Yeast cells having NADP(H)-dependent reductive TCA pathway from pyruvate to succinate

Abstract

Recombinant yeast cells contain a reductive TCA pathway from phosphoenolpyruvate or pyruvate to succinate. At least one metabolic step in the pathway includes a reaction of NADPH to produce NADP+. The yeast cell contains at least one exogenous NADPH-dependent gene in the pathway from phosphoenolpyruvate or pyruvate to succinate, preferably an NADPH-dependent malate dehydrogensase or fumarate reducase gene (or both).

Claims

1. A recombinant I. orientalis cell having an active reductive TCA pathway from pyruvate to succinate and which further metabolizes succinate to one or more succinate metabolization products, which reductive TCA pathway includes a reaction that oxidizes NADPH to NADP+ comprising a conversion of fumarate to succinate catalyzed by an NADPH-dependent fumarate reductase enzyme and the recombinant cell has integrated into its genome an heterologous fumarate reductase gene that encodes for the NADPH-dependent fumarate reductase enzyme.

2. The recombinant cell of claim 1, wherein the recombinant cell further includes a reaction that oxidizes NADPH to NADP+comprising a conversion of oxaloacetate to malate catalyzed by an NADPH-dependent malate dehydrogenase enzyme and the recombinant cell has integrated into its genome an heterologous malate dehydrogenase gene that encodes for the NADPH-dependent malate dehydrogenase enzyme.

3. The recombinant cell of claim 1 wherein the recombinant cell further includes a step of converting pyruvate or phosphoenolpyruvate to oxaloacetate, a step of converting oxaloacetate to malate, and a step of converting malate to fumarate.

4. The recombinant I. orientalis cell of claim 1, wherein the succinate metabolization product is one or more of 1,4-butanediol, 1,3-butadiene, propionic acid, and 3-hydroxyisobutryic acid.

Description

EXAMPLE 1

Example 1A

Changing the Co-Factor Preference of the L.mexicana FRD to a NADPH-Dependent Enzyme

(1) A codon-optimized L. mexicana FRD gene having nucleotide sequence SEQ ID NO: 10 is used as a template to modify the coding sequence in five separate reactions to introduce substitutions to the amino acid residues of the putative NADH binding domain of the enzyme.

(2) A mutated L. mexicana FRD gene having nucleotide sequence SEQ ID NO. 15 is prepared by performing site-directed substitutions at amino acids 219 (glutamic acid) and 220 (tryptophan).

(3) A mutated L. mexicana FRD gene having nucleotide sequence SEQ ID NO. 16 is prepared by performing site-directed substitution at amino acid 417 (glutamic acid).

(4) A mutated L. mexicana FRD gene having nucleotide sequence SEQ ID NO. 17 is prepared by performing site-directed substitutions at amino acid 641 (aspartic acid).

(5) A mutated L. mexicana FRD gene having nucleotide sequence SEQ ID NO. 18 is prepared by performing site-directed substitutions at amino acids 861 (glutamic acid) and 862 (cysteine).

(6) A mutated L. mexicana FRD gene having nucleotide sequence SEQ ID NO. 19 is prepared by performing site-directed substitutions at amino acids 1035 (aspartic acid) and 1036 (serine).

Example 1B

Changing the Co-Factor Preference of the T.brucei FRD to a NADPH-Dependent Enzyme

(7) A codon-optimized T. brucei FRD gene having nucleotide sequence SEQ ID NO: 13 is used as a template to modify the coding sequence to introduce an amino acid substitution in the putative NADH binding domain of the enzyme. The site-directed mutgenesis is performed to target amino acid 411 (glutamic acid). The resulting mutated gene encodes for a mutated T. brucei FRD enzyme having amino acid sequence SEQ ID NO: 20.

Example 1C

FRD Enzyme Assay Method

(8) The FRD genes having SEQ ID NOs: 10 and 15-20 are each modified at 5 end by the addition of a short DNA sequence having SEQ ID NO: 21, immediately downstream of the initiation codon. This short nucleotide sequence encodes a peptide consisting of six histidine residues, followed by a single methionine, four aspartate residues and a single lysine residue, and effectively adds a 6His affinity tag and an enterokinase cleavage site to the FRD sequence. The expression of the FRD gene is driven by the IPTG inducible T5 promoter. The construct in each case also contains an optimized Shine-Dalgarno sequence followed by 8 by of AT rich spacer sequence upstream of the initiation codon as shown in SEQ ID NO: 42. Each of these constructs is separately transformed into E. coli Top10 (Invitrogen) according to manufacturer's instructions. Successful transformants are cultured and lysed, and clarified lysate is separated from insoluble material. The FRD enzymes in each case, with attached 6His affinity tag and enterokinase cleavage site, are purified using standard methods. Enterokinase (NEB) is added to protein to cleave the 6His affinity tag, and the resulting FRD enzyme is purified and collected.

(9) The activities (v.sub.0) of the purified enzymes are measured in an assay with and without 2 mM fumarate in 100 mM NaPO.sub.4 buffer (pH7.5) and either 250 M NADH or 250 M NADPH as the co-factor. The reaction is carried out in a cuvette containing a total volume of 0.8 mL assay solution. The initial velocity of the enzyme is determined by monitoring the change in A.sub.340 nm over the course of 10 minutes; assays are run using a Beckman DU-800 spectrophotometer and cuvettes with a 1 cm pathway length. V.sub.max and K.sub.M of the enzymes to NADH and NADPH is determined from the v.sub.o using a Lineweaver-Burk plot (Lineweaver, H and Burk, D. (1934), The Determination of Enzyme Dissociation Constants. Journal of the American Chemical Society 56 (3): 658-666) where the concentration of NADH or NADPH is varied from 25 to 400 M.

(10) Protein concentration ([E].sub.T) is determined against a BSA standard curve by measuring the absorbance at OD.sub.595 nm using the Advanced Protein Assay (Cytoskeleton, Inc.). k.sub.cat for both cofactors is calculated V.sub.max/[E].sub.T, and the specificity constant is given as k.sub.cat/K.sub.M. The enzyme is NADPH-dependent when k.sub.cat/K.sub.M for NADPH is greater than k.sub.cat/K.sub.M for NADH.

Example 1D

Integration of NADPH-Dependent FRD Producing Strains

(11) Two integration fragments are made from each of the mutated L. mexicana FRD genes (SEQ ID NOs: 15, 16, 17, 18 and 19). The first fragment in each case is made by digesting the gene with XbaI and PacI and subcloning it into SEQ ID NO: 9, replacing the wild-type FRD gene with the mutated gene. The second fragment in each case is made by digesting the gene with XbaI and PacI and subcloning it into SEQ ID NO: 11, again replacing the wild-type FRD gene with the mutated gene.

(12) Two integration fragments are made from the mutated T. brucei FRD gene (SEQ ID NO: 20). The first fragment is made by digesting the gene with XbaI and PacI and subcloning it into SEQ ID NO: 12, replacing the wild-type FRD gene with the mutated gene. The second fragment in each case is made by digesting the gene with XbaI and PacI and subcloning it into SEQ ID NO: 14, again replacing the wild-type FRD gene with the mutated gene.

(13) Separate samples of strain P-4 are separately transformed with the first of the integration constructs for each FRD gene, using the LiOAc transformation method described in previous examples, to integrate the respective NADPH-dependent FRD genes (as shown in Table 2) at the native CYB2b locus. After correct integration is confirmed by PCR using primers having nucleotide sequences SEQ ID NOs: 79 and 81 to confirm the 5-crossover and SEQ ID NOs: 82 and 83 to confirm the 3-crossover, an isolate from each transformation is grown and plated until the URA3 marker has looped out, and in each case the resulting isolate is transformed again with the second of the integration fragments for each FRD gene to introduce a second copy of the same NADPH-dependent FRD gene at the CYB2b locus. Loopout of the second URA3 marker is confirmed by PCR using primers having nucleotide sequences SEQ ID NOs: 82 and 81 to confirm the 5-crossover and SEQ ID NOs: 79 and 83 to confirm the 3-crossover. Retention of the first integration for each FRD gene is also reconfirmed by repeating the PCR reactions used to verify proper integration of the first of the integration constructs for each FRD gene as above. The resulting strains are designated Examples 1-1, 1-2, 1-3, 1-4, 1-5 and 1-6 respectively.

(14) TABLE-US-00002 TABLE 2 FRD Site Directed Mutants Description Example (in addition to transformations as Designation indicated for strain C-4) Parent strain 1-1 Mutated L. mexicana FRD SEQ ID P-4 NO. 15 insertion at CYB2b (2) 1-2 Mutated L. mexicana FRD SEQ ID P-4 NO. 16 insertion at CYB2b (2) 1-3 Mutated L. mexicana FRD SEQ ID P-4 NO. 17 insertion at CYB2b (2) 1-4 Mutated L. mexicana FRD SEQ ID P-4 NO. 18 insertion at CYB2b (2) 1-5 Mutated L. mexicana FRD SEQ ID P-4 NO. 19 insertion at CYB2b (2) 1-6 Mutated T. brucei FRD SEQ ID NO. 20 P-4 insertion at CYB2b (2)

EXAMPLE 2

Integration of Malate Dehydrogenase (MDH) and Fumarase (FUM) Genes

Example 2A

RdMDH Integration Fragment

(15) Integration fragment 2A (SEQ ID NO: 22) contains the Rhizopus delemar MDH gene (having nucleotide sequence SEQ ID NO: 153, (which is similar to SEQ ID NO: 23, except for a T to A substitution at by 438, both encoding for the amino acid of SEQ ID NO: 149), ADHb upstream integration arm, ENO promoter, RKI terminator, URA3 promoter and first 583 base pairs of the URA3 marker.

Example 2B

Cytosolic Kluyveromyces marxianus MDH (KmMDH3) Integration Fragment

(16) Integration fragment 2B (SEQ ID NO: 24) contains the cytosolic Kluyveromyces marxianus MDH (KmMDH3) gene (having nucleotide sequence SEQ ID NO: 25), ADHb upstream integration arm, ENO promoter, RKI terminator, URA3 promoter and first 583 base pairs of the URA3 marker.

Example 2C

Cytosolic Zygosaccharomyces rouxii MDH (ZrMDH) Integration Fragment

(17) Integration fragment 2C (SEQ ID NO: 26) contains the cytosolic Zygosaccharomyces rouxii MDH (ZrMDH) gene (having nucleotide sequence SEQ ID NO: 27), ADHb upstream integration arm, ENO promoter, RKI terminator, URA3 promoter and first 583 base pairs of the URA3 marker.

Example 2D

Sorghum bicolor NADPH-Dependent MDH (SbMDH) Integration Fragment

(18) Integration fragment 2D (SEQ ID NO: 28) contains the Sorghum bicolor NADPH-dependent MDH (SbMDH) gene (having nucleotide sequence SEQ ID NO: 29), ADHb upstream integration arm, ENO promoter, RKI terminator, URA3 promoter and first 583 base pairs of the URA3 marker.

Example 2E:

Chlamydomonas reinhardtii NADPH-Dependent MDH (CrMDH) Integration Fragment

(19) Integration fragment 2E (SEQ ID NO: 30) contains the Chlamydomonas reinhardtii NADPH-dependent MDH (CrMDH) gene (having nucleotide sequence SEQ ID NO: 31), ADHb upstream integration arm, ENO promoter, RKI terminator, URA3 promoter and first 583 base pairs of the URA3 marker.

Example 2F

Engineered NADPH-Dependent Rhizopus delemar MDH (RdPMDH) Integration Fragment

(20) A R. delemar MDH gene having the nucleotide sequence SEQ ID NO: 153 (which is similar to SEQ ID NO: 23, except for T to A substitution at by 438, both encoding for the amino acid of SEQ ID NO: 149) is used as a template to modify the coding sequence to introduce substitutions of amino acid residues of the putative NADH binding domain of the enzyme. A mutated R. delemar MDH gene having the nucleotide sequence SEQ ID NO. 32 is prepared by performing site-directed substitutions.

(21) Integration fragment 2F (SEQ ID NO: 33) contains the NADPH-dependent engineered variant of the Rhizopus delemar MDH (RdPMDH) gene, ADHb upstream integration arm, ENO promoter, URA3 promoter and first 583 base pairs of the URA3 marker.

Example 2G

I. orientalis FUM (IoFUM) Integration Fragment

(22) Integration fragment 2G (having SEQ ID NO: 34) contains the I. orientalis FUM (IoFUM) gene (having nucleotide sequence, SEQ ID NO: 35), the last 568 base pairs of the URA3 marker, URA3 promoter, PGK promoter, PDC terminator and ADHb downstream integration arm.

Example 2H

Actinobacillus succinogenes FUM (AsFUM) Integration Fragment

(23) Integration fragment 2H (having SEQ ID NO: 36) contains the Actinobacillus succinogenes FUM (AsFUM) gene (having nucleotide sequence SEQ ID NO: 37), the last 568 base pairs of the URA3 marker, URA3 promoter, PGK promoter, PDC terminator and ADHb downstream integration arm.

Example 2I

Rhizopus delemar MDH (RdMDH) Reverse Integration Fragment

(24) Second R. delemar MDH (RdMDH) integration fragment 21 (having SEQ ID NO: 38) contains the R. delemar MDH (RdMDH) gene (having nucleotide sequence SEQ ID NO: 23), ADHb downstream integration arm, ENO promoter, URA3 promoter and first 583 base pairs of the URA3 marker.

Example 2J

Engineered Rhizopus delemar MDH (RdPMDH) Reverse Integration Fragment

(25) Reverse Rhizopus delemar MDH (RdPMDH) integration fragment 2J (having SEQ ID NO: 39) contains the engineered NADPH-dependent RdMDH gene (having nucleotide sequence SEQ ID NO: 32), ADHb downstream integration arm, ENO promoter, URA3 promoter and first 583 base pairs of the URA3 marker.

Example 2K

I. orientalis FUM (IoFUM) Reverse Integration Fragment

(26) Second I. orientalis FUM (IoFUM) integration fragment 2K (having SEQ ID NO: 40) contains the IoFUM gene (having SEQ ID NO: 35), the last 568 base pairs of the URA3 marker, URA3 promoter, PGK promoter, PDC terminator and ADHb upstream integration arm.

Example 2L

A. succinogenes FUM (AsFUM) Reverse Integration Fragment

(27) A. succinogenes FUM (AsFUM) reverse integration fragment (having SEQ ID NO: 41) contains the truncated AsFUM gene (having nucleotide sequence SEQ ID NO: 37), the last 568 base pairs of the URA3 marker, URA3 promoter, PGK promoter, PDC terminator and ADHb upstream integration arm.

Example 2M

Preparation of Examples 2-1 through 2-12

(28) To produce Example 2-1, Example 1-4 is simultaneously transformed with each of integration fragments 2A and 2G using the standard lithium acetate process described before. Successful transformants are selected on selection plates lacking uracil and confirmed by PCR using primers having nucleotide sequences SEQ ID NOs: 84 and 85 to confirm the 5-crossover, SEQ ID NOs: 107 and 108 to confirm the junction of integration fragments 2A and 2G, and SEQ ID NOs: 91 and 93 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. This strain, which contains the NADPH-dependent L. Mexicana FRD gene having SEQ. ID. NO: 18, is designated as Example 2-1.

(29) Examples 2-2 and 2-12 are made in the same general manner. The integration fragments used to make those strains, and their respective genotypes, are identified in Table 3.

(30) TABLE-US-00003 TABLE 3 I. orientalis MDH/FUM Insertion Strains Integration Description (in addition to transformations as Ex. No. Fragments indicated for Example 1-4) 2-1 2A/2G R. delemar MDH insertion at ADHb (1) I. orientalis FUM insertion at ADHb (1) 2-2 2B/2G K. marxianus MDH insertion at ADHb (1) I. orientalis FUM insertion at ADHb (1) 2-3 2C/2K Z. rouxii MDH insertion at ADHb (1) I. orientalis FUM insertion at ADHb (1) 2-4 2D/2K S. bicolor MDH insertion at ADHb (1) I. orientalis FUM insertion at ADHb (1) 2-5 2E/2K C. reinhardtii MDH insertion at ADHb (1) I. orientalis FUM insertion at ADHb (1) 2-6 2F/2K Engineered R. delemar MDH insertion at ADHb (1) I. orientalis FUM insertion at ADHb (1) 2-7 2A/2H R. delemar MDH insertion at ADHb (1) A. succinogenes FUM insertion at ADHb (1) 2-8 2B/2H K. marxianus MDH3 insertion at ADHb (1) A. succinogenes FUM insertion at ADHb (1) 2-9 2C/2H Z. rouxii MDH insertion at ADHb (1) A. succinogenes FUM insertion at ADHb (1) 2-10 2D/2H S. bicolor MDH insertion at ADHb (1) A. succinogenes FUM insertion at ADHb (1) 2-11 2E/2H C. reinhardtii MDH insertion at ADHb (1) A. succinogenes FUM insertion at ADHb (1) 2-12 2F/2H Engineered R. delemar MDH insertion at ADHb (1) A. succinogenes FUM insertion at ADHb (1)

Example 2N

Preparation of Examples 2-13 through 2-15

(31) To produce Example 2-13, Example 2-2 is simultaneously transformed with each of fragments 21 and 2K using the lithium acetate process described before. Successful transformants are identified by PCR using primers having nucleotide sequences SEQ ID NOs: 84 and 85 to confirm the 5-crossover, SEQ ID NOs: 107 and 108 to confirm the junction between the integration fragments and SEQ ID NOs: 91 and 93 to confirm the 3-crossover. Retention of the first integration at ADHb is also reconfirmed by repeating the PCR reactions used to verify the 5- and 3-crossovers as in Example 2M above. The successful tranformants are grown and plated until a strain in which the URA3 marker has looped out is identified as before as confirmed by PCR using primers having nucleotide sequences SEQ ID NOs: 84 and 91 to confirm the 5-crossover and SEQ ID NOs: 85 and 93 to confirm the 3-crossover. This strain is designated as Example 2-13.

(32) Examples 2-14 and 2-15 are made in the same general manner by integrating Examples 2-6 and 2-7, respectively. The integration fragments used to make those strains, and their respective genotypes, are identified in Table 4.

(33) TABLE-US-00004 TABLE 4 Ex. Parent Integration Description (in addition to No. Strain Fragments transformations as indicated for strain 1-4) 2-13 2-2 2I/2K R. delemar MDH insertion at ADHb (2) I. orientalis FUM insertion at ADHb (2) 2-14 2-6 2J/2K Engineered R. delemar MDH insertion at ADHb (2) I. orientalis FUM insertion at ADHb (2) 2-15 2-7 2K/2L R. delemar MDH insertion at ADHb (2) A. succinogenes FUM insertion at ADHb (2)

Example 2O

Preparation of Examples 2-16 through 2-30

(34) Examples 2-16 through 2-30 are produced in the same manner as Examples 2-1 through 2-15, respectively, except that the host strain is Example 1-1 rather than Example 1-4. The genotypes of Examples 2-16 through 2-30 are the same as described in Tables 3 and 4 above for Examples 2-1 through 2-15, respectively, except the FRD gene is the mutated L. mexicana FRD gene having SEQ ID NO: 15.

Example 2P

Preparation of Examples 2-31 through 2-45

(35) Examples 2-31 through 2-45 are produced in the same manner as Examples 2-1 through 2-15, respectively, except that the host strain is Example 1-2 rather than Example 1-4. The genotypes of Examples 2-31 through 2-45 are the same as described in Tables 3 and 4 above for Examples 2-31 through 2-45, respectively, except the FRD gene is the mutated L. mexicana FRD gene having SEQ ID NO: 16.

Example 2Q

Preparation of Examples 2-46 through 2-60

(36) Examples 2-46 through 2-60 are produced in the same manner as Examples 2-1 through 2-15, respectively, except that the host strain is Example 1-3 rather than Example 1-4. The genotypes of Examples 2-46 through 2-60 are the same as described in Tables 3 and 4 above for Examples 2-46 through 2-60, respectively, except the FRD gene is the mutated L. mexicana FRD gene having SEQ ID NO: 17.

Example 2R

Preparation of Examples 2-61 through 2-75

(37) Examples 2-61 through 2-75 are produced in the same manner as Examples 2-1 through 2-15, respectively, except that the host strain is Example 1-5 rather than Example 1-4. The genotypes of Examples 2-61 through 2-75 are the same as described in Tables 3 and 4 above for stains 2-61 through 2-75, respectively, except the FRD gene is the mutated L. mexicana FRD gene having SEQ ID NO: 19.

Example 2S

Preparation of Examples 2-76 through 2-90

(38) Examples 2-76 through 2-90 are produced in the same manner as Examples 2-1 through 2-15, respectively, except that the host strain is Example 1-6 rather than Example 1-4. The genotypes of Examples 2-76 through 2-90 are the same as described in Tables 3 and 4 above for Examples 2-76 through 2-90, respectively, except the FRD gene is the mutated T. brucei gene having SEQ ID NO: 20.

Example 2T

(39) The MDH genes having SEQ ID NOs 29, 31 and 32 are each modified at 5 end by the addition of a short DNA sequence having SEQ ID NO: 21, immediately downstream of the initiation codon. This short nucleotide sequence encodes a peptide consisting of six histidine residues, followed by a single methionine, four aspartate residues and a single lysine residue, and effectively adds a 6His affinity tag and an enterokinase cleavage site to the MDH sequence. The expression of the MDH gene is driven by the IPTG inducible T5 promoter. The construct in each case also contains an optimized Shine-Dalgarno sequence followed by 8 by of AT rich spacer sequence upstream of the initiation codon as shown in SEQ ID NO: 42. Each of these constructs is separately transformed into E. coli Top10 (Invitrogen) according to manufacturer's instructions. Successful transformants are cultured and lysed, and clarified lysate is separated from insoluble material. The MDH enzymes in each case, with attached 6His affinity tag and enterokinase cleavage, are purified using standard methods. Enterokinase (NEB) is added to protein to cleave the 6His affinity tag, and the resulting MDH enzyme is purified and collected.

(40) The activities (v.sub.0) of the purified enzymes are measured in an assay with and without 100 micromoles M OAA in Tris-HCl (pH 8.0) and either 400 M NADH or 400 M NADPH as the co-factor. The reaction is carried out in a cuvette containing a total volume of 0.8 mL assay solution. The initial velocity of the enzyme is determined by monitoring the change in A.sub.340 nm over the course of 10 minutes; assays are run using a Beckman DU-800 spectrophotometer and cuvettes with a 1 cm pathway length. V.sub.max and K.sub.M of the enzymes to NADH and NADPH is determined from the v.sub.o using a Lineweaver-Burk plot (Lineweaver, H and Burk, D. (1934), The Determination of Enzyme Dissociation Constants. Journal of the American Chemical Society 56 (3): 658-666) where the concentration of NADH or NADPH is varied from 25 to 400 M.

(41) Protein concentration ([E].sub.T) is determined against a BSA standard curve by measuring the absorbance at OD.sub.595 nm using the Advanced Protein Assay (Cytoskeleton, Inc.). k.sub.cat for both cofactors is calculated V.sub.max/[E].sub.T, and the specificity constant is given as k.sub.cat/.sub.Mm. The enzyme is NADPH-dependent when k.sub.cat/K.sub.M for NADPH is greater than k.sub.cat/K.sub.M for NADH.

EXAMPLE 3

Integration of Soluble Transhydrogenase

(42) Integration Fragment 3A: Right Hand Integration FragmentMarker Only

(43) Integration fragment 3A having nucleotide sequence SEQ ID NO: 43, contains the following elements, 5 to 3: a DNA fragment corresponding to the last 567 by of the I. orientalis URA3 open reading frame, the I. orientalis URA3 terminator, the I. orientalis URA3 promoter, the I. orientalis TDH3 promoter, the I. orientalis TKL terminator, the I. orientalis PGK promoter, the I. orientalis PDC terminator and a DNA fragment with homology for integration corresponding to the region immediately downstream of the I. orientalis MAE1 open reading frame.

(44) Integration Fragment 3B: Left Hand Integration Fragment with the E. coli SthA Gene

(45) Integration fragment 3B, having nucleotide sequence SEQ ID NO: 44, contains the following elements, 5 to 3: a DNA fragment with homology for integration corresponding to the region immediately upstream of the I. orientalis MAE1 open reading frame, an I. orientalis PDC1 promoter, the I. orientalis TAL terminator, the I. orientalis ENO promoter, the E. coli SthA gene (having the nucleotide sequence SEQ ID NO: 45), I. orientalis RKI terminator, URA3 promoter, and the first 582 by of the I. orientalis URA3 open reading frame.

(46) Integration Fragment 3C: Left Hand Integration Fragment with a Codon Optimized E. coli SthA Gene

(47) Integration fragment 3C, having nucleotide sequence SEQ ID NO: 46, contains the following elements, 5 to 3: a DNA fragment with homology for integration corresponding to the region immediately upstream of the I. orientalis MAE1 open reading frame, an I. orientalis PDC1 promoter, the I. orientalis TAL terminator, the I. orientalis ENO promoter, the codon-optimized E. coli SthA gene (having the nucleotide sequence SEQ ID NO: 47), I. orientalis RKI terminator, URA3 promoter, and the first 582 by of the I. orientalis URA3 open reading frame.

(48) Integration Fragment 3D: Left Hand Integration Fragment with the A. vinelandii SthA Gene

(49) Integration fragment 3D, having nucleotide sequence SEQ ID NO: 48, contains the following elements, 5 to 3: a DNA fragment with homology for integration corresponding to the region immediately upstream of the I. orientalis MAE1 open reading frame, an I. orientalis PDC1 promoter, the I. orientalis TAL terminator, the I. orientalis ENO promoter, the A.vinelandii SthA gene (having the nucleotide sequence, SEQ ID NO: 49), I. orientalis RKI terminator, URA3 promoter, and the first 582 by of the I. orientalis URA3 open reading frame.

(50) To produce Example 3-1, Example 1-4 is simultaneously transformed with each of integration fragments 3B and 3A using the lithium acetate process described before. Successful transformants are selected on selection plates lacking uracil and confirmed by PCR using primers having nucleotide sequences SEQ ID NOs: 96 and 98 to confirm the 5-crossover and SEQ ID NOs: 100 and 101 to confirm the 3-crossover. Successful transformants are grown and plated on FOA as before until a strain in which the URA3 marker has looped out is identified. This strain is designated as Example 3-1. Examples 3-2 and 3-3 are made in the same general manner. The integration fragments used to make those strains, and their respective genotypes, are identified in Table 4.

(51) To produce Example 3-4, Example 2-13 is simultaneously transformed with each of integration fragments 3B and 3A using the standard lithium acetate process described before. Successful transformants are selected on selection plates lacking uracil, confirmed by PCR using primers having nucleotide sequences SEQ ID NOs: 96 and 98 to confirm the 5-crossover and SEQ ID NOs: 100 and 101 to confirm the 3-crossover. Successful tranformants are grown and plated on FOA as before until a strain in which the URA3 marker has looped out is identified. This strain is designated as Example 3-4. Examples 3-5 and 3-6 are made in the same general manner. The integration fragments used to make those strains, and their respective genotypes, are identified in Table 5.

(52) TABLE-US-00005 TABLE 5 I. orientalis Insertion Strains Ex. Integration Description (in addition to transformations Parent No. Fragments as indicated for parent strain) strain 3-1 3A, 3B E. coli SthA insertion at MAE1 (1) Ex. 1-4 3-2 3A, 3C Codon optimized E. coli SthA Ex. 1-4 insertion at MAE1 (1) 3-3 3A, 3D A. vinelandii SthA insertion at MAE1 (1) Ex. 1-4 3-4 3A, 3B E. coli SthA insertion at MAE1 (1) Ex. 2-13 3-5 3A, 3C Codon optimized E. coli SthA Ex. 2-13 insertion at MAE1 (1) 3-6 3A, 3D A. vinelandii SthA insertion at MAE1 (1) Ex. 2-13

(53) Examples 3-7 through 3-100 are produced by separately transforming Examples 1-1, 1-2, 1-3, 1-5, 1-6 and 2-1 through 2-12 and 2-14 through 2-90 with integration fragments 3A and 3B in the same manner described with regard to Example 3-1. All of Examples 3-9 through 3-100 have the E. coli SthA gene inserted at the locus of the MAE1 gene, which is deleted, as well as the modifications indicated in Tables 1-4, as applicable.

(54) Examples 3-101 through 3-194 are produced by separately transforming Examples 1-1, 1-2, 1-3, 1-5, 1-6 and 2-1 through 2-12 and 2-14 through 2-90 with integration fragments 3A and 3C in the same manner described with regard to Example 3-1. All of Examples 3-101 through 3-194 have the codon optimized E. coli SthA gene inserted at the locus of the MAE1 gene, which is deleted, as well as the modifications indicated in Tables 1-4, as applicable.

(55) Examples 3-195 through 3-288 are produced by separately transforming Examples 1-1, 1-2, 1-3, 1-5, 1-6 and 2-1 through 2-11 and 2-13 through 2-90 with integration fragments 3A and 3D in the same manner described with regard to Example 3-1. All of Examples 3-195 through 3-288 have the A. vinelandii SthA gene inserted at the locus of the MAE1 gene, which is deleted, as well as the modifications indicated in Tables 1-4, as applicable.

EXAMPLE 4

Integration of Stb5p Gene

(56) Integration Fragment 4A: Left Hand Integration FragmentMarker Only

(57) Integration fragment 4A, having the nucleotide sequence SEQ ID NO: 109, contains the following elements, 5 to 3: a DNA fragment with homology for integration corresponding to the region immediately upstream of the I. orientalis MAE1 open reading frame, an I. orientalis PDC1 promoter, the I. orientalis TAL terminator, the I. orientalis ENO promoter, I. orientalis RKI terminator, URA3 promoter, and the first 582 by of the I. orientalis URA3 open reading frame.

(58) Integration Fragment 4B: Right Hand Integration Fragment with the S. cerevisiae Stb5p Gene

(59) Integration fragment 4B, having nucleotide sequence SEQ ID NO: 50, contains the following elements, 5 to 3: a DNA fragment corresponding to the last 567 by 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. cerevisiae Stb5p gene (having nucleotide sequence SEQ ID NO: 51), the I. orientalis TKL terminator, the I. orientalis PGK promoter, the I. orientalis PDC terminator and a DNA fragment with homology for integration corresponding to the region immediately downstream of the I. orientalis MAE1 open reading frame.

(60) Example 4-1 is produced by simultaneously transforming Example 1-1 with integration fragments 4A and 4B using the lithium acetate methods described before. Successful tranformants having the S. cerevisiae Stb5p gene integrated at the MAE1 locus are selected.

(61) Examples 4-2 through 4-6 are produced in the same manner by transforming Examples 1-2 through 1-6, respectively, with integration fragments 4A and 4B. Examples 4-7 through 4-96 are produced in the same manner by transforming Examples 2-1 through 2-90, respectively, with integration fragments 4A and 4B. All of Examples 4-2 through 4-96 have the S. cerevisiae Stb5p gene integrated at the MAE1 locus, as well as the modifications indicated in Tables 1-4, as applicable.

(62) Examples 4-1 through 4-96 each are transformed with integration fragments 3B and 4B in the manner described in Example 3 to produce Examples 4-97 through 4-192, respectively. Examples 4-97 through 4-192 contain the E. coli SthA gene and the S. cerevisiae Stb5p gene integrated at the MAE1 locus, as well as the modifications indicated in Tables 1-4, as applicable.

(63) Examples 4-1 through 4-96 each are transformed with integration fragments 3C and 4B in the manner described in Example 3 to produce Examples 4-193 through 4-288, respectively. Examples 4-193 through 4-288 contain the codon optimized E. coli SthA gene and the S. cerevisiae Stb5p gene integrated at the MAE1 locus, as well as the modifications indicated in Tables 1-4, as applicable.

(64) Examples 4-1 through 4-96 each are transformed with integration fragments 3D and 4B in the manner described in Example 3 to produce Examples 4-289 through 4-384, respectively. Examples 4-289 through 4-384 contain the A.vinelandii SthA gene and the S. cerevisiae Stb5p gene integrated at the MAE1 locus, as well as the modifications indicated in Tables 1-4, as applicable.

EXAMPLE 5

Deletion of Native GPD Gene

(65) Example 1-1 is transformed with integration fragment 5 (having nucleotide sequence SEQ ID NO: 52) using lithium acetate methods as described before. This integration fragment contains the following elements, 5 to 3: a DNA fragment with homology for integration corresponding to the region immediately upstream of the I. orientalis GPD1 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 GPD1 open reading frame. Successful transformants are selected on selection plates lacking uracil, confirmed by PCR using primers having nucleotide sequences SEQ ID NOs: 150 and 151 to confirm the 5-crossover and SEQ ID NOs: 152 and 101 to confirm the 3-crossover, and grown and plated on FOA as before until a strain in which the URA3 marker has looped out is identified. This strain is then transformed with an integration fragment having nucleotide sequence SEQ ID NO: 53. This integration fragment contains the following elements, 5 to 3: a DNA fragment with homology for integration corresponding to the region immediately upstream of the I. orientalis GPD1 open reading frame, the URA3 promoter, the I. orientalis URA3 gene, an additional URA3 promoter direct repeat for marker recycling a PDC transcriptional terminator, and a DNA fragment with homology for integration corresponding to the region directly downstream of the I. orientalis GPD1 open reading frame. Successful transformants are again selected on selection plates lacking uracil, and integration of the second GPD1 deletion construct confirmed by PCR using primers having nucleotide sequences SEQ ID NOs: 151 and 101 to confirm the 5-crossover and SEQ ID NOs: 150 and 152 to confirm the 3-crossover. Retention of the first GPD1 deletion construct is also reconfirmed by repeating the PCR reactions used to verify proper integration of integration fragment 5 above. Confirmed isolates are grown and plated until a strain in which the URA3 marker has looped out is identified as before. One such transformant which has a deletion of both native GPD genes, is designated Example 5-1.

(66) Examples 5-2 through 5-6 are made in the same manner by transforming Examples 1-1 through 1-6, respectively.

(67) Examples 5-7 through 5-96 are made in the same manner by transforming Examples 2-1 through 2-12, respectively.

(68) Examples 5-97 through 5-384 are made in the same manner by transforming Examples 3-1 through 3-288, respectively.

(69) Examples 5-385 through 5-768 are made in the same manner by transforming Examples 4-1 through 4-384, respectively.

(70) All of Examples 5-1 through 5-768 have a deletion of a native GPD gene in addition to the modification indicated before in relation to the respective parent strains.

EXAMPLE 6

Deletion of Phosphoglucoisomerase (PGI) Gene

(71) Integration fragment 6-1 (having SEQ ID NO: 54) for the deletion of the first copy of the I. orientalis PGI gene, contains the following elements, 5 to 3: a DNA fragment with homology for integration corresponding to the region immediately upstream of the I. orientalis PGI open reading frame, a PDC1 transcriptional 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 PGI open reading frame.

(72) Integration fragment 6-2 (having SEQ ID NO: 55) for the deletion of the second copy of the I. orientalis PGI gene contains the following elements, 5 to 3: a DNA fragment with homology for integration corresponding to the region immediately downstream of the I. orientalis PGI open reading frame, a PDC1 transcriptional 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 PGI open reading frame.

(73) Example 1-1 is transformed with integration fragment 6-1 using the lithium acetate process described before. Successful transformants are selected on PGI deletion selection plates lacking uracil (SC ura, +20 g/L fructose, +0.5 g/L glucose) incubated 3-5 days and confirmed by PCR using primers having nucleotide sequences SEQ ID NOs: 104 and 105 to confirm the 5-crossover and SEQ ID NOs: 79 and 106 to confirm the 3-crossover. Successful 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 then transformed with integration fragment 6-2 in the same manner, and a successful deletant is identified using primers having nucleotide sequences SEQ ID NOs: 79 and 104 to confirm the 5-crossover and SEQ ID NOs: 104 and 106 to confirm the 3-crossover. A strain in which the URA3 marker has looped out as before is designated Example 6-1-1.

(74) Examples 6-1-2 through 6-1-6 are prepared in the same manner as strain 6-1-1, by transforming each of strains 1-2 through 1-6 sequentially with integration fragments 6-1 and 6-2.

(75) Strains 6-2-1 through 6-2-90 are prepared in the same manner as strain 6-1-1, by transforming each of strains 2-1 through 2-90 sequentially with integration fragments 6-1 and 6-2.

(76) Strains 6-3-1 through 6-3-288 are prepared in the same manner as strain 6-1-1, by transforming each of strains 3-1 through 3-288 sequentially with integration fragments 6-1 and 6-2.

(77) Strains 6-4-1 through 6-4-384 are prepared in the same manner as strain 6-1-1, by transforming each of strains 4-1 through 4-384 sequentially with integration fragments 6-1 and 6-2.

(78) Strains 6-5-1 through 6-5-768 are prepared in the same manner as strain 6-1-1, by transforming each of strains 5-1 through 5-768 sequentially with integration fragments 6-1 and 6-2.

EXAMPLE 7

7A: Construction of Preparatory Strains

(79) Preparatory Strains P-2sc through P-4sc are engineered in a manner analogous to Strains P-2 through P-4 above, starting from a wild-type Saccharomyces cerevisiae strain (CEN.PK 113-7D, on deposit in the CBS culture collection as CBS 8340). The wild-type strain is transformed with an integration fragment designed to disrupt the URA3 gene. A successful deletent strain is then transformed with integration fragment P3 as described in P-3 above, modified to target the S. cerevisiae YLR044C gene by replacing first 855bp of the fragment with 855bp of DNA from immediately downstream of the target YLR044C gene and replacing the final 803bp of the fragment with 803bp from immediately upstream of the target YLR044C gene. The resultant PDC deletant strain is designated strain P-3sc.

(80) One copy of each of the PYC and MAE genes are inserted into Strain P-3sc in a manner analogous to that described in P-4 above. The integration is performed using the integration fragments P4-1 and P4-2 described in P-4 above, modified in each case to target the S. cerevisiae YOL086C locus. Integration fragment P4-1 is modified by replacing the first 855 bp with 855 bp from immediately upstream of the ATG start codon for the YOL086C locus and integration fragment P4-2 is modified by replacing the last 1003p with the 1003 bp from immediately downstream of the TAA stop codon for the YOL086C locus. This strain, after loopout of the URA3 marker, is designated strain P-4sc.

(81) TABLE-US-00006 TABLE 6 S. cerevisiae URA and PDC Deletion Strains Strain name Description Parent strain P-1sc Wild-type strain Wild-type P-2sc URA3 deletion (1) P-1sc P-3sc URA3 deletion (1) P-2sc PDC deletion (1) P-4sc URA deletion (1) P-3sc PDC deletion (1) I. orientalis PYC1 insertion at ADH1 (1) S. pombe MAE insertion at ADH1 (1)
6B. Insertion of NADPH-Dependent FRD Gene

(82) Six separate transformations of strain P-4sc are performed to insert one copy of the mutated NADPH-dependent FRD genes, analogously to Example 1D. The integration constructs in each case are the same as described in Examples 1-1 through 1-6, respectively, except in each case the 5 and 3 flanks of the fragments are replaced with a 300-1200 bp upstream (5) sequence or downstream (3) sequence of the target CYB2b gene. Successful transformants, after loopout of the URA3 marker, are designated Examples 7-1, 7-2, 7-3, 7-4, 7-5 and 7-6 respectively.

(83) TABLE-US-00007 TABLE 7 Description Ex. (in addition to transformations as indicated for Parent No. strain P-1sc) strain 7-1 L. mexicana FRD SEQ ID NO: 15 insertion at CYB2 (1) P-4sc 7-2 L. mexicana FRD SEQ ID NO: 16 insertion at CYB2 (1) P-4sc 7-3 L. mexicana FRD SEQ ID NO: 17 insertion at CYB2 (1 P-4sc 7-4 L. mexicana FRD SEQ ID NO: 18 insertion at CYB2 (1) P-4sc 7-5 L. mexicana SEQ ID NO: 19 insertion at CYB2 (1) P-4sc 7-6 T. brucei FRD SEQ ID NO: 20 insertion at CYB2 (1) P-4sc

EXAMPLE 8

(84) Integration fragments analogous to those described in Examples 2A through 2E above are prepared by replacing the first 769 by of each of integration fragments 2A through 2E with the 769bp from immediately upstream of the ATG start codon for the S. cerevisiae YMR303C gene.

(85) Integration fragments analogous to those described in Examples 2G, 2H, 21 and 2K above are prepared by replacing the last 615 by of each of integration fragments 2G, 2H, 21 and 2K above with the 615 by from immediately downstream of the stop codon for the S. cerevisiae YMR303C gene.

(86) Examples 8-1 through 8-12 are prepared in the same manner as Examples 2-1 through 2-12, respectively, using the corresponding modified integration fragments and Example 7-1 as the parent strain. Successful transformants are selected as before and grown and plated on FOA as before until the URA3 marker has looped out. The genotypes of those respective Examples are identified in Table 8.

(87) TABLE-US-00008 TABLE 8 S. cerevisiae MDH/FUM Insertion Strains Description Ex. (in addition to transformations as indicated for Parent No. the indicated parent strain) Strain 8-1 R. delemar MDH insertion at YMR303C (1) Ex. 7-1 I. orientalis FUM insertion at YMR303C (1) 8-2 K. marxianus MDH3 insertion at YMR303C (1) Ex. 7-1 I. orientalis FUM insertion at YMR303C (1) 8-3 Z. rouxii MDH insertion at YMR303C (1) Ex. 7-1 I. orientalis FUM insertion at YMR303C (1) 8-4 S. bicolor MDH insertion at YMR303C (1) Ex. 7-1 I. orientalis FUM insertion at YMR303C (1) 8-5 C. reinhardtii MDH insertion at YMR303C (1) Ex. 7-1 I. orientalis FUM insertion at YMR303C (1) 8-6 Engineered R. delemar MDH insertion at YMR303C (1) Ex. 7-1 I. orientalis FUM insertion at YMR303C (1) 8-7 R. delemar MDH insertion at YMR303C (1) Ex. 7-1 A. succinogenes FUM insertion at YMR303C (1) 8-8 K. marxianus MDH3 insertion at YMR303C (1) Ex. 7-1 A. succinogenes FUM insertion at YMR303C (1) 8-9 Z. rouxii MDH insertion at YMR303C (1) Ex. 7-1 A. succinogenes FUM insertion at YMR303C (1) 8-10 S. bicolor MDH insertion at YMR303C (1) Ex. 7-1 A. succinogenes FUM insertion at YMR303C (1) 8-11 C. reinhardtii MDH insertion at YMR303C (1) Ex. 7-1 A. succinogenes FUM insertion at YMR303C (1) 8-12 Engineered R. delemar MDH insertion at YMR303C (1) Ex. 7-1 A. succinogenes FUM insertion at YMR303C (1)

(88) Examples 8-13 through 8-24 are produced in the same manner as Examples 8-1 through 8-12, respectively, except that the host strain is Example 7-2 rather than Example 7-1. The genotypes of Examples 8-13 through 8-24 are the same as described in Table 8 above for Examples 8-1 through 8-12, respectively, except the FRD gene is the mutated L. mexicana FRD gene having nucleotide sequence no. 16.

(89) Examples 8-25 through 8-36 are produced in the same manner as Examples 8-1 through 8-12, respectively, except that the host strain is Example 7-3 rather than Example 7-1. The genotypes of Examples 8-25 through 8-36 are the same as described in Table 8 above for Examples 8-1 through 8-12, respectively, except the FRD gene is the mutated L. mexicana FRD gene having nucleotide sequence no. 17.

(90) Examples 8-37 through 8-48 are produced in the same manner as Examples 8-1 through 8-12, respectively, except that the host strain is Example 7-4 rather than Example 7-1. The genotypes of Examples 8-37 through 8-48 are the same as described in Table 8 above for Examples 8-1 through 8-12, respectively, except the FRD gene is the mutated L. mexicana FRD gene having nucleotide sequence no. 18.

(91) Examples 8-49 through 8-60 are produced in the same manner as Examples 8-1 through 8-12, respectively, except that the host strain is Example 7-5 rather than Example 7-1. The genotypes of Examples 8-49 through 8-60 are the same as described in Table 8 above for Examples 8-1 through 8-12, respectively, except the FRD gene is the mutated L. mexicana FRD gene having nucleotide sequence no. 19.

(92) Examples 8-61 through 8-72 are produced in the same manner as Examples 8-1 through 8-12, respectively, except that the host strain is Example 7-6 rather than Example 7-1. The genotypes of Examples 8-61 through 8-72 are the same as described in Table 8 above for Examples 8-1 through 8-12, respectively, except the FRD gene is the mutated T. brucei FRD gene having nucleotide sequence no. 20.

EXAMPLE 9

(93) Integration fragments 3A through 3D are modified as follows:

(94) Integration fragment 3A is modified for insertion at the S. cerevisiae YKL029C locus by replacing the last 377 by with the 377 by from immediately upstream of the ATG start codon of the native S. cerevisiae YKL029C gene.

(95) Integration fragments 3B, 3C and 3D each are separately modified for insertion at the S. cerevisiae YKL029C locus by replacing the first 361 by with the 361 by from immediately upstream of the ATG start codon of the native S. cerevisiae YKL029C gene.

(96) To produce Example 9-1, Example 7-1 is simultaneously transformed with each of the modified integration fragments 3B and 3A using the standard lithium acetate process described before. Successful transformants are selected on selection plates lacking uracil, confirmed by PCR, and grown and plated until a strain in which the URA3 marker has looped out is identified as before. This strain is designated as Example 9-1. Examples 9-2 and 9-3 are made in the same general manner. The integration fragments used to make those strains, and their respective genotypes, are identified in Table 9.

(97) TABLE-US-00009 TABLE 9 S. cerevisiae Insertion Strains Integration Description Example Fragments (in addition to transformations Parent No. (modified) as indicated for parent strain) strain 9-1 3A, 3B E. coli SthA insertion at MAE1 (1) Ex. 1-4 9-2 3A, 3C Codon optimized E. coli SthA insertion Ex. 1-4 at MAE1 (1) 9-3 3A, 3D A. vinelandii SthA insertion at MAE1 Ex. 1-4 (1)

(98) Examples 9-4 through 9-80 are produced by separately transforming Examples 7-2 through 7-6 and 8-1 through 8-72, respectively, with modified integration fragments 3A and 3B in the same manner described with regard to Example 9-1. Examples 9-4 through 9-80 all contain the E. coli SthA gene at the native YKL029C locus, with deletion of the native gene.

(99) Examples 9-81 through 9-157 are produced by separately transforming Examples 7-2 through 7-6 and 8-1 through 8-72 with modified integration fragments 3A and 3C in the same manner described with regard to Example 9-2. Examples 9-81 through 9-157 all contain the codon-optimized E. coli SthA gene at the native YKL029C locus, with deletion of the native gene.

(100) Examples 9-158 through 9-234 are produced by separately transforming Examples 7-2 through 7-6 and 8-1 through 8-72 with modified integration fragments 3A and 3D in the same manner described with regard to Example 9-3. Examples 9-158 through 9-234 all contain the A. vinelandii SthA gene at the native YKL029C locus, with deletion of the native gene.

EXAMPLE 10

(101) Integration fragment 5 is modified for insertion at the native S. cerevisiae YDL022W gene by replacing the first 853 by with 853 by of DNA from immediately upstream of the YDL022W gene and the final 1004 by with 1004 by of DNA from immediately downstream of the YDL022W gene. Example 7-1 is transformed with the modified integration fragment using methods as described before. A successful transformant in which the native GPD gene is deleted and the URA3 marker has looped out is designated Example 10-7-1.

(102) Examples 10-7-2 through 10-7-6 are made in the same manner by transforming Examples 7-2 through 7-6, respectively. Examples 10-7-2 through 10-7-6 have a deletion of a native GPD gene in addition to the genetic modifications indicated earlier with respect to the respective parent strains.

(103) Examples 10-8-1 through 10-8-72 are made in the same manner by transforming strains 8-1 through 8-72, respectively. Examples 10-8-1 through 10-8-72 have a deletion of a native GPD gene in addition to the genetic modifications indicated earlier with respect to the respective parent strains.

(104) Examples 10-9-1 through 10-9-234 are made in the same manner by transforming strains 9-1 through 9-234, respectively. Examples 10-9-1 through 10-9-234 have a deletion of a native GPD gene in addition to the genetic modifications indicated earlier with respect to the respective parent strains.

EXAMPLE 11

Construction of Preparatory Strains P1wtIo through P6wtIo

(105) Preparatory Strains P-2wtIo through P-6wtIo are made in the same manner as Preparatory Strains P-2 through P6, except the starting strain is I. orientalis strain ATCC PTA-6658.

(106) Examples 11-1 through 11-6 are made in the same way as Examples 1-1 through 1-6, respectively, by transforming Preparatory Strain P-4wtIo.

EXAMPLE 12

(107) Examples 12-1 through 12-15 are made in the same way as Examples 2-1 through 2-15, respectively, by transforming strain 11-4 instead of strain 1-4.

(108) Examples 12-16 through 12-30 are made in the same way as Examples 2-16 through 2-30, respectively, by transforming strain 11-1 instead of strain 1-1.

(109) Examples 12-31 through 12-45 are made in the same way as Examples 2-31 through 2-45, respectively, by transforming strain 11-2 instead of strain 1-2.

(110) Examples 12-46 through 12-60 are made in the same way as Examples 2-46 through 2-60, respectively, by transforming strain 11-3 instead of strain 1-3.

(111) Examples 12-61 through 12-75 are made in the same way as Examples 2-61 through 2-75, respectively, by transforming strain 11-5 instead of strain 1-5.

(112) Examples 12-76 through 12-90 are made in the same way as Examples 2-76 through 2-90, respectively, by transforming strain 11-6 instead of strain 1-6.

EXAMPLE 13

(113) Examples 13-1 through 12-3 are made in the same way as Examples 3-1 through 3-3, respectively, by transforming strain 11-4 instead of Strain 1-4.

(114) Examples 13-4 through 13-6 are made in the same way as Examples 3-4 through 3-6, respectively, by transforming strain 12-13 instead of Strain 2-13.

(115) Examples 13-7 through 13-100 are produced by separately transforming Examples 11-1, 11-2, 11-3, 11-5, 11-6, 12-1 through 12-12 and 12-14 through 12-90 with integration fragments 3A and 3B in the same manner described with regard to Example 3-1. All of Examples 13-7 through 13-100 have the E. coli SthA gene inserted at the locus of the MAE1 gene, which is deleted, as well as modifications analogous to those indicated in Tables 1-4, as applicable.

(116) Examples 13-101 through 13-194 are produced by separately transforming Examples 11-1, 11-2, 11-3, 11-5, 11-6, 12-1 through 12-12 and 12-14 through 12-90 with integration fragments 3A and 3C in the same manner described with regard to Example 3-1. All of Examples 13-101 through 13-194 have the codon optimized E. coli SthA gene inserted at the locus of the MAE1 gene, which is deleted, as well as modifications analogous to those indicated in Tables 1-4, as applicable.

(117) Examples 13-195 through 13-288 are produced by separately transforming Examples 11-1, 11-2, 11-3, 11-5, 11-6, 12-1 through 12-12 and 12-14 through 12-90 with integration fragments 3A and 3D in the same manner described with regard to Example 3-1. All of Examples 13-195 through 13-288 have the A. vinelandii SthA gene inserted at the locus of the MAE1 gene, which is deleted, as well as modifications analogous to those indicated in Tables 1-4, as applicable.

EXAMPLE 14

Integration of Stb5p Gene

(118) Example 14-1 is produced by simultaneously transforming Example 11-1 with integration fragments 4A and 4B using the lithium acetate methods described before. Successful tranformants having the S. cerevisiae Stb5p gene integrated at the MAE1 locus are selected.

(119) Examples 14-2 through 14-6 are produced in the same manner by transforming Examples 11-2 through 11-6, respectively, with integration fragments 4A and 4B. Examples 14-7 through 14-96 are produced in the same manner by transforming Examples 12-1 through 12-90, respectively, with integration fragments 4A and 4B. All of Examples 14-2 through 14-96 have the S. cerevisiae Stb5p gene integrated at the MAE1 locus, as well as the modifications indicated in Tables 1-4, as applicable.

(120) Examples 14-1 through 14-96 each are transformed with integration fragments 3B and 4B in the manner described in Example 3 to produce Examples 14-97 through 14-192, respectively. Examples 14-97 through 14-192 contain the E. coli SthA gene and the S. cerevisiae Stb5p gene integrated at the MAE1 locus, as well as the modifications indicated in Tables 1-4, as applicable.

(121) Examples 14-1 through 14-96 each are transformed with integration fragments 3C and 4B in the manner described in Example 3 to produce Examples 14-193 through 14-288, respectively. Examples 14-193 through 14-288 contain the codon optimized E. coli SthA gene and the S. cerevisiae Stb5p gene integrated at the MAE1 locus, as well as the modifications indicated in Tables 1-4, as applicable.

(122) Examples 14-1 through 14-96 each are transformed with integration fragments 3D and 4B in the manner described in Example 3 to produce Examples 14-289 through 14-384, respectively. Examples 14-289 through 14-384 contain the A. vinelandii SthA gene and the S. cerevisiae Stb5p gene integrated at the MAE1 locus, as well as the modifications indicated in Tables 1-4, as applicable.

EXAMPLE 15

(123) Example 11-1 is transformed with integration fragment 5 in the manner described in Example 5 to delete the native GPD gene. A successful transformant having the looped out marker is designated Example 15-1. Examples 15-2 through 15-6 are made in the same manner by transforming Examples 11-2 through 11-6, respectively.

(124) Examples 15-7 through 15-96 are made in the same manner by transforming Examples 12-1 through 12-90, respectively.

(125) Examples 15-97 through 15-384 are made in the same manner by transforming Examples 13-1 through 13-288, respectively.

(126) Examples 15-385 through 15-768 are made in the same manner by transforming Examples 14-1 through 14-384, respectively.

(127) All of Examples 15-1 through 15-768 have a deletion of a native GPD gene in addition to the modification indicated before in relation to the respective parent strains.

EXAMPLE 16

(128) Both alleles of the phosphoglucoisomerase (PGI) gene are deleted in each of Examples 11-1 through 11-6, 12-1 through 12-90, 13-1 through 13-288, 14-1 through 14-384 and 15-1 through 15-768 by transforming the cells sequentially with integration fragments 6-1 and 6-2 as described in Example 6. The resulting cells are designated Examples 16-11-1 through 16-11-6, 16-12-1 through 16-12-90, 16-13-1 through 16-13-288, 16-14-1 through 16-14-384 and 16-15-1 through 16-15-768, respectively.

EXAMPLE 17

Shake Flask Evaluation for Succinate Production

(129) Example 1-1 is streaked out for single colonies on URA selection plates and incubated at 30 C. until colonies are visible (1-2 days). Cells from plates are scraped into sterile growth medium and the optical density (OD.sub.600) is measured. Optical density is measured at wavelength of 600 nm with a 1 cm pathlength using a model Genesys20 spectrophotometer (Thermo Scientific). Dry cell mass is calculated from the measured OD.sub.600 value using an experimentally derived conversion factor of 1.7 OD.sub.600 units per 1 g dry cell mass.

(130) A shake flask is inoculated with the cell slurry to reach an initial OD.sub.600 of 0.1-0.3. Prior to incoculation, the 250 mL baffled shake flasks containing 1.75 g/L dry CaCO.sub.3 are sterilized by autoclave at 121 C. for 15 minutes. Immediately prior to inoculating, 50 mL of shake flask medium is added to the dry calcium carbonate. The shake flask medium is a sterilized, 5.5 pH aqueous solution of urea (2.3 g/L), magnesium sulfate heptahydrate (0.5 g/L), potassium phosphate monobasic (3.0 g/L), trace element solution (1 mL/L) and vitamin solution (1 mL/L), glucose (120.0 g/L), glycerol (0.1 g/L), 2-(N-Morpholino) ethanesulfonic acid (MES) (4.0 g/L). For strains lacking the URA3 gene (URA-) 20 mg/L uracil is added to the media. The trace element solution is an aqueous solution of EDTA (15.0 g/L), zinc sulfate heptahydrate (4.5 g/L), manganese chloride dehydrate (1.0 g/L), cobalt(II) chloride hexahydrate (0.3 g/L), copper(II)sulfate pentahydrate (0.3 g/L), disodium molybdenum dehydrate (0.4 g/L), calcium chloride dehydrate (4.5 g/L), iron sulphate heptahydrate (3 g/L), boric acid (1.0 g/L), and potassium iodide (0.1 g/L). The vitamin solution is an aqueous solution of biotin (D-; 0.05 g/L), calcium pantothenate (D+; 1 g/L), nicotinic acid (5 g/L), myo-inositol (25 g/L), pyridoxine hydrochloride (1 g/L), p-aminobenzoic acid (0.2 g/L).

(131) The inoculated flask is incubated at 30 C. with shaking at 150 rpm for 72 hours and taken to analysis. Succinate concentration in the broth at the end of 72 hours fermentation is determined by gas chromatography with flame ionization detector and glucose by high performance liquid chromatography with refractive index detector.

(132) Examples 1-2 through 1-6, 2-1 through 2-90, 3-1 through 3-288, 4-1 through 4-384, 5-1 through 5-768, 6-1-1 through 6-1-6, 6-2-1 through 6-290, 6-3-1 through 6-3-288, 6-4-1 through 6-4-384, 6-5-1 through 6-5-768, are made in the same manner by transforming Examples 7-1 through 7-6, 8-1 through 8-72, 9-1 through 9-234, 10-7-1 through 10-7-6, 10-8-1 through 10-8-72, 10-9-1 through 10-9-234,11-1 through 11-6, 12-1 through 12-90, 13-1 through 13-288, 14-1 through 14-384 and 15-1 through 15-768, 16-11-1 through 16-11-6, 16-12-1 through 16-12-90, 16-13-1 through 16-13-288, 16-14-1 through 16-14-384 and 16-15-1 through 16-15-768, are separately cultured in shake flasks in similar manner and found to produce succinate. The succinate concentration in the broth is measured and yield and titer are calculated.

Further Specific Embodiments

(133) The invention includes but is not limited to the following specific embodiments: 1. A recombinant cell having an active reductive TCA pathway from pyruvate to succinate which reductive TCA pathway includes at least one reaction that oxidizes NADPH to NADP.sup.+. 2. The recombinant cell of embodiment 1, wherein the reaction that oxidizes NADPH to NADP+is a conversion of oxaloacetate to malate catalyzed by an NADPH-dependent malate dehydrogenase enzyme. 3. The recombinant cell of embodiment 2, which overexpresses the NADPH-dependent malate dehydrogenase enzyme. 4. The recombinant cell of embodiment 2 or 3, which has integrated into its genome at least one exogenous malate dehydrogenase gene that encodes for the overexpressed NADPH-dependent malate dehydrogenase enzyme. 5. The recombinant cell of embodiment 4, wherein the malate dehydrogenase gene is non-native to the yeast cell. 6. The recombinant cell of any of embodiments 3-5, wherein the NADPH-dependent malate dehydrogenase enzyme has an amino acid sequence at least 80% identical to either of SEQ. ID. NOs: 143 or 144. 7. The recombinant cell of any of embodiments 3-5, wherein the NADPH-dependent malate dehydrogenase enzyme has either of amino acid sequences SEQ. ID. NOs: 143 or 144. 8. The recombinant cell of any of embodiments 4-7, wherein the malate dehydrogenase gene has a nucleotide sequence at least 80% identical to any of SEQ. ID. NO: 29, 31 or 32. 9. The recombinant cell of any of embodiments 4-7, wherein the malate dehydrogenase gene has nucleotide sequence SEQ. ID. NO: 29, 31 or 32. 10. The recombinant cell of embodiment 1, wherein the reaction that oxidizes NADPH to NADP+ is a conversion of fumarate to succinate catalyzed by an NADPH-dependent fumarate reductase enzyme. 11. The recombinant cell of embodiment 10, which overexpresses the NADPH-dependent fumarate reductase enzyme. 12. The recombinant cell of embodiment 11 or 12, which has integrated into its genome at least one exogenous fumarate reductase gene that encodes for the overexpressed NADPH-dependent fumarate reductase enzyme. 13. The recombinant cell of embodiment 12, wherein the fumarate reductase gene is non-native to the yeast cell. 14. The recombinant cell of any of embodiments 10-13, wherein the NADPH-dependent fumarate reductase enzyme has an amino acid sequence at least 80% identical to any of SEQ. ID. NOs: 110, 111, 112, 113, 114 or 115. 15. The recombinant cell of any of embodiments 10-13, wherein the NADPH-dependent fumarate reductase enzyme has any of amino acid sequences SEQ. ID. NOs: 110, 111, 112, 113, 114 or 115. 16. The recombinant cell of any of embodiments 10-15, wherein the fumarate reductase gene has a nucleotide sequence at least 80% identical to any of SEQ. ID. NOs: 15, 16, 17, 18, 19 or 20. 17. The recombinant cell of any of embodiments 10-15, wherein the fumarate reductase gene has any of nucleotide sequences SEQ. ID. NOs: 15, 16, 17, 18, 19 or 20. 18. The recombinant cell of any of embodiments 1-17 wherein the active reductive TCA pathway from pyruvate to succinate includes a step of converting pyruvate or phosphoenolpyruvate to oxaloacetate, a step of converting oxaloacetate to malate, a step of converting malate to fumarate, and a step of converting fumarate to succinate. 19. A recombinant yeast cell that overexpresses an NADPH-dependent malate dehydrogenase enzyme. 20. The recombinant cell of embodiment 19 having integrated into its genome an exogenous malate dehydrogenase gene that encodes for the NADPH-dependent malate dehydrogenase enzyme. 21. The recombinant cell of embodiment 19 or 20, wherein the NADPH-dependent malate dehydrogenase enzyme has an amino acid sequence at least 80% identical to either of SEQ. ID. NOs: 143 or 144. 22. The recombinant cell of embodiment 19 or 20, wherein the NADPH-dependent malate dehydrogenase enzyme has either of amino acid sequences SEQ. ID. NOs: 143 or 144. 23. The recombinant cell of any of embodiments 19-22, wherein the malate dehydrogenase gene has a nucleotide sequence at least 80% identical to any of SEQ. ID. NO: 29, 31 or 32. 24. The recombinant cell of any of embodiments 19-22, wherein the malate dehydrogenase gene has any of nucleotide sequences SEQ. ID. NO: 29, 31 or 32. 25. The recombinant cell of any of embodiments 19-24 which expresses a NADPH-dependent fumarate reductase enzyme. 26. A recombinant yeast cell that overexpresses an NADPH-dependent fumarate reductase enzyme. 27. The recombinant cell of embodiment 26 having integrated into its genome an exogenous fumarase reductase gene that encodes for the NADPH-dependent fumarate reductase enzyme. 28. The recombinant cell of embodiment 26 or 27, wherein the NADPH-dependent fumarate reductase enzyme has an amino acid sequence at least 80% identical to any of SEQ. ID. NOs: 110, 111, 112, 113, 114 or 115. 29. The recombinant cell embodiment 26 or 27, wherein the NADPH-dependent fumarate reductase enzyme has any of amino acid sequences SEQ. ID. NOs: 110, 111, 112, 113, 114 or 115. 30. The recombinant cell of any of embodiments 26-29, wherein the fumarate reductase gene has a nucleotide sequence at least 80% identical to any of SEQ. ID. NO.: 15, 16, 17, 18, 19 or 20. 31. The recombinant cell of any of embodiments 26-29, wherein the fumarate reductase gene has any of nucleotide sequences SEQ. ID. NO.: 15, 16, 17, 18, 19 or 20. 32. The recombinant cell of any of embodiments 26-31 which expresses a NADPH-dependent malate dehydrogenase enzyme. 33. The recombinant cell of any preceding embodiment which has integrated into its genome one or more of (i) an exogenous pyruvate carboxylase gene that encodes for an enzyme which catalyzes the conversion of pyruvate to oxaloacetate, (ii) an exogenous malate dehydrogenase gene which encodes for an enzyme that catalyzes the conversion of oxaloacetate to malate, (iii) an exogenous fumarase gene that encodes for an enzyme which catalyzes the conversion of malate to fumarate and (iv) an exogenous fumarate reductase gene which encodes for an enzyme which catalyzes the conversion of fumarate to succinate. 34. The recombinant cell of any preceding embodiment which has integrated into its genome one or more of (i) a non-native pyruvate carboxylase gene that encodes for an enzyme which catalyzes the conversion of pyruvate to oxaloacetate, (ii) a non-native malate dehydrogenase gene which encodes for an enzyme that catalyzes the conversion of oxaloacetate to malate, (iii) a non-native exogenous fumarase gene that encodes for an enzyme which catalyzes the conversion of malate to fumarate and (iv) a non-native exogenous fumarate reductase gene which encodes for an enzyme which catalyzes the conversion of fumarate to succinate. 35. The recombinant cell of any preceding embodiment which overexpresses at least one enzyme which catalyzes a reaction that includes the reduction of NADP+ to NADPH. 36. The recombinant cell of embodiment 35, wherein the overexpressed enzyme which catalyzes a reaction that includes the reduction of NADP+ to NADPH is in the pentose phosphate pathway. 37. The recombinant cell of embodiment 35 or 36, wherein the overexpressed enzyme which catalyzes a reaction that includes the reduction of NADP+ to NADPH is a 6-phosphogluconate dehydrogenase enzyme. 38. The recombinant cell of embodiment 37, which has integrated into its genome at least one exogenous 6-phosphogluconate dehydrogenase gene that encodes for the overexpressed 6-phosphogluconate dehydrogenase enzyme. 39. The recombinant cell of embodiment 38, wherein the exogenous 6-phosphogluconate dehydrogenase gene is native to the cell. 40. The recombinant cell of embodiment 38, wherein the 6-phosphogluconate dehydrogenase enzyme has an amino acid sequence at least 80% identical to SEQ. ID. NO: 140. 41. The recombinant cell of embodiment 19 or 20, wherein the 6-phosphogluconate dehydrogenase enzyme has amino acid sequence SEQ. ID. NO: 140. 42. The recombinant cell of embodiment 38, wherein the exogenous 6-phosphogluconate dehydrogenase gene has a nucleotide sequence at least 80% identical to SEQ. ID. NO: 99. 43. The recombinant cell of embodiment 38, wherein the exogenous 6-phosphogluconate dehydrogenase gene has the nucleotide sequence SEQ. ID. NO: 99. 44. The recombinant cell of embodiment 35 or 36, wherein the overexpressed enzyme which catalyzes a reaction that includes the reduction of NADP+ to NADPH is a glucose 6-phosphate dehydrogenase enzyme. 45. The recombinant cell of embodiment 44, which has integrated into its genome at least one exogenous glucose 6-phosphate dehydrogenase gene that encodes for the overexpressed glucose 6-phosphate dehydrogenase enzyme. 46. The recombinant cell of embodiment 45, wherein the exogenous glucose 6-phosphate dehydrogenase gene is native to the cell. 47. The recombinant cell of embodiment 44 or 45, wherein the glucose 6-phosphate dehydrogenase enzyme has an amino acid sequence at least 80% identical to SEQ ID NO: 139. 48. The recombinant cell of embodiment 44 or 45, wherein the glucose 6-phosphate dehydrogenaseenzyme has amino acid sequence SEQ ID NO: 139. 49. The recombinant cell of embodiment 45, wherein the exogenous glucose 6-phosphate dehydrogenase gene has a nucleotide sequence at least 80% identical to SEQ. ID. NO: 97. 50. The recombinant cell of any of embodiments 19-22, wherein the exogenous glucose 6-phosphate dehydrogenase gene has the nucleotide sequence SEQ. ID. NO: 97. 51. The recombinant cell of any preceding embodiment, which overexpresses at least one Stb5p enzyme. 52. The recombinant cell of embodiment 51, which has integrated into its genome at least one exogenous Stb5p gene that encodes for the overexpressed Stb5p enzyme. 53. The recombinant cell of embodiment 52, wherein the exogenous Stb5p gene is non-native to the cell. 54. The recombinant cell of any of embodiments 51-54, wherein the Stb5p enzyme has an amino acid sequence at least 80% identical to SEQ. ID. NO: 148. 55. The recombinant cell of embodiment 52, wherein the Stb5p enzyme has amino acid sequence SEQ. ID. NO: 148. 56. The recombinant cell of embodiment 52, wherein the exogenous Stb5p gene has a nucleotide sequence at least 80% identical to any of SEQ. ID. NO: 51. 57. The recombinant cell of embodiment 52, wherein the exogenous Stb5p gene has nucleotide sequence SEQ. ID. NO: 51. 58. The recombinant cell of any preceding embodiment, which further overexpresses a NAD(P).sup.+transhydrogenase enzyme. 59. The recombinant yeast cell of embodiment 58 which has integrated into its genome an exogenous NAD(P)+ transhydrogenase gene that encodes for the NAD(P)+ transhydrogenase enzyme. 60. The recombinant yeast cell of embodiment 58 or 59 wherein the exogenous NAD(P)+ transhydrogenase enzyme has an amino acid sequence at least 80% identical to any of SEQ. ID. NOs: 145, 146 or 147. 61. The recombinant cell of embodiment 58 or 59, wherein the NAD(P)+ transhydrogenase enzyme has any of amino acid sequences SEQ. ID. NOs: 145, 146 or 147. 62. The recombinant cell of embodiment 58, wherein the exogenous NAD(P)+ transhydrogenase gene has a nucleotide sequence at least 80% identical to any of SEQ. ID. NO: 45, 47 or 49. 63. The recombinant cell of embodiment 59, wherein the exogenous NAD(P)+ transhydrogenase gene has nucleotide sequence SEQ. ID. NO: 45, 47 or 49. 64. The recombinant cell of any preceding embodiment, which has a deletion or disruption of a native phosphoglucose isomerase gene. 65. The recombinant cell of any preceding embodiment, which has a deletion or disruption of a native pyruvate decarboxylase gene. 66. A malate dehydrogenase gene having nucleotide sequence SEQ. ID. NO: 32. 67. The malate dehydrogenase gene of embodiment 66 which is produced by converting an NADH-dependent malate dehydrogenase gene to a NADPH-dependent malate dehydrogenase gene. 68. A fumarate reductase gene having any of nucleotide sequences SEQ. ID. NOs: 15, 16, 17, 18, 19 or 20. 69. The fumarate reductase gene of embodiment 68 which is produced by converting an NADH- dependent fumarate reductase gene to a NADPH-dependent fumarate reductase gene. 70. A recombinant yeast having a deletion or disruption of a native phosphoglucose isomerase gene, has an active reductive TCA pathway from pyruvate to succinate and has integrated into its genome one or more of (i) an exogenous pyruvate carboxylate gene that encodes for an enzyme which catalyzes the conversion of pyruvate to oxaloacetate, (ii) an exogenous malate dehydrogenase gene which encodes for an enzyme that catalyzes the conversion of oxaloacetate to malate (iii) an exogenous fumarase gene that encodes for an enzyme which catalyzes the conversion of malate to fumarate and (iv) an exogenous fumarate reductase gene which encodes for an enzyme which catalyzes the conversion of fumarate to succinate. 71. A recombinant yeast having an active reductive TCA pathway from pyruvate to succinate and which has integrated into its genome at least one exogenous Stb5p gene and one or more of (i) an exogenous pyruvate carboxylate gene that encodes for an enzyme which catalyzes the conversion of pyruvate to oxaloacetate, (ii) an exogenous malate dehydrogenase gene which encodes for an enzyme that catalyzes the conversion of oxaloacetate to malate (iii) an exogenous fumarase gene that encodes for an enzyme which catalyzes the conversion of malate to fumarate and (iv) an exogenous fumarate reductase gene which encodes for an enzyme which catalyzes the conversion of fumarate to succinate 72. The recombinant cell of embodiment 71, wherein the exogenous Stb5p gene is non-native to the cell. 73. The recombinant cell of any of embodiments 70-72 which overexpresses at least one enzyme which catalyzes a reaction that includes the reduction of NADP+ to NADPH. 74. The recombinant cell of embodiment 73, wherein the overexpressed enzyme which catalyzes a reaction that includes the reduction of NADP+ to NADPH is in the pentose phosphate pathway. 75. The recombinant cell of embodiment 73 or 74, wherein the overexpressed enzyme which catalyzes a reaction that includes the reduction of NADP+ to NADPH is a 6-phosphogluconate dehydrogenase enzyme. 76. The recombinant cell of embodiment 75, which has integrated into its genome at least one exogenous 6-phosphogluconate dehydrogenase gene that encodes for the overexpressed 6-phosphogluconate dehydrogenase enzyme. 77. The recombinant cell of embodiment 73 or 74, wherein the overexpressed enzyme which catalyzes a reaction that includes the reduction of NADP+ to NADPH is a glucose 6-phosphate dehydrogenase enzyme. 78. The recombinant cell of embodiment 77, which has integrated into its genome at least one exogenous glucose 6-phosphate dehydrogenase gene that encodes for the overexpressed glucose 6-phosphate dehydrogenase enzyme. 79. The recombinant cell of any of embodiments 70-78, which has a deletion or disruption of a native phosphoglucose isomerase gene. 80. The recombinant cell of any of embodiments 70-79, which further overexpresses a NAD(P).sup.+ transhydrogenase enzyme. 81. The recombinant yeast cell of embodiment 80 which has integrated into its genome an exogenous NAD(P)+ transhydrogenase gene that encodes for the NAD(P)+ transhydrogenase enzyme. 82. The recombinant cell of any of embodiments 70-81, which has a deletion or disruption of a native pyruvate decarboxylase gene. 83. The recombinant cell of any of embodiments 1-65 and 70-82, wherein the host cell is a yeast cells classified under one or more of the genera Candida, Pichia, Saccharomyces, Schizosaccharomyces, Zygosaccharomyces, Kluyveromyces, Debaryomyces, Pichia, Issatchenkia, and Hansenula. 84. The recombinant cell of any of embodiments 1-65 and 70-82, wherein the host cell is selected from C. sonorensis, K. marxianus, K. thermotolerans, C. methanesorbosa, S. bulderi, I. orientalis, C. lambica, C. sorboxylosa, C. zemplinina, C. geochares, P. membranifaciens, Z. kombuchaensis, C. sorbosivorans, C. vanderwaltii, C. sorbophila, Z. bisporus, Z. lentus, S. bayanus, D. castellii, C, boidinii, C. etchellsii, K. lactis, P. jadinii, P. anomala, Saccharomyces cerevisae and Saccharomycopsis crataegensis. 85. The recombinant cell of any of embodiments 1-65 and 70-82, wherein the host cell is selected from Issatchenkia orientalis, Pichia galeiformis, Pichia sp. YB-4149 (NRRL designation), Candida ethanolica, P. deserticola, P. membranifaciens and P. fermentans. 86. The recombinant cell of any of embodiments 1-65 and 70-82, wherein the host cell is I. orientalis. 87. The recombinant cell of any of embodiments 1-65 and 70-82, wherein the host cell is S. cerevisiae. 88. The recombinant cell of any of embodiments 1-65 and 70-86, wherein the host cell is Crabtree negative as a wild-type strain. 89. The recombinant cell of any of embodiments 1-65 and 70-88, wherein the host cell is succinate-resistant as a wild-type strain. 90. The recombinant cell of any of embodiments 1-65 and 70-89, wherein the host cell exhibits a specific glucose consumption rate of at least 0.5 gram of glucose per gram dry weight of cells per hour, as a wild-type strain. 91. The recombinant cell of embodiment 90, wherein the host cell exhibits a specific glucose consumption rate of at least 1.0 gram of glucose per gram dry weight of cells per hour, as a wild-type strain. 92. The recombinant cell of embodiment 91, wherein the host cell exhibits a specific glucose consumption rate of at least 1.5 gram of glucose per gram dry weight of cells per hour, as a wild-type strain. 93. The recombinant cell of any of embodiments 1-65 and 70-89, wherein the host cell exhibits a volumetric glucose consumption rate of at least 3 gram of glucose per liter per hour, as a wild-type strain. 94. The recombinant cell of embodiment 93, wherein the host cell exhibits a volumetric glucose consumption rate of at least 5 gram of glucose per liter per hour, as a wild-type strain. 95. The recombinant cell of embodiment 91, wherein the host cell exhibits a volumetric glucose consumption rate of at least 8 gram of glucose per liter per hour, as a wild-type strain. 96. The recombinant cell of any of embodiments 1-65 and 70-95, which exhibits a volumetric glucose consumption rate of at least 0.5 gram of glucose per liter per hour. 97. The recombinant cell of any embodiment 96, which exhibits a glucose consumption rate of at least 0.75 gram of glucose per liter per hour. 98. The recombinant cell of any embodiment 97, which exhibits a glucose consumption rate of at least 0.9 gram of glucose per minute per liter per hour. 99. The recombinant cell of any embodiments 1-65 and 70-98 which produces succinate and transports succinate out of the cell. 100. The recombinant cell of any embodiments 1-65 and 70-99, which further metabolizes succinate to one or more succinate metabolization products. 101. The recombinant cell of embodiment 100, which transports at least one said succinate metabolization product out of the cell. 102. The recombinant cell of embodiment 100 or 101, wherein the succinate metabolization product is one or more of 1,4-butanediol, 1,3-butadiene, propionic acid, and 3-hydroxyisobutryic acid. 103. A process for producing succinate or a succinate metabolization product of succinate, comprising culturing the recombinant cell of any of embodiments 1-65 and 70-102 under fermentation conditions in a fermentation broth that includes a sugar that is fermentable by the cell. 104. The process of embodiment 103, wherein the recombinant cell produces succcinate and transports succinate out of the cell. 105. The process of embodiment 103, wherein the recombinant cell further metabolizes succinate to one or more succinate metabolization products, and the recombinant cell transports at least one of said succinate metabolization product out of the cell. 106. The process of embodiment 105, wherein the succinate metabolization product is one or more of 1,4-butanediol, 1,3-butadiene, propionic acid, and 3-hydroxyisobutryic acid.