Modified microorganism with improved biomass separation behaviour

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 wcaJ-gene. The present invention also relates to a method for producing an organic compound and to the use of a modified microorganism.

Claims

1. A modified microorganism having, compared to its wild-type, a deletion of the wcaJ-gene or an introduction of at least one mutation into the wcaJ-gene that leads to the expression of a truncated enzyme encoded by the wcaJ-gene in which at least 100 amino acids of the wild-type enzyme encoded by the wcaJ-gene are deleted from the C-terminal end; wherein the wild-type from which the modified microorganism has been derived belongs to the family of Pasteurellaceae and wherein the wild-type refers to the naturally occurring microorganism that has not been genetically modified, wherein the wcaJ-gene in the wildtype comprises a nucleic acid encoding an amino acid sequence which is at least 95% identical to SEQ ID NO: 4 over the total length of the amino acid sequence of SEQ ID NO: 4.

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

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

4. 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. 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. Modified microorganism according to claim 1, wherein the microorganism further has, compared to its wild-type, i) a reduced pyruvate formate lyase activity, ii) a reduced lactate dehydrogenase activity, or iii) a reduced pyruvate formate lyase activity and a reduced lactate dehydrogenase activity.

7. Modified microorganism according to claim 6, wherein the microorganism comprises: A) a deletion of the ldhA-gene or at least a part thereof, a deletion of a regulatory element of the ldhA-gene or at least a part thereof or an introduction of at least one mutation into the ldhA-gene; B) a deletion of the pflD-gene or at least a part thereof, a deletion of a regulatory element of the pflD-gene or at least a part thereof or an introduction of at least one mutation into the pflD-gene; C) a deletion of the pflA-gene or at least a part thereof, a deletion of a regulatory element of the pflA-gene or at least a part thereof or an introduction of at least one mutation into the pflA-gene; D) a deletion of the ldhA-gene or at least a part thereof, a deletion of a regulatory element of the ldhA-gene or at least a part thereof or an introduction of at least one mutation into the ldhA-gene and a deletion of the pflD-gene or at least a part thereof, a deletion of a regulatory element of the pflD-gene or at least a part thereof or an introduction of at least one mutation into the pflD-gene; or E) a deletion of the ldhA-gene or at least a part thereof, a deletion of a regulatory element of the ldhA-gene or at least a part thereof or an introduction of at least one mutation into the ldhA-gene and a deletion of the pflA-gene or at least a part thereof, a deletion of a regulatory element of the pflA-gene or at least a part thereof an introduction of at least one mutation into the pflA-gene.

8. A method of producing an organic compound comprising: I) cultivating the modified microorganism of claim 1 in a culture medium comprising at least one assimilable carbon source to allow the modified microorganism to produce the organic compound, thereby obtaining a fermentation broth comprising the organic compound; II) recovering the organic compound from the fermentation broth obtained in process step I), wherein the organic compound is a carboxylic, dicarboxylic or tricarboxylic acid.

9. Method according to claim 8, wherein the organic compound is succinic acid.

10. 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.

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

12. Method according to claim 11, wherein the organic compound is succinic acid and 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 ldhA (SEQ ID NO: 6).

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

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

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

(7) FIG. 6 shows a pellet of cells as obtained by sedimentation of different culture media.

EXAMPLES

Example 1: General Method for the Transformation of Basfia succiniciproducens

(8) 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 wcaJ DD1 ldhA DD1 ldhA pflD DD1 ldhA pflD wcaJ DD1 ldhA pflA DD1 ldhA pflA wcaJ

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

(10) 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.).

(11) 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.

(12) 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 Constructs

(13) Mutation/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 ldhA, pSacB_delta_ldhA (SEQ ID NO: 6), the pyruvate formate lyase activating enzyme pflA, pSacB_delta_pflA (SEQ ID No: 7), the putative fructose-specific transporter wcaJ, pSacB_delta_wcaJ (SEQ ID No: 8) and pyruvate formate lyase pflD, pSacB_delta.sub. pflD (SEQ ID No: 9) were constructed. FIGS. 2, 3, 4 and 5 show schematic maps of plasmid pSacB_delta_ldhA, pSacB_delta_pflA, pSacB_delta_wcaJ and pSacB_delta_pflD, respectively.

(14) 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).

(15) In the plasmid sequence of pSacB_delta_ldhA (SEQ ID NO: 6) the 5 flanking region of the ldhA gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 1519-2850, while the 3 flanking region of the ldhA-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 62-1518. The sacB-gene 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).

(16) In the plasmid sequence of pSacB_delta_pflA (SEQ ID NO: 7) the 5 flanking region of the OA-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 1506-3005, while the 3 flanking region of the pflA-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).

(17) In the plasmid sequence of pSacB_delta_wcaJ (SEQ ID NO: 8) the 5 flanking region of the wcaJ gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 1506-3122, while the 3 flanking region of the wcaJ-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 6-1505. The sacB-gene is contained from bases 5395-6816. The sacB-promoter is contained from bases 6817-7279. The chloramphenicol gene is contained from base 3541-3999. The origin of replication for E. coli (ori EC) is contained from base 4492-5352 (see FIG. 4).

(18) In the plasmid sequence of pSacB_delta_pflD (SEQ ID NO: 9) the 5 flanking region of the pflD-gene, which is homologous to the genome of Basfia succiniciproducens, is contained from bases 1533-2955, while the 3 flanking region of the pflD-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. 5).

Example 3: Generation of Improved Succinate Producing Strains

(19) a) Basfia succiniciproducens DD1 was transformed as described above with the pSacB_delta_ldhA 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 integrational 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-evan-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 the mutation/deletion of the ldhA-gene were confirmed by chloramphenicol sensitivity. The mutation/deletion mutants among these strains were identified and confirmed by PCR analysis. This led to the ldhA-deletion mutant Basfia succiniciproducens DD1 ldhA. b) Basfia succiniciproducens DD1 ldhA was transformed with pSacB_delta_pflD 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 ldhA pflD-double deletion mutant Basfia succiniciproducens DD1 ldhA pflD. c) Basfia succiniciproducens DD1 ldhA pflD was transformed with pSacB_delta_wcaJ 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 ldhA pflD wcaJ-triple deletion mutant Basfia succiniciproducens DD1 ldhA pflD wcaJ. d) Basfia succiniciproducens DD1 ldhA was transformed with pSacB_delta_pflA 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 ldhA pflA-double deletion mutant Basfia succiniciproducens DD1 ldhA pflA. e) Basfia succiniciproducens DD1 ldhA pflA was transformed with pSacB_delta_wcaJ 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 ldhA pflA wcaJ-triple deletion mutant Basfia succiniciproducens DD1 ldhA pflA wcaJ. f) Basfia succiniciproducens DD1 was transformed with pSacB_delta_wcaJ 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 wcaJ-deletion mutant Basfia succiniciproducens DD1 wcaJ.

Example 4: Cultivation of Various DD1-Strains on Glucose and Sucrose

(20) The productivity of the DD1 was compared with the productivity of the mutant strain DD1 wcaJ in the presence of glucose or sucrose as a carbon source.

(21) The productivity of the DD1 ldhA pflD was compared with the productivity of the mutant strain DD1 ldhA pflD wcaJ in the presence of glucose or sucrose as a carbon source.

(22) The productivity of the DD1 ldhA pflA was compared with the productivity of the mutant strain DD1 ldhA pflA wcaJ in the presence of glucose or sucrose as a carbon source.

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

(24) 1. Medium Preparation The composition and preparation of the cultivation medium is as described in the following table 2, 3, 4 and 5.

(25) TABLE-US-00002 TABLE 2 Composition of trace element solution. Trace element solution Final Compound concentration citric acid 3.5 g/L ZnSO.sub.4 7 H.sub.2O 1851 mg/L CaSO.sub.4 2 H.sub.2O 10 mg/L FeSO.sub.4 7 H.sub.2O 2040 mg/L CaCl.sub.2 2 H.sub.2O 12460 mg/L MnCl.sub.2 4 H.sub.2O 1200 mg/L Na.sub.2MoO.sub.4 2 H.sub.2O 38 mg/L CuCl.sub.2 2 H.sub.2O 188 mg/L NiCl.sub.2 6 H.sub.2O 32 mg/L CoCl.sub.2 6 H.sub.2O 101 mg/L

(26) TABLE-US-00003 TABLE 3 Composition of vitamin solution. Vitamin solution Final Compound concentration Thiamin HCl (B1) 500 mg/L Nicotinic acid (B3) 500 mg/L Riboflavin (B2) 20 mg/L Biotin (B7) 5 mg/L Pantothenic acid (B5) 100 mg/L Pyridoxine (B6) 500 mg/L Cyanocobalamin (B12) 5 mg/L Lipoic acid 5 mg/L

(27) TABLE-US-00004 TABLE 4 Composition of LSM medium for cultivation on glucose. Volume/ Stock Final Compound Mass concentration concentration Medium 1 MgCO.sub.3 2.5 g 100% 50.00 g/L Water 28 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 0.25 mL 500 g/L 2.50 g/L (NH.sub.4).sub.2HPO.sub.4 0.5 mL 200 g/L 2.00 g/L K.sub.2CO.sub.3 0.50 mL 200 g/L 2.00 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 0.50 mL 25 g/L 0.25 g/L trace element solution 0.50 mL 21 g/L 0.21 g/L

(28) TABLE-US-00005 TABLE 5 Composition of LSM medium for cultivation on sucrose. Volume/ Stock Final Compound Mass concentration concentration Medium 1 MgCO.sub.3 2.5 g 100% 50.00 g/L Water 28 mL Medium 2 Succinic acid 2.5 mL 50 g/L 2.50 g/L Sucrose 3.85 mL 650 g/L 50.00 g/L (NH.sub.4).sub.2SO.sub.4 0.25 mL 500 g/L 2.50 g/L (NH.sub.4).sub.2HPO.sub.4 0.5 mL 200 g/L 2.00 g/L K.sub.2CO.sub.3 0.50 mL 200 g/L 2.00 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 0.50 mL 25 g/L 0.25 g/L trace element 0.50 mL 21 g/L 0.21 g/L solution

(29) 2. Cultivations and Analytics For growing the main culture bacteria from a freshly grown BHI-agar plate 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 2 and 3 with a CO.sub.2-atmosphere. 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 table 10 and 11) after 24 h or 48 h. Cell growth was measured by measuring the absorbance at 600 nm (OD600) using a spectrophotometer (Ultrospec3000, Amersham Biosciences, Uppsala Sweden).

(30) 3. Results The results of the cultivation experiments with different DD1-strains are shown in table 6, 7 and 8. As can be seen from these results, a reduction of the activity of the enzyme that is encoded by the wcaJ-cell leads to increased production of succinic acid. Moreover, samples obtained by cultivation of the DD1 ldhA pflD/DD1 ldhA pflA-strain and the DD1ldhA pflD wcaJ/DD1ldhA pflA wcaJ-strain after 24 h or 48 h of incubation have been transferred into a 15 ml tube to measure supernatant volumes obtained after centrifugation. As shown in table 9, a reduction of the activity of the enzyme that is encoded by the wcaJ-cell leads to modified microorganisms that show a significantly improved sedimentation behavior (a denser cell pellet is obtained after centrifugation) and that can thus easier be removed from the culture medium in the subsequent purification process.

(31) TABLE-US-00006 TABLE 6 Cultivation of the DD1 and the DD1 wcaJ-strain on glucose and sucrose. DD1 DD1wcaJ DD1 DD1wcaJ substrate glucose glucose sucrose sucrose tc [h].sup.a 24 24 48 48 C.sub.substrate [g/l].sup.b 48.4 48.4 48.6 48.1 C.sub.SA[g/l].sup.c 26.1 28.2 22.0 24.3 (succinic acid) C.sub.LA [g/L].sup.c,h 5.1 3.3 11.8 10.0 (lactic acid) C.sub.FA[g/l].sup.c,h 4.8 4.2 4.1 4.2 (formic acid) C.sub.AA[g/l].sup.c,h 7.2 7.2 6.2 6.9 (acetic acid) C.sub.PA[g/l].sup.c,h 0.0 0.0 0.0 0.0 (pyruvic acid) C.sub.P[g/l].sup.c,h 0.0 0.0 0.0 0.0 (propionic acid) C.sub.E[g/l].sup.c 0.3 0.2 0.3 0.2 (ethanol) SA Yield 0.54 0.58 0.45 0.50 (SA/S) [g/g].sup.g .sup.acultivation time .sup.bconsumption of substrate (glucose or sucrose) .sup.cformation of succinic acid, lactic acid, formic acid, acetic acid, pyruvic acid, propionic 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

(32) TABLE-US-00007 TABLE 7 Cultivation of the DD1 ldhA pflD-strain and the DD1 ldhA pflD wcaJ-strain on glucose and sucrose. DD1 DD1 DDl ldhA DD1 ldhA ldhApflD pflDwcaJ ldhApflD pflDwcaJ substrate glucose glucose sucrose sucrose tc [h].sup.a 24 24 48 48 C.sub.substrate [g/l].sup.b 49.95 49.95 50.40 50.40 C.sub.SA[g/l].sup.c 31.45 32.80 28.82 36.94 (succinic acid) C.sub.LA [g/L].sup.c,h 0.22 0.16 0.35 0.22 (lactic acid) C.sub.FA[g/l].sup.c,h 0.00 0.00 0.00 0.00 (formic acid) C.sub.AA[g/l].sup.c,h 2.89 4.48 1.39 5.45 (acetic acid) C.sub.PA[g/l].sup.c,h 1.87 0.94 2.70 0.23 (pyruvic acid) C.sub.P[g/l].sup.c,h 0.00 0.00 0.00 0.00 (propionic acid) C.sub.E[g/l].sup.c 0.00 0.00 0.00 0.00 (ethanol) SA Yield 0.63 0.66 0.57 0.73 (SA/S) [g/g].sup.g .sup.acultivation time .sup.bconsumption of substrate (glucose or sucrose) .sup.cformation of succinic acid, lactic acid, formic acid, acetic acid, pyruvic acid, propionic 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

(33) TABLE-US-00008 TABLE 8 Cultivation of the DD1 ldhA pflA-strain and the DD1 ldhA pflA wcaJ-strain on glucose and sucrose DD1 DD1 DD1 ldhA DD1 ldhA ldhApflA pflAwcaJ ldhApflA pflAwcaJ substrate glucose glucose sucrose sucrose tc [h].sup.a 24 24 48 48 C.sub.substrate [g/l].sup.b 49.95 49.95 50.40 50.40 C.sub.SA[g/l].sup.c 32.37 31.77 29.36 31.36 (succinic acid) C.sub.LA [g/L].sup.c,h 0.23 0.28 0.37 0.37 (lactic acid) C.sub.FA[g/l].sup.c,h 0.00 0.00 0.00 0.00 (formic acid) C.sub.AA[g/l].sup.c,h 2.68 2.40 0.90 1.87 (acetic acid) C.sub.PA[g/l].sup.c,h 1.96 2.05 3.16 2.49 (pyruvic acid) C.sub.P[g/l].sup.c,h 0.00 0.00 0.00 0.00 (propionic acid) C.sub.E[g/l].sup.c 0.00 0.00 0.00 0.00 (ethanol) SA Yield 0.65 0.64 0.58 0.62 (SA/S) [g/g].sup.g .sup.acultivation time .sup.bconsumption of substrate (glucose or sucrose) .sup.cformation of succinic acid, lactic acid, formic acid, acetic acid, pyruvic acid, propionic 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

(34) TABLE-US-00009 TABLE 9 Supernatant volumes obtained after centrifugation (4,600 rpm, 10 min) of 10 mL of bacterial cultures. Cultivation Supernatant Strain Substrate time volume DD1 ldhA pflD glucose 24 h 8.0 mL DD1 ldhA pflD wcaJ glucose 24 h 8.8 mL DD1 ldhA pflA glucose 24 h 8.0 mL DD1 ldhA pflA wcaJ glucose 24 h 8.4 mL DD1 ldhA pflD sucrose 48 h 8.3 mL DD1 ldhA pflD wcaJ sucrose 48 h 9.0 mL DD1 ldhA pflA sucrose 48 h 8.3 mL DD1 ldhA pflA wcaJ sucrose 48 h 8.6 mL

(35) TABLE-US-00010 TABLE 10 HPLC method (ZX-THF50) for analysis of glucose, 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 Gradient Time [min] A[%] B[%] Flow [ml/min] 0.0 50 50 0.50 28.0 50 50 0.50

(36) TABLE-US-00011 TABLE 11 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 Gradient Time [min] A[%] B[%] Flow [ml/min] 0.0 50 50 1.00 8.0 50 50 1.00

SEQUENCES

(37) TABLE-US-00012 (nucleotidesequenceof16SrDNAofstrainDD1) SEQIDNO:1 tttgatcctggctcagattgaacgctggcggcaggcttaacacatgcaagtcgaacggtagcgggaggaaagcttgctttctttgccga cgagtggcggacgggtgagtaatgcttggggatctggcttatggagggggataacgacgggaaactgtcgctaataccgcgtaatat cttcggattaaagggtgggactttcgggccacccgccataagatgagcccaagtgggattaggtagttggtggggtaaaggcctacc aagccgacgatctctagctggtctgagaggatgaccagccacactggaactgagacacggtccagactcctacgggaggcagca gtggggaatattgcacaatggggggaaccctgatgcagccatgccgcgtgaatgaagaaggccttcgggttgtaaagttctttcggtg acgaggaaggtgtttgttttaataggacaagcaattgacgttaatcacagaagaagcaccggctaactccgtgccagcagccgcggt aatacggagggtgcgagcgttaatcggaataactgggcgtaaagggcatgcaggcggacttttaagtgagatgtgaaagccccgg gcttaacctgggaattgcatttcagactgggagtctagagtactttagggaggggtagaattccacgtgtagcggtgaaatgcgtagag atgtggaggaataccgaaggcgaaggcagccccttgggaagatactgacgctcatatgcgaaagcgtggggagcaaacaggatt agataccctggtagtccacgcggtaaacgctgtcgatttggggattgggctttaggcctggtgctcgtagctaacgtgataaatcgacc gcctggggagtacggccgcaaggttaaaactcaaatgaattgacgggggcccgcacaagcggtggagcatgtggtttaattcgatg caacgcgaagaaccttacctactcttgacatccagagaatcctgtagagatacgggagtgccttcgggagctctgagacaggtgctg catggctgtcgtcagctcgtgttgtgaaatgttgggttaagtcccgcaacgagcgcaacccttatcctttgttgccagcatgtaaagatgg gaactcaaaggagactgccggtgacaaaccggaggaaggtggggatgacgtcaagtcatcatggcccttacgagtagggctaca cacgtgctacaatggtgcatacagagggcggcgataccgcgaggtagagcgaatctcagaaagtgcatcgtagtccggattggagt ctgcaactcgactccatgaagtcggaatcgctagtaatcgcaaatcagaatgttgcggtgaatacgttcccgggccttgtacacaccg cccgtcacaccatgggagtgggttgtaccagaagtagatagcttaaccttcggggggggcgtttaccacggtatgattcatgactggg gtgaagtcgtaacaaggtaaccgtaggggaacctgcgg (nucleotidesequenceof23SrDNAofstrainDD1) SEQIDNO:2 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 (nucleotidesequenceofwcaJ-genefromstrainDD1) SEQIDNO:3 atgataaaacgccttttcgatattgttgtcgcattgatagcattgattttgttttcgcccttatatttgtttgtggcttataaggtaaaacaaaattt gggatcaccggtgttatttaaacaaacccgccccggattgcatggtaaaccctttgagatgattaagttcagaacaatgaaagacggc gcagatgaaaacggtaatattttgccggatgcggagcgcttaacacctttcggcaaaatgttgcgcgctaccagtctggacgagttgcc ggaactttggaatgtattaaaaggtgatatgagtctggtggggccgcgtcctctactgatggaatatttgccgctgtataacgaaagaca ggctaagcgccatgaagtgaaacccggaattaccggttatgcacaggtaaacggtcgcaatgccatcagttgggagcagaaatttg aattggatgcctggtatgttgaacatcaatccttgtggctggatttgaaaattatcgcaaagaccatccaaaaagtgatcgcaaaagac gatattaatgcggcagatgatgccaccatgcctaaatttgaagggaataaaaaatcatga (aminoacidsequenceoftheenzymeencodedbytheabovewcaJ-gene) SEQIDNO:4 MIKRLFDIVVALIALILFSPLYLFVAYKVKQNLGSPVLFKQTRPGLHGKPFEMIKFRTMKDGADEN GNILPDAERLTPFGKMLRATSLDELPELWNVLKGDMSLVGPRPLLMEYLPLYNERQAKRHEVK PGITGYAQVNGRNAISWEQKFELDAWYVEHQSLWLDLKIIAKTIQKVIAKDDINAADDATMPKFE GNKKS (completenucleotidesequenceofplasmidpSacB) SEQIDNO:5 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 (completenucleotidesequenceofplasmidpSacB_delta_ldhA) SEQIDNO:6 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 (completenucleotidesequenceofplasmidpSacB_delta_pflA) SEQIDNO:7 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 (completenucleotidesequenceofplasmidpSacB_wcaJ) SEQIDNO:8 tcgagtaagccgattcagctgatccgccacatggggaaaaagcctaatctgcggaatatgaaaccgataccagtccagtaaagttg acaaatcgacatcatattgctcaaccaagtattgaaaagcgttttcaccgcgatgatacaattcgaccagccggttaaataacgtttcac tccgttccggtgccaaacgagacgcaatatgcttataggcggaatacagaaaatcgatttccgcataaagcgtatcgtccaaatctaa aaccaacgctttatttttcataatgatgagccagtacttccgcgtcataccgcaacattaataaatccgcttcccagtcttcgaacgcagg aatcggctgattaaacaaatattcctgaatcaaccaacggggataattggctcccgccagataactcaacggataaccgccgccga acctagggttaatttcaataccaagaatttcagcggtggattccttataaaatacttggattgttaagcaaccgcgcgcccccggtaaac gggacaatttttccgataattgcgtcacgatggcattttttctggtcacacctttgttaatttcccccgctctgacaaaaattctctttctcggta ccgcacttttcagttcggaatttttatcaaaataacaatccacggtatattcgtcgtattccgccggcgaaatatattgcataaacattaattc gggattttccaattgctccggtgaaatatcttccggtttctccgccacaaaaattcctttacttaaactaccgttgtaaggcttcacaaaaac aggatattcaaattgacctttttcaaactgcttcggtaccgcaatattatgttcaataaacagttgattggttaatcgtttgtcgcgacattttct gacaaactctgtatcactaacggaaataaaaatacctttttctttaaaccgttgcagatgttcgcttaaaataagcaattccgtatcaatag tcggaataatcaatttcacgttattttcttcacagattttaagtaaggtcggaatatactccgcatcagtgacccggggtacaggaaaatgt ccgtcggccacataacaagccggcgccaactcgggatttaaatctacggttaacacttttccgtcacttactaactgcgataattcctatt aaacgcctgaacgagagaaacacgttgtccggccgatgtaacaagaatattcatgattttttattcccttcaaatttaggcatggtggcat catctgccgcattaatatcgtcttttgcgatcactttttggatggtctttgcgataattttcaaatccagccacaaggattgatgttcaacatac caggcatccaattcaaatttctgctcccaactgatggcattgcgaccgtttacctgtgcataaccggtaattccgggtttcacttcatggcg cttagcctgtctttcgttatacagcggcaaatattccatcacacaaacaaatataagggcgaaaacaaaatcaatgctatcaatgcgac aacaatatcgaaaaggcgttttatcatgaaaatctcctacgaccgaccaatttggggctgacaaaagtgccgtttttcaccagaaccgt ataaactaaaaccaggaaaagcggataccagactaacggcagtcttaatatggaagacggcacccaaacgatccaacaaaaatt aataaaaataaataaaataaatatacgttctttccacgccaacaattgggtattcatcacatagataattgaatttaataaaaaatatata aatgccaaaaatgcaaaaatgccaaatgccaaataaagttctataaagaagctatgcggattagtgtaacccaaagggaaacttaa ctttatttgatcgaaatactgaatgtagtcccgcggtccataacccaaccataaaattttaaaattatctaaaaatgtcgtataaatttccgt ccggtaacctacggacttatcatcgcccatagaaaatattaccaatgaaaaacgttcaatcggacgctccagccaatcaattttggcg agcagaataaacacttcctgtaaccaagaaagattaaatataaataatgcgatcacgcaggcgaaaaacagatataccgccttaaa ataggtagatgcgtttaaaaacaagatcagcatcaacataatcaaatagctcagtaataccgaacgggaggcactgatcacaatag ctaaccccataataaaaataagagcatagccgattaacttaattttccagttgttttctctgatgatgtaaaaaaatcccaccgccaccgc aagagaaatcataattacggactggtcattggtattaaagaaaaaacctttaaacgccttatcagttacggttaattcttcattacccgaa accaactggaaccccaataaagcctcaataaaaaagcccgccagcacaattaatgatattcccaacaaaaggtgcctaatccctgc ttctccatcaccccggttaaacgtcaaaaaagaataatgaaataaaaacatcacaattccgaagaaaaacaaatcaactaatttttct gtagaaaacgcatttaataccgataaaaagccgaagaaaaacaacacgtacaccggaaactgtaatttaaaaaaatcagtttccat atcccttaaaaaaggggttattaccgctaagaaaaagaacaaaaaacacaacgcactatctaacctcggcactcctatttgtgtcgat agtgcgggagaaagtatcaccagtcccaacgcaaagagtaatagcaacttaaaaatgctgataacattaatattcatatcaaataat atttttgattaatttctcaatttctttataagaacgctcgcgcagaaacttctcttttgccagcgataaattcacttgcgacattttgtctaaaacc gttctgtcttcggccaatttattcaacgtctcagccaactcccgataatctcccgccgtatattgaattccaccgcctttcgccagtagtttttc cacttcaggatgtttctgacagcttacaatcggtaatgcgcaacagatataatcggatctagactccataggccgctttcctggctttgctt ccagatgtatgctctcctccggagagtaccgtgactttattttcggcacaaatacaggggtcgatggataaatacggcgatagtttcctg acggatgatccgtatgtaccggcggaagacaagctgcaaacctgtcagatggagattgatttaatggcggatgtgctgagagcaccg ccccgtgaatccgcagaactgatccgctatgtgtttgcggatgattggccggaataaataaagccgggcttaatacagattaagcccgt atagggtattattactgaataccaaacagcttacggaggacggaatgttacccattgagacaaccagactgccttctgattattaatatttt tcactattaatcagaaggaataaccatgaattttacccggattgacctgaatacctggaatcgcagggaacactttgccctttatcgtcag cagattaaatgcggattcagcctgaccaccaaactcgatattaccgctttgcgtaccgcactggcggagacaggttataagttttatccg ctgatgatttacctgatctcccgggctgttaatcagtttccggagttccggatggcactgaaagacaatgaacttatttactgggaccagtc agacccggtctttactgtctttcataaagaaaccgaaacattctctgcactgtcctgccgttattttccggatctcagtgagtttatggcaggt tataatgcggtaacggcagaatatcagcatgataccagattgtttccgcagggaaatttaccggagaatcacctgaatatatcatcatta ccgtgggtgagttttgacgggatttaacctgaacatcaccggaaatgatgattattttgccccggtttttacgatggcaaagtttcagcagg aaggtgaccgcgtattattacctgtttctgtacaggttcatcatgcagtctgtgatggctttcatgcagcacggtttattaatacacttcagct gatgtgtgataacatactgaaataaattaattaattctgtatttaagccaccgtatccggcaggaatggtggctttttttttatattttaaccgta atctgtaatttcgtttcagactggttcaggatgagctcgcttggactcctgttgatagatccagtaatgacctcagaactccatctggatttgt tcagaacgctcggttgccgccgggcgttttttattggtgagaatccaagcactagcggcgcgccggccggcccggtgtgaaataccgc acagatgcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcg agcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaagg ccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatc gacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctg ttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagtt cggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttg agtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgct acagagttcttgaagtggtggcctaactacggctacactagaaggacagtatttggtatctgcgctctgctgaagccagttaccttcgga aaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaa aaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgag attatcaaaaaggatcttcacctagatccttttaaaggccggccgcggccgccatcggcattttcttttgcgtttttatttgttaactgttaattgt ccttgttcaaggatgctgtctttgacaacagatgttttcttgcctttgatgttcagcaggaagctcggcgcaaacgttgattgtttgtctgcgta gaatcctctgtttgtcatatagcttgtaatcacgacattgtttcctttcgcttgaggtacagcgaagtgtgagtaagtaaaggttacatcgtta ggatcaagatccatttttaacacaaggccagttttgttcagcggcttgtatgggccagttaaagaattagaaacataaccaagcatgtaa atatcgttagacgtaatgccgtcaatcgtcatttttgatccgcgggagtcagtgaacaggtaccatttgccgttcattttaaagacgttcgc gcgttcaatttcatctgttactgtgttagatgcaatcagcggtttcatcacttttttcagtgtgtaatcatcgtttagctcaatcataccgagagc gccgtttgctaactcagccgtgcgttttttatcgctttgcagaagtttttgactttcttgacggaagaatgatgtgcttttgccatagtatgctttgt taaataaagattcttcgccttggtagccatcttcagttccagtgtttgcttcaaatactaagtatttgtggcctttatcttctacgtagtgaggat ctctcagcgtatggttgtcgcctgagctgtagttgccttcatcgatgaactgctgtacattttgatacgtttttccgtcaccgtcaaagattgatt tataatcctctacaccgttgatgttcaaagagctgtctgatgctgatacgttaacttgtgcagttgtcagtgtttgtttgccgtaatgtttaccgg agaaatcagtgtagaataaacggatttttccgtcagatgtaaatgtggctgaacctgaccattcttgtgtttggtatttaggatagaatcatt tgcatcgaatttgtcgctgtctttaaagacgcggccagcgtttttccagctgtcaatagaagtttcgccgactttttgatagaacatgtaaat cgatgtgtcatccgcatttttaggatctccggctaatgcaaagacgatgtggtagccgtgatagtttgcgacagtgccgtcagcgttttgta atggccagctgtcccaaacgtccaggccttttgcagaagagatatttttaattgtggacgaatcaaattcagaaacttgatatttttcatttttt tgctgttcagggatttgcagcatatcatggcgtgtaatatgggaaatgccgtatgtttccttatatggcttttggttcgtttctttcgcaaacgctt gagttgcgcctcctgccagcagtgcggtagtaaaggttaatactgttgcttgttttgcaaactttttgatgttcatcgttcatgtctccttttttatg tactgtgttagcggtctgcttcttccagccctcctgtttgaagatggcaagttagttacgcacaataaaaaaagacctaaaatatgtaagg ggtgacgccaaagtatacactttgccctttacacattttaggtcttgcctgctttatcagtaacaaacccgcgcgatttacttttcgacctcatt ctattagactctcgtttggattgcaactggtctattttcctcttttgtttgatagaaaatcataaaaggatttgcagactacgggcctaaagaa ctaaaaaatctatctgtttcttttcattctctgtattttttatagtttctgttgcatgggcataaagttgcctttttaatcacaattcagaaaatatcat aatatctcatttcactaaataatagtgaacggcaggtatatgtgatgggttaaaaaggatcggcggccgctcgatttaaatc (completenucleotidesequenceofplasmidpSacB_pflD) SEQIDNO:9 tcgagaggcctgacgtcgggcccggtaccacgcgtcatatgactagttcggacctagggatgggatcgagctcttttccttgccgaca aggcggaagctttaggggaaattcccgtaggtgccgtattggtggatgaacggggcaatatcattggtgaaggctggaacctctctatt gtgaactcggatcccaccgcccatgccgaaattattgcgttgcgtaacgccgcgcagaaaatccaaaattaccgcctgctcaatacc actttatacgtgactttagaaccctgcaccatgtgcgccggcgcgattttacacagccgaatcaaacgcttggtattcggggcgtccgat tacaaaaccggtgcggtgggttccagatttcatttttttgaggattataaaatgaatcatggggttgagatcacaagcggtgtcttatagga tcaatgcagtcagaagttaagccgctttttccaaaagcgcagggaacagaaaaaacaacaaaaagctaccgcacttttacaacacc cccggcttaactcctctgaaaaatagtgacaaaaaaaccgtcataatgtttacgacggtttttttatttcttctaatatgtcacattaagcccg tagcctgcaagcaaccccttaacatgctccattaattcttttgtcggcggttttacatcttcaagctcgtatttatcgccgagtacttcccattta tgggcgcctagacggtgataaggtaataattccactttttcgatattcttcatatctttaatgaaattccccagcatgtgcaaatcttcgtcact atctgtataacccggcactacaacatggcggatccaggtacgctgatttcgatccgctaaatattttgcgaattcgagcactcttttattcg gcacgccaatcaggctttcgtgaacccgttcattcatttctttcaggtcaagcaacacaagatccgtgtcatcaatcaattcatcaataat atgatcatgatgacggacgaaaccgttggtatccaagcaagtattaattccttctttatggcaggctctgaaccagtcccgtacaaattcc gcctgtaaaatagcttcaccgccggaagcggtaactccgccgcccgaggcgttcataaaatggcgataggtcaccacttctttcattaa ttcttcaacggaaatttctttaccgccgtgcaaatcccaggtgtctctgttatggcaatatttacaacgcattaagcagccttgtaaaaataa aataaagcggattcccggcccgtcaactgtcccgcaggtttcaaatgaatgaattcgtcctaaaaccgacataatatgcccttaaataa tcaacaaaatatagcaagaagattatagcaaagaatttcgtttttttcagagaatagtcaaatcttcgcaaaaaactaccgcacttttatc 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 cctgaccattcttgtgtttggtatttaggatagaatcatttgcatcgaatttgtcgctgtctttaaagacgcggccagcgtttttccagctgtca atagaagtttcgccgactttttgatagaacatgtaaatcgatgtgtcatccgcatttttaggatctccggctaatgcaaagacgatgtggta gccgtgatagtttgcgacagtgccgtcagcgttttgtaatggccagctgtcccaaacgtccaggccttttgcagaagagatatttttaattg tggacgaatcaaattcagaaacttgatatttttcatttttttgctgttcagggatttgcagcatatcatggcgtgtaatatgggaaatgccgtat gtttccttatatggcttttggttcgtttctttcgcaaacgcttgagttgcgcctcctgccagcagtgcggtagtaaaggttaatactgttgcttgtt ttgcaaactttttgatgttcatcgttcatgtctccttttttatgtactgtgttagcggtctgcttcttccagccctcctgtttgaagatggcaagttagt tacgcacaataaaaaaagacctaaaatatgtaaggggtgacgccaaagtatacactttgccctttacacattttaggtcttgcctgcttta tcagtaacaaacccgcgcgatttacttttcgacctcattctattagactctcgtttggattgcaactggtctattttcctcttttgtttgatagaaa atcataaaaggatttgcagactacgggcctaaagaactaaaaaatctatctgtttcttttcattctctgtattttttatagtttctgttgcatgggc ataaagttgcctttttaatcacaattcagaaaatatcataatatctcatttcactaaataatagtgaacggcaggtatatgtgatgggttaaa aaggatcggcggccgctcgatttaaatc (nucleotidesequenceofldhA-genefromstrainDD1) SEQIDNO:10 ttgacaaaatcagtatgtttaaataaggagctaactatgaaagttgccgtttacagtactaaaaattatgatcgcaaacatctggatttgg cgaataaaaaatttaattttgagcttcatttctttgattttttacttgatgaacaaaccgcgaaaatggcggagggcgccgatgccgtctgta ttttcgtcaatgatgatgcgagccgcccggtgttaacaaagttggcgcaaatcggagtgaaaattatcgctttacgttgtgccggttttaat aatgtggatttggaggcggcaaaagagctgggattaaaagtcgtacgggtgcctgcgtattcgccggaagccgttgccgagcatgcg atcggattaatgctgactttaaaccgccgtatccataaggcttatcagcgtacccgcgatgcgaatttttctctggaaggattggtcggtttt aatatgttcggcaaaaccgccggagtgattggtacgggaaaaatcggcttggcggctattcgcattttaaaaggcttcggtatggacgtt ctggcgtttgatccttttaaaaatccggcggcggaagcgttgggcgcaaaatatgtcggtttagacgagctttatgcaaaatcccatgtta tcactttgcattgcccggctacggcggataattatcatttattaaatgaagcggcttttaataaaatgcgcgacggtgtaatgattattaata ccagccgcggcgttttaattgacagccgggcggcaatcgaagcgttaaaacggcagaaaatcggcgctctcggtatggatgtttatg aaaatgaacgggatttgtttttcgaggataaatctaacgatgttattacggatgatgtattccgtcgcctttcttcctgtcataatgtgctttttac cggtcatcaggcgtttttaacggaagaagcgctgaataatatcgccgatgtgactttatcgaatattcaggcggtttccaaaaatgcaac gtgcgaaaatagcgttgaaggctaa (aminoacidsequenceofLdhAfromstrainDD1) SEQIDNO:11 MTKSVCLNKELTMKVAVYSTKNYDRKHLDLANKKFNFELHFFDFLLDEQTAKMAEGADAVCIFV NDDASRPVLTKLAQIGVKIIALRCAGFNNVDLEAAKELGLKVVRVPAYSPEAVAEHAIGLMLTLN RRIHKAYQRTRDANFSLEGLVGFNMFGKTAGVIGTGKIGLAAIRILKGFGMDVLAFDPFKNPAAE ALGAKYVGLDELYAKSHVITLHCPATADNYHLLNEAAFNKMRDGVMIINTSRGVLIDSRAAIEAL KRQKIGALGMDVYENERDLFFEDKSNDVITDDVFRRLSSCHNVLFTGHQAFLTEEALNNIADVT LSNIQAVSKNATCENSVEG (nucleotidesequenceofpflA-genefromstrainDD1) SEQIDNO:12 atgtcggttttaggacgaattcattcatttgaaacctgcgggacagttgacgggccgggaatccgctttattttatttttacaaggctgcttaa tgcgttgtaaatactgccataatagagacacctgggatttgcacggcggtaaagaaatttccgttgaagaattaatgaaagaagtggtg acctatcgccattttatgaacgcctcgggcggcggagttaccgcttccggcggtgaagctattttacaggcggaatttgtacgggactgg ttcagagcctgccataaagaaggaattaatacttgcttggataccaacggtttcgtccgtcatcatgatcatattattgatgaattgattgat gacacggatcttgtgttgcttgacctgaaagaaatgaatgaacgggttcacgaaagcctgattggcgtgccgaataaaagagtgctcg aattcgcaaaatatttagcggatcgaaatcagcgtacctggatccgccatgttgtagtgccgggttatacagatagtgacgaagatttgc acatgctggggaatttcattaaagatatgaagaatatcgaaaaagtggaattattaccttatcaccgtctaggcgcccataaatgggaa gtactcggcgataaatacgagcttgaagatgtaaaaccgccgacaaaagaattaatggagcatgttaaggggttgcttgcaggctac gggcttaatgtgacatattag (aminoacidsequenceofPflAfromstrainDD1) SEQIDNO:13 MSVLGRIHSFETCGTVDGPGIRFILFLQGCLMRCKYCHNRDTWDLHGGKEISVEELMKEVVTY RHFMNASGGGVTASGGEAILQAEFVRDWFRACHKEGINTCLDTNGFVRHHDHIIDELIDDTDLV LLDLKEMNERVHESLIGVPNKRVLEFAKYLADRNQRTWIRHVVVPGYTDSDEDLHMLGNFIKD MKNIEKVELLPYHRLGAHKWEVLGDKYELEDVKPPTKELMEHVKGLLAGYGLNVTY (nucleotidesequenceofpflD-genefromstrainDD1) SEQIDNO:14 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 (aminoacidofPflDfromstrainDD1) SEQIDNO:15 MAELTEAQKKAWEGFVPGEWONGVNLRDFIQKNYTPYEGDESFLADATPATSELWNSVMEGI KIENKTHAPLDFDEHTPSTITSHKPGYINKDLEKIVGLQTDAPLKRAIMPYGGIKMIKGSCEVYGR KLDPQVEFIFTEYRKTHNQGVFDVYTPDILRCRKSGVLTGLPDAYGRGRIIGDYRRLAVYGIDYL MKDKKAQFDSLQPRLEAGEDIQATIQLREEIAEQHRALGKIKEMAASYGYDISGPATNAQEAIQ WTYFAYLAAVKSQNGAAMSFGRTSTFLDIYIERDLKRGLITEQQAQELMDHLVMKLRMVRFLRT PEYDQLFSGDPMWATETIAGMGLDGRPLVTKNSFRVLHTLYTMGTSPEPNLTILWSEQLPEAF KRFCAKVSIDTSSVQYENDDLMRPDFNNDDYAIACCVSPMVVGKQMQFFGARANLAKTMLYAI NGGIDEKNGMQVGPKTAPITDEVLNFDTVIERMDSFMDWLATQYVTALNIIHFMHDKYAYEAAL MAFHDRDVFRTMACGIAGLSVAADSLSAIKYAKVKPIRGDIKDKDGNVVASNVAIDFEIEGEYPQ FGNNDPRVDDLAVDLVERFMKKVQKHKTYRNATPTQSILTITSNVVYGKKTGNTPDGRRAGAP FGPGANPMHGRDQKGAVASLTSVAKLPFAYAKDGISYTFSIVPNALGKDDEAQKRNLAGLMDG YFHHEATVEGGQHLNVNVLNREMLLDAMENPEKYPQLTIRVSGYAVRFNSLTKEQQQDVITRT FTQSM (nucleotidesequenceofwcaJ-genefromstrainDD1withinsertion ofcytosinebetweennucleotides81and82) SEQIDNO:16 Atgataaaacgccttttcgatattgttgtcgcattgatagcattgattttgttttcgcccttatatttgtttgtggcttatcaaggtaaaacaaaatt tgggatcaccggtgttatttaaacaaacccgccccggattgcatggtaaaccctttgagatgattaagttcagaacaatgaaagacgg cgcagatgaaaacggtaatattttgccggatgcggagcgcttaacacctttcggcaaaatgttgcgcgctaccagtctggacgagttgc cggaactttggaatgtattaaaaggtgatatgagtctggtggggccgcgtcctctactgatggaatatttgccgctgtataacgaaagac aggctaagcgccatgaagtgaaacccggaattaccggttatgcacaggtaaacggtcgcaatgccatcagttgggagcagaaattt gaattggatgcctggtatgttgaacatcaatccttgtggctggatttgaaaattatcgcaaagaccatccaaaaagtgatcgcaaaaga cgatattaatgcggcagatgatgccaccatgcctaaatttgaagggaataaaaaatcatga