, 5′ to 3′: a DNA fragment with homology for integration corresponding to the region immediately downstream of the I. orientalis ADHa open reading frame, an I. orientalis PDC1 promoter, the I. orientalis PYC gene (SEQ ID NO:7), the I. orientalis TAL terminator, the I. orientalis URA3 promoter, and the first 530 bp of the I. orientalis URA3 open reading frame.
Integration fragment P4-4 (SEQ ID NO:163) contains the following elements, 5′ to 3′: a DNA fragment corresponding to the last 568 bp of the I. orientalis URA3 open reading frame, the I. orientalis URA3 terminator, the I. orientalis URA3 promoter, the I. orientalis TDH3 promoter, the S. pombe MAE gene (SEQ ID NO:45), the I. orientalis TKL terminator, and a DNA fragment with homology for integration corresponding to the region immediately upstream of the I. orientalis ADHa open reading frame.
Integration again occurs via three crossover events. Transformants are streaked to isolates and screened by PCR to identify a strain containing both copies of the I. orientalis PYC and S. pombe MAE genes at the ADHa locus. PCR screening to confirm the first copy is performed using the primers of SEQ ID NOs:111 and 112) to confirm the 5′-crossover and the primers of SEQ ID NOs:159 and 114 to confirm the 3′-crossover. PCR screening to confirm the second copy is performed using the primers of SEQ ID NOs:111 and 159 to confirm the 5′-crossover and the primers of SEQ ID NOs:112 and 114 to confirm the 3′-crossover. The resultant strain is grown and replated on FOA until a strain in which the URA3 marker has looped out is identified. That strain is designated strain 11-4.
Strain 11-4 is transformed with integration fragment P5-1 (SEQ ID NO:165) using LiOAc transformation methods as described in previous examples to integrate the L. mexicana FRD gene at the locus of the native CYB2B open reading frame. The integration fragment P5-1 contains the following elements, 5′ to 3′: a DNA fragment with homology for integration corresponding to the region immediately downstream of the I. orientalis CYB2B open reading frame, an I. orientalis PDC1 promoter, the L. mexicana FRD gene (SEQ ID NO:43), the I. orientalis PDC1 terminator, the I. orientalis URA3 promoter, gene, and terminator in succession, followed by an additional URA3 promoter which serves as a direct repeat for marker recycling, and a region immediately upstream of the I. orientalis CYB2B open reading frame.
Successful integration of a single copy of the L. mexicana FRD gene in one isolate is identified by selection on selection plates lacking uracil and confirmed by PCR using the primers of SEQ ID NOs:152 and 129 to confirm the 5′-crossover and the primers of SEQ ID NOs:112 and 130 to confirm the 3′-crossover. That isolate is grown and plated on FOA as before until a strain in which the URA3 marker has looped out is identified by PCR. That isolate is transformed with the integration fragment P5-2 in the same manner as before to integrate a second copy of the L. mexicana FRD gene downstream of the CYB2 open reading frame.
Integration fragment P5-2 (SEQ ID NO:169) contains the following elements, 5′ to 3′: a DNA fragment with homology for integration corresponding to the region immediately upstream of the I. orientalis CYB2B open reading frame, an I. orientalis PDC1 promoter, the L. mexicana FRD gene (SEQ ID NO:43), the I. orientalis PDC1 terminator, the I. orientalis URA3 promoter, gene, and terminator in succession, followed by an additional URA3 promoter which serves as a direct repeat for marker recycling, and a region immediately downstream of the I. orientalis CYB2B open reading frame.
PCR is performed using the primers of SEQ ID NOs:112 and 129 to confirm the 5′-crossover and the primers of SEQ ID NOs:152 and 130 to confirm the 3′-crossover to confirm correct integration of both copies of the L. mexicana FRD gene in one isolate. That isolate is grown and plated on FOA as before until the loop out of the URA3 marker is confirmed by PCR in one isolate. That isolate is designated strain 11-5.







TABLE 18







I. orientalis URA and PDC Deletion Strains









Strain name
Description
Parent strain





11-1
Organic acid tolerant isolate
Wild-type


11-2
URA3 deletion (2)
11-1


11-3
URA3 deletion (2)
11-2



PDC deletion (2)


11-4
URA deletion (2)
11-3



PDC deletion (2)



I. orientalis PYC1 insertion at ADHa (2)



S. pombe MAE insertion at ADHa (2)


11-5
URA deletion (2)
11-4



PDC deletion (2)



I. orientalis PYC1 insertion at ADHa (2)



S. pombe MAE insertion at ADHa (2)



L. mexicana FRD insertion at CYB2B (2)









Example 12: Integration of MDH and FUM Genes
Example 12A: First Copy R. delemar MDH Integration Fragment
Integration fragment 12A (SEQ ID NO:168) contains the R. delemar MDH gene (SEQ ID NO:166), ADHb upstream integration arm, ENO promoter, URA3 promoter, and first 583 base pairs of the URA3 marker.
Example 12B: First Copy I. orientalis FUM Integration Fragment
Integration fragment 12B (SEQ ID NO:164) contains the I. orientalis FUM gene (SEQ ID NO:1), the last 568 base pairs of the URA3 marker, URA3 promoter, PGK promoter, and ADHb downstream integration arm.
Example 12C: Second Copy R. delemar MDH Integration Fragment
Integration fragment 12C (SEQ ID NO:162) contains the R. delemar MDH gene (SEQ ID NO:166), ADHb downstream integration arm, ENO promoter, URA3 promoter, and first 583 base pairs of the URA3 marker.
Example 12D: Second Copy I. orientalis FUM Integration Fragment
Integration fragment 12D (SEQ ID NO:160) contains the I. orientalis FUM gene (SEQ ID NO:1) the last 568 base pairs of the URA3 marker, URA3 promoter, PGK promoter, and ADHb upstream integration arm.
Example 12E: Preparation of Strain 13723
Strain 11-5 is simultaneously transformed with integration fragments 12A and 12B using the standard lithium acetate process described before. Successful transformants are selected on selection plates lacking uracil and confirmed by PCR using the primers of SEQ ID NOs:141 and 158 to confirm the 5′-crossover, the primers of SEQ ID NOs:82 and 157 to confirm the junction of integration fragments 12A and 12B, and the primers of SEQ ID NOs:136 and 142 to confirm the 3′-crossover. These transformants are grown and plated on FOA as before until a strain in which the URA3 marker has looped out is identified. That strain is simultaneously transformed with each of integration fragments 12C and 12D using the standard lithium acetate process described before. Successful transformants are selected on selection plates lacking uracil and confirmed by PCR using the primers of SEQ ID NOs:141 and 154 to confirm the 5′-crossover, the primers of SEQ ID NOs:82 and 157 to confirm the junction of integration fragments 12C and 12D, and the primers of SEQ ID NOs:142 and 156 to confirm the 3′-crossover. The resultant strain, which contains the L. Mexicana FRD gene (SEQ. ID. NO:43), is designated strain 13723.







TABLE 19







I. orientalis MDH/FUM Insertion Strains









Strain
Integration
Description (in addition to transformations as


No
Fragments
indicated for Examples 1-4)





13723
12A/12B
R. delemar MDH insertion at ADHb (2)




I. orientalis FUM insertion at ADHb (2)









Example 13: Succinate Production of Strain 13723
Strain 13723 was characterized across a range of oxygen transfer rates from 2.6 to 25.6 mmol/L/h using the straight batch fermentation method described in Example 10. Oxygen transfer rate was varied by vessel agitation as described in Example 10. An experimentally derived conversion factor for strain 13723 of 1.51 OD600 units per 1 g dry cell mass is used to estimate cell dry weight using methods and instruments as described in Example 10. Samples are taken at 90 hours batch time or when glucose concentration in the batch is >5 g/L and <15 g/L if this occurs prior to 90 hours batch time. Table 20 shows the cell dry weight at inoculation and at the end of fermentation.











TABLE 20







Cell dry






weight at



Cell dry weight at
end of


OUR
inoculation
fermentation

Mode of


(mmol/L/h)
(g/L)
(g/L)
Strain
operation



















2.6
0.14
1.19
13723
Straight Batch


4.1
0.14
1.52
13723
Straight Batch


6.7
0.10
2.98
13723
Straight Batch


9.5
0.15
3.91
13723
Straight Batch


18.0
0.10
8.41
13723
Straight Batch


25.6
0.11
16.29
13723
Straight Batch









Succinate production is summarized in Table 21 and FIG. 42. Table 21 shows total succinate production (g/L), rate of succinate production (g/L/h), and total succinate yield per gram of glucose (g/g) for strain 13723. Total succinate yield decreases slowly from an OUR of about 9.5 mmol/L/h to an OUR of about 25.6 mmol/L/h. However, the product of succinate production rate times succinate yield times total succinate titer produced increases as the OUR increases from about 5 mmol/L/h to about 18 mmol/L/h (or about 20 mmol/L/h). This confirms that the strains and fermentation processes disclosed herein can be used to produce succinate with a high production rate, high final titer of succinate in the fermentation broth, and an acceptable yield.












TABLE 21








Rate of





Succinate
Yield (g
succinate
Rate ×
Cells


OUR
titer
succinate/
production
yield ×
(g/L - 1.5


(mmol/L/h)
(g/L)
g glucose)
(g/L/h)
titer
conversion)




















2.6
30.3
0.57
0.33
5.7
1.19


4.1
41.5
0.55
0.46
10.5
1.52


6.7
45.4
0.54
0.5
12.3
2.98


9.5
47.4
0.55
0.52
13.5
3.91


18.0
48.2
0.45
0.97
20.3
8.41


25.6
45.0
0.4
1.03
18.6
16.29

## Claims

1. A method of producing a succinate, succinic acid, or succinate/succinic acid-containing fermentation broth, the method comprising:
(i) culturing in a fermenter a recombinant yeast in the presence of at least one carbon source to produce succinate, wherein the recombinant yeast is genetically engineered to produce succinate through a reductive tricarboxylic acid (TCA) active succinate fermentation pathway from phosphoenolpyruvate or pyruvate to succinate, wherein the active succinate fermentation pathway comprises the reactions:
(a) pyruvate to oxaloacetate;
(b) oxaloacetate to malate;
(c) malate to fumarate;
(d) fumarate to succinate; and
(e) export of succinate from inside the cell to the extracellular environment, and

(ii) introducing into the fermenter sufficient oxygen to provide an oxygen uptake rate (OUR) of greater than 8 mmol/L/h,

wherein the recombinant yeast produces more succinate when cultured under an OUR of greater than 8 mmol/L/h as compared to when the recombinant yeast is cultured at an OUR of 5 mmol/L/h, and
wherein a final concentration of succinate, succinic acid, or succinate/succinic acid in the fermentation broth is greater than 20 g/L.

2. The method of claim 1, wherein the recombinant yeast is a yeast of the species: Issatchenkia orientalis (Pichia kudriavzevii or Candida krusei), Candida lambica, Candida sorboxylosa, Candida zemplinina, Candida geochares, Pichia membranifaciens, Zygosaccharomyces kombuchaensis, Candida sorbosivorans, Kluyveromyces 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, Saccharomycopsis crataegensis, or Pichia jadinii.

3. The method of claim 1, wherein a production rate of succinate, succinic acid, or succinate/succinic acid is greater than 0.25 g/L/h, or a specific production rate of succinate, succinic acid, or succinate/succinic acid is greater than 0.1 g/g/h.

4. The method of claim 1, wherein the product yield of succinate, succinic acid, or succinate/succinic acid is greater than 25%.

5. The method of claim 1, wherein the cell dry weight at the end of fermentation is less than 10 g/L.

6. The method of claim 1, wherein the recombinant yeast dry weight at inoculation is less than 5 g/L.

7. The method of claim 1, wherein the recombinant yeast comprises one or more endogenous genes that encode an enzyme that catalyzes any one of reactions (a) though (e).

8. The method of claim 7, wherein the recombinant yeast comprises one or more copies of one or more endogenous genes encoding pyruvate carboxylase, malate dehydrogenase, fumarase, fumarate reductase, or a succinate exporter.

9. The method of claim 8, wherein the one or more of the endogenous genes are operatively linked to an exogenous regulatory element which is an exogenous promoter or an exogenous terminator.

10. The method of claim 1, wherein the recombinant yeast is genetically engineered to comprise one or more exogenous genes that encode an enzyme that catalyzes any one of reactions (a) through (e).

11. The method of claim 10, wherein the recombinant yeast comprises one or more copies of one or more exogenous genes encoding pyruvate carboxylase, malate dehydrogenase, fumarase, fumarate reductase, or a succinate exporter.

12. The method of claim 1, wherein the recombinant yeast is from a species which is Issatchenkia orientalis (Pichia kudriavzevii or Candida krusei) or Candida lambica.

13. The method of claim 1, wherein the recombinant yeast is from the Pichia fermentans/Issatchenkia orientalis clade.

14. The method of claim 1, wherein the carbon source is glucose, xylose, arabinose, sucrose, fructose, cellulose, glucose oligomers, or glycerol.

15. The method of claim 1, wherein a final pH of the fermentation broth is less than 6.0.

16. The method of claim 1, wherein a final concentration of succinic acid in the fermentation broth is greater than 20 g/L.

17. The method of claim 1, wherein the recombinant yeast comprises a genetic modification to enhance succinate export.

18. The method of claim 1, wherein the recombinant yeast has a deletion or disruption in a native pyruvate decarboxylase (PDC) gene.

19. The method of claim 1, wherein the OUR is between 8 mmol/L/h and 25 mmol/L/h.

20. The method of claim 1, wherein the OUR is greater than 10 mmol/L/h.

21. The method of claim 20, wherein the OUR is greater than 12 mmol/L/h.

22. A method of producing succinate, succinic acid, or succinate/succinic acid, the method comprising:
culturing in a fermenter a recombinant yeast in the presence of at least one carbon source to produce succinate, wherein the recombinant yeast is from the species Issatchenkia orientalis (Pichia kudriavzevii or Candida krusei) and is genetically engineered to produce succinate through a reductive tricarboxylic acid (TCA) active succinate fermentation pathway from phosphoenolpyruvate or pyruvate to succinate; and
providing sufficient oxygen to the fermenter to obtain an oxygen uptake rate (OUR) of 8 mmol/L/h to 25 mmol/L/h,
wherein the recombinant yeast produces more succinate when cultured under an OUR of between 8 mmol/L/h and 25 mmol/L/h, as compared to when the recombinant yeast is cultured at an OUR of 5 mmol/L/h.

23. The method of claim 22, wherein a final pH of the fermentation broth is less than 6.0.

24. The method of claim 22, wherein the OUR is from 8 mmol/L/h to 20 mmol/L/h.

25. The method of claim 22, wherein the production rate of succinate, succinic acid, or succinate/succinic acid is greater than 1 g/L/h.

26. The method of claim 22, wherein the product yield of succinate, succinic acid, or succinate/succinic acid is greater than 50%.

27. The method of claim 1, wherein the dissolved oxygen in the fermentation broth is maintained at less than 10% of air saturation at one atmosphere for greater than 10 hours during the batch time.

28. The method of claim 22, wherein the dissolved oxygen in the fermentation broth is maintained at less than 10% of air saturation at one atmosphere for greater than 10 hours during the batch time.

29. The method of claim 1, wherein a final pH of the fermentation broth is less than 5.5.

30. The method of claim 1, wherein a final pH of the fermentation broth is less than 5.0.

31. The method of claim 1, wherein a final pH of the fermentation broth is less than 4.0.

32. The method of claim 1, wherein a final pH of the fermentation broth is less than 3.5.