Microorganisms for succinic acid production

Abstract

The present invention relates to a modified microorganism having, compared to its wild-type, a reduced activity of the enzyme that is encoded by the pykA-gene. The present invention also relates to a method for producing an organic compound and to the use of modified microorganisms.

Claims

1. A modified microorganism having, compared to its wild-type, a reduced activity of the enzyme that is encoded by the pykA-gene, wherein the activity of the enzyme encoded by the pykA-gene is reduced by introducing at least one mutation into the pykA-gene, wherein the reduction of the activity of the enzyme encoded by the pykA-gene is in the range of 15 to 99%, wherein the wild-type from which the modified microorganism has been derived belongs to the family of Pasteurellaceae, wherein the wildtype refers to the naturally occurring microorganism that has not been genetically modified and wherein the wild-type-pykA-gene comprises a nucleic acid selected from the group consisting of: a) a nucleic acid having the nucleotide sequence of SEQ ID NO: 3; b) a nucleic acid encoding the amino acid sequence of SEQ ID NO: 4; c) a nucleic acid which is at least 90% identical to the nucleic acid of a) or b), the identity being the identity over the total length of the nucleic acid of a) or b); and d) a nucleic acid encoding an amino acid sequence which is at least 90% identical to the amino acid sequence encoded by the nucleic acid of a) or b), the identity being the identity over the total length of amino acid sequence encoded by the nucleic acid of a) or b), wherein the modified microorganism, compared to the wild-type microorganism in which the activity of the enzyme encoded by the pykA-gene has not been reduced, is characterized by an increased yield of succinic acid and by faster growth when cultivated under anaerobic conditions with at least one assimilable carbon source.

2. The modified microorganism according to claim 1, wherein the wild-type from which the modified microorganism has been derived has a 16 S rDNA of SEQ ID NO: 1 or a sequence, which shows a sequence homology of at least 96% with SEQ ID NO: 1.

3. The modified microorganism according to claim 1, wherein the wild-type from which the modified microorganism has been derived belongs to the genus Basfia.

4. The modified microorganism according to claim 3, wherein the wild-type from which the modified microorganism has been derived belongs to the species Basfia succiniciproducens.

5. The modified microorganism according to claim 4, wherein the wild-type from which the modified microorganism has been derived is Basfia succiniciproducens strain DD1 as deposited under DSM 18541 with the DSMZ, Germany.

6. The modified microorganism according to claim 1, wherein the at least one mutation leads to a modification of the nucleic acid sequence of the pykA-gene, such that the amino acid sequence of the enzyme encoded by the modified gene differs from the amino acid sequence of the enzyme encoded by the wild-type pykA-gene in at least one amino acid.

7. The modified microorganism according to claim 1, wherein the microorganism further comprises: A) a deletion of the IdhA-gene or at least a part thereof, a deletion of a regulatory element of the IdhA-gene or at least a part thereof or an introduction of at least one mutation into the IdhA-gene; B) a deletion of the pfID-gene or at least a part thereof, a deletion of a regulatory element of the pfID-gene or at least a part thereof or an introduction of at least one mutation into the pfID-gene; C) a deletion of the pfIA-gene or at least a part thereof, a deletion of a regulatory element of the pfIA-gene or at least a part thereof or an introduction of at least one mutation into the pfIA-gene; D) a deletion of the IdhA-gene or at least a part thereof, a deletion of a regulatory element of the IdhA-gene or at least a part thereof or an introduction of at least one mutation into the IdhA-gene and a deletion of the pfID-gene or at least a part thereof, a deletion of a regulatory element of the pfID-gene or at least a part thereof or an introduction of at least one mutation into the pfID-gene; or E) a deletion of the IdhA-gene or at least a part thereof, a deletion of a regulatory element of the IdhA-gene or at least a part thereof or an introduction of at least one mutation into the IdhA-gene and a deletion of the pfIA-gene or at least a part thereof, a deletion of a regulatory element of the pfIA-gene or at least a part thereof or an introduction of at least one mutation into the pfIA-gene.

8. A method of producing succinic acid comprising: I) cultivating the modified microorganism according to claim 1 in a culture medium comprising at least one assimilable carbon source to allow the modified microorganism to produce succinic acid, thereby obtaining a fermentation broth comprising succinic acid; II) recovering succinic acid from the fermentation broth obtained in process step I).

9. Method according to claim 8, wherein the assimilable carbon source is selected from the group consisting of sucrose, maltose, D-glucose, glycerol, mixtures of glycerol and D-glucose, mixtures of glycerol and sucrose, mixtures of glycerol and D-xylose, mixtures of glycerol and mixtures of maltose and D-glucose and fructose.

10. Method according to claim 8, wherein the process further comprises the process step: III) conversion of succinic acid contained in the fermentation broth obtained in process step I) or conversion of the recovered succinic acid obtained in process step II) into a secondary organic product being different from succinic acid by at least one chemical reaction.

11. Method according to claim 10, wherein the secondary organic product is selected from the group consisting of succinic acid esters or polymers thereof, tetrahydrofuran (THF), 1,4-butanediol (BDO), gamma-butyrolactone (GBL) and pyrrolidones.

Description

(1) The invention is now explained in more detail with the aid of figures and non-limiting examples.

(2) FIG. 1 shows a schematic map of plasmid pSacB (SEQ ID NO: 5).

(3) FIG. 2 shows a schematic map of plasmid pSacB IdhA (SEQ ID NO: 6).

(4) FIG. 3 shows a schematic map of plasmid pSacB pfIA (SEQ ID NO: 7).

(5) FIG. 4 shows a schematic map of plasmid pSacB pfID (SEQ ID NO: 8).

(6) FIG. 5 shows a schematic map of plasmid pSacB pykA1 (SEQ ID NO: 9).

(7) FIG. 6 shows a schematic map of plasmid pSacB pykA2 (SEQ ID NO: 10).

(8) FIG. 7 shows a schematic map of plasmid pSacB pykA3 (SEQ ID NO: 11).

(9) FIG. 8 shows a schematic map of plasmid pSacB pykA4 (SEQ ID NO: 12).

(10) FIG. 9 shows a schematic map of plasmid pSacB pykA5 (SEQ ID NO: 13).

(11) FIG. 10 shows a schematic map of plasmid pSacB pykA6 (SEQ ID NO: 14).

(12) FIG. 11 shows the amino acid sequence alignment of pyruvate kinases (pykA wild-type: amino acid sequence of pyruvate kinase from the wild-type strain Basfia succiniciproducens DD1; pykA2: amino acid sequence of pyruvate kinase PykA2 from the DD1 IdhA pfIA pykA2-strain; pykA4: amino acid sequence of pyruvate kinase PykA4 from the DD1 IdhA pfID pykA4-strain; pykA5: amino acid sequence of pyruvate kinase PykA5 from the DD1 IdhA pfID pykA5-strain; pykA6: amino acid sequence of pyruvate kinase PykA6 from the DD1 IdhA pfID pykA6-strain).

EXAMPLES

Example 1

General Method for the Transformation of Basfia Succiniciproducens

(13) TABLE-US-00001 TABLE 1 Nomenclature of the DD1-wild-type and mutants referred to in the examples Strain Wild-type DD1 (deposit DSM18541) DD1 ldhA DD1 ldhA pflA DD1 ldhA pflA pykA2 DD1 ldhA pflD DD1 ldhA pflD pykA4 DD1 ldhA pflD pykA5 DD1 ldhA pflD pykA5

(14) Basfia succiniciproducens DD1 (wild-type) was transformed with DNA by electroporation using the following protocol:

(15) For preparing a pre-culture DD1 was inoculated from frozen stock into 40 ml BHI (brain heart infusion; Becton, Dickinson and Company) in 100 ml shake flask. Incubation was performed over night at 37 C.; 200 rpm. For preparing the main-culture 100 ml BHI were placed in a 250 ml shake flask and inoculated to a final OD (600 nm) of 0.2 with the pre-culture. Incubation was performed at 37 C., 200 rpm. The cells were harvested at an OD of approximately 0.5, 0.6 and 0.7, pellet was washed once with 10% cold glycerol at 4 C. and re-suspended in 2 ml 10% glycerol (4 C.).

(16) 100 l of competent cells were the mixed with 2-8 g Plasmid-DNA and kept on ice for 2 min in an electroporation cuvette with a width of 0.2 cm. Electroporation under the following conditions: 400 ; 25 F; 2.5 kV (Gene Pulser, Bio-Rad). 1 ml of chilled BHI was added immediately after electroporation and incubation was performed for approximately 2 h at 37 C.

(17) Cells were plated on BHI with 5 mg/L chloramphenicol and incubated for 2-5 d at 37 C. until the colonies of the transformants were visible. Clones were isolated and restreaked onto BHI with 5 mg/l chloramphenicol until purity of clones was obtained.

Example 2

Generation of Deletion/Mutation Constructs

(18) 1. Generation of Deletions Constructs

(19) Deletion plasmids were constructed based on the vector pSacB (SEQ ID NO: 5). FIG. 1 shows a schematic map of plasmid pSacB. 5- and 3-flanking regions (approx. 1500 bp each) of the chromosomal fragment, which should be deleted were amplified by PCR from chromosomal DNA of Basfia succiniciproducens and introduced into said vector using standard techniques. Normally, at least 80% of the ORF were targeted for a deletion. In such a way, the deletion plasmids for the lactate dehydrogenase IdhA, pSacB_delta_IdhA (SEQ ID NO: 6), the pyruvate formate lyase activating enzyme pfIA, pSacB_delta.sub. pfIA (SEQ ID NO: 7) and the pyruvate formate lyase pfID, pSacB_delta_pfID (SEQ ID NO: 8) were constructed. FIGS. 2, 3 and 4 show schematic maps of plasmid pSacB_delta_IdhA, pSacB_delta_pfIA and pSacB_delta_pfID, respectively.

(20) In the plasmid sequence of pSacB (SEQ ID NO: 5) the sacB-gene is contained from bases 2380-3801. The sacB-promotor is contained from bases 3802-4264. The chloramphenicol gene is contained from base 526-984. The origin of replication for E. coli (ori EC) is contained from base 1477-2337 (see FIG. 1).

(21) In the plasmid sequence of pSacB_delta_IdhA (SEQ ID NO: 6) the 5 flanking region of the IdhA gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 1519-2850, while the 3 flanking region of the IdhA-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 62-1518. The sacBgene is contained from bases 5169-6590. The sacB-promoter is contained from bases 6591-7053. The chloramphenicol gene is contained from base 3315-3773. The origin of replication for E. coli (ori EC) is contained from base 4266-5126 (see FIG. 2).

(22) In the plasmid sequence of pSacB_delta_pfIA (SEQ ID NO: 7) the 5 flanking region of the pfIA-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 1506-3005, while the 3 flanking region of the pfIA-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 6-1505. The sacB-gene is contained from bases 5278-6699. The sacB-promoter is contained from bases 6700-7162. The chloramphenicol gene is contained from base 3424-3882. The origin of replication for E. coli (ori EC) is contained from base 4375-5235 (see FIG. 3).

(23) In the plasmid sequence of pSacB_delta_pfID (SEQ ID NO: 8) the 5 flanking region of the pfID-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 1533-2955, while the 3 flanking region of the pfID-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 62-1532. The sacB-gene is contained from bases 5256-6677. The sacB-promoter is contained from bases 6678-7140. The chloramphenicol gene is contained from base 3402-3860. The origin of replication for E. coli (ori EC) is contained from base 4353-5213 (see FIG. 4).

(24) 2. Generation of constructs used for introduction of point mutations into the pykA-gene

(25) In the plasmid sequence of pSacB_pykA1 (SEQ ID NO: 9) the part of the pykA-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 6-1185. The sacB-gene is contained from bases 3458-4879. The sacB-promoter is contained from bases 4880-5342. The chloramphenicol gene is contained from bases 1604-2062. The origin of replication for E. coli (ori EC) is contained from bases 2555-3415 (see FIG. 5).

(26) The plasmid pSacB_pykA2 was generated by site-directed mutagenesis from the pSac_pykA1 plasmid. The introduced G to T mutation in the pykA-gene will finally result in exchange of G (glycine) to C (cysteine) at position 167 in the PykA-protein (see FIG. 6). The nucleotide sequence of the pykA-gene in pSacB_pykA2 is shown in SEQ ID NO: 15, the amino acid sequence of the enzyme encoded by this gene in SEQ ID NO: 16.

(27) In the plasmid sequence of pSacB_delta_pykA2 (SEQ ID NO: 10) the part of the pykA-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 6-1185. The sacB-gene is contained from bases 3458-4879. The sacB-promoter is contained from bases 4880-5342. The chloramphenicol gene is contained from base 1604-2062. The origin of replication for E. coli (ori EC) is contained from base 2555-3415 (see FIG. 7).

(28) In the plasmid sequence of pSacB_pykA3 (SEQ ID NO: 11) the part of the pykA-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 6-909. The sacB-gene is contained from bases 3729-5150. The sacB-promoter is contained from bases 5151-5613. The chloramphenicol gene is contained from bases 1875-2333. The origin of replication for E. coli (ori EC) is contained from bases 2826-3686 (see FIG. 8).

(29) The plasmid pSacB_pykA4 was generated by site-directed mutagenesis from the pSac_pykA3 plasmid. The introduced G to A mutation in the pykA-gene will finally result in exchange of C (cysteine) to Y (tyrosine) at position 417 in the PykA-protein (see FIG. 8). The nucleotide sequence of the pykA-gene in pSacB_pykA4 is shown in SEQ ID NO: 17, the amino acid sequence of the enzyme encoded by this gene in SEQ ID NO: 18. In addition a DraI-restriction site was generated by silent mutation. This restriction site will help to identify correct transformants. In the plasmid sequence of pSacB_delta_pykA4 (SEQ ID NO: 19) the part of the pykA-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 6-909. The sacB-gene is contained from bases 3729-5150. The sacB-promoter is contained from bases 5151-5613. The chloramphenicol gene is contained from bases 1875-2333. The origin of replication for E. coli (ori EC) is contained from bases 2826-3686 (see FIG. 9).

(30) The plasmid pSacB_pykA5 was generated by site-directed mutagenesis from the pSac_pykA1-plasmid. The introduced C to G mutation in the pykA-gene will finally result in exchange of A (alanine) to G (glycine) at position 171 in the PykA-protein (see FIG. 9). The nucleotide sequence of the pykA-gene in pSacB_pykA5 is shown in SEQ ID NO: 19, the amino acid sequence of the enzyme encoded by this gene in SEQ ID NO: 20. In addition a HpaI-restriction site was generated by silent mutation. This restriction site will help to identify correct transformants. In the plasmid sequence of pSacB_pykA5 (SEQ ID NO: 13) the part of the pykA-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 6-1185. The sacB-gene is contained from bases 3458-4879. The sacB-promoter is contained from bases 4880-5342. The chloramphenicol gene is contained from bases 1604-2062. The origin of replication for E. coli (ori EC) is contained from bases 2555-3415.

(31) The plasmid pSacB_pykA6 was generated by site-directed mutagenesis from the pSac_pykA1 plasmid. The introduced G to T mutation in the pykA-gene will finally result in exchange of G (glycine) to C (cysteine) at position 167 in the PykA-protein (see FIG. 10). The nucleotide sequence of the pykA-gene in pSacB_pykA6 is shown in SEQ ID NO: 21, the amino acid sequence of the enzyme encoded by this gene in SEQ ID NO: 22. In addition a HpaI-restriction site was generated by silent mutation. This restriction site will help to identify correct transformants. In the plasmid sequence of pSacB_pykA6 (SEQ ID NO: 14) the part of the pykA-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 6-1185. The sacB-gene is contained from bases 3458-4879. The sacB-promoter is contained from bases 4880-5342. The chloramphenicol gene is contained from bases 1604-2062. The origin of replication for E. coli (ori EC) is contained from bases 2555-3415.

Example 3

Generation of Improved Succinate Producing Strains

(32) 1. Generation of Deletion Mutants

(33) a) Basfia succiniciproducens DD1 was transformed as described above with the pSacB_delta_IdhA and Campbelled in to yield a Campbell in strain. Transformation and integration into the genome of Basfia succiniciproducens was confirmed by PCR yielding bands for the integration event of the plasmid into the genome of Basfia succiniciproducens. The Campbell in strain was then Campbelled out using agar plates containing sucrose as a counter selection medium, selecting for the loss (of function) of the sacB-gene. Therefore, the Campbell in strains were incubated in 25-35 ml of non selective medium (BHI containing no antibiotic) at 37 C., 220 rpm over night. The overnight culture was then streaked onto freshly prepared BHI containing sucrose plates (10%, no antibiotics) and incubated overnight at 37 C. (first sucrose transfer). Single colony obtained from first transfer were again streaked onto freshly prepared BHI containing sucrose plates (10%) and incubated overnight at 37 C. (second sucrose transfer). This procedure was repeated until a minimal completion of five transfers (third, forth, fifth sucrose transfer) in sucrose. The term first to fifth sucrose transfer refers to the transfer of a strain after chromosomal integration of a vector containing a sacB-levan-sucrase gene onto sucrose and growth medium containing agar plates for the purpose of selecting for strains with the loss of the sacB-gene and the surrounding plasmid sequences. Single colony from the fifth transfer plates were inoculated onto 25-35 ml of non selective medium (BHI containing no antibiotic) and incubated at 37 C., 220 rpm over night. The overnight culture was serially diluted and plated onto BHI plates to obtain isolated single colonies. The Campbelled out strains containing either the wild-type situation of the IdhAlocus or the mutation/deletion of the IdhA-gene were confirmed by chloramphenicol sensitivity. The mutation/deletion mutants among these strains were identified and confirmed by PCR analysis. This led to the IdhA-deletion mutant Basfia succiniciproducens DD1 LIdhA.

(34) b) Basfia succiniciproducens DD1 IdhA was transformed with pSacB_delta_pfIA as described above and Campbelled in to yield a Campbell in strain. Transformation and integration was confirmed by PCR. The Campbell in strain was then Campbelled out as described previously. The deletion mutants among these strains were identified and confirmed by PCR analysis. This led to the IdhA pfID-double deletion mutant Basfia succiniciproducens DD1 IdhA pfIA. c) Basfia succiniciproducens IdhA was transformed with pSacB_delta_pfID as described above and Campbelled in to yield a Campbell in strain. Transformation and integration was confirmed by PCR. The Campbell in strain was then Campbelled out as described previously. The deletion mutants among these strains were identified and confirmed by PCR analysis. This led to the IdhA pfID-double deletion mutant Basfia succiniciproducens DD1 IdhA pfID.
2. Generation of Mutants Carrying Point Mutations in the pykA-gene a) Basfia succiniciproducens DD1 IdhA pfIA was transformed with pSacB_pykA2 as described above and Campbelled in to yield a Campbell in strain. The Campbell in strain was then Campbelled out as described previously. The pykA-coding region was amplified by means of PCR and sequenced to identify the Campbell out clones carrying a mutation within the pykA-gene. This led to the mutant Basfia succiniciproducens DD1 IdhA pfIA pykA2. b) Basfia succiniciproducens DD1 IdhA pfID was transformed with pSacB_pykA4 as described above and Campbelled in to yield a Campbell in strain. The Campbell in strain was then Campbelled out as described previously. To identify the Campbell out clones carrying a mutation within the pykA-gene a part of the pykA-locus was amplified by means of PCR with primers pykA4_fw (SEQ ID NO: 23) and pykA4_ry (SEQ ID NO: 24). In the pre-screen resulted DNA fragment was digested with DraI restriction enzyme. Fragments which showed the additional DraI-restriction site were sequenced to confirm the expected point mutation close to the introduced DraI-restriction site within the pykA-gene. This led to the mutant Basfia succiniciproducens DD1 IdhA pfID pykA4. c) Basfia succiniciproducens IdhA pfID was transformed with pSacB_pykA5 as described above and Campbelled in to yield a Campbell in strain. The Campbell in strain was then Campbelled out as described previously. To identify the Campbell out clones carrying a mutation within the pykA-gene a part of the pykA-locus was amplified by means of PCR with primers pykA5/6_fw (SEQ ID NO: 25) and pykA5/6_rv (SEQ ID NO: 26). In the pre-screen resulted DNA fragment was digested with HpaI-restriction enzyme. Fragments which showed the additional HpaI-restriction site were sequenced to confirm the expected point mutation close to the introduced HpaI-restriction site within the pykA-gene. This led to the mutant Basfia succiniciproducens DD1 IdhA pfID pykA5. d) Basfia succiniciproducens IdhA pfID was transformed with pSacB_pykA6 as described above and Campbelled in to yield a Campbell in strain. The Campbell in strain was then Campbelled out as described previously. To identify the Campbell out clones carrying a mutation within the pykA-gene a part of the pykA-locus was amplified by means of PCR with primers pykA5/6_fw (SEQ ID NO: 25) and pykA5/6 ry (SEQ ID NO: 26). In the pre-screen resulted DNA fragment was digested with HpaI-restriction enzyme. Fragments which showed the additional HpaI-restriction site were sequenced to confirm the expected point mutation close to the introduced HpaI-restriction site within the pykA-gene. This led to the mutant Basfia succiniciproducens DD1 IdhA pfID pykA6.

Example 4

Pyruvate Kinase Activity

(35) Basfia strains were grown anaerobically in serum bottles in the medium as follows (BHI Medium (Becton Dickinsion, containing the following additions MOPS: 9.4 g/l, Mg(OH).sub.2: 0.625 g/l, BISTRIS: 5.8 g/l, NaHCO.sub.3: 1.8 g/l) for 18h at 37 C. Cells were harvested by centrifugation and frozen. Pellets of cells were resuspended in 60 mM HEPES-Na, 60 mM KCl, 8.5 mM MgCl.sub.2 pH 7.5. Cells were lysed using the Ribolyser (ThermoHybaid) machine and blue matrix tubes. Extracts were centrifuged for 10 minutes at 14000 rpm in an eppedorf centrifufge at 4 C. and kept on ice until the assay was performed. Supernatants were used to assay pyruvate kinase activity using the following assay: Enzyme activity of PykA was determined according to the Methods of Enzymatic Analysis, 2nd English ed., Vol. 1, Bergmeyer, H. U., ed., Academic Press (New York, N.Y.: 1974), pp. 509-511. Protein concentrations in cell extracts were determined by the Biorad protein blue assay using IgG as the protein standard for calibration. Enzyme activities are expressed as specific activities (U/mg of protein and min) and relatively as in percent of the activity of the strain DD1 IdhA pfID carrying an unmutated pykA-allele. The results are shown in table 2.

(36) TABLE-US-00002 TABLE 2 Results of the PykA-enzyme activity test Specific activity .sub.activity Strain [mU/mg protein] [%] DD1 ldhA pflD 32.4 0 DD1 ldhA pflA pykA2 2.25 93.1 DD1 ldhA pflD pykA4 2.40 92.7 DD1 ldhA pflD pykA5 9.70 90.3

Example 5

Cultivation of Various DD1-Strains on Glucose, Sucrose and Maltose/Glycerol

(37) The productivity of the DD1 IdhA pfIA strain was compared with the productivity of the mutant strain DD1 IdhA pfIA pykA2 in the presence of glucose, or sucrose, or maltose and glycerol as a carbon source (table 6, 7 and 8).

(38) Productivity was analyzed utilizing media and incubation conditions described below.

(39) 1. Medium Preparation

(40) The composition and preparation of the cultivation medium is as described in the following table 3, 4 and 5.

(41) TABLE-US-00003 TABLE 3 Medium composition for cultivation on glucose (medium P) Compound Concentration [g/L] Yeast extract 10.0 (Bio Springer) CaCl.sub.2 2H.sub.2O 0.2 MgCl.sub.2 6H.sub.2O 0.2 (NH.sub.4).sub.2SO.sub.4 2.0 NaCl 1.0 K.sub.2HPO.sub.4 3.0 MgCO.sub.3 50.0 NaHCO.sub.3 8.4 glucose 52.0

(42) TABLE-US-00004 TABLE 4 Medium composition for cultivation on sucrose (medium P) Compound Concentration [g/L] Yeast extract 10.0 (Bio Springer) CaCl.sub.2 2H.sub.2O 0.2 MgCl.sub.2 6H.sub.2O 0.2 (NH.sub.4).sub.2SO.sub.4 2.0 NaCl 1.0 K.sub.2HPO.sub.4 3.0 MgCO.sub.3 50.0 NaHCO.sub.3 8.4 sucrose 50.0

(43) TABLE-US-00005 TABLE 5 Medium composition for cultivation on maltose/glycerol (medium P) Compound Concentration [g/L] Yeast extract (Bio Springer) 10.0 CaCl.sub.2 2H.sub.2O 0.2 MgCl.sub.2 6H.sub.2O 0.2 (NH.sub.4).sub.2SO.sub.4 2.0 NaCl 1.0 K.sub.2HPO.sub.4 3.0 MgCO.sub.3 70.0 NaHCO.sub.3 8.4 maltose 22.5 glycerol 50.0
2. Cultivations and Analytics

(44) For growing the main culture bacteria from a freshly grown BHI-agar plate (incubated overnight at 37 C. under anaerobic conditions) was used to inoculate to OD600=0.75 a 100 ml-serum bottle with gas tight butyl rubber stopper containing 50 ml of the liquid medium described in table 3, 4 and 5 with a 002-atmosphere with 0.8 bar overpressure. The bottles were incubated at 37 C. and 160 rpm (shaking diameter: 2.5 cm). Consumption of the C-sources and production of carboxylic acids was quantified via HPLC (HPLC methods are described in tables 9 and 10) after 24h. Cell growth was measured by measuring the absorbance at 600 nm (OD600) using a spectrophotometer (Ultrospec3000, Amersham Biosciences, Uppsala Sweden).

(45) 3. Results

(46) The results of the cultivation experiments with for different DD1-strains are shown in tables 6 to 8.

(47) TABLE-US-00006 TABLE 6 Cultivation of the DD1 ldhA pflA-strain and the DD1 ldhA pflA pykA2-strain on glucose DD1 ldhApflA DD1 ldhA pflA pykA2 substrate glucose glucose tc [h].sup.a 24 24 C [g/l].sup.b 54.00 54.00 C.sub.SA[g/l].sup.c (succinic acid) 35.27 38.08 C.sub.LA [g/L].sup.c,h 0.21 0.32 (lactic acid) C.sup.FA[g/l].sup.c,h (formic acid) 0.00 0.00 C.sub.AA[g/l].sup.c,h (acetic acid) 1.10 2.30 C.sub.PA[g/l].sup.c,h 2.76 2.43 (pyruvic acid) C.sub.P[g/l].sup.c,h 0.00 0.00 (propionic acid) C.sub.E[g/l].sup.c (ethanol) 0.00 0.00 Carbon Yield (YP/S) 0.73 0.80 [g/g] SA Yield (SA/S) [g/g].sup.g 0.65 0.71 .sup.acultivation time .sup.bconsumption of substrate (glucose) .sup.cformation of succinic acid, lactic acid, formic acid, acetic acid, pyruvic acid and ethanol .sup.gSA yield (ration of SA per consumed substrate) .sup.hdetection limits for acetic acid, lactic acid, malic acid, and formic acid were found to be lower than 0.01 g/l in the given HPLC method

(48) TABLE-US-00007 TABLE 7 Cultivation of the DD1 ldhA pflA-strain and the DD1 ldhA, pflA pykA2-strain on sucrose DD1 ldhA DD1 ldhA pflA pflA pykA2 substrate sucrose sucrose tc [h].sup.a 16 16 C [g/l].sup.b 18.55 50.90 C.sub.SA[g/l].sup.c 10.51 32.98 (succinic acid) .sup.acultivation time .sup.bconsumption of substrate (sucrose) .sup.cformation of succinic acid

(49) A reduction of the activity of the enzyme encoded by the pykA-gene leads to a faster growth of the cells on sucrose. This gets evident when comparing the values for the sucrose consumption in table 7: within 16 h the DD1 IdhA pfIA-strain consumed 18.55 g/L of sucrose; during the same time (also 16 h) the DD1 IdhA pfIA pykA2-strain consumed already 50.90 g/L of sucrose.

(50) TABLE-US-00008 TABLE 8 Cultivation of the DD1 ldhA pflA-strain and the DD1 ldhA pflA pykA2-strain on maltose/glycerol DD1 ldhA pflA DD1 ldhA pflA pykA2 substrate maltose/ maltose/ glycerol glycerol tc [h].sup.a 16 16 C [g/l].sup.b 7.50 9.92 (maltose) (maltose) 41.76 53.39 (glycerol) (glycerol) C.sub.SA[g/l].sup.c (succinic 52.58 66.04 acid) .sup.acultivation time .sup.bconsumption of substrate (maltose and glycerol) .sup.cformation of succinic acid

(51) TABLE-US-00009 TABLE 9 HPLC method (ZX-THF50) for analysis of glucose, maltose, glycerol, succinic acid, formic acid, lactic acid, acetic acid, pyruvic acid and ethanol HPLC column Aminex HPX-87 H, 300 7.8 mm (BioRad) Precolumn Cation H Temperature 50 C. Eluent flow rate 0.50 ml/min Injection volume 5.0 l Diode array detector RI-Detector Runtime 28 min max. pressure 140 bar Eluent A 5 mM H.sub.2SO.sub.4 Eluent B 5 mM H.sub.2SO.sub.4 Time [min] A [%] B [%] Flow [ml/min] Gradient 0.0 50 50 0.50 28.0 50 50 0.50

(52) TABLE-US-00010 TABLE 10 HPLC method (Fast-CH) for analysis of glucose and sucrose HPLC column Fast Carbohydrate, 100 7.8 mm (Biorad) Precolumn Deashing Refill Cartridges (30 C.) Temperature 75 C. Eluent flow rate 1.00 ml/min Injection volume 1.0 l Diode array detector RI-Detector Runtime 8 min max. pressure 150 bar Eluent A water Eluent B water Time [min] A [%] B [%] Flow [ml/min] Gradient 0.0 50 50 1.00 8.0 50 50 1.00

Example 6

Cultivation of DD1 IdhA pfID pykA4 in the Presence of Glucose, or Sucrose, or Glycerol and Glucose

(53) The productivity of the DD1 IdhA pfID strain was compared with the productivity of the mutant strain DD1 IdhA pfID pykA4 in the presence of glucose, or sucrose, or glucose and glycerol as a carbon source.

(54) Productivity was analyzed utilizing media and incubation conditions described below.

(55) 1. Medium Preparation

(56) The composition and preparation of the cultivation medium CGM is as described in the following table 11.

(57) TABLE-US-00011 TABLE 11 The composition of the cultivation medium CGM. Medium CGM Compound Concentration [g/L] Yeast extract (Bio Springer) 12.5 Succinic acid 2.5 (NH.sub.4).sub.2SO.sub.4 0.5 KH.sub.2PO.sub.4 1.0 MgCO.sub.3 50.0 Na.sub.2CO.sub.3 2.0 Glucose 52

(58) The composition of the cultivation medium LSM_3_glucose, medium LSM_3_sucrose, and medium LSM_3_glycerol_glucose is as described in the following table 14, 15 and 16.

(59) TABLE-US-00012 TABLE 12 Composition of trace element solution 5. Trace element solution 5 Compound Final conc. citric acid 10 g/L ZnSO.sub.4 7H2O 1851 mg/L CaSO.sub.4 2H.sub.2O 10 mg/L FeSO.sub.4 7H.sub.2O 2040 mg/L CaCl.sub.2 2H.sub.2O 12460 mg/L MnCl.sub.2 4H.sub.2O 1200 mg/L Na.sub.2MoO.sub.4 2H.sub.2O 38 mg/L CuCl.sub.2 2H.sub.2O 188 mg/L NiCl.sub.2 6H.sub.2O 32 mg/L CoCl.sub.2 6H.sub.2O 101 mg/L

(60) TABLE-US-00013 TABLE 13 Composition of vitamin solution_9. Vitamin solution 9 Compound Final conc. Thiamin HCl (B1) 1.0 g/L Nicotinic acid (B3) 1.0 g/L Riboflavin (B2) 20 mg/L Biotin (B7) 50 mg/L Pantothenic acid (B5) 1.0 g/L Pyridoxine (B6) 1.0 g/L Cyanocobalamin (B12) 50 mg/L Lipoic acid 5 mg/L

(61) TABLE-US-00014 TABLE 14 Composition of LSM_3_glucose medium. Medium LSM_3_glucose Compound Volume/Mass Stock conc. Final conc. Medium 1 MgCO.sub.3 2.5 g.sup. 100% 50.00 g/L Water 38.45 mL Medium 2 Succinic acid 2.5 mL 50 g/L 2.50 g/L Glucose 4.00 mL 650 g/L 52.00 g/L (NH.sub.4).sub.2SO.sub.4 0.5 mL 500 g/L 5.00 g/L Betain 0.5 mL 23 g/L 0.23 g/L KH.sub.2PO.sub.4 0.50 mL 100 g/L 1.00 g/L Na.sub.2CO.sub.3 0.50 mL 200 g/L 2.00 g/L vitamin solution 9 0.50 mL 4 g/L 0.04 g/L (conc. 100x) trace element solution 5 0.05 mL 21 g/L 0.02 g/L

(62) TABLE-US-00015 TABLE 15 Composition of LSM_3_sucrose medium. Medium LSM_3_sucrose Compound Volume/Mass Stock conc. Final conc. Medium 1 MgCO.sub.3 2.5 g.sup. 100% 50.00 g/L Water 38.45 mL Medium 2 Succinic acid 2.5 mL 50 g/L 2.50 g/L Sucrose 4.00 mL 650 g/L 52.00 g/L (NH.sub.4).sub.2SO.sub.4 0.5 mL 500 g/L 5.00 g/L Betain 0.5 mL 23 g/L 0.23 g/L KH.sub.2PO.sub.4 0.50 mL 100 g/L 1.00 g/L Na.sub.2CO.sub.3 0.50 mL 200 g/L 2.00 g/L vitamin solution 9 0.50 mL 4 g/L 0.04 g/L (conc. 100x) trace element solution 5 0.05 mL 21 g/L 0.02 g/L

(63) TABLE-US-00016 TABLE 16 Composition of LSM_3_glycerol_glucose medium. Medium LSM_3_glycerol_glucose Compound Volume/Mass Stock conc. Final conc. Medium 1 MgCO.sub.3 2.5 g.sup. 100% 50.00 g/L Water 37.87 mL Medium 2 Succinic acid 2.5 mL 50 g/L 2.50 g/L Glucose 3.08 mL 650 g/L 40.00 g/L Glycerol 1.50 mL 500 g/L 15.00 g/L (NH.sub.4).sub.2SO.sub.4 0.5 mL 500 g/L 5.00 g/L Betain 0.5 mL 23 g/L 0.23 g/L KH.sub.2PO.sub.4 0.50 mL 100 g/L 1.00 g/L Na.sub.2CO.sub.3 0.50 mL 200 g/L 2.00 g/L vitamin solution 9 0.50 mL 4 g/L 0.04 g/L (conc. 100x) trace element solution 5 0.05 mL 21 g/L 0.02 g/L
2. Cultivations and Analytics

(64) For growing the pre-culture bacteria from a freshly grown BHI-agar plate (incubated overnight at 37 C. under anaerobic conditions) was used to inoculate a 100 ml-serum bottle with gas tight butyl rubber stopper containing 50 ml of the CGM liquid medium described in table 11 with a CO.sub.2-atmosphere. The bottles were incubated at 37 C. and 170 rpm (shaking diameter: 2.5 cm). For growing the main culture 2.5 ml of the bacterial culture in the CGM medium (after 10 hours of incubation) was used to inoculate a 100 ml-serum bottle with gas tight butyl rubber stopper containing 50 ml of the LSM_glucose liquid medium (or the LSM_3_sucrose medium, or the LSM_3_glycerol_glucose medium) described in table 14 (LSM_3_sucrose medium: Table 15, LSM_3_glycerol_glucose medium: Table 16) with a 002-atmosphere. Consumption of C-source and production of carboxylic acids was quantified via HPLC (HPLC methods are described in tables 9 and 10) after 6h and 30h. Cell growth was measured by measuring the absorbance at 600 nm (OD600) using a spectrophotometer (Ultrospec3000, Amersham Biosciences, Uppsala Sweden).

(65) 3. Results

(66) The results of the cultivation experiments with the DD1 IdhA pfID pykA4 strain on glucose, sucrose and maltose/glycerol are shown in tables 17, 18 and 19.

(67) TABLE-US-00017 TABLE 17 Cultivation of the DD1 ldhA pflD strain, and the DD1 ldhA pflD pykA4 strain on glucose. DD1 ldhA DD1 ldhA DD1 ldhA DD1 ldhA pflD pflD pykA4 pflD pflD pykA4 substrate glucose glucose glucose glucose tc [h].sup.a 6 h 6 h 30 h 30 h C [g/L].sup.b 25.2 28.5 51.6 51.6 C [g/L].sup.c 19.2 20.5 38.3 38.2 (succinic acid) C [g/L].sup.c (lactic acid) 0.1 0.1 0.4 0.3 C [g/L].sup.c (formic acid) 0.0 0.0 0.0 0.0 C [g/L].sup.c (acetic acid) 0.8 1.1 2.6 3.1 C [g/L].sup.c (pyruvic acid) 2.1 2.1 1.3 0.5 .sup.acultivation time .sup.bconsumption of substrate (glucose) .sup.cformation of succinic acid, lactic acid, formic acid, acetic acid, pyruvic acid

(68) TABLE-US-00018 TABLE 18 Cultivation of the DD1 ldhA pflD strain, and the DD1 ldhA pflD pykA4 strain on sucrose. DD1 ldhA DD1 ldhA DD1 ldhA DD1 ldhA pflD pflD pykA4 pflD pflD pykA4 substrate sucrose sucrose sucrose sucrose tc [h].sup.a 6 h 6 h 30 h 30 h C [g/L].sup.b 13.9 34.7 32.4 50.5 C [g/L].sup.c 10.6 25.4 19.3 37.4 (succinic acid) C [g/L].sup.c (lactic acid) 0.1 0.1 0.3 0.4 C [g/L].sup.c (formic acid) 0.0 0.0 0.0 0.0 C [g/L].sup.c (acetic acid) 0.1 0.1 0.4 2.0 C [g/L].sup.c (pyruvic acid) 0.0 0.0 0.0 2.2 .sup.acultivation time .sup.bconsumption of substrate (sucrose) .sup.cformation of succinic acid, lactic acid, formic acid, acetic acid, pyruvic acid

(69) TABLE-US-00019 TABLE 19 Cultivation of the DD1 ldhA pflD strain, and the DD1 ldhA pflD pykA4 strain on glycerol and glucose. DD1 ldhA DD1 ldhA DD1 ldhA DD1 ldhA pflD pflD pykA4 pflD pflD pykA4 substrate glycerol/ glycerol/ glycerol/ glycerol/ glucose glucose glucose glucose tc [h].sup.a 6 h 6 h 30 h 30 h C [g/L].sup.b (glycerol) 9.8 1.6 16.0 16.0 C [g/L].sup.b (glucose) 23.9 25.9 37.1 37.1 C [g/L].sup.c 28.8 31.6 46.3 47.5 (succinic acid) C [g/L].sup.c (lactic acid) 0.1 0.1 0.3 0.3 C [g/L].sup.c (formic acid) 0.0 0.0 0.0 0.0 C [g/L].sup.c (acetic acid) 0.5 0.8 1.9 2.1 C [g/L].sup.c (pyruvic acid) 2.0 1.7 0.6 0.2 .sup.acultivation time .sup.bconsumption of substrate (glycerol, glucose) .sup.cformation of succinic acid, lactic acid, formic acid, acetic acid, pyruvic acid

(70) A reduction of the activity of the enzyme encoded by the pykA-gene leads to a faster growth of the cells on glucose, sucrose, and also glycerol/glucose mix. This gets evident when comparing the DD1 IdhA pfID strain with the DD1 IdhA pfID pykA4 strain growing on glucose (Table 17), on sucrose (Table 18), and on glycerol/glucose (Table 19).

(71) TABLE-US-00020 SEQUENCES SEQIDNO:1(nucleotidesequenceof16SrDNAofstrainDD1) tttgatcctggctcagattgaacgctggcggcaggcttaacacatgcaagtcgaacggtagcgggaggaaagcttgctttctttgccga cgagtggcggacgggtgagtaatgcttggggatctggcttatggagggggataacgacgggaaactgtcgctaataccgcgtaatat cttcggattaaagggtgggactttcgggccacccgccataagatgagcccaagtgggattaggtagttggtggggtaaaggcctacc aagccgacgatctctagctggtctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagca gtggggaatattgcacaatggggggaaccctgatgcagccatgccgcgtgaatgaagaaggccttcgggttgtaaagttctttcggtg acgaggaaggtgtttgttttaataggacaagcaattgacgttaatcacagaagaagcaccggctaactccgtgccagcagccgcggt aatacggagggtgcgagcgttaatcggaataactgggcgtaaagggcatgcaggcggacttttaagtgagatgtgaaagccccgg gcttaacctgggaattgcatttcagactgggagtctagagtactttagggaggggtagaattccacgtgtagcggtgaaatgcgtagag atgtggaggaataccgaaggcgaaggcagccccttgggaagatactgacgctcatatgcgaaagcgtggggagcaaacaggatt agataccctggtagtccacgcggtaaacgctgtcgatttggggattgggctttaggcctggtgctcgtagctaacgtgataaatcgacc gcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatg caacgcgaagaaccttacctactcttgacatccagagaatcctgtagagatacgggagtgccttcgggagctctgagacaggtgctg catggctgtcgtcagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcatgtaaagatgg gaactcaaaggagactgccggtgacaaaccggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtagggctaca cacgtgctacaatggtgcatacagagggcggcgataccgcgaggtagagcgaatctcagaaagtgcatcgtagtccggattggagt ctgcaactcgactccatgaagtcggaatcgctagtaatcgcaaatcagaatgttgcggtgaatacgttcccgggccttgtacacaccg cccgtcacaccatgggagtgggttgtaccagaagtagatagcttaaccttcggggggggcgtttaccacggtatgattcatgactggg gtgaagtcgtaacaaggtaaccgtaggggaacctgcgg SEQIDNO:2(nucleotidesequenceof23SrDNAofstrainDD1) agtaataacgaacgacacaggtataagaatacttgaggttgtatggttaagtgactaagcgtacaaggtggatgccttggcaatcaga ggcgaagaaggacgtgctaatctgcgaaaagcttgggtgagttgataagaagcgtctaacccaagatatccgaatggggcaaccc agtagatgaagaatctactatcaataaccgaatccataggttattgaggcaaaccgggagaactgaaacatctaagtaccccgagg aaaagaaatcaaccgagattacgtcagtagcggcgagcgaaagcgtaagagccggcaagtgatagcatgaggattagaggaat cggctgggaagccgggcggcacagggtgatagccccgtacttgaaaatcattgtgtggtactgagcttgcgagaagtagggcggga cacgagaaatcctgtttgaagaaggggggaccatcctccaaggctaaatactcctgattgaccgatagtgaaccagtactgtgaagg aaaggcgaaaagaaccccggtgaggggagtgaaatagaacctgaaaccttgtacgtacaagcagtgggagcccgcgagggtga ctgcgtaccttttgtataatgggtcagcgacttatattatgtagcgaggttaaccgaataggggagccgaagggaaaccgagtcttaact gggcgtcgagttgcatgatatagacccgaaacccggtgatctagccatgggcaggttgaaggttgggtaacactaactggaggacc gaaccgactaatgttgaaaaattagcggatgacctgtggctgggggtgaaaggccaatcaaaccgggagatagctggttctccccg aaatctatttaggtagagccttatgtgaataccttcgggggtagagcactgtttcggctagggggccatcccggcttaccaacccgatgc aaactgcgaataccgaagagtaatgcataggagacacacggcgggtgctaacgttcgtcgtggagagggaaacaacccagacc gccagctaaggtcccaaagtttatattaagtgggaaacgaagtgggaaggcttagacagctaggatgttggcttagaagcagccatc atttaaagaaagcgtaatagctcactagtcgagtcggcctgcgcggaagatgtaacggggctcaaatatagcaccgaagctgcggc atcaggcgtaagcctgttgggtaggggagcgtcgtgtaagcggaagaaggtggttcgagagggctgctggacgtatcacgagtgcg aatgctgacataagtaacgataaaacgggtgaaaaacccgttcgccggaagaccaagggttcctgtccaacgttaatcggggcag ggtgagtcggcccctaaggcgaggctgaagagcgtagtcgatgggaaacgggttaatattcccgtacttgttataattgcgatgtggg gacggagtaggttaggttatcgacctgttggaaaaggtcgtttaagttggtaggtggagcgtttaggcaaatccggacgcttatcaaca ccgagagatgatgacgaggcgctaaggtgccgaagtaaccgataccacacttccaggaaaagccactaagcgtcagattataata aaccgtactataaaccgacacaggtggtcaggtagagaatactcaggcgcttgagagaactcgggtgaaggaactaggcaaaata gcaccgtaacttcgggagaaggtgcgccggcgtagattgtagaggtatacccttgaaggttgaaccggtcgaagtgacccgctggct gcaactgtttattaaaaacacagcactctgcaaacacgaaagtggacgtatagggtgtgatgcctgcccggtgctggaaggttaattg atggcgttatcgcaagagaagcgcctgatcgaagccccagtaaacggcggccgtaactataacggtcctaaggtagcgaaattcctt gtcgggtaagttccgacctgcacgaatggcataatgatggccaggctgtctccacccgagactcagtgaaattgaaatcgccgtgaa gatgcggtgtacccgcggctagacggaaagaccccgtgaacctttactatagcttgacactgaaccttgaattttgatgtgtaggatag gtgggaggctttgaagcggtaacgccagttatcgtggagccatccttgaaataccaccctttaacgtttgatgttctaacgaagtgcccg gaacgggtactcggacagtgtctggtgggtagtttgactggggcggtctcctcccaaagagtaacggaggagcacgaaggtttgcta atgacggtcggacatcgtcaggttagtgcaatggtataagcaagcttaactgcgagacggacaagtcgagcaggtgcgaaagcag gtcatagtgatccggtggttctgaatggaagggccatcgctcaacggataaaaggtactccggggataacaggctgataccgccca agagttcatatcgacggcggtgtttggcacctcgatgtcggctcatcacatcctggggctgaagtaggtcccaagggtatggctgttcgc catttaaagtggtacgcgagctgggtttaaaacgtcgtgagacagtttggtccctatctgccgtgggcgttggagaattgagaggggct gctcctagtacgagaggaccggagtggacgcatcactggtgttccggttgtgtcgccagacgcattgccgggtagctacatgcggaa gagataagtgctgaaagcatctaagcacgaaacttgcctcgagatgagttctcccagtatttaatactgtaagggttgttggagacgac gacgtagataggccgggtgtgtaagcgttgcgagacgttgagctaaccggtactaattgcccgagaggcttagccatacaacgctca agtgtttttggtagtgaaagttattacggaataagtaagtagtcagggaatcggct SEQIDNO:3(nucleotidesequenceofpykA-genefromstrainDD1) atgtccagaagattaagaagaacgaaaatcgtatgtacaatggggcctgcaacagacaaaggcaataatttagaaaaaatcattgc tgccggtgcaaacgttgtacgtatgaacttctcccacggtacgcccgaagatcatatcggtcgtgctgaaaaagtacgtgaaatcgctc ataaattaggtaaacacgtagcaatcttaggtgacttacaaggccctaaaatccgtgtttctacttttaaagaaggcaaaattttcttaaat atcggtgataaattcattttagacgcagagatgcctaaaggtgaaggtaaccaggaagcggttggtttagactataaaacattaccgc aagatgtggttccgggcgatatcttattattagatgacggtcgagttcaattgaaagtattggcaaccgaaggtgcaaaagtattcaccg aagtaacggtcggtggcccactatcaaataataaaggcattaacaaattaggcggcggtttatctgccgatgcattaaccgaaaaag ataaagcggatatcattactgcggcgcgtatcggtgtggattaccttgccgtatctttcccgcgttcaagcgcggatttaaactacgcccg tcaattagcaaaagatgcgggcttggatgcgaaaatcgttgcgaaagtagaacgtgccgaaacagttgaaacggacgaagcaatg gacgatatcatcaatgcggcggacgtaatcatggttgcgcgcggtgacttaggtgttgaaatcggtgatccggaattagtcggtgttcag aaaaaattaatccgtcgttcacgtcagttaaatcgtgttgttattaccgcaactcaaatgatggaatcaatgattagtaatcctatgccgac tcgtgcggaagtaatggacgtagctaacgcagtattggacggtaccgatgcggtaatgctttctgctgaaaccgcggctggtcaatatc cggcggaaactgttgcggcgatggcgaaagttgcgttaggtgcggagaaaatgccaagcattaatgtgtctaaacaccgtatgaacg ttcaattcgagtctattgaagaatctgttgcgatgtctgcaatgtatgcggcaaaccacatgagaggcgtagcggcgattatcacattaa caagtagcggtcgtactgctcgtttaatgtctcgcattagttccggtttaccaatctttgcattgtcacgtaacgaatctacattaaacttatgc gcattatatcgtggtgtgacaccggttcattttgataaagacagccgtacctcagaaggtgcgacagcggcggttcaattattaaaaga cgaaggtttcttagtgtctggcgatttagtgttattaactcagggcgacgcaagcagttctagcggtactaacctttgccgtacattgattgtt gaataa SEQIDNO:4(aminoacidsequenceofPykA fromstrainDD1) MSRRLRRTKIVCTMGPATDKGNNLEKIIAAGANVVRMNFSHGTPEDHIGR AEKVREIAHKLGKHVAILGDLQGPKIRVSTFKEGKIFLNIGDKFILDAEMPK GEGNQEAVGLDYKTLPQDVVPGDILLLDDGRVQLKVLATEGAKVFTEVTV GGPLSNNKGINKLGGGLSADALTEKDKADIITAARIGVDYLAVSFPRSSAD LNYARQLAKDAGLDAKIVAKVERAETVETDEAMDDIINAADVIMVARGDL GVEIGDPELVGVQKKLIRRSRQLNRVVITATQMMESMISNPMPTRAEVMD VANAVLDGTDAVMLSAETAAGQYPAETVAAMAKVALGAEKMPSINVSKH RMNVQFESIEESVAMSAMYAANHMRGVAAIITLTSSGRTARLMSRISSGL PIFALSRNESTLNLCALYRGVTPVHFDKDSRTSEGATAAVQLLKDEGFLV SGDLVLLTQGDASSSSGTNLCRTLIVE SEQIDNO:5(completenucleotidesequenceofplasmidpSacB) tcgagaggcctgacgtcgggcccggtaccacgcgtcatatgactagttcggacctagggatatcgtcgacatcgatgctcttctgcgtt aattaacaattgggatcctctagactccataggccgctttcctggctttgcttccagatgtatgctctcctccggagagtaccgtgactttatt ttcggcacaaatacaggggtcgatggataaatacggcgatagtttcctgacggatgatccgtatgtaccggcggaagacaagctgca aacctgtcagatggagattgatttaatggcggatgtgctgagagcaccgccccgtgaatccgcagaactgatccgctatgtgtttgcgg atgattggccggaataaataaagccgggcttaatacagattaagcccgtatagggtattattactgaataccaaacagcttacggagg acggaatgttacccattgagacaaccagactgccttctgattattaatatttttcactattaatcagaaggaataaccatgaattttacccg gattgacctgaatacctggaatcgcagggaacactttgccctttatcgtcagcagattaaatgcggattcagcctgaccaccaaactcg atattaccgctttgcgtaccgcactggcggagacaggttataagttttatccgctgatgatttacctgatctcccgggctgttaatcagtttcc ggagttccggatggcactgaaagacaatgaacttatttactgggaccagtcagacccggtctttactgtctttcataaagaaaccgaaa cattctctgcactgtcctgccgttattttccggatctcagtgagtttatggcaggttataatgcggtaacggcagaatatcagcatgatacca gattgtttccgcagggaaatttaccggagaatcacctgaatatatcatcattaccgtgggtgagttttgacgggatttaacctgaacatca ccggaaatgatgattattttgccccggtttttacgatggcaaagtttcagcaggaaggtgaccgcgtattattacctgtttctgtacaggttc atcatgcagtctgtgatggctttcatgcagcacggtttattaatacacttcagctgatgtgtgataacatactgaaataaattaattaattctg tatttaagccaccgtatccggcaggaatggtggctttttttttatattttaaccgtaatctgtaatttcgtttcagactggttcaggatgagctcg cttggactcctgttgatagatccagtaatgacctcagaactccatctggatttgttcagaacgctcggttgccgccgggcgttttttattggtg agaatccaagcactagcggcgcgccggccggcccggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcg ctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggtt atccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgc gttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacagg actataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttc tcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcac gaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactgg cagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctaca ctagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaacc accgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacgg ggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaagg ccggccgcggccgccatcggcattttcttttgcgtttttatttgttaactgttaattgtccttgttcaaggatgctgtctttgacaacagatgttttct tgcctttgatgttcagcaggaagctcggcgcaaacgttgattgtttgtctgcgtagaatcctctgtttgtcatatagcttgtaatcacgacatt gtttcctttcgcttgaggtacagcgaagtgtgagtaagtaaaggttacatcgttaggatcaagatccatttttaacacaaggccagttttgtt cagcggcttgtatgggccagttaaagaattagaaacataaccaagcatgtaaatatcgttagacgtaatgccgtcaatcgtcatttttgat ccgcgggagtcagtgaacaggtaccatttgccgttcattttaaagacgttcgcgcgttcaatttcatctgttactgtgttagatgcaatcagc ggtttcatcacttttttcagtgtgtaatcatcgtttagctcaatcataccgagagcgccgtttgctaactcagccgtgcgttttttatcgctttgca gaagtttttgactttcttgacggaagaatgatgtgcttttgccatagtatgctttgttaaataaagattcttcgccttggtagccatcttcagttcc agtgtttgcttcaaatactaagtatttgtggcctttatcttctacgtagtgaggatctctcagcgtatggttgtcgcctgagctgtagttgccttc atcgatgaactgctgtacattttgatacgtttttccgtcaccgtcaaagattgatttataatcctctacaccgttgatgttcaaagagctgtctg atgctgatacgttaacttgtgcagttgtcagtgtttgtttgccgtaatgtttaccggagaaatcagtgtagaataaacggatttttccgtcaga tgtaaatgtggctgaacctgaccattcttgtgtttggtcttttaggatagaatcatttgcatcgaatttgtcgctgtctttaaagacgcggccag cgtttttccagctgtcaatagaagtttcgccgactttttgatagaacatgtaaatcgatgtgtcatccgcatttttaggatctccggctaatgc aaagacgatgtggtagccgtgatagtttgcgacagtgccgtcagcgttttgtaatggccagctgtcccaaacgtccaggccttttgcaga agagatatttttaattgtggacgaatcaaattcagaaacttgatatttttcatttttttgctgttcagggatttgcagcatatcatggcgtgtaata tgggaaatgccgtatgtttccttatatggcttttggttcgtttctttcgcaaacgcttgagttgcgcctcctgccagcagtgcggtagtaaagg ttaatactgttgcttgttttgcaaactttttgatgttcatcgttcatgtctccttttttatgtactgtgttagcggtctgcttcttccagccctcctgtttga agatggcaagttagttacgcacaataaaaaaagacctaaaatatgtaaggggtgacgccaaagtatacactttgccctttacacatttt aggtcttgcctgctttatcagtaacaaacccgcgcgatttacttttcgacctcattctattagactctcgtttggattgcaactggtctattttcct cttttgtttgatagaaaatcataaaaggatttgcagactacgggcctaaagaactaaaaaatctatctgtttcttttcattctctgtattttttata gtttctgttgcatgggcataaagttgcctttttaatcacaattcagaaaatatcataatatctcatttcactaaataatagtgaacggcaggt atatgtgatgggttaaaaaggatcggcggccgctcgatttaaatc SEQIDNO:6(completenucleotidesequenceofplasmidpSacB_delta_ldhA) tcgagaggcctgacgtcgggcccggtaccacgcgtcatatgactagttcggacctagggatgggtcagcctgaacgaaccgcactt gtatgtaggtagttttgaccgcccgaatattcgttataccttggtggaaaaattcaaaccgatggagcaattatacaattttgtggcggcgc aaaaaggtaaaagcggtatcgtctattgcaacagccgtagcaaagtggagcgcattgcggaagccctgaagaaaagaggcatttc cgcagccgcttatcatgcgggcatggagccgtcgcagcgggaagcggtgcaacaggcgtttcaacgggataatattcaagtggtgg tggcgaccattgcttttggtatggggatcaacaaatctaatgtgcgttttgtggcgcattttgatttatctcgcagcattgaggcgtattatcag gaaaccgggcgcgcggggcgggacgacctgccggcggaagcggtactgttttacgagccggcggattatgcctggttgcataaaat tttattggaagagccggaaagcccgcaacgggatattaaacggcataagctggaagccatcggcgaatttgccgaaagccagacc tgccgtcgtttagtgctgttaaattatttcggcgaaaaccgccaaacgccatgtaataactgtgatatctgcctcgatccgccgaaaaaat atgacggattattagacgcgcagaaaatcctttcgaccatttatcgcaccgggcaacgtttcggcacgcaatacgtaatcggcgtaatg cgcggtttgcagaatcagaaaataaaagaaaatcaacatgatgagttgaaagtctacggaattggcaaagataaaagcaaagaat actggcaatcggtaattcgtcagctgattcatttgggctttgtgcaacaaatcatcagcgatttcggcatggggaccagattacagctcac cgaaagcgcgcgtcccgtgctgcgcggcgaagtgtctttggaactggccatgccgagattatcttccattaccatggtacaggctccgc aacgcaatgcggtaaccaactacgacaaagatttatttgcccgcctgcgtttcctgcgcaaacagattgccgacaaagaaaacattc cgccttatattgtgttcagtgacgcgaccttgcaggaaatgtcgttgtatcagccgaccagcaaagtggaaatgctgcaaatcaacggt gtcggcgccatcaaatggcagcgcttcggacagccttttatggcgattattaaagaacatcaggctttgcgtaaagcgggtaagaatc cgttggaattgcaatcttaaaatttttaactttttgaccgcacttttaaggttagcaaattccaataaaaagtgcggtgggttttcgggaattttt aacgcgctgatttcctcgtcttttcaatttyttcgyctccatttgttcggyggttgccggatcctttcttgactgagatccataagagagtagaa tagcgccgcttatatttttaatagcgtacctaatcgggtacgctttttttatgcggaaaatccatatttttctaccgcactttttctttaaagatttat acttaagtctgtttgattcaatttatttggaggttttatgcaacacattcaactggctcccgatttaacattcagtcgcttaattcaaggattctg gcggttaaaaagctggcggaaatcgccgcaggaattgcttacattcgttaagcaaggattagaattaggcgttgatacgctggatcat gccgcttgttacggggcttttacttccgaggcggaattcggacgggcgctggcgctggataaatccttgcgcgcacagcttactttggtg accaaatgcgggattttgtatcctaatgaagaattacccgatataaaatcccatcactatgacaacagctaccgccatattatgtggtcg gcgcaacgttccattgaaaaactgcaatgcgactatttagatgtattgctgattcaccgwctttctccctgtgcggatcccgaacaaatcg cgcgggcttttgatgaactttatcaaaccggraaagtacgttatttcggggtatctaactatacgccggctaagttcgccatgttgcaatctt atgtgaatcagccgttaatcactaatcaaattgagatttcgcctcttcatcgtcaggcttttgatgacggtaccctggattttttactggaaaa acgtattcaaccgatggcatggtcgccacttgccggcggtcgtttattcaatcaggatgagaacagtcgggcggtgcaaaaaacatta ctcgaaatcggtgaaacgaaaggagaaacccgtttagatacattggcttatgcctggttattggcgcatccggcaaaaattatgccggt tatggggtccggtaaaattgaacgggtaaaaagcgcggcggatgcgttacgaatttccttcactgaggaagaatggattaaggtttatg ttgccgcacagggacgggatattccgtaacatcatccgtctaatcctgcgtatctggggaaagatgcgtcatcgtaagaggtctataat attcgtcgttttgataagggtgccatatccggcacccgttaaaatcacattgcgttcgcaacaaaattattccttacgaatagcattcacct cttttaacagatgttgaatatccgtatcggcaaaaatatcctctatatttgcggttaaacggcgccgccagttagcatattgagtgctggttc ccggaatattgacgggttcggtcataccgagccagtcttcaggttggaatccccatcgtcgacatcgatgctcttctgcgttaattaacaa ttgggatcctctagactccataggccgctttcctggctttgcttccagatgtatgctctcctccggagagtaccgtgactttattttcggcaca aatacaggggtcgatggataaatacggcgatagtttcctgacggatgatccgtatgtaccggcggaagacaagctgcaaacctgtca gatggagattgatttaatggcggatgtgctgagagcaccgccccgtgaatccgcagaactgatccgctatgtgtttgcggatgattggc cggaataaataaagccgggcttaatacagattaagcccgtatagggtattattactgaataccaaacagcttacggaggacggaatg ttacccattgagacaaccagactgccttctgattattaatatttttcactattaatcagaaggaataaccatgaattttacccggattgacct gaatacctggaatcgcagggaacactttgccctttatcgtcagcagattaaatgcggattcagcctgaccaccaaactcgatattaccg ctttgcgtaccgcactggcggagacaggttataagttttatccgctgatgatttacctgatctcccgggctgttaatcagtttccggagttcc ggatggcactgaaagacaatgaacttatttactgggaccagtcagacccggtctttactgtctttcataaagaaaccgaaacattctctg cactgtcctgccgttattttccggatctcagtgagtttatggcaggttataatgcggtaacggcagaatatcagcatgataccagattgtttc cgcagggaaatttaccggagaatcacctgaatatatcatcattaccgtgggtgagttttgacgggatttaacctgaacatcaccggaaa tgatgattattttgccccggtttttacgatggcaaagtttcagcaggaaggtgaccgcgtattattacctgtttctgtacaggttcatcatgca gtctgtgatggctttcatgcagcacggtttattaatacacttcagctgatgtgtgataacatactgaaataaattaattaattctgtatttaagc caccgtatccggcaggaatggtggctttttttttatattttaaccgtaatctgtaatttcgtttcagactggttcaggatgagctcgcttggactc ctgttgatagatccagtaatgacctcagaactccatctggatttgttcagaacgctcggttgccgccgggcgttttttattggtgagaatcca agcactagcggcgcgccggccggcccggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgct tcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacaga atcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcg tttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaag ataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgg gaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccc cgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagcc actggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagg acagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggt agcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacg ctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaaggccggccgc ggccgccatcggcattttcttttgcgtttttatttgttaactgttaattgtccttgttcaaggatgctgtctttgacaacagatgttttcttgcctttgat gttcagcaggaagctcggcgcaaacgttgattgtttgtctgcgtagaatcctctgtttgtcatatagcttgtaatcacgacattgtttcctttcg cttgaggtacagcgaagtgtgagtaagtaaaggttacatcgttaggatcaagatccatttttaacacaaggccagttttgttcagcggctt gtatgggccagttaaagaattagaaacataaccaagcatgtaaatatcgttagacgtaatgccgtcaatcgtcatttttgatccgcggga gtcagtgaacaggtaccatttgccgttcattttaaagacgttcgcgcgttcaatttcatctgttactgtgttagatgcaatcagcggtttcatc acttttttcagtgtgtaatcatcgtttagctcaatcataccgagagcgccgtttgctaactcagccgtgcgttttttatcgctttgcagaagttttt gactttcttgacggaagaatgatgtgcttttgccatagtatgctttgttaaataaagattcttcgccttggtagccatcttcagttccagtgtttg cttcaaatactaagtatttgtggcctttatcttctacgtagtgaggatctctcagcgtatggttgtcgcctgagctgtagttgccttcatcgatg aactgctgtacattttgatacgtttttccgtcaccgtcaaagattgatttataatcctctacaccgttgatgttcaaagagctgtctgatgctga tacgttaacttgtgcagttgtcagtgtttgtttgccgtaatgtttaccggagaaatcagtgtagaataaacggatttttccgtcagatgtaaat gtggctgaacctgaccattcttgtgtttggtcttttaggatagaatcatttgcatcgaatttgtcgctgtctttaaagacgcggccagcgtttttc cagctgtcaatagaagtttcgccgactttttgatagaacatgtaaatcgatgtgtcatccgcatttttaggatctccggctaatgcaaagac gatgtggtagccgtgatagtttgcgacagtgccgtcagcgttttgtaatggccagctgtcccaaacgtccaggccttttgcagaagagat atttttaattgtggacgaatcaaattcagaaacttgatatttttcatttttttgctgttcagggatttgcagcatatcatggcgtgtaatatgggaa atgccgtatgtttccttatatggcttttggttcgtttctttcgcaaacgcttgagttgcgcctcctgccagcagtgcggtagtaaaggttaatact gttgcttgttttgcaaactttttgatgttcatcgttcatgtctccttttttatgtactgtgttagcggtctgcttcttccagccctcctgtttgaagatgg caagttagttacgcacaataaaaaaagacctaaaatatgtaaggggtgacgccaaagtatacactttgccctttacacattttaggtctt gcctgctttatcagtaacaaacccgcgcgatttacttttcgacctcattctattagactctcgtttggattgcaactggtctattttcctcttttgttt gatagaaaatcataaaaggatttgcagactacgggcctaaagaactaaaaaatctatctgtttcttttcattctctgtattttttatagtttctgt tgcatgggcataaagttgcctttttaatcacaattcagaaaatatcataatatctcatttcactaaataatagtgaacggcaggtatatgtg atgggttaaaaaggatcggcggccgctcgatttaaatc SEQIDNO:7(completenucleotidesequenceofplasmidpSacB_delta_pflA) tcgagtcaatgcggatttgacttatgatgtggcaaacaaccgatttccgattattactacacgtaaaagttattggaaagcggcgattgcg gagtttctgggttatatccgcggctacgataatgcggcggatttccgtaaattaggagcaaaaacctgggatgccaacgctaatgaaa atcaggtatggctgaataaccctcatcgcaaaggcaccgacgacatggggcgcgtttacggcgtacagggcagagcctggcgtaa gcctaacggcgaaaccgttgatcaattacgcaaaattgtcaacaatttaagtcgcggcattgatgatcgcggcgaaattctgaccttttt aaacccgggcgaattcgatctcggttgtctgcgcccttgtatgtacaatcacacgttttctttgctgggcgatacgctttatttaaccagttat caacgctcctgtgacgtacctttaggcttgaatttcaatcaaattcaagtatttacattcttagctttaatggcgcagattaccggtaaaaaa gccggtcaggcatatcacaaaatcgtcaatgcgcatatttacgaagaccagctggaactaatgcgcgacgtgcagttaaaacgcga accgttcccgtcgccaaaactggaaattaatccggacattaaaacccttgaagatttagaaacctgggtaaccatggatgatttcaacg tcgttggttaccaatgccacgaaccgataaaatatccgttctcggtataaaccgacaaaagtgcggtcaaaaatttaatattttcatctgtt atagaaaatatttttcaacataaaatctagggatgcctgtttggcgtccgtaaatacgcagaaaaatattaaatttttgaccgcacttttttc atctcaattaacagcctgataattcttatggatcaacaaattagctttgacgaaaaaatgatgaatcgagctcttttccttgccgacaagg cggaagctttaggggaaattcccgtaggtgccgtattggtggatgaacggggcaatatcattggtgaaggctggaacctctctattgtg aactcggatcccaccgcccatgccgaaattattgcgttgcgtaacgccgcgcagaaaatccaaaattaccgcctgctcaataccactt tatacgtgactttagaaccctgcaccatgtgcgccggcgcgattttacacagccgaatcaaacgcttggtattcggggcgtccgattac aaaaccggtgcggtgggttccagatttcatttttttgaggattataaaatgaatcatggggttgagatcacaagcggtgtcttacaggatc aatgcagtcagaagttaagccgctttttccaaaagcgcagggaacagaaaaaacaacaaaaagctaccgcacttttacaacaccc ccggcttaactcctctgaaaaatagtgacaaaaaaaccgtcataatgtttacgacggtttttttatttcttaatatgcccttaaataatcaac aaaatatagcaagaagattatagcaaagaatttcgtttttttcagagaatagtcaaatcttcgcaaaaaactaccgcacttttatccgcttt aatcaggggaattaaaacaaaaaaattccgcctattgaggcggaatttattaagcaataagacaaactctcaattacattgattgtgta aacgtacgagtgatgacgtcttgttgttgctctttagttaatgagttgaaacgaaccgcgtaacctgaaacacgaatggttaattgcgggt atttttccggattttccatcgcgtctaacaacatttcacggttaagaacgttaacattcaagtgttgaccgccttccactgtcgcttcatgatg gaaataaccgtccattaaaccggcaaggttgcgtttttgcgcttcgtcatctttacctaatgcgttcggtacgatagagaaggtatatgaa ataccgtctttcgcgtaagcgaacggaagtttagccacagaagtaagtgaagcaaccgcacctttttggtcacgaccgtgcattgggttt gcacccggtccgaatggcgcgcctgctcgacgaccgtccggagtattaccggttttcttaccgtataccacgttagaagtgatagtcag gatagattgtgtcggagttgcgttgcggtaagttttgtgtttttgaacttttttcatgaaacgttcaactaagtctaccgctaaatcatcaacac gcggatcattgttaccgaattgcggatattcgccttcaatttcgaagtcgatagcaacattcgaggccacgacattaccgtctttatctttga tgtcgccgcgaatcggtttaactttcgcatatttgattgcggataatgagtccgcagccacggaaagacccgcgataccgcaagccatt gtacggaatacgtcgcgatcgtggaacgccatcaatgccgcttcatatgcatatttatcgtgcatgaagtggatgatgttcaatgcggtta catattgagtcgccaaccagtccatgaaactgtccatacgttcgattacggtatcgaaattcaatacttcgtctgtaatcggcgcagtttta ggaccgacttgcataccatttttctcatcgataccgccgttaattgcgtataacatagttttagctaagtttgcgcgcgcaccgaagaattg catttgtttacctacgaccatcggtgatacgcagcatgcgattgcatagtcatcgttgttgaagtcaggacgcattaagtcatcattttcgta ttgtacggaggaagtatcaatagatactttcgcacagaaacgtttgaacgcttcaggtaattgttcggaccaaagaatagttaagtttggt tccggagaagtacccatagtgtataaagtatgtaatacgcggaagctgtttttagttaccaacggacgaccgtctaagcccataccggc gatagtttcggttgccctctagactccataggccgctttcctggctttgcttccagatgtatgctctcctccggagagtaccgtgactttattttc ggcacaaatacaggggtcgatggataaatacggcgatagtttcctgacggatgatccgtatgtaccggcggaagacaagctgcaaa cctgtcagatggagattgatttaatggcggatgtgctgagagcaccgccccgtgaatccgcagaactgatccgctatgtgtttgcggat gattggccggaataaataaagccgggcttaatacagattaagcccgtatagggtattattactgaataccaaacagcttacggaggac ggaatgttacccattgagacaaccagactgccttctgattattaatatttttcactattaatcagaaggaataaccatgaattttacccggat tgacctgaatacctggaatcgcagggaacactttgccctttatcgtcagcagattaaatgcggattcagcctgaccaccaaactcgata ttaccgctttgcgtaccgcactggcggagacaggttataagttttatccgctgatgatttacctgatctcccgggctgttaatcagtttccgg agttccggatggcactgaaagacaatgaacttatttactgggaccagtcagacccggtctttactgtctttcataaagaaaccgaaaca ttctctgcactgtcctgccgttattttccggatctcagtgagtttatggcaggttataatgcggtaacggcagaatatcagcatgataccag attgtttccgcagggaaatttaccggagaatcacctgaatatatcatcattaccgtgggtgagttttgacgggatttaacctgaacatcac cggaaatgatgattattttgccccggtttttacgatggcaaagtttcagcaggaaggtgaccgcgtattattacctgtttctgtacaggttcat catgcagtctgtgatggctttcatgcagcacggtttattaatacacttcagctgatgtgtgataacatactgaaataaattaattaattctgta tttaagccaccgtatccggcaggaatggtggctttttttttatattttaaccgtaatctgtaatttcgtttcagactggttcaggatgagctcgctt ggactcctgttgatagatccagtaatgacctcagaactccatctggatttgttcagaacgctcggttgccgccgggcgttttttattggtgag aatccaagcactagcggcgcgccggccggcccggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctc ttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatc cacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgtt gctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggac tataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctc ccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacg aaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggc agcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacac tagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaacca ccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggg gtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaaggc cggccgcggccgccatcggcattttcttttgcgtttttatttgttaactgttaattgtccttgttcaaggatgctgtctttgacaacagatgttttctt gcctttgatgttcagcaggaagctcggcgcaaacgttgattgtttgtctgcgtagaatcctctgtttgtcatatagcttgtaatcacgacattg tttcctttcgcttgaggtacagcgaagtgtgagtaagtaaaggttacatcgttaggatcaagatccatttttaacacaaggccagttttgttc agcggcttgtatgggccagttaaagaattagaaacataaccaagcatgtaaatatcgttagacgtaatgccgtcaatcgtcatttttgatc cgcgggagtcagtgaacaggtaccatttgccgttcattttaaagacgttcgcgcgttcaatttcatctgttactgtgttagatgcaatcagc ggtttcatcacttttttcagtgtgtaatcatcgtttagctcaatcataccgagagcgccgtttgctaactcagccgtgcgttttttatcgctttgca gaagtttttgactttcttgacggaagaatgatgtgcttttgccatagtatgctttgttaaataaagattcttcgccttggtagccatcttcagttcc agtgtttgcttcaaatactaagtatttgtggcctttatcttctacgtagtgaggatctctcagcgtatggttgtcgcctgagctgtagttgccttc atcgatgaactgctgtacattttgatacgtttttccgtcaccgtcaaagattgatttataatcctctacaccgttgatgttcaaagagctgtctg atgctgatacgttaacttgtgcagttgtcagtgtttgtttgccgtaatgtttaccggagaaatcagtgtagaataaacggatttttccgtcaga tgtaaatgtggctgaacctgaccattcttgtgtttggtcttttaggatagaatcatttgcatcgaatttgtcgctgtctttaaagacgcggccag cgtttttccagctgtcaatagaagtttcgccgactttttgatagaacatgtaaatcgatgtgtcatccgcatttttaggatctccggctaatgc aaagacgatgtggtagccgtgatagtttgcgacagtgccgtcagcgttttgtaatggccagctgtcccaaacgtccaggccttttgcaga agagatatttttaattgtggacgaatcaaattcagaaacttgatatttttcatttttttgctgttcagggatttgcagcatatcatggcgtgtaata tgggaaatgccgtatgtttccttatatggcttttggttcgtttctttcgcaaacgcttgagttgcgcctcctgccagcagtgcggtagtaaagg ttaatactgttgcttgttttgcaaactttttgatgttcatcgttcatgtctccttttttatgtactgtgttagcggtctgcttcttccagccctcctgtttga agatggcaagttagttacgcacaataaaaaaagacctaaaatatgtaaggggtgacgccaaagtatacactttgccctttacacatttt aggtcttgcctgctttatcagtaacaaacccgcgcgatttacttttcgacctcattctattagactctcgtttggattgcaactggtctattttcct cttttgtttgatagaaaatcataaaaggatttgcagactacgggcctaaagaactaaaaaatctatctgtttcttttcattctctgtattttttata gtttctgttgcatgggcataaagttgcctttttaatcacaattcagaaaatatcataatatctcatttcactaaataatagtgaacggcaggt atatgtgatgggttaaaaaggatcggcggccgctcgatttaaatc SEQIDNO:8(completenucleotidesequenceofplasmidpSacB_delta_pflD) tcgagaggcctgacgtcgggcccggtaccacgcgtcatatgactagttcggacctagggatgggatcgagctcttttccttgccgaca aggcggaagctttaggggaaattcccgtaggtgccgtattggtggatgaacggggcaatatcattggtgaaggctggaacctctctatt gtgaactcggatcccaccgcccatgccgaaattattgcgttgcgtaacgccgcgcagaaaatccaaaattaccgcctgctcaatacc actttatacgtgactttagaaccctgcaccatgtgcgccggcgcgattttacacagccgaatcaaacgcttggtattcggggcgtccgat tacaaaaccggtgcggtgggttccagatttcatttttttgaggattataaaatgaatcatggggttgagatcacaagcggtgtcttatagga tcaatgcagtcagaagttaagccgctttttccaaaagcgcagggaacagaaaaaacaacaaaaagctaccgcacttttacaacacc cccggcttaactcctctgaaaaatagtgacaaaaaaaccgtcataatgtttacgacggtttttttatttcttctaatatgtcacattaagcccg tagcctgcaagcaaccccttaacatgctccattaattcttttgtcggcggttttacatcttcaagctcgtatttatcgccgagtacttcccattta tgggcgcctagacggtgataaggtaataattccactttttcgatattcttcatatctttaatgaaattccccagcatgtgcaaatcttcgtcact atctgtataacccggcactacaacatggcggatccaggtacgctgatttcgatccgctaaatattttgcgaattcgagcactcttttattcg gcacgccaatcaggctttcgtgaacccgttcattcatttctttcaggtcaagcaacacaagatccgtgtcatcaatcaattcatcaataat atgatcatgatgacggacgaaaccgttggtatccaagcaagtattaattccttctttatggcaggctctgaaccagtcccgtacaaattcc gcctgtaaaatagcttcaccgccggaagcggtaactccgccgcccgaggcgttcataaaatggcgataggtcaccacttctttcattaa ttcttcaacggaaatttctttaccgccgtgcaaatcccaggtgtctctgttatggcaatatttacaacgcattaagcagccttgtaaaaataa aataaagcggattcccggcccgtcaactgtcccgcaggtttcaaatgaatgaattcgtcctaaaaccgacataatatgcccttaaataa tcaacaaaatatagcaagaagattatagcaaagaatttcgtffttttcagagaatagtcaaatcttcgcaaaaaactaccgcacttttatc cgctttaatcaggggaattaaaacaaaaaaattccgcctattgaggcggaatttattaagcaataagacaaactctcaattttaatacttc cttcttttctagtattgataagattgaaaccttgcaaggatgacggcggatttgccgtcactctcacccaactaatgtggacgactggtaa accattgcattagaccaatgcaaacaccaccaccgacgatgttacctaaagtaacaggaattaaatttttaattactaaatggtacatat ctaaatttgcaaactgctcggcatttaaacccgttgcctgccagaattccggcgatgcgaaatttgcaattaccatgcccatagggatca taaacatatttgctacgcagtgttcaaagcctgaagcgacaaayaacccgatcggcaggatcataataaaagctttatccgttagagt yttgccggcataggccatccaaacggcaatacataccataatgttgcaaagaatacctaaacagaaggcttcaayccaggtatgttct attttatgttgtgccgtatttaaaatggttaatccccactgaccgtttgccgccatgatctgaccggaaaaccaaattaatgcaacaataa ataaaccgccgacaaaattaccgaartaaaccacaatccagttacgtaacatctgaattgttgtaattttactctcaaagcgggcaata gtcgataaagttgatgaagtaaatagttcacagccgcaaaccgccaccataattaccccgagagagaacaccaaaccgccgacc agtttagttaatccccaaggcgctcccgcagaggctgtttgagttgttgtataaaaaacgaatgcaagagcaataaacataccggcag agatcgccgataaaaatgaataggcttgttttttcgtagctttataaacgccgacgtctaacccggtttgagccatctcggttggcgaagc catccaagccaatttaaaatcttccgatttcattgagctttccttagtaataaaactactcggaaatgagtagaactgccttaaagcataa atgatagattaaaaaatccaaaattgttgaatattatttaacggggggattataaaagattcataaattagataatagctaatttgagtgat ccatatcaccttttacagattttttgacctaaatcaaaattacccaaatagagtaataataccattataaagggtgtggatttattcctttggttt acgagataaattgctatttaagctgatttctgataaaaagtgcggtagatttttcccaaaaataaggaaacacaaaatggcagaagaa acaattttcagtaaaattattcgtaaagaaattcccgccgacattatatatcaagacgatcttgtcaccgcatttcgcgatattgcgccgca ggcaaaaactcatattttaattattccgaataaattgattccgacagtaaacgacgtaaccgcccatcgtcgacatcgatgctcttctgcg ttaattaacaattgggatcctctagactttgcttccagatgtatgctctcctccggagagtaccgtgactttattttcggcacaaatacaggg gtcgatggataaatacggcgatagtttcctgacggatgatccgtatgtaccggcggaagacaagctgcaaacctgtcagatggagatt gatttaatggcggatgtgctgagagcaccgccccgtgaatccgcagaactgatccgctatgtgtttgcggatgattggccggaataaat aaagccgggcttaatacagattaagcccgtatagggtattattactgaataccaaacagcttacggaggacggaatgttacccattga gacaaccagactgccttctgattattaatatttttcactattaatcagaaggaataaccatgaattttacccggattgacctgaatacctgg aatcgcagggaacactttgccctttatcgtcagcagattaaatgcggattcagcctgaccaccaaactcgatattaccgctttgcgtacc gcactggcggagacaggttataagttttatccgctgatgatttacctgatctcccgggctgttaatcagtttccggagttccggatggcact gaaagacaatgaacttatttactgggaccagtcagacccggtctttactgtctttcataaagaaaccgaaacattctctgcactgtcctgc cgttattttccggatctcagtgagtttatggcaggttataatgcggtaacggcagaatatcagcatgataccagattgtttccgcagggaa atttaccggagaatcacctgaatatatcatcattaccgtgggtgagttttgacgggatttaacctgaacatcaccggaaatgatgattatttt gccccggtttttacgatggcaaagtttcagcaggaaggtgaccgcgtattattacctgtttctgtacaggttcatcatgcagtctgtgatgg ctttcatgcagcacggtttattaatacacttcagctgatgtgtgataacatactgaaataaattaattaattctgtatttaagccaccgtatcc ggcaggaatggtggctttttttttatattttaaccgtaatctgtaatttcgtttcagactggttcaggatgagctcgcttggactcctgttgatag atccagtaatgacctcagaactccatctggatttgttcagaacgctcggttgccgccgggcgttttttattggtgagaatccaagcactag cggcgcgccggccggcccggtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctc actgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggg gataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccat aggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccag gcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgt ggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcag cccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggta acaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtat ttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggt ggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtg gaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaaggccggccgcggccgcc atcggcattttcttttgcgtttttatttgttaactgttaattgtccttgttcaaggatgctgtctttgacaacagatgttttcttgcctttgatgttcagca ggaagctcggcgcaaacgttgattgtttgtctgcgtagaatcctctgtttgtcatatagcttgtaatcacgacattgtttcctttcgcttgaggt acagcgaagtgtgagtaagtaaaggttacatcgttaggatcaagatccatttttaacacaaggccagttttgttcagcggcttgtatggg ccagttaaagaattagaaacataaccaagcatgtaaatatcgttagacgtaatgccgtcaatcgtcatttttgatccgcgggagtcagtg aacaggtaccatttgccgttcattttaaagacgttcgcgcgttcaatttcatctgttactgtgttagatgcaatcagcggtttcatcacttttttca gtgtgtaatcatcgtttagctcaatcataccgagagcgccgtttgctaactcagccgtgcgttttttatcgctttgcagaagtttttgactttcttg acggaagaatgatgtgcttttgccatagtatgctttgttaaataaagattcttcgccttggtagccatcttcagttccagtgtttgcttcaaata ctaagtatttgtggcctttatcttctacgtagtgaggatctctcagcgtatggttgtcgcctgagctgtagttgccttcatcgatgaactgctgt acattttgatacgtttttccgtcaccgtcaaagattgatttataatcctctacaccgttgatgttcaaagagctgtctgatgctgatacgttaac ttgtgcagttgtcagtgtttgtttgccgtaatgtttaccggagaaatcagtgtagaataaacggatttttccgtcagatgtaaatgtggctgaa cctgaccattcttgtgtttggtcttttaggatagaatcatttgcatcgaatttgtcgctgtctttaaagacgcggccagcgtttttccagctgtca atagaagtttcgccgactttttgatagaacatgtaaatcgatgtgtcatccgcatttttaggatctccggctaatgcaaagacgatgtggta gccgtgatagtttgcgacagtgccgtcagcgttttgtaatggccagctgtcccaaacgtccaggccttttgcagaagagatatttttaattg tggacgaatcaaattcagaaacttgatatttttcatttttttgctgttcagggatttgcagcatatcatggcgtgtaatatgggaaatgccgtat gtttccttatatggcttttggttcgtttctttcgcaaacgcttgagttgcgcctcctgccagcagtgcggtagtaaaggttaatactgttgcttgtt ttgcaaactttttgatgttcatcgttcatgtctccttttttatgtactgtgttagcggtctgcttcttccagccctcctgtttgaagatggcaagttagt tacgcacaataaaaaaagacctaaaatatgtaaggggtgacgccaaagtatacactttgccctttacacattttaggtcttgcctgcttta tcagtaacaaacccgcgcgatttacttttcgacctcattctattagactctcgtttggattgcaactggtctattttcctcttttgtttgatagaaa atcataaaaggatttgcagactacgggcctaaagaactaaaaaatctatctgtttcttttcattctctgtattttttatagtttctgttgcatgggc ataaagttgcctttttaatcacaattcagaaaatatcataatatctcatttcactaaataatagtgaacggcaggtatatgtgatgggttaaa aaggatcggcggccgctcgatttaaatc SEQIDNO:9(completenucleotidesequenceofplasmidpSacB_pykA1) tcgagcagaagattaagaagaacgaaaatcgtatgtacaatggggcctgcaacagacaaaggcaataatttagaaaaaatcattg ctgccggtgcaaacgttgtacgtatgaacttctcccacggtacgcccgaagatcatatcggtcgtgctgaaaaagtacgtgaaatcgct cataaattaggtaaacacgtagcaatcttaggtgacttacaaggccctaaaatccgtgtttctacttttaaagaaggcaaaattttcttaaa tatcggtgataaattcattttagacgcagagatgcctaaaggtgaaggtaaccaggaagcggttggtttagactataaaacattaccgc aagatgtggttccgggcgatatcttattattagatgacggtcgagttcaattgaaagtattggcaaccgaaggtgcaaaagtattcaccg aagtaacggtcggtggcccactatcaaataataaaggcattaacaaattaggcggcggtttatctgccgatgcattaaccgaaaaag ataaagcggatatcattactgcggcgcgtatcggtgtggattaccttgccgtatctttcccgcgttcaagcgcggatttaaactacgcccg tcaattagcaaaagatgcgggcttggatgcgaaaatcgttgcgaaagtagaacgtgccgaaacagttgaaacggacgaagcaatg gacgatatcatcaatgcggcggacgtaatcatggttgcgcgcggtgacttaggtgttgaaatcggtgatccggaattagtcggtgttcag aaaaaattaatccgtcgttcacgtcagttaaatcgtgttgttattaccgcaactcaaatgatggaatcaatgattagtaatcctatgccgac tcgtgcggaagtaatggacgtagctaacgcagtattggacggtaccgatgcggtaatgctttctgctgaaaccgcggctggtcaatatc cggcggaaactgttgcggcgatggcgaaagttgcgttaggtgcggagaaaatgccaagcattaatgtgtctaaacaccgtatgaacg ttcaattcgagtctattgaagaatctgttgcgatgtctgcaatgtatgcggcaaaccacatgagaggcgtagcggcgattatcacattaa caagtagcggtcgtactgctcgtttaatgtctagactccataggccgctttcctggctttgcttccagatgtatgctctcctccggagagtac cgtgactttattttcggcacaaatacaggggtcgatggataaatacggcgatagtttcctgacggatgatccgtatgtaccggcggaag acaagctgcaaacctgtcagatggagattgatttaatggcggatgtgctgagagcaccgccccgtgaatccgcagaactgatccgct atgtgtttgcggatgattggccggaataaataaagccgggcttaatacagattaagcccgtatagggtattattactgaataccaaaca gcttacggaggacggaatgttacccattgagacaaccagactgccttctgattattaatatttttcactattaatcagaaggaataaccat gaattttacccggattgacctgaatacctggaatcgcagggaacactttgccctttatcgtcagcagattaaatgcggattcagcctgac caccaaactcgatattaccgctttgcgtaccgcactggcggagacaggttataagttttatccgctgatgatttacctgatctcccgggct gttaatcagtttccggagttccggatggcactgaaagacaatgaacttatttactgggaccagtcagacccggtctttactgtctttcataa agaaaccgaaacattctctgcactgtcctgccgttattttccggatctcagtgagtttatggcaggttataatgcggtaacggcagaatat cagcatgataccagattgtttccgcagggaaatttaccggagaatcacctgaatatatcatcattaccgtgggtgagttttgacgggattt aacctgaacatcaccggaaatgatgattattttgccccggtttttacgatggcaaagtttcagcaggaaggtgaccgcgtattattacctg tttctgtacaggttcatcatgcagtctgtgatggctttcatgcagcacggtttattaatacacttcagctgatgtgtgataacatactgaaata aattaattaattctgtatttaagccaccgtatccggcaggaatggtggctttttttttatattttaaccgtaatctgtaatttcgtttcagactggttc aggatgagctcgcttggactcctgttgatagatccagtaatgacctcagaactccatctggatttgttcagaacgctcggttgccgccgg gcgttttttattggtgagaatccaagcactagcggcgcgccggccggcccggtgtgaaataccgcacagatgcgtaaggagaaaat accgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaag gcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaacc gtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggc gaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccgg atacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaa gctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacac gacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtgg cctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttga tccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagat cctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcac ctagatccttttaaaggccggccgcggccgccatcggcattttcttttgcgtttttatttgttaactgttaattgtccttgttcaaggatgctgtcttt gacaacagatgttttcttgcctttgatgttcagcaggaagctcggcgcaaacgttgattgtttgtctgcgtagaatcctctgtttgtcatatag cttgtaatcacgacattgtttcctttcgcttgaggtacagcgaagtgtgagtaagtaaaggttacatcgttaggatcaagatccatttttaac acaaggccagttttgttcagcggcttgtatgggccagttaaagaattagaaacataaccaagcatgtaaatatcgttagacgtaatgcc gtcaatcgtcatttttgatccgcgggagtcagtgaacaggtaccatttgccgttcattttaaagacgttcgcgcgttcaatttcatctgttactg tgttagatgcaatcagcggtttcatcacttttttcagtgtgtaatcatcgtttagctcaatcataccgagagcgccgtttgctaactcagccgt gcgttttttatcgctttgcagaagtttttgactttcttgacggaagaatgatgtgcttttgccatagtatgctttgttaaataaagattcttcgcctt ggtagccatcttcagttccagtgtttgcttcaaatactaagtatttgtggcctttatcttctacgtagtgaggatctctcagcgtatggttgtcgc ctgagctgtagttgccttcatcgatgaactgctgtacattttgatacgtttttccgtcaccgtcaaagattgatttataatcctctacaccgttga tgttcaaagagctgtctgatgctgatacgttaacttgtgcagttgtcagtgtttgtttgccgtaatgtttaccggagaaatcagtgtagaataa acggatttttccgtcagatgtaaatgtggctgaacctgaccattcttgtgtttggtcttttaggatagaatcatttgcatcgaatttgtcgctgtct ttaaagacgcggccagcgtttttccagctgtcaatagaagtttcgccgactttttgatagaacatgtaaatcgatgtgtcatccgcattttta ggatctccggctaatgcaaagacgatgtggtagccgtgatagtttgcgacagtgccgtcagcgttttgtaatggccagctgtcccaaac gtccaggccttttgcagaagagatatttttaattgtggacgaatcaaattcagaaacttgatatttttcatttttttgctgttcagggatttgcag catatcatggcgtgtaatatgggaaatgccgtatgtttccttatatggcttttggttcgtttctttcgcaaacgcttgagttgcgcctcctgccag cagtgcggtagtaaaggttaatactgttgcttgttttgcaaactttttgatgttcatcgttcatgtctccttttttatgtactgtgttagcggtctgctt cttccagccctcctgtttgaagatggcaagttagttacgcacaataaaaaaagacctaaaatatgtaaggggtgacgccaaagtatac actttgccctttacacattttaggtcttgcctgctttatcagtaacaaacccgcgcgatttacttttcgacctcattctattagactctcgtttggat tgcaactggtctattttcctcttttgtttgatagaaaatcataaaaggatttgcagactacgggcctaaagaactaaaaaatctatctgtttctt ttcattctctgtattttttatagtttctgttgcatgggcataaagttgcctttttaatcacaattcagaaaatatcataatatctcatttcactaaata atagtgaacggcaggtatatgtgatgggttaaaaaggatcggcggccgctcgatttaaatc SEQIDNO:10(completenucleotidesequenceofplasmidpSacB_pykA2) tcgagcagaagattaagaagaacgaaaatcgtatgtacaatggggcctgcaacagacaaaggcaataatttagaaaaaatcattg ctgccggtgcaaacgttgtacgtatgaacttctcccacggtacgcccgaagatcatatcggtcgtgctgaaaaagtacgtgaaatcgct cataaattaggtaaacacgtagcaatcttaggtgacttacaaggccctaaaatccgtgtttctacttttaaagaaggcaaaattttcttaaa tatcggtgataaattcattttagacgcagagatgcctaaaggtgaaggtaaccaggaagcggttggtttagactataaaacattaccgc aagatgtggttccgggcgatatcttattattagatgacggtcgagttcaattgaaagtattggcaaccgaaggtgcaaaagtattcaccg aagtaacggtcggtggcccactatcaaataataaaggcattaacaaattaggctgcggtttatctgccgatgcattaaccgaaaaaga taaagcggatatcattactgcggcgcgtatcggtgtggattaccttgccgtatctttcccgcgttcaagcgcggatttaaactacgcccgt caattagcaaaagatgcgggcttggatgcgaaaatcgttgcgaaagtagaacgtgccgaaacagttgaaacggacgaagcaatg gacgatatcatcaatgcggcggacgtaatcatggttgcgcgcggtgacttaggtgttgaaatcggtgatccggaattagtcggtgttcag aaaaaattaatccgtcgttcacgtcagttaaatcgtgttgttattaccgcaactcaaatgatggaatcaatgattagtaatcctatgccgac tcgtgcggaagtaatggacgtagctaacgcagtattggacggtaccgatgcggtaatgctttctgctgaaaccgcggctggtcaatatc cggcggaaactgttgcggcgatggcgaaagttgcgttaggtgcggagaaaatgccaagcattaatgtgtctaaacaccgtatgaacg ttcaattcgagtctattgaagaatctgttgcgatgtctgcaatgtatgcggcaaaccacatgagaggcgtagcggcgattatcacattaa caagtagcggtcgtactgctcgtttaatgtctagactccataggccgctttcctggctttgcttccagatgtatgctctcctccggagagtac cgtgactttattttcggcacaaatacaggggtcgatggataaatacggcgatagtttcctgacggatgatccgtatgtaccggcggaag acaagctgcaaacctgtcagatggagattgatttaatggcggatgtgctgagagcaccgccccgtgaatccgcagaactgatccgct atgtgtttgcggatgattggccggaataaataaagccgggcttaatacagattaagcccgtatagggtattattactgaataccaaaca gcttacggaggacggaatgttacccattgagacaaccagactgccttctgattattaatatttttcactattaatcagaaggaataaccat gaattttacccggattgacctgaatacctggaatcgcagggaacactttgccctttatcgtcagcagattaaatgcggattcagcctgac caccaaactcgatattaccgctttgcgtaccgcactggcggagacaggttataagttttatccgctgatgatttacctgatctcccgggct gttaatcagtttccggagttccggatggcactgaaagacaatgaacttatttactgggaccagtcagacccggtctttactgtctttcataa agaaaccgaaacattctctgcactgtcctgccgttattttccggatctcagtgagtttatggcaggttataatgcggtaacggcagaatat cagcatgataccagattgtttccgcagggaaatttaccggagaatcacctgaatatatcatcattaccgtgggtgagttttgacgggattt aacctgaacatcaccggaaatgatgattattttgccccggtttttacgatggcaaagtttcagcaggaaggtgaccgcgtattattacctg tttctgtacaggttcatcatgcagtctgtgatggctttcatgcagcacggtttattaatacacttcagctgatgtgtgataacatactgaaata aattaattaattctgtatttaagccaccgtatccggcaggaatggtggctttttttttatattttaaccgtaatctgtaatttcgtttcagactggttc aggatgagctcgcttggactcctgttgatagatccagtaatgacctcagaactccatctggatttgttcagaacgctcggttgccgccgg gcgttttttattggtgagaatccaagcactagcggcgcgccggccggcccggtgtgaaataccgcacagatgcgtaaggagaaaat accgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaag gcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaacc gtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggc gaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccgg atacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaa gctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacac gacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtgg cctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttga tccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagat cctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcac ctagatccttttaaaggccggccgcggccgccatcggcattttcttttgcgtttttatttgttaactgttaattgtccttgttcaaggatgctgtcttt gacaacagatgttttcttgcctttgatgttcagcaggaagctcggcgcaaacgttgattgtttgtctgcgtagaatcctctgtttgtcatatag cttgtaatcacgacattgtttcctttcgcttgaggtacagcgaagtgtgagtaagtaaaggttacatcgttaggatcaagatccatttttaac acaaggccagttttgttcagcggcttgtatgggccagttaaagaattagaaacataaccaagcatgtaaatatcgttagacgtaatgcc gtcaatcgtcatttttgatccgcgggagtcagtgaacaggtaccatttgccgttcattttaaagacgttcgcgcgttcaatttcatctgttactg tgttagatgcaatcagcggtttcatcacttttttcagtgtgtaatcatcgtttagctcaatcataccgagagcgccgtttgctaactcagccgt gcgttttttatcgctttgcagaagtttttgactttcttgacggaagaatgatgtgcttttgccatagtatgctttgttaaataaagattcttcgcctt ggtagccatcttcagttccagtgtttgcttcaaatactaagtatttgtggcctttatcttctacgtagtgaggatctctcagcgtatggttgtcgc ctgagctgtagttgccttcatcgatgaactgctgtacattttgatacgtttttccgtcaccgtcaaagattgatttataatcctctacaccgttga tgttcaaagagctgtctgatgctgatacgttaacttgtgcagttgtcagtgtttgtttgccgtaatgtttaccggagaaatcagtgtagaataa acggatttttccgtcagatgtaaatgtggctgaacctgaccattcttgtgtttggtcttttaggatagaatcatttgcatcgaatttgtcgctgtct ttaaagacgcggccagcgtttttccagctgtcaatagaagtttcgccgactttttgatagaacatgtaaatcgatgtgtcatccgcattttta ggatctccggctaatgcaaagacgatgtggtagccgtgatagtttgcgacagtgccgtcagcgttttgtaatggccagctgtcccaaac gtccaggccttttgcagaagagatatttttaattgtggacgaatcaaattcagaaacttgatatttttcatttttttgctgttcagggatttgcag catatcatggcgtgtaatatgggaaatgccgtatgtttccttatatggcttttggttcgtttctttcgcaaacgcttgagttgcgcctcctgccag cagtgcggtagtaaaggttaatactgttgcttgttttgcaaactttttgatgttcatcgttcatgtctccttttttatgtactgtgttagcggtctgctt cttccagccctcctgtttgaagatggcaagttagttacgcacaataaaaaaagacctaaaatatgtaaggggtgacgccaaagtatac actttgccctttacacattttaggtcttgcctgctttatcagtaacaaacccgcgcgatttacttttcgacctcattctattagactctcgtttggat tgcaactggtctattttcctcttttgtttgatagaaaatcataaaaggatttgcagactacgggcctaaagaactaaaaaatctatctgtttctt ttcattctctgtattttttatagtttctgttgcatgggcataaagttgcctttttaatcacaattcagaaaatatcataatatctcatttcactaaata atagtgaacggcaggtatatgtgatgggttaaaaaggatcggcggccgctcgatttaaatc SEQIDNO:11(completenucleotidesequenceofplasmidpSacB_pykA3) tcgagagcggatatcattactgcggcgcgtatcggtgtggattaccttgccgtatctttcccgcgttcaagcgcggatttaaactacgccc gtcaattagcaaaagatgcgggcttggatgcgaaaatcgttgcgaaagtagaacgtgccgaaacagttgaaacggacgaagcaat ggacgatatcatcaatgcggcggacgtaatcatggttgcgcgcggtgacttaggtgttgaaatcggtgatccggaattagtcggtgttca gaaaaaattaatccgtcgttcacgtcagttaaatcgtgttgttattaccgcaactcaaatgatggaatcaatgattagtaatcctatgccga ctcgtgcggaagtaatggacgtagctaacgcagtattggacggtaccgatgcggtaatgctttctgctgaaaccgcggctggtcaatat ccggcggaaactgttgcggcgatggcgaaagttgcgttaggtgcggagaaaatgccaagcattaatgtgtctaaacaccgtatgaac gttcaattcgagtctattgaagaatctgttgcgatgtctgcaatgtatgcggcaaaccacatgagaggcgtagcggcgattatcacatta acaagtagcggtcgtactgctcgtttaatgtctcgcattagttccggtttaccaatctttgcattgtcacgtaacgaatctacattaaacttatg cgcattatatcgtggtgtgacaccggttcattttgataaagacagccgtacctcagaaggtgcgacagcggcggttcaattattaaaag acgaaggtttcttagtgtctggcgatttagtgttattaactcagggcgacgcaagcagttctagcggtactaacctttgccgtacattgattg ttgaataataggcaatgaacaaaaaaacgatgatttaagtcatcgttttttttttttgcttttctataaaaattcggaaaaatgcaccgcacta tttgtttaacagatcttttaagcccgccttcattgccgacatctggctttcatacaacgctttgctttcccgctcgtcgatcatataggtgatggt ttcggaaagtgtcattttcatctttttcgaatatttggaaaggcgtaaccaaaccccatattctaaatcaatagattttttcttcgtggataattttt ccgcgttaaagaaacgtttacgtctggctcgaatcgcctgatctaatttgataatcaacgattcagccatatgattggctatccattcttcaa ttttttcaggataattctggctttctaataaatcatgcactttgctttgctgcaaactgcgttcttcataacgggtaatattctcgccttcacgatttt ttttaattaaatacaaccatttccaatgcgcttcttgattttctaatttttgatacttcatggataggttctccatctagactccataggccgctttc ctggctttgcttccagatgtatgctctcctccggagagtaccgtgactttattttcggcacaaatacaggggtcgatggataaatacggcg atagtttcctgacggatgatccgtatgtaccggcggaagacaagctgcaaacctgtcagatggagattgatttaatggcggatgtgctg agagcaccgccccgtgaatccgcagaactgatccgctatgtgtttgcggatgattggccggaataaataaagccgggcttaatacag attaagcccgtatagggtattattactgaataccaaacagcttacggaggacggaatgttacccattgagacaaccagactgccttctg attattaatatttttcactattaatcagaaggaataaccatgaattttacccggattgacctgaatacctggaatcgcagggaacactttgc cctttatcgtcagcagattaaatgcggattcagcctgaccaccaaactcgatattaccgctttgcgtaccgcactggcggagacaggtt ataagttttatccgctgatgatttacctgatctcccgggctgttaatcagtttccggagttccggatggcactgaaagacaatgaacttattt actgggaccagtcagacccggtctttactgtctttcataaagaaaccgaaacattctctgcactgtcctgccgttattttccggatctcagtg agtttatggcaggttataatgcggtaacggcagaatatcagcatgataccagattgtttccgcagggaaatttaccggagaatcacctg aatatatcatcattaccgtgggtgagttttgacgggatttaacctgaacatcaccggaaatgatgattattttgccccggtttttacgatggc aaagtttcagcaggaaggtgaccgcgtattattacctgtttctgtacaggttcatcatgcagtctgtgatggctttcatgcagcacggtttatt aatacacttcagctgatgtgtgataacatactgaaataaattaattaattctgtatttaagccaccgtatccggcaggaatggtggcttttttt ttatattttaaccgtaatctgtaatttcgtttcagactggttcaggatgagctcgcttggactcctgttgatagatccagtaatgacctcagaa ctccatctggatttgttcagaacgctcggttgccgccgggcgttttttattggtgagaatccaagcactagcggcgcgccggccggcccg gtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtc gttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaaca tgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacga gcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctcc ctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgc tgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatcc ggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgag gtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaa gccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagca gattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaa gggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaaggccggccgcggccgccatcggcattttcttttgcgttttt atttgttaactgttaattgtccttgttcaaggatgctgtctttgacaacagatgttttcttgcctttgatgttcagcaggaagctcggcgcaaac gttgattgtttgtctgcgtagaatcctctgtttgtcatatagcttgtaatcacgacattgtttcctttcgcttgaggtacagcgaagtgtgagtaa gtaaaggttacatcgttaggatcaagatccatttttaacacaaggccagttttgttcagcggcttgtatgggccagttaaagaattagaaa cataaccaagcatgtaaatatcgttagacgtaatgccgtcaatcgtcatttttgatccgcgggagtcagtgaacaggtaccatttgccgtt cattttaaagacgttcgcgcgttcaatttcatctgttactgtgttagatgcaatcagcggtttcatcacttttttcagtgtgtaatcatcgtttagct caatcataccgagagcgccgtttgctaactcagccgtgcgttttttatcgctttgcagaagtttttgactttcttgacggaagaatgatgtgct tttgccatagtatgctttgttaaataaagattcttcgccttggtagccatcttcagttccagtgtttgcttcaaatactaagtatttgtggcctttat cttctacgtagtgaggatctctcagcgtatggttgtcgcctgagctgtagttgccttcatcgatgaactgctgtacattttgatacgtttttccgt caccgtcaaagattgatttataatcctctacaccgttgatgttcaaagagctgtctgatgctgatacgttaacttgtgcagttgtcagtgtttgt ttgccgtaatgtttaccggagaaatcagtgtagaataaacggatttttccgtcagatgtaaatgtggctgaacctgaccattcttgtgtttgg tcttttaggatagaatcatttgcatcgaatttgtcgctgtctttaaagacgcggccagcgtttttccagctgtcaatagaagtttcgccgacttt ttgatagaacatgtaaatcgatgtgtcatccgcatttttaggatctccggctaatgcaaagacgatgtggtagccgtgatagtttgcgaca gtgccgtcagcgttttgtaatggccagctgtcccaaacgtccaggccttttgcagaagagatatttttaattgtggacgaatcaaattcag aaacttgatatttttcatttttttgctgttcagggatttgcagcatatcatggcgtgtaatatgggaaatgccgtatgtttccttatatggcttttggt tcgtttctttcgcaaacgcttgagttgcgcctcctgccagcagtgcggtagtaaaggttaatactgttgcttgttttgcaaactttttgatgttca tcgttcatgtctccttttttatgtactgtgttagcggtctgcttcttccagccctcctgtttgaagatggcaagttagttacgcacaataaaaaaa gacctaaaatatgtaaggggtgacgccaaagtatacactttgccctttacacattttaggtcttgcctgctttatcagtaacaaacccgcg cgatttacttttcgacctcattctattagactctcgtttggattgcaactggtctattttcctcttttgtttgatagaaaatcataaaaggatttgca gactacgggcctaaagaactaaaaaatctatctgtttcttttcattctctgtattttttatagtttctgttgcatgggcataaagttgcctttttaatc acaattcagaaaatatcataatatctcatttcactaaataatagtgaacggcaggtatatgtgatgggttaaaaaggatcggcggccgc tcgatttaaatc SEQIDNO:12(completenucleotidesequenceofplasmidpSacB_pykA4) tcgagagcggatatcattactgcggcgcgtatcggtgtggattaccttgccgtatctttcccgcgttcaagcgcggatttaaactacgccc gtcaattagcaaaagatgcgggcttggatgcgaaaatcgttgcgaaagtagaacgtgccgaaacagttgaaacggacgaagcaat ggacgatatcatcaatgcggcggacgtaatcatggttgcgcgcggtgacttaggtgttgaaatcggtgatccggaattagtcggtgttca gaaaaaattaatccgtcgttcacgtcagttaaatcgtgttgttattaccgcaactcaaatgatggaatcaatgattagtaatcctatgccga ctcgtgcggaagtaatggacgtagctaacgcagtattggacggtaccgatgcggtaatgctttctgctgaaaccgcggctggtcaatat ccggcggaaactgttgcggcgatggcgaaagttgcgttaggtgcggagaaaatgccaagcattaatgtgtctaaacaccgtatgaac gttcaattcgagtctattgaagaatctgttgcgatgtctgcaatgtatgcggcaaaccacatgagaggcgtagcggcgattatcacatta acaagtagcggtcgtactgctcgtttaatgtctcgcattagttccggtttaccaatctttgcattgtcacgtaacgaatctactttaaacttata cgcattatatcgtggtgtgacaccggttcattttgataaagacagccgtacctcagaaggtgcgacagcggcggttcaattattaaaag acgaaggtttcttagtgtctggcgatttagtgttattaactcagggcgacgcaagcagttctagcggtactaacctttgccgtacattgattg ttgaataataggcaatgaacaaaaaaacgatgatttaagtcatcgttttttttttttgcttttctataaaaattcggaaaaatgcaccgcacta tttgtttaacagatcttttaagcccgccttcattgccgacatctggctttcatacaacgctttgctttcccgctcgtcgatcatataggtgatggt ttcggaaagtgtcattttcatctttttcgaatatttggaaaggcgtaaccaaaccccatattctaaatcaatagattttttcttcgtggataattttt ccgcgttaaagaaacgtttacgtctggctcgaatcgcctgatctaatttgataatcaacgattcagccatatgattggctatccattcttcaa ttttttcaggataattctggctttctaataaatcatgcactttgctttgctgcaaactgcgttcttcataacgggtaatattctcgccttcacgatttt ttttaattaaatacaaccatttccaatgcgcttcttgattttctaatttttgatacttcatggataggttctccatctagactccataggccgctttc ctggctttgcttccagatgtatgctctcctccggagagtaccgtgactttattttcggcacaaatacaggggtcgatggataaatacggcg atagtttcctgacggatgatccgtatgtaccggcggaagacaagctgcaaacctgtcagatggagattgatttaatggcggatgtgctg agagcaccgccccgtgaatccgcagaactgatccgctatgtgtttgcggatgattggccggaataaataaagccgggcttaatacag attaagcccgtatagggtattattactgaataccaaacagcttacggaggacggaatgttacccattgagacaaccagactgccttctg attattaatatttttcactattaatcagaaggaataaccatgaattttacccggattgacctgaatacctggaatcgcagggaacactttgc cctttatcgtcagcagattaaatgcggattcagcctgaccaccaaactcgatattaccgctttgcgtaccgcactggcggagacaggtt ataagttttatccgctgatgatttacctgatctcccgggctgttaatcagtttccggagttccggatggcactgaaagacaatgaacttattt actgggaccagtcagacccggtctttactgtctttcataaagaaaccgaaacattctctgcactgtcctgccgttattttccggatctcagtg agtttatggcaggttataatgcggtaacggcagaatatcagcatgataccagattgtttccgcagggaaatttaccggagaatcacctg aatatatcatcattaccgtgggtgagttttgacgggatttaacctgaacatcaccggaaatgatgattattttgccccggtttttacgatggc aaagtttcagcaggaaggtgaccgcgtattattacctgtttctgtacaggttcatcatgcagtctgtgatggctttcatgcagcacggtttatt aatacacttcagctgatgtgtgataacatactgaaataaattaattaattctgtatttaagccaccgtatccggcaggaatggtggcttttttt ttatattttaaccgtaatctgtaatttcgtttcagactggttcaggatgagctcgcttggactcctgttgatagatccagtaatgacctcagaa ctccatctggatttgttcagaacgctcggttgccgccgggcgttttttattggtgagaatccaagcactagcggcgcgccggccggcccg gtgtgaaataccgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtc gttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaaca tgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacga gcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctcc ctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgc tgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatcc ggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgag gtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaa gccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagca gattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaa gggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaaggccggccgcggccgccatcggcattttcttttgcgttttt atttgttaactgttaattgtccttgttcaaggatgctgtctttgacaacagatgttttcttgcctttgatgttcagcaggaagctcggcgcaaac gttgattgtttgtctgcgtagaatcctctgtttgtcatatagcttgtaatcacgacattgtttcctttcgcttgaggtacagcgaagtgtgagtaa gtaaaggttacatcgttaggatcaagatccatttttaacacaaggccagttttgttcagcggcttgtatgggccagttaaagaattagaaa cataaccaagcatgtaaatatcgttagacgtaatgccgtcaatcgtcatttttgatccgcgggagtcagtgaacaggtaccatttgccgtt cattttaaagacgttcgcgcgttcaatttcatctgttactgtgttagatgcaatcagcggtttcatcacttttttcagtgtgtaatcatcgtttagct caatcataccgagagcgccgtttgctaactcagccgtgcgttttttatcgctttgcagaagtttttgactttcttgacggaagaatgatgtgct tttgccatagtatgctttgttaaataaagattcttcgccttggtagccatcttcagttccagtgtttgcttcaaatactaagtatttgtggcctttat cttctacgtagtgaggatctctcagcgtatggttgtcgcctgagctgtagttgccttcatcgatgaactgctgtacattttgatacgtttttccgt caccgtcaaagattgatttataatcctctacaccgttgatgttcaaagagctgtctgatgctgatacgttaacttgtgcagttgtcagtgtttgt ttgccgtaatgtttaccggagaaatcagtgtagaataaacggatttttccgtcagatgtaaatgtggctgaacctgaccattcttgtgtttgg tcttttaggatagaatcatttgcatcgaatttgtcgctgtctttaaagacgcggccagcgtttttccagctgtcaatagaagtttcgccgacttt ttgatagaacatgtaaatcgatgtgtcatccgcatttttaggatctccggctaatgcaaagacgatgtggtagccgtgatagtttgcgaca gtgccgtcagcgttttgtaatggccagctgtcccaaacgtccaggccttttgcagaagagatatttttaattgtggacgaatcaaattcag aaacttgatatttttcatttttttgctgttcagggatttgcagcatatcatggcgtgtaatatgggaaatgccgtatgtttccttatatggcttttggt tcgtttctttcgcaaacgcttgagttgcgcctcctgccagcagtgcggtagtaaaggttaatactgttgcttgttttgcaaactttttgatgttca tcgttcatgtctccttttttatgtactgtgttagcggtctgcttcttccagccctcctgtttgaagatggcaagttagttacgcacaataaaaaaa gacctaaaatatgtaaggggtgacgccaaagtatacactttgccctttacacattttaggtcttgcctgctttatcagtaacaaacccgcg cgatttacttttcgacctcattctattagactctcgtttggattgcaactggtctattttcctcttttgtttgatagaaaatcataaaaggatttgca gactacgggcctaaagaactaaaaaatctatctgtttcttttcattctctgtattttttatagtttctgttgcatgggcataaagttgcctttttaatc acaattcagaaaatatcataatatctcatttcactaaataatagtgaacggcaggtatatgtgatgggttaaaaaggatcggcggccgc tcgatttaaatc SEQIDNO:13(completenucleotidsequenceofplasmidpSacB_pykA5) tcgagcagaagattaagaagaacgaaaatcgtatgtacaatggggcctgcaacagacaaaggcaataatttagaaaaaatcattg ctgccggtgcaaacgttgtacgtatgaacttctcccacggtacgcccgaagatcatatcggtcgtgctgaaaaagtacgtgaaatcgct cataaattaggtaaacacgtagcaatcttaggtgacttacaaggccctaaaatccgtgtttctacttttaaagaaggcaaaattttcttaaa tatcggtgataaattcattttagacgcagagatgcctaaaggtgaaggtaaccaggaagcggttggtttagactataaaacattaccgc aagatgtggttccgggcgatatcttattattagatgacggtcgagttcaattgaaagtattggcaaccgaaggtgcaaaagtattcaccg aagtaacggtcggtggcccactatcaaataataaaggcattaacaaattaggcggcggtttatctggcgatgcgttaaccgaaaaag ataaagcggatatcattactgcggcgcgtatcggtgtggattaccttgccgtatctttcccgcgttcaagcgcggatttaaactacgcccg tcaattagcaaaagatgcgggcttggatgcgaaaatcgttgcgaaagtagaacgtgccgaaacagttgaaacggacgaagcaatg gacgatatcatcaatgcggcggacgtaatcatggttgcgcgcggtgacttaggtgttgaaatcggtgatccggaattagtcggtgttcag aaaaaattaatccgtcgttcacgtcagttaaatcgtgttgttattaccgcaactcaaatgatggaatcaatgattagtaatcctatgccgac tcgtgcggaagtaatggacgtagctaacgcagtattggacggtaccgatgcggtaatgctttctgctgaaaccgcggctggtcaatatc cggcggaaactgttgcggcgatggcgaaagttgcgttaggtgcggagaaaatgccaagcattaatgtgtctaaacaccgtatgaacg ttcaattcgagtctattgaagaatctgttgcgatgtctgcaatgtatgcggcaaaccacatgagaggcgtagcggcgattatcacattaa caagtagcggtcgtactgctcgtttaatgtctagactccataggccgctttcctggctttgcttccagatgtatgctctcctccggagagtac cgtgactttattttcggcacaaatacaggggtcgatggataaatacggcgatagtttcctgacggatgatccgtatgtaccggcggaag acaagctgcaaacctgtcagatggagattgatttaatggcggatgtgctgagagcaccgccccgtgaatccgcagaactgatccgct atgtgtttgcggatgattggccggaataaataaagccgggcttaatacagattaagcccgtatagggtattattactgaataccaaaca gcttacggaggacggaatgttacccattgagacaaccagactgccttctgattattaatatttttcactattaatcagaaggaataaccat gaattttacccggattgacctgaatacctggaatcgcagggaacactttgccctttatcgtcagcagattaaatgcggattcagcctgac caccaaactcgatattaccgctttgcgtaccgcactggcggagacaggttataagttttatccgctgatgatttacctgatctcccgggct gttaatcagtttccggagttccggatggcactgaaagacaatgaacttatttactgggaccagtcagacccggtctttactgtctttcataa agaaaccgaaacattctctgcactgtcctgccgttattttccggatctcagtgagtttatggcaggttataatgcggtaacggcagaatat cagcatgataccagattgtttccgcagggaaatttaccggagaatcacctgaatatatcatcattaccgtgggtgagttttgacgggattt aacctgaacatcaccggaaatgatgattattttgccccggtttttacgatggcaaagtttcagcaggaaggtgaccgcgtattattacctg tttctgtacaggttcatcatgcagtctgtgatggctttcatgcagcacggtttattaatacacttcagctgatgtgtgataacatactgaaata aattaattaattctgtatttaagccaccgtatccggcaggaatggtggctttttttttatattttaaccgtaatctgtaatttcgtttcagactggttc aggatgagctcgcttggactcctgttgatagatccagtaatgacctcagaactccatctggatttgttcagaacgctcggttgccgccgg gcgttttttattggtgagaatccaagcactagcggcgcgccggccggcccggtgtgaaataccgcacagatgcgtaaggagaaaat accgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaag gcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaacc gtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggc gaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccgg atacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaa gctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacac gacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtgg cctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttga tccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagat cctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcac ctagatccttttaaaggccggccgcggccgccatcggcattttcttttgcgtttttatttgttaactgttaattgtccttgttcaaggatgctgtcttt gacaacagatgttttcttgcctttgatgttcagcaggaagctcggcgcaaacgttgattgtttgtctgcgtagaatcctctgtttgtcatatag cttgtaatcacgacattgtttcctttcgcttgaggtacagcgaagtgtgagtaagtaaaggttacatcgttaggatcaagatccatttttaac acaaggccagttttgttcagcggcttgtatgggccagttaaagaattagaaacataaccaagcatgtaaatatcgttagacgtaatgcc gtcaatcgtcatttttgatccgcgggagtcagtgaacaggtaccatttgccgttcattttaaagacgttcgcgcgttcaatttcatctgttactg tgttagatgcaatcagcggtttcatcacttttttcagtgtgtaatcatcgtttagctcaatcataccgagagcgccgtttgctaactcagccgt gcgttttttatcgctttgcagaagtttttgactttcttgacggaagaatgatgtgcttttgccatagtatgctttgttaaataaagattcttcgcctt ggtagccatcttcagttccagtgtttgcttcaaatactaagtatttgtggcctttatcttctacgtagtgaggatctctcagcgtatggttgtcgc ctgagctgtagttgccttcatcgatgaactgctgtacattttgatacgtttttccgtcaccgtcaaagattgatttataatcctctacaccgttga tgttcaaagagctgtctgatgctgatacgttaacttgtgcagttgtcagtgtttgtttgccgtaatgtttaccggagaaatcagtgtagaataa acggatttttccgtcagatgtaaatgtggctgaacctgaccattcttgtgtttggtcttttaggatagaatcatttgcatcgaatttgtcgctgtct ttaaagacgcggccagcgtttttccagctgtcaatagaagtttcgccgactttttgatagaacatgtaaatcgatgtgtcatccgcattttta ggatctccggctaatgcaaagacgatgtggtagccgtgatagtttgcgacagtgccgtcagcgttttgtaatggccagctgtcccaaac gtccaggccttttgcagaagagatatttttaattgtggacgaatcaaattcagaaacttgatatttttcatttttttgctgttcagggatttgcag catatcatggcgtgtaatatgggaaatgccgtatgtttccttatatggcttttggttcgtttctttcgcaaacgcttgagttgcgcctcctgccag cagtgcggtagtaaaggttaatactgttgcttgttttgcaaactttttgatgttcatcgttcatgtctccttttttatgtactgtgttagcggtctgctt cttccagccctcctgtttgaagatggcaagttagttacgcacaataaaaaaagacctaaaatatgtaaggggtgacgccaaagtatac actttgccctttacacattttaggtcttgcctgctttatcagtaacaaacccgcgcgatttacttttcgacctcattctattagactctcgtttggat tgcaactggtctattttcctcttttgtttgatagaaaatcataaaaggatttgcagactacgggcctaaagaactaaaaaatctatctgtttctt ttcattctctgtattttttatagtttctgttgcatgggcataaagttgcctttttaatcacaattcagaaaatatcataatatctcatttcactaaata atagtgaacggcaggtatatgtgatgggttaaaaaggatcggcggccgctcgatttaaatc SEQIDNO:14(completenucleotidesequenceofplasmidpSacB_pykA6) tcgagcagaagattaagaagaacgaaaatcgtatgtacaatggggcctgcaacagacaaaggcaataatttagaaaaaatcattg ctgccggtgcaaacgttgtacgtatgaacttctcccacggtacgcccgaagatcatatcggtcgtgctgaaaaagtacgtgaaatcgct cataaattaggtaaacacgtagcaatcttaggtgacttacaaggccctaaaatccgtgtttctacttttaaagaaggcaaaattttcttaaa tatcggtgataaattcattttagacgcagagatgcctaaaggtgaaggtaaccaggaagcggttggtttagactataaaacattaccgc aagatgtggttccgggcgatatcttattattagatgacggtcgagttcaattgaaagtattggcaaccgaaggtgcaaaagtattcaccg aagtaacggtcggtggcccactatcaaataataaaggcattaacaaattaggctgcggtttatctgccgatgcgttaaccgaaaaaga taaagcggatatcattactgcggcgcgtatcggtgtggattaccttgccgtatctttcccgcgttcaagcgcggatttaaactacgcccgt caattagcaaaagatgcgggcttggatgcgaaaatcgttgcgaaagtagaacgtgccgaaacagttgaaacggacgaagcaatg gacgatatcatcaatgcggcggacgtaatcatggttgcgcgcggtgacttaggtgttgaaatcggtgatccggaattagtcggtgttcag aaaaaattaatccgtcgttcacgtcagttaaatcgtgttgttattaccgcaactcaaatgatggaatcaatgattagtaatcctatgccgac tcgtgcggaagtaatggacgtagctaacgcagtattggacggtaccgatgcggtaatgctttctgctgaaaccgcggctggtcaatatc cggcggaaactgttgcggcgatggcgaaagttgcgttaggtgcggagaaaatgccaagcattaatgtgtctaaacaccgtatgaacg ttcaattcgagtctattgaagaatctgttgcgatgtctgcaatgtatgcggcaaaccacatgagaggcgtagcggcgattatcacattaa caagtagcggtcgtactgctcgtttaatgtctagactccataggccgctttcctggctttgcttccagatgtatgctctcctccggagagtac cgtgactttattttcggcacaaatacaggggtcgatggataaatacggcgatagtttcctgacggatgatccgtatgtaccggcggaag acaagctgcaaacctgtcagatggagattgatttaatggcggatgtgctgagagcaccgccccgtgaatccgcagaactgatccgct atgtgtttgcggatgattggccggaataaataaagccgggcttaatacagattaagcccgtatagggtattattactgaataccaaaca gcttacggaggacggaatgttacccattgagacaaccagactgccttctgattattaatatttttcactattaatcagaaggaataaccat gaattttacccggattgacctgaatacctggaatcgcagggaacactttgccctttatcgtcagcagattaaatgcggattcagcctgac caccaaactcgatattaccgctttgcgtaccgcactggcggagacaggttataagttttatccgctgatgatttacctgatctcccgggct gttaatcagtttccggagttccggatggcactgaaagacaatgaacttatttactgggaccagtcagacccggtctttactgtctttcataa agaaaccgaaacattctctgcactgtcctgccgttattttccggatctcagtgagtttatggcaggttataatgcggtaacggcagaatat cagcatgataccagattgtttccgcagggaaatttaccggagaatcacctgaatatatcatcattaccgtgggtgagttttgacgggattt aacctgaacatcaccggaaatgatgattattttgccccggtttttacgatggcaaagtttcagcaggaaggtgaccgcgtattattacctg tttctgtacaggttcatcatgcagtctgtgatggctttcatgcagcacggtttattaatacacttcagctgatgtgtgataacatactgaaata aattaattaattctgtatttaagccaccgtatccggcaggaatggtggctttttttttatattttaaccgtaatctgtaatttcgtttcagactggttc aggatgagctcgcttggactcctgttgatagatccagtaatgacctcagaactccatctggatttgttcagaacgctcggttgccgccgg gcgttttttattggtgagaatccaagcactagcggcgcgccggccggcccggtgtgaaataccgcacagatgcgtaaggagaaaat accgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaag gcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaacc gtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggc gaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccgg atacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaa gctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacac gacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtgg cctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttga tccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagat cctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcac ctagatccttttaaaggccggccgcggccgccatcggcattttcttttgcgtttttatttgttaactgttaattgtccttgttcaaggatgctgtcttt gacaacagatgttttcttgcctttgatgttcagcaggaagctcggcgcaaacgttgattgtttgtctgcgtagaatcctctgtttgtcatatag cttgtaatcacgacattgtttcctttcgcttgaggtacagcgaagtgtgagtaagtaaaggttacatcgttaggatcaagatccatttttaac acaaggccagttttgttcagcggcttgtatgggccagttaaagaattagaaacataaccaagcatgtaaatatcgttagacgtaatgcc gtcaatcgtcatttttgatccgcgggagtcagtgaacaggtaccatttgccgttcattttaaagacgttcgcgcgttcaatttcatctgttactg tgttagatgcaatcagcggtttcatcacttttttcagtgtgtaatcatcgtttagctcaatcataccgagagcgccgtttgctaactcagccgt gcgttttttatcgctttgcagaagtttttgactttcttgacggaagaatgatgtgcttttgccatagtatgctttgttaaataaagattcttcgcctt ggtagccatcttcagttccagtgtttgcttcaaatactaagtatttgtggcctttatcttctacgtagtgaggatctctcagcgtatggttgtcgc ctgagctgtagttgccttcatcgatgaactgctgtacattttgatacgtttttccgtcaccgtcaaagattgatttataatcctctacaccgttga tgttcaaagagctgtctgatgctgatacgttaacttgtgcagttgtcagtgtttgtttgccgtaatgtttaccggagaaatcagtgtagaataa acggatttttccgtcagatgtaaatgtggctgaacctgaccattcttgtgtttggtcttttaggatagaatcatttgcatcgaatttgtcgctgtct ttaaagacgcggccagcgtttttccagctgtcaatagaagtttcgccgactttttgatagaacatgtaaatcgatgtgtcatccgcattttta ggatctccggctaatgcaaagacgatgtggtagccgtgatagtttgcgacagtgccgtcagcgttttgtaatggccagctgtcccaaac gtccaggccttttgcagaagagatatttttaattgtggacgaatcaaattcagaaacttgatatttttcatttttttgctgttcagggatttgcag catatcatggcgtgtaatatgggaaatgccgtatgtttccttatatggcttttggttcgtttctttcgcaaacgcttgagttgcgcctcctgccag cagtgcggtagtaaaggttaatactgttgcttgttttgcaaactttttgatgttcatcgttcatgtctccttttttatgtactgtgttagcggtctgctt cttccagccctcctgtttgaagatggcaagttagttacgcacaataaaaaaagacctaaaatatgtaaggggtgacgccaaagtatac actttgccctttacacattttaggtcttgcctgctttatcagtaacaaacccgcgcgatttacttttcgacctcattctattagactctcgtttggat tgcaactggtctattttcctcttttgtttgatagaaaatcataaaaggatttgcagactacgggcctaaagaactaaaaaatctatctgtttctt ttcattctctgtattttttatagtttctgttgcatgggcataaagttgcctttttaatcacaattcagaaaatatcataatatctcatttcactaaata atagtgaacggcaggtatatgtgatgggttaaaaaggatcggcggccgctcgatttaaatc SEQIDNO:15(nucleotidesequenceofthepykA-genefromtheDD1ldhA pflApykA2-strain) atgtccagaagattaagaagaacgaaaatcgtatgtacaatggggcctgcaacagacaaaggcaataatttagaaaaaatcattgc tgccggtgcaaacgttgtacgtatgaacttctcccacggtacgcccgaagatcatatcggtcgtgctgaaaaagtacgtgaaatcgctc ataaattaggtaaacacgtagcaatcttaggtgacttacaaggccctaaaatccgtgtttctacttttaaagaaggcaaaattttcttaaat atcggtgataaattcattttagacgcagagatgcctaaaggtgaaggtaaccaggaagcggttggtttagactataaaacattaccgc aagatgtggttccgggcgatatcttattattagatgacggtcgagttcaattgaaagtattggcaaccgaaggtgcaaaagtattcaccg aagtaacggtcggtggcccactatcaaataataaaggcattaacaaattaggcTgcggtttatctgccgatgcattaaccgaaaaag ataaagcggatatcattactgcggcgcgtatcggtgtggattaccttgccgtatctttcccgcgttcaagcgcggatttaaactacgcccg tcaattagcaaaagatgcgggcttggatgcgaaaatcgttgcgaaagtagaacgtgccgaaacagttgaaacggacgaagcaatg gacgatatcatcaatgcggcggacgtaatcatggttgcgcgcggtgacttaggtgttgaaatcggtgatccggaattagtcggtgttcag aaaaaattaatccgtcgttcacgtcagttaaatcgtgttgttattaccgcaactcaaatgatggaatcaatgattagtaatcctatgccgac tcgtgcggaagtaatggacgtagctaacgcagtattggacggtaccgatgcggtaatgctttctgctgaaaccgcggctggtcaatatc cggcggaaactgttgcggcgatggcgaaagttgcgttaggtgcggagaaaatgccaagcattaatgtgtctaaacaccgtatgaacg ttcaattcgagtctattgaagaatctgttgcgatgtctgcaatgtatgcggcaaaccacatgagaggcgtagcggcgattatcacattaa caagtagcggtcgtactgctcgtttaatgtctcgcattagttccggtttaccaatctttgcattgtcacgtaacgaatctacattaaacttatgc gcattatatcgtggtgtgacaccggttcattttgataaagacagccgtacctcagaaggtgcgacagcggcggttcaattattaaaaga cgaaggtttcttagtgtctggcgatttagtgttattaactcagggcgacgcaagcagttctagcggtactaacctttgccgtacattgattgtt gaataa SEQIDNO:16(aminoacidsequenceofPykA2fromtheDD1 ldhApflApykA2-strain) MSRRLRRTKIVCTMGPATDKGNNLEKIIAAGANVVRMNFSHGTPEDHIGRAEKVREIAHKLGKH VAILGDLQGPKIRVSTFKEGKIFLNIGDKFILDAEMPKGEGNQEAVGLDYKTLPQDVVPGDILLLD DGRVQLKVLATEGAKVFTEVTVGGPLSNNKGINKLGCGLSADALTEKDKADIITAARIGVDYLAV SFPRSSADLNYARQLAKDAGLDAKIVAKVERAETVETDEAMDDIINAADVIMVARGDLGVEIGDP ELVGVQKKLIRRSRQLNRVVITATQMMESMISNPMPTRAEVMDVANAVLDGTDAVMLSAETAA GQYPAETVAAMAKVALGAEKMPSINVSKHRMNVQFESIEESVAMSAMYAANHMRGVAAIITLT SSGRTARLMSRISSGLPIFALSRNESTLNLCALYRGVTPVHFDKDSRTSEGATAAVQLLKDEGF LVSGDLVLLTQGDASSSSGTNLCRTLIVE SEQIDNO:17(nucleotidesequenceofthepykA-genefromtheDD1ldhApflDpykA4- strain) atgtccagaagattaagaagaacgaaaatcgtatgtacaatggggcctgcaacagacaaaggcaataatttagaaaaaatcattgc tgccggtgcaaacgttgtacgtatgaacttctcccacggtacgcccgaagatcatatcggtcgtgctgaaaaagtacgtgaaatcgctc ataaattaggtaaacacgtagcaatcttaggtgacttacaaggccctaaaatccgtgtttctacttttaaagaaggcaaaattttcttaaat atcggtgataaattcattttagacgcagagatgcctaaaggtgaaggtaaccaggaagcggttggtttagactataaaacattaccgc aagatgtggttccgggcgatatcttattattagatgacggtcgagttcaattgaaagtattggcaaccgaaggtgcaaaagtattcaccg aagtaacggtcggtggcccactatcaaataataaaggcattaacaaattaggcggcggtttatctgccgatgcattaaccgaaaaag ataaagcggatatcattactgcggcgcgtatcggtgtggattaccttgccgtatctttcccgcgttcaagcgcggatttaaactacgcccg tcaattagcaaaagatgcgggcttggatgcgaaaatcgttgcgaaagtagaacgtgccgaaacagttgaaacggacgaagcaatg gacgatatcatcaatgcggcggacgtaatcatggttgcgcgcggtgacttaggtgttgaaatcggtgatccggaattagtcggtgttcag aaaaaattaatccgtcgttcacgtcagttaaatcgtgttgttattaccgcaactcaaatgatggaatcaatgattagtaatcctatgccgac tcgtgcggaagtaatggacgtagctaacgcagtattggacggtaccgatgcggtaatgctttctgctgaaaccgcggctggtcaatatc cggcggaaactgttgcggcgatggcgaaagttgcgttaggtgcggagaaaatgccaagcattaatgtgtctaaacaccgtatgaacg ttcaattcgagtctattgaagaatctgttgcgatgtctgcaatgtatgcggcaaaccacatgagaggcgtagcggcgattatcacattaa caagtagcggtcgtactgctcgtttaatgtctcgcattagttccggtttaccaatctttgcattgtcacgtaacgaatctacTttaaacttatac gcattatatcgtggtgtgacaccggttcattttgataaagacagccgtacctcagaaggtgcgacagcggcggttcaattattaaaaga cgaaggtttcttagtgtctggcgatttagtgttattaactcagggcgacgcaagcagttctagcggtactaacctttgccgtacattgattgtt gaataa SEQIDNO:18(aminoacidsequenceofPykA4fromtheDD1ldhA pflDpykA4-strain) MSRRLRRTKIVCTMGPATDKGNNLEKIIAAGANVVRMNFSHGTPEDHIGRAEKVREIAHKLGKH VAILGDLQGPKIRVSTFKEGKIFLNIGDKFILDAEMPKGEGNQEAVGLDYKTLPQDVVPGDILLLD DGRVQLKVLATEGAKVFTEVTVGGPLSNNKGINKLGGGLSADALTEKDKADIITAARIGVDYLAV SFPRSSADLNYARQLAKDAGLDAKIVAKVERAETVETDEAMDDIINAADVIMVARGDLGVEIGDP ELVGVQKKLIRRSRQLNRVVITATQMMESMISNPMPTRAEVMDVANAVLDGTDAVMLSAETAA GQYPAETVAAMAKVALGAEKMPSINVSKHRMNVQFESIEESVAMSAMYAANHMRGVAAIITLT SSGRTARLMSRISSGLPIFALSRNESTLNLYALYRGVTPVHFDKDSRTSEGATAAVQLLKDEGF LVSGDLVLLTQGDASSSSGTNLCRTLIVE SEQIDNO:19(nucleotidesequenceofthepykA-genefromtheDD1ldhApflDpykA5- strain) atgtccagaagattaagaagaacgaaaatcgtatgtacaatggggcctgcaacagacaaaggcaataatttagaaaaaatcattgc tgccggtgcaaacgttgtacgtatgaacttctcccacggtacgcccgaagatcatatcggtcgtgctgaaaaagtacgtgaaatcgctc ataaattaggtaaacacgtagcaatcttaggtgacttacaaggccctaaaatccgtgtttctacttttaaagaaggcaaaattttcttaaat atcggtgataaattcattttagacgcagagatgcctaaaggtgaaggtaaccaggaagcggttggtttagactataaaacattaccgc aagatgtggttccgggcgatatcttattattagatgacggtcgagttcaattgaaagtattggcaaccgaaggtgcaaaagtattcaccg aagtaacggtcggtggcccactatcaaataataaaggcattaacaaattaggcggcggtttatctggcgatgcGttaaccgaaaaag ataaagcggatatcattactgcggcgcgtatcggtgtggattaccttgccgtatctttcccgcgttcaagcgcggatttaaactacgcccg tcaattagcaaaagatgcgggcttggatgcgaaaatcgttgcgaaagtagaacgtgccgaaacagttgaaacggacgaagcaatg gacgatatcatcaatgcggcggacgtaatcatggttgcgcgcggtgacttaggtgttgaaatcggtgatccggaattagtcggtgttcag aaaaaattaatccgtcgttcacgtcagttaaatcgtgttgttattaccgcaactcaaatgatggaatcaatgattagtaatcctatgccgac tcgtgcggaagtaatggacgtagctaacgcagtattggacggtaccgatgcggtaatgctttctgctgaaaccgcggctggtcaatatc cggcggaaactgttgcggcgatggcgaaagttgcgttaggtgcggagaaaatgccaagcattaatgtgtctaaacaccgtatgaacg ttcaattcgagtctattgaagaatctgttgcgatgtctgcaatgtatgcggcaaaccacatgagaggcgtagcggcgattatcacattaa caagtagcggtcgtactgctcgtttaatgtctcgcattagttccggtttaccaatctttgcattgtcacgtaacgaatctacattaaacttatgc gcattatatcgtggtgtgacaccggttcattttgataaagacagccgtacctcagaaggtgcgacagcggcggttcaattattaaaaga cgaaggtttcttagtgtctggcgatttagtgttattaactcagggcgacgcaagcagttctagcggtactaacctttgccgtacattgattgtt gaataa SEQIDNO:20(aminoacidsequenceofPykA5fromtheDD1ldhA pflDpykA5-strain) MSRRLRRTKIVCTMGPATDKGNNLEKIIAAGANVVRMNFSHGTPEDHIGRAEKVREIAHKLGKH VAILGDLQGPKIRVSTFKEGKIFLNIGDKFILDAEMPKGEGNQEAVGLDYKTLPQDVVPGDILLLD DGRVQLKVLATEGAKVFTEVTVGGPLSNNKGINKLGGGLSGDALTEKDKADIITAARIGVDYLAV SFPRSSADLNYARQLAKDAGLDAKIVAKVERAETVETDEAMDDIINAADVIMVARGDLGVEIGDP ELVGVQKKLIRRSRQLNRVVITATQMMESMISNPMPTRAEVMDVANAVLDGTDAVMLSAETAA GQYPAETVAAMAKVALGAEKMPSINVSKHRMNVQFESIEESVAMSAMYAANHMRGVAAIITLT SSGRTARLMSRISSGLPIFALSRNESTLNLCALYRGVTPVHFDKDSRTSEGATAAVQLLKDEGF LVSGDLVLLTQGDASSSSGTNLCRTLIVE SEQIDNO:21(nucleotidesequenceofthepykA-genefromtheDD1ldhApflD pykA6-strain) atgtccagaagattaagaagaacgaaaatcgtatgtacaatggggcctgcaacagacaaaggcaataatttagaaaaaatcattgc tgccggtgcaaacgttgtacgtatgaacttctcccacggtacgcccgaagatcatatcggtcgtgctgaaaaagtacgtgaaatcgctc ataaattaggtaaacacgtagcaatcttaggtgacttacaaggccctaaaatccgtgtttctacttttaaagaaggcaaaattttcttaaat atcggtgataaattcattttagacgcagagatgcctaaaggtgaaggtaaccaggaagcggttggtttagactataaaacattaccgc aagatgtggttccgggcgatatcttattattagatgacggtcgagttcaattgaaagtattggcaaccgaaggtgcaaaagtattcaccg aagtaacggtcggtggcccactatcaaataataaaggcattaacaaattaggctgcggtttatctgccgatgcGttaaccgaaaaag ataaagcggatatcattactgcggcgcgtatcggtgtggattaccttgccgtatctttcccgcgttcaagcgcggatttaaactacgcccg tcaattagcaaaagatgcgggcttggatgcgaaaatcgttgcgaaagtagaacgtgccgaaacagttgaaacggacgaagcaatg gacgatatcatcaatgcggcggacgtaatcatggttgcgcgcggtgacttaggtgttgaaatcggtgatccggaattagtcggtgttcag aaaaaattaatccgtcgttcacgtcagttaaatcgtgttgttattaccgcaactcaaatgatggaatcaatgattagtaatcctatgccgac tcgtgcggaagtaatggacgtagctaacgcagtattggacggtaccgatgcggtaatgctttctgctgaaaccgcggctggtcaatatc cggcggaaactgttgcggcgatggcgaaagttgcgttaggtgcggagaaaatgccaagcattaatgtgtctaaacaccgtatgaacg ttcaattcgagtctattgaagaatctgttgcgatgtctgcaatgtatgcggcaaaccacatgagaggcgtagcggcgattatcacattaa caagtagcggtcgtactgctcgtttaatgtctcgcattagttccggtttaccaatctttgcattgtcacgtaacgaatctacattaaacttatgc gcattatatcgtggtgtgacaccggttcattttgataaagacagccgtacctcagaaggtgcgacagcggcggttcaattattaaaaga cgaaggtttcttagtgtctggcgatttagtgttattaactcagggcgacgcaagcagttctagcggtactaacctttgccgtacattgattgtt gaataa SEQIDNO:22(aminoacidsequenceofPykA6fromtheDD1ldhA pflDpykA6-strain) MSRRLRRTKIVCTMGPATDKGNNLEKIIAAGANVVRMNFSHGTPEDHIGRAEKVREIAHKLGKH VAILGDLQGPKIRVSTFKEGKIFLNIGDKFILDAEMPKGEGNQEAVGLDYKTLPQDVVPGDILLLD DGRVQLKVLATEGAKVFTEVTVGGPLSNNKGINKLGCGLSADALTEKDKADIITAARIGVDYLAV SFPRSSADLNYARQLAKDAGLDAKIVAKVERAETVETDEAMDDIINAADVIMVARGDLGVEIGDP ELVGVQKKLIRRSRQLNRVVITATQMMESMISNPMPTRAEVMDVANAVLDGTDAVMLSAETAA GQYPAETVAAMAKVALGAEKMPSINVSKHRMNVQFESIEESVAMSAMYAANHMRGVAAIITLT SSGRTARLMSRISSGLPIFALSRNESTLNLCALYRGVTPVHFDKDSRTSEGATAAVQLLKDEGF LVSGDLVLLTQGDASSSSGTNLCRTLIVE SEQIDNO:23(sequenceofthepykA4_fwprimer) aatgcggcggacgtaatcat SEQIDNO:24(sequenceofthepykA4_rvprimer) tggagaacctatccatgaagtatca SEQIDNO:25(sequenceofthepykA5/6_fwprimer) tggggcctgcaacagacaaa SEQIDNO:26(sequenceofthepykA5/6_rvprimer) aaacgagcagtacgaccgct SEQIDNO:27(nucleotidesequenceofldhA-genefromstrainDD1) ttgacaaaatcagtatgtttaaataaggagctaactatgaaagttgccgtttacagtactaaaaattatgatcgcaaacatctggatttgg cgaataaaaaatttaattttgagcttcatttctttgattttttacttgatgaacaaaccgcgaaaatggcggagggcgccgatgccgtctgta ttttcgtcaatgatgatgcgagccgcccggtgttaacaaagttggcgcaaatcggagtgaaaattatcgctttacgttgtgccggttttaat aatgtggatttggaggcggcaaaagagctgggattaaaagtcgtacgggtgcctgcgtattcgccggaagccgttgccgagcatgcg atcggattaatgctgactttaaaccgccgtatccataaggcttatcagcgtacccgcgatgcgaatttttctctggaaggattggtcggtttt aatatgttcggcaaaaccgccggagtgattggtacgggaaaaatcggcttggcggctattcgcattttaaaaggcttcggtatggacgtt ctggcgtttgatccttttaaaaatccggcggcggaagcgttgggcgcaaaatatgtcggtttagacgagctttatgcaaaatcccatgtta tcactttgcattgcccggctacggcggataattatcatttattaaatgaagcggcttttaataaaatgcgcgacggtgtaatgattattaata ccagccgcggcgttttaattgacagccgggcggcaatcgaagcgttaaaacggcagaaaatcggcgctctcggtatggatgtttatg aaaatgaacgggatttgtttttcgaggataaatctaacgatgttattacggatgatgtattccgtcgcctttcttcctgtcataatgtgctttttac cggtcatcaggcgtttttaacggaagaagcgctgaataatatcgccgatgtgactttatcgaatattcaggcggtttccaaaaatgcaac gtgcgaaaatagcgttgaaggctaa SEQIDNO:28(aminoacidsequenceofLdhAfromstrainDD1) MTKSVCLNKELTMKVAVYSTKNYDRKHLDLANKKFNFELHFFDFLLDEQTAKMAEGADAVCIFV NDDASRPVLTKLAQIGVKIIALRCAGFNNVDLEAAKELGLKVVRVPAYSPEAVAEHAIGLMLTLN RRIHKAYQRTRDANFSLEGLVGFNMFGKTAGVIGTGKIGLAAIRILKGFGMDVLAFDPFKNPAAE ALGAKYVGLDELYAKSHVITLHCPATADNYHLLNEAAFNKMRDGVMIINTSRGVLIDSRAAIEAL KRQKIGALGMDVYENERDLFFEDKSNDVITDDVFRRLSSCHNVLFTGHQAFLTEEALNNIADVT LSNIQAVSKNATCENSVEG SEQIDNO:29(nucleotidesequenceofpflA-genefromstrainDD1) atgtcggttttaggacgaattcattcatttgaaacctgcgggacagttgacgggccgggaatccgctttattttatttttacaaggctgcttaa tgcgttgtaaatactgccataatagagacacctgggatttgcacggcggtaaagaaatttccgttgaagaattaatgaaagaagtggtg acctatcgccattttatgaacgcctcgggcggcggagttaccgcttccggcggtgaagctattttacaggcggaatttgtacgggactgg ttcagagcctgccataaagaaggaattaatacttgcttggataccaacggtttcgtccgtcatcatgatcatattattgatgaattgattgat gacacggatcttgtgttgcttgacctgaaagaaatgaatgaacgggttcacgaaagcctgattggcgtgccgaataaaagagtgctcg aattcgcaaaatatttagcggatcgaaatcagcgtacctggatccgccatgttgtagtgccgggttatacagatagtgacgaagatttgc acatgctggggaatttcattaaagatatgaagaatatcgaaaaagtggaattattaccttatcaccgtctaggcgcccataaatgggaa gtactcggcgataaatacgagcttgaagatgtaaaaccgccgacaaaagaattaatggagcatgttaaggggttgcttgcaggctac gggcttaatgtgacatattag SEQIDNO:30(aminoacidsequenceofPflAfromstrainDD1) MSVLGRIHSFETCGTVDGPGIRFILFLQGCLMRCKYCHNRDTWDLHGGKEISVEELMKEVVTY RHFMNASGGGVTASGGEAILQAEFVRDWFRACHKEGINTCLDTNGFVRHHDHIIDELIDDTDLV LLDLKEMNERVHESLIGVPNKRVLEFAKYLADRNQRTWIRHVVVPGYTDSDEDLHMLGNFIKD MKNIEKVELLPYHRLGAHKWEVLGDKYELEDVKPPTKELMEHVKGLLAGYGLNVTY SEQIDNO:31(nucleotidesequenceofpflD-genefromstrainDD1) atggctgaattaacagaagctcaaaaaaaagcatgggaaggattcgttcccggtgaatggcaaaacggcgtaaatttacgtgacttt atccaaaaaaactatactccgtatgaaggtgacgaatcattcttagctgatgcgactcctgcaaccagcgagttgtggaacagcgtga tggaaggcatcaaaatcgaaaacaaaactcacgcacctttagatttcgacgaacatactccgtcaactatcacttctcacaagcctgg ttatatcaataaagatttagaaaaaatcgttggtcttcaaacagacgctccgttaaaacgtgcaattatgccgtacggcggtatcaaaat gatcaaaggttcttgcgaagtttacggtcgtaaattagatccgcaagtagaatttattttcaccgaatatcgtaaaacccataaccaagg cgtattcgacgtttatacgccggatattttacgctgccgtaaatcaggcgtgttaaccggtttaccggatgcttacggtcgtggtcgtattatc ggtgactaccgtcgtttagcggtatacggtattgattacctgatgaaagataaaaaagcccaattcgattcattacaaccgcgtttggaa gcgggcgaagacattcaggcaactatccaattacgtgaagaaattgccgaacaacaccgcgctttaggcaaaatcaaagaaatgg cggcatcttacggttacgacatttccggccctgcgacaaacgcacaggaagcaatccaatggacatattttgcttatctggcagcggtt aaatcacaaaacggtgcggcaatgtcattcggtcgtacgtctacattcttagatatctatatcgaacgtgacttaaaacgcggtttaatca ctgaacaacaggcgcaggaattaatggaccacttagtaatgaaattacgtatggttcgtttcttacgtacgccggaatacgatcaattatt ctcaggcgacccgatgtgggcaaccgaaactatcgccggtatgggcttagacggtcgtccgttggtaactaaaaacagcttccgcgt attacatactttatacactatgggtacttctccggaaccaaacttaactattctttggtccgaacaattacctgaagcgttcaaacgtttctgt gcgaaagtatctattgatacttcctccgtacaatacgaaaatgatgacttaatgcgtcctgacttcaacaacgatgactatgcaatcgcat gctgcgtatcaccgatggtcgtaggtaaacaaatgcaattcttcggtgcgcgcgcaaacttagctaaaactatgttatacgcaattaac ggcggtatcgatgagaaaaatggtatgcaagtcggtcctaaaactgcgccgattacagacgaagtattgaatttcgataccgtaatcg aacgtatggacagtttcatggactggttggcgactcaatatgtaaccgcattgaacatcatccacttcatgcacgataaatatgcatatg aagcggcattgatggcgttccacgatcgcgacgtattccgtacaatggcttgcggtatcgcgggtctttccgtggctgcggactcattatc cgcaatcaaatatgcgaaagttaaaccgattcgcggcgacatcaaagataaagacggtaatgtcgtggcctcgaatgttgctatcga cttcgaaattgaaggcgaatatccgcaattcggtaacaatgatccgcgtgttgatgatttagcggtagacttagttgaacgtttcatgaaa aaagttcaaaaacacaaaacttaccgcaacgcaactccgacacaatctatcctgactatcacttctaacgtggtatacggtaagaaa accggtaatactccggacggtcgtcgagcaggcgcgccattcggaccgggtgcaaacccaatgcacggtcgtgaccaaaaaggt gcggttgcttcacttacttctgtggctaaacttccgttcgcttacgcgaaagacggtatttcatataccttctctatcgtaccgaacgcattag gtaaagatgacgaagcgcaaaaacgcaaccttgccggtttaatggacggttatttccatcatgaagcgacagtggaaggcggtcaa cacttgaatgttaacgttcttaaccgtgaaatgttgttagacgcgatggaaaatccggaaaaatacccgcaattaaccattcgtgtttcag gttacgcggttcgtttcaactcattaactaaagagcaacaacaagacgtcatcactcgtacgtttacacaatcaatgtaa SEQIDNO:32(aminoacidofPflDfromstrainDD1) MAELTEAQKKAWEGFVPGEWQNGVNLRDFIQKNYTPYEGDESFLADATPATSELWNSVMEGI KIENKTHAPLDFDEHTPSTITSHKPGYINKDLEKIVGLQTDAPLKRAIMPYGGIKMIKGSCEVYGR KLDPQVEFIFTEYRKTHNQGVFDVYTPDILRCRKSGVLTGLPDAYGRGRIIGDYRRLAVYGIDYL MKDKKAQFDSLQPRLEAGEDIQATIQLREEIAEQHRALGKIKEMAASYGYDISGPATNAQEAIQ WTYFAYLAAVKSQNGAAMSFGRTSTFLDIYIERDLKRGLITEQQAQELMDHLVMKLRMVRFLRT PEYDQLFSGDPMWATETIAGMGLDGRPLVTKNSFRVLHTLYTMGTSPEPNLTILWSEQLPEAF KRFCAKVSIDTSSVQYENDDLMRPDFNNDDYAIACCVSPMVVGKQMQFFGARANLAKTMLYAI NGGIDEKNGMQVGPKTAPITDEVLNFDTVIERMDSFMDWLATQYVTALNIIHFMHDKYAYEAAL MAFHDRDVFRTMACGIAGLSVAADSLSAIKYAKVKPIRGDIKDKDGNVVASNVAIDFEIEGEYPQ FGNNDPRVDDLAVDLVERFMKKVQKHKTYRNATPTQSILTITSNVVYGKKTGNTPDGRRAGAP FGPGANPMHGRDQKGAVASLTSVAKLPFAYAKDGISYTFSIVPNALGKDDEAQKRNLAGLMDG YFHHEATVEGGQHLNVNVLNREMLLDAMENPEKYPQLTIRVSGYAVRFNSLTKEQQQDVITRT FTQSM