Use of glycerol with limited feed of carbohydrates for fermentation

Abstract

The present invention relates to a process for producing an organic acid by fermentation, comprising the process steps: I) cultivating microorganisms in a culture medium to which are fed, as assimilable carbon sources, glycerol and a further carbonaceous compound, to allow the microorganisms to produce the organic acid, thereby obtaining a fermentation broth comprising the organic acid; II) recovering the organic acid or the salt thereof from the fermentation broth obtained in process step I); wherein, at least for a certain period of time in process step I), the consumption rate of the further carbonaceous compound (C.sub.Rc.c.; in g per liter per hour) is lower than the maximum theoretical consumption rate of the further carbonaceous compound (CR.sub.c.c. max; in g per liter per hour).

Claims

1. A process for producing an organic acid by fermentation, comprising the process steps: I) cultivating a modified microorganism in a culture medium to which is fed, as assimilable carbon sources, glycerol and a further carbonaceous compound, to allow the microorganism to produce the organic acid, thereby obtaining a fermentation broth comprising the organic acid; II) recovering the organic acid or the salt thereof from the fermentation broth obtained in process step I); wherein further carbonaceous compound is fed to the culture medium such that the consumption rate of the further carbonaceous compound (CR.sub.c.c.; in g per liter per hour) is lower than the maximum theoretical consumption rate of the further carbonaceous compound (CR.sub.c.c. max; in g per liter per hour), wherein the organic acid is succinic acid, wherein the further carbonaceous compound is a carbohydrate, and wherein the wildtype from which the modified microorganism has been derived belongs to the family of Pasteurellaceae, wherein the modified microorganism has, compared to its wildtype, 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, and wherein the modified 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 or an introduction of at least one mutation into the pflA-gene.

2. The process according to claim 1, wherein for a cultivation period of at least 30 minutes the consumption rate of the further carbonaceous compound (CR.sub.c.c.; in g per liter per hour) is lower than the maximum theoretical consumption rate of the further carbonaceous compound (CR.sub.c.c. max; in g per liter per hour).

3. The process according to claim 1, wherein the consumption rate of the further carbonaceous compound (CR.sub.c.c.; in g per liter per hour) is not more than 50% of the maximum theoretical consumption rate of the further carbonaceous compound (CR.sub.c.c. max; in g per liter per hour).

4. The process according to claim 1, wherein in process step I) glycerol and the further carbonaceous compound are fed into the culture medium in a total weight ratio glycerol:further carbonaceous compound of at least 5:1.

5. The process according to claim 1, wherein the carbohydrate is selected from the group consisting of sucrose, D-glucose or mixtures thereof.

6. The process according to claim 1, wherein the wildtype from which the modified microorganism has been derived belongs to the genus Basfia.

7. The process according to claim 6, wherein the microorganism used in process step I) belongs to the species Basfia succiniciproducens.

8. The process according to claim 7, wherein the wildtype 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.

9. The process according to claim 1, wherein the process further comprises the process step: III) converting the organic acid contained in the fermentation broth obtained in process step I) or converting the recovered organic acid obtained in process step II) into a secondary organic product being different from the organic acid by at least one chemical reaction.

10. The process according to claim 9, wherein the organic acid 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: 9).

(3) FIG. 2 shows a schematic map of plasmid pSacB ldhA (SEQ ID NO: 10).

(4) FIG. 3 shows a schematic map of plasmid pSacB pflA (SEQ ID NO: 11).

(5) FIG. 4 shows a schematic map of plasmid pSacB pflD (SEQ ID NO: 12).

EXAMPLES

Example 1: General Method for the Transformation of Basfia succiniciproducens

(6) TABLE-US-00001 TABLE 1 Nomenclature of the DD1-wildtype and mutants referred to in the examples Strain Wildtype DD1 (deposit DSM18541) DD1 ldhA pflA DD1 ldhA pflD

(7) Basfia succiniciproducens DD1 (wildtype) was transformed with DNA by electroporation using the following protocol:

(8) 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.).

(9) 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.

(10) 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

(11) Generation of Deletions Constructs:

(12) Deletion plasmids were constructed based on the vector pSacB (SEQ ID NO: 9). 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: 10), the pyruvate formate lyase activating enzyme pflA, pSacB_delta_pflA (SEQ ID NO: 11) and the pyruvate formate lyase pflD, pSacB_delta_pflD (SEQ ID NO: 12) were constructed. FIGS. 2, 3 and 4 show schematic maps of plasmid pSacB_delta_ldhA, pSacB_delta_pflA and pSacB_delta_pflD, respectively.

(13) In the plasmid sequence of pSacB (SEQ ID NO: 9) 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).

(14) In the plasmid sequence of pSacB_delta_ldhA (SEQ ID NO: 10) 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).

(15) In the plasmid sequence of pSacB_delta_pflA (SEQ ID NO: 11) the 5 flanking region of the pflA-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).

(16) In the plasmid sequence of pSacB_delta_pflD (SEQ ID NO: 12) 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. 4).

Example 3: Generation of Improved Succinate Producing Strains

(17) Generation of Deletion Mutants:

(18) 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 integration event of the plasmid into the genome of Basfia succiniciproducens. The Campbell in strain was then Campbelled our 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 our strains containing either the wildtype situation of the ldhA-locus or 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_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 our 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 pflA. c) Basfia succiniciproducens 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 our 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.

Example 4: Determination of Glucose or Glycerol Consumption Rate for DD1 ldhA pflA

(19) 1. Medium Preparation

(20) The composition of the cultivation medium used for the seed culture is described in table 1. For the main culture fermentation, the medium used is described in table 2.

(21) TABLE-US-00002 TABLE 1 Medium composition for cultivation of the seed culture Compound Concentration [g/L] Yeast extract (Bio Springer) 12.5 (NH.sub.4).sub.2SO.sub.4 0.05 succinic acid 2.5 Na.sub.2CO.sub.3 2 KH.sub.2PO.sub.4 1 MgCO.sub.3 50 glucose 50

(22) TABLE-US-00003 TABLE 2 Medium composition for cultivation of the main culture Compound Concentration [g/L] Yeast extract (Bio Springer) 12.5 (NH.sub.4).sub.2SO.sub.4 0.05 betaine 0.23 Na.sub.2CO.sub.3 2 KH.sub.2PO.sub.4 1 Polypropylene glycol (antifoam) 50 glucose 50
2. Cultivations and Analytics The main culture was inoculated after one seed culture step. For the seed culture, the medium described in table 1 was prepared by autoclaving the water, MgCO.sub.3 and Na.sub.2CO.sub.3 in a 2 L bottle. The other components were prepared and sterilized separated and added to the bottle afterwards in a sterile manner. 1% of cryo stocks were inoculated in a 2 L bottle containing 1800 mL of the liquid medium described above. A CO.sub.2 atmosphere was applied in the bottle. The starting pH of the medium was in the range of 7.5 to 8.0 due to the presence of MgCO.sub.3 and the CO.sub.2 atmosphere. The incubation was performed at 37 C., 170 rpm (shaking diameter: 2.5 cm) under anaerobic conditions for 12 hours. The culture reached OD.sub.600 nm of 21.

(23) A total of 5% of the seed culture described above was used to inoculate the main culture. The main culture was performed in 1 L-fermenters containing an initial volume of 500 mL of the liquid medium described in table 2. The medium was prepared by autoclaving the water, Na.sub.2CO.sub.3 and antifoam in the fermenter. The other components were prepared separated as solutions and added to the fermenter afterwards in a sterile manner. Glucose was used as the carbon source for these fermentations in order to determine its consumption rate by the succinic acid producing strain. 45 g/L of glucose was batched in the medium and also provided along the fermentation by feeding which was added at a rate of 2 g/L/h. The feeding started 4 h after the start of the fermentation. Glucose was in an excess amount during the entire fermentation time and it was measured by HPLC as described in the next section (an excess amount of glucose was confirmed by the fact the that detectable glucose concentration in the fermentation was always above 1 g/L). A pH 6.5 was kept constant during the fermentation and it was controlled by the addition of magnesium hydroxide 15 wt-%. CO.sub.2 was applied in the fermenter at flow of 0.1 vvm and the steering rate was 500 rpm. The analytics of the seed culture and the main culture are described in the next section.

(24) 3. Analytics

(25) The production of carboxylic acids was quantified via HPLC. The details about the HPLC method applied are described in table 3 and 4. Cell growth was measured by measuring the absorbance at 600 nm (OD.sub.600 nm) using a spectrophotometer (Ultrospec3000, Amersham Biosciences, Uppsala Sweden).

(26) TABLE-US-00004 TABLE 3 HPLC method (ZX-THF50) for analysis of glycerol, glucose and succinic acid 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
4. Results To calculate the glucose consumption rate, fermentation with batched glucose was performed. In this fermentation, glucose was in excess to allow for the calculation of the glucose consumption rate over time. The concentration of glucose was checked offline by HPLC during the fermentation and glucose was always detected in the medium. The measurements confirmed that glucose was always in excess in the medium. In this experiment 45 g/L of glucose was batched and a feeding rate of 2 g/L/h was applied after 4 hours.

(27) TABLE-US-00005 TABLE 5 Glucose consumption rate for different time intervals of the succinic acid fermentation with Basfia DD1 ldhA pflD described above Time interval [h] 0-9 9-15 15-23 23-40 Glucose consumption rate 2.76 3.87 2.22 1.61 [g/L/h] In order to calculate the consumption rate, the amount of glucose consumed in certain time interval was determined.

Example 5: Comparison of Fermentations with DD1 ldhA pflA Using Glycerol in Combination with Limited or Unlimited Glucose

(28) 5. Medium Preparation

(29) The composition of the cultivation media used for the seed culture and for the main culture are described in the previous section (example 4).
6. Cultivations and Analytics The main culture is inoculated from a seed train consisting of one seed culture. For the seed culture 1% of cryo stocks were inoculated in a 2 L bottle with containing 1800 ml of the liquid medium described in table 1 with a CO.sub.2 atmosphere. The starting pH of the medium was in the range of 7.5 to 8.0 due to the presence of MgCO.sub.3 and the CO.sub.2 atmosphere. The incubation was overnight at 37 C. and 170 rpm (shaking diameter: 2.5 cm) under anaerobic conditions for 12 hours (OD.sub.600 nm of 21). A total of 5% of the seed culture was used to inoculate the main culture in 1 L-fermenters containing an initial volume of 500 mL of the liquid medium described in table 2. Glycerol and glucose were the carbon sources. 54 g/L of glycerol and 8 g/L of glucose were provided by batch before the start of the fermentation. Glycerol was kept in excess during the entire fermentation and glucose was fed in rates that provided either limited or non-limited amounts of glucose for the cells. The feed is considered limited when the feeding rate is lower than the consumption rate of glucose that was previously determined. The feed started after 4 hours of fermentation and the feeding rate of glucose was either 2.5 g/L/h (unlimited) or 0.25 g/L/h (limited). The base utilized was magnesium hydroxide 15 wt.-%. CO.sub.2 was applied in the fermenter at flow of 0.1 vvm and the steering rate was 500 rpm. The analytics of the seed culture and the main culture have been performed as described in the previous section (table 3). The concentration of glucose was checked offline by HPLC during the fermentation and glucose was always detected in the medium when fed with 2.5 g/L/h. When the limited feed of glucose was applied (0.25 g/L/h), the sugar was not detected in the culture supernatant during a certain time interval.
7. Results
7a. Determination of Glucose Consumption Rate In this experiment 8 g/L of glucose and 54 g/L of glycerol were batched before the start of the fermentation and a feeding rate of 2.5 g/L/h of glucose were applied after 4 hours of fermentation. The glucose consumption rate was calculated as described in example 4 and the results for each time interval for this specific experimental set up are described in table 6. In this experiment the detectable glucose concentration in the fermentation was always above 1 g/L. The consumption rate for glycose given in table 6 thus represent the maximum theoretical consumption rate for the further carbonaceous compound CR.sub.c.c. max under the given culture conditions.

(30) TABLE-US-00006 TABLE 6 Glucose consumption rate for different time intervals of the succinic acid fermentation with B. succiniciproducens DD1 ldhA pflD Time interval [h] 0-9 9-15 15-23 23-40 Glucose consumption rate 0.94 2.48 2.09 1.29 [g/L/h]
7b. Comparison Between Limited and Unlimited Feed of Glucose in the Succinic Acid Titer and Space Time Yield of Fermentations with B. Succiniciproducens DD1 ldhA pflD After determination of the glucose consumption rate described above, either limited or unlimited feed of glucose were applied in fermentations. The different feeding rates to provide the limited and unlimited glucose concentrations are shown in table 7. The succinic acid titer and space time yield are also shown in table 7.

(31) TABLE-US-00007 TABLE 7 Comparison between limited and unlimited feed of glucose in the production of succinic acid Feeding rate [g/L/h] Unlimited Feed Limited Feed [Concentration (g/L)], time (h) 2.5 g/L/h 0.25 g/L/h [Glycerol] t = 0 54 54 [Glucose] t = 0 8 8 [Glucose] t = 23 h 25 0 [Succinic acid] t = 0 1.5 1.5 [Succinic acid] t = 23 h 25.3 50.6 Space time yield [g/L/h] 1.035 2.135 When feeding glucose with a rate of 0.25 g/L/h (limited feed), the glucose concentration in the fermentation was always below the detection limit. The results show an increase in the titer of succinic acid produced and a higher space time yield when glucose is added in a limited way.

Example 6: Comparison Between Different Limited Feeds of Glucose in a Fermentation with B. succiniciproducens DD1 ldhA pflD

(32) 8. Medium Preparation

(33) The compositions of the cultivation media used for the seed culture and for the main culture are described in the section 1 (example 4).
9. Cultivations and Analytics The main culture is inoculated from a seed train consisting of one seed culture. For the seed culture 1% of cryo stocks were inoculated in a 2 L bottle with containing 1800 ml of the liquid medium described in table 1 with a CO.sub.2 atmosphere. The starting pH of the medium was in the range of 7.5 to 8.0 due to the presence of MgCO.sub.3 and the CO.sub.2 atmosphere. The incubation was overnight at 37 C. and 170 rpm (shaking diameter: 2.5 cm) under anaerobic conditions for 12 hours (OD.sub.600 nm of 21). A total of 5% of the seed culture was used to inoculate the main culture in 1 L-fermenters containing an initial volume of 500 mL of the liquid medium described in table 2. Glycerol and glucose were the carbon sources. 50 g/L of glycerol and 8 g/L of glucose were provided by batch before the start of the fermentation. Glycerol was kept in excess during the entire fermentation and glucose was fed in rates that provided limited amounts of glucose for the cells. The feed is considered limited when the feeding rate is lower than the consumption rate of glucose that was previously determined. The feed started after 4 hours of fermentation and the feeding rates of glucose are described in table 8. The base utilized was magnesium hydroxide 15 wt-%. CO.sub.2 was applied in the fermenter at flow of 0.1 vvm and the steering rate was 500 rpm. The analytics of the seed culture and the main culture have been performed as described in the previous section (table 3). The concentration of glucose was checked offline by HPLC during the fermentation and glucose not detected in the culture supernatant during a certain time interval.
10. Results After calculating the glucose consumption rate as described in example 1, fermentations with feeding rates of glucose lower than the consumption rate (limited feed) were performed. Glycerol is in excess in the fermentation. The succinic acid titer and the space time yield are directly influenced by the amount of glucose fed into the fermenter (table 8). The results observed in table 8 confirm that in fermentations where glycerol is the main carbon source, the amount of succinic acid and the space time yield are improved with limited amounts of glucose. The lower the feeding rate of glucose, the higher is the succinic acid titer and the space time yield.

(34) TABLE-US-00008 TABLE 8 Influence of limited feeding rates of glucose in combination with an excess of glycerol on the titer and the space time yield of succinic acid fermentation by Basfia succiniciproducens DD1 ldhA pflA. Feed rate glucose Succinic acid titer Space time yield [g/L/h] [%] [g succinic acid/L/time (h)] 0.93 100 2.5 0.56 105 2.62 0.37 112 2.76 The examples show that limited amounts of glucose are beneficial when using glycerol as the main C-source for succinic acid fermentations.

(35) TABLE-US-00009 SEQUENCES SEQIDNO:1(nucleottdesequenceof16SrDNA ofstratnDD1) tttgatcctggctcagattgaacgctggcggcaggcttaacacat gcaagtcgaacggtagcgggaggaaagcttgctttctttgccga cgagtggcggacgggtgagtaatgcttggggatctggcttatgga gggggataacgacgggaaactgtcgctaataccgcgtaatat cttcggattaaagggtgggactttcgggccacccgccataagatg agcccaagtgggattaggtagttggtggggtaaaggcctacc aagccgacgatctctagctggtctgagaggatgaccagccacact ggaactgagacacggtccagactcctacgggaggcagca gtggggaatattgcacaatggggggaaccctgatgcagccatgcc gcgtgaatgaagaaggccttcgggttgtaaagttctttcggtg acgaggaaggtgtttgttttaataggacaagcaattgacgttaat cacagaagaagcaccggctaactccgtgccagcagccgcggt aatacggagggtgcgagcgttaatcggaataactgggcgtaaagg gcatgcaggcggacttttaagtgagatgtgaaagccccgg gcttaacctgggaattgcatttcagactgggagtctagagtactt tagggaggggtagaattccacgtgtagcggtgaaatgcgtagag atgtggaggaataccgaaggcgaaggcagccccttgggaagatac tgacgctcatatgcgaaagcgtggggagcaaacaggatt agataccctggtagtccacgcggtaaacgctgtcgatttggggat tgggctttaggcctggtgctcgtagctaacgtgataaatcgacc gcctggggagtacggccgcaaggttaaaactcaaatgaattgacg ggggcccgcacaagcggtggagcatgtggtttaattcgatg caacgcgaagaaccttacctactcttgacatccagagaatcctgt agagatacgggagtgccttcgggagctctgagacaggtgctg catggctgtcgtcagctcgtgttgtgaaatgttgggttaagtccc gcaacgagcgcaacccttatcctttgttgccagcatgtaaagatgg gaactcaaaggagactgccggtgacaaaccggaggaaggtggggat gacgtcaagtcatcatggcccttacgagtagggctaca cacgtgctacaatggtgcatacagagggcggcgataccgcgaggta gagcgaatctcagaaagtgcatcgtagtccggattggagt ctgcaactcgactccatgaagtcggaatcgctagtaatcgcaaatc agaatgttgcggtgaatacgttcccgggccttgtacacaccg cccgtcacaccatgggagtgggttgtaccagaagtagatagcttaa ccttcggggggggcgtttaccacggtatgattcatgactggg gtgaagtcgtaacaaggtaaccgtaggggaacctgcgg SEQIDNO:2(nucleottdesequenceof23SrDNAof stratnDD1) agtaataacgaacgacacaggtataagaatacttgaggttgtat ggttaagtgactaagcgtacaaggtggatgccttggcaatcaga ggcgaagaaggacgtgctaatctgcgaaaagcttgggtgagttg ataagaagcgtctaacccaagatatccgaatggggcaaccc agtagatgaagaatctactatcaataaccgaatccataggttat tgaggcaaaccgggagaactgaaacatctaagtaccccgagg aaaagaaatcaaccgagattacgtcagtagcggcgagcgaaagc gtaagagccggcaagtgatagcatgaggattagaggaat cggctgggaagccgggcggcacagggtgatagccccgtacttga aaatcattgtgtggtactgagcttgcgagaagtagggcggga cacgagaaatcctgtttgaagaaggggggaccatcctccaaggc taaatactcctgattgaccgatagtgaaccagtactgtgaagg aaaggcgaaaagaaccccggtgaggggagtgaaatagaacctga aaccttgtacgtacaagcagtgggagcccgcgagggtga ctgcgtaccttttgtataatgggtcagcgacttatattatgtag cgaggttaaccgaataggggagccgaagggaaaccgagtcttaact gggcgtcgagttgcatgatatagacccgaaacccggtgatctagcc atgggcaggttgaaggttgggtaacactaactggaggacc gaaccgactaatgttgaaaaattagcggatgacctgtggctggggg tgaaaggccaatcaaaccgggagatagctggttctccccg aaatctatttaggtagagccttatgtgaataccttcgggggtagag cactgtttcggctagggggccatcccggcttaccaacccgatgc aaactgcgaataccgaagagtaatgcataggagacacacggcgggt gctaacgttcgtcgtggagagggaaacaacccagacc gccagctaaggtcccaaagtttatattaagtgggaaacgaagtggg aaggcttagacagctaggatgttggcttagaagcagccatc atttaaagaaagcgtaatagctcactagtcgagtcggcctgcgcgg aagatgtaacggggctcaaatatagcaccgaagctgcggc atcaggcgtaagcctgttgggtaggggagcgtcgtgtaagcggaag aaggtggttcgagagggctgctggacgtatcacgagtgcg aatgctgacataagtaacgataaaacgggtgaaaaacccgttcgcc ggaagaccaagggttcctgtccaacgttaatcggggcag ggtgagtcggcccctaaggcgaggctgaagagcgtagtcgatggga aacgggttaatattcccgtacttgttataattgcgatgtggg gacggagtaggttaggttatcgacctgttggaaaaggtcgtttaag ttggtaggtggagcgtttaggcaaatccggacgcttatcaaca ccgagagatgatgacgaggcgctaaggtgccgaagtaaccgatacc acacttccaggaaaagccactaagcgtcagattataata aaccgtactataaaccgacacaggtggtcaggtagagaatactcag gcgcttgagagaactcgggtgaaggaactaggcaaaata gcaccgtaacttcgggagaaggtgcgccggcgtagattgtagaggt atacccttgaaggttgaaccggtcgaagtgacccgctggct gcaactgtttattaaaaacacagcactctgcaaacacgaaagtgga cgtatagggtgtgatgcctgcccggtgctggaaggttaattg atggcgttatcgcaagagaagcgcctgatcgaagccccagtaaacg gcggccgtaactataacggtcctaaggtagcgaaattcctt gtcgggtaagttccgacctgcacgaatggcataatgatggccaggc tgtctccacccgagactcagtgaaattgaaatcgccgtgaa gatgcggtgtacccgcggctagacggaaagaccccgtgaaccttta ctatagcttgacactgaaccttgaattttgatgtgtaggatag gtgggaggctttgaagcggtaacgccagttatcgtggagccatcctt gaaataccaccctttaacgtttgatgttctaacgaagtgcccg gaacgggtactcggacagtgtctggtgggtagtttgactggggcggt ctcctcccaaagagtaacggaggagcacgaaggtttgcta atgacggtcggacatcgtcaggttagtgcaatggtataagcaagctt aactgcgagacggacaagtcgagcaggtgcgaaagcag gtcatagtgatccggtggttctgaatggaagggccatcgctcaacgg ataaaaggtactccggggataacaggctgataccgccca agagttcatatcgacggcggtgtttggcacctcgatgtcggctcatc acatcctggggctgaagtaggtcccaagggtatggctgttcgc catttaaagtggtacgcgagctgggtttaaaacgtcgtgagacagtt tggtccctatctgccgtgggcgttggagaattgagaggggct gctcctagtacgagaggaccggagtggacgcatcactggtgttccgg ttgtgtcgccagacgcattgccgggtagctacatgcggaa gagataagtgctgaaagcatctaagcacgaaacttgcctcgagatga gttctcccagtatttaatactgtaagggttgttggagacgac gacgtagataggccgggtgtgtaagcgttgcgagacgttgagctaac cggtactaattgcccgagaggcttagccatacaacgctca agtgtttttggtagtgaaagttattacggaataagtaagtagtcagg gaatcggct SEQIDNO:3(nucleottdesequenceofIdhA-gene fromstratnDD1) ttgacaaaatcagtatgtttaaataaggagctaactatgaaagttgc cgtttacagtactaaaaattatgatcgcaaacatctggatttgg cgaataaaaaatttaattttgagcttcatttctttgattttttactt gatgaacaaaccgcgaaaatggcggagggcgccgatgccgtctgta ttttcgtcaatgatgatgcgagccgcccggtgttaacaaagttggcg caaatcggagtgaaaattatcgctttacgttgtgccggttttaat aatgtggatttggaggcggcaaaagagctgggattaaaagtcgtacg ggtgcctgcgtattcgccggaagccgttgccgagcatgcg atcggattaatgctgactttaaaccgccgtatccataaggcttatca gcgtacccgcgatgcgaatttttctctggaaggattggtcggtttt aatatgttcggcaaaaccgccggagtgattggtacgggaaaaatcgg cttggcggctattcgcattttaaaaggcttcggtatggacgtt ctggcgtttgatccttttaaaaatccggcggcggaagcgttgggcgc aaaatatgtcggtttagacgagctttatgcaaaatcccatgtta tcactttgcattgcccggctacggcggataattatcatttattaaat gaagcggcttttaataaaatgcgcgacggtgtaatgattattaata ccagccgcggcgttttaattgacagccgggcggcaatcgaagcgtta aaacggcagaaaatcggcgctctcggtatggatgtttatg aaaatgaacgggatttgtttttcgaggataaatctaacgatgttatta cggatgatgtattccgtcgcctttcttcctgtcataatgtgcttttta ccggtcatcaggcgtttttaacggaagaagcgctgaataatatcgccg atgtgactttatcgaatattcaggcggtttccaaaaatgcaac gtgcgaaaatagcgttgaaggctaa SEQIDNO:4(aminoacidsequenceofIdhAfrom strainDD1) MTKSVCLNKELTMKVAVYSTKNYDRKHLDLANKKFNFELHFFDFLLDEQ TAKMAEGADAVCIFVNDDASRPVLTKLAQIGVKIIALRCAGFNNVDLE AAKELGLKVVRVPAYSPEAVAEHAIGLMLTLNRRIHKAYQRTRDANFSL EGLVGFNMFGKTAGVIGTGKIGLAAIRILKGFGMDVLAFDPFKNPAAE ALGAKYVGLDELYAKSHVITLHCPATADNYHLLNEAAFNKMRDGVMIIN TSRGVLIDSRAAIEALKRQKIGALGMDVYENERDLFFEDKSNDVITDDV FRRLSSCHNVLFTGHQAFLTEEALNNIADVT LSNIQAVSKNATCENSVEG SEQIDNO:5(nucleottdesequenceofpflA-gene fromstrainDD1) atgtcggttttaggacgaattcattcatttgaaacctgcgggacagt tgacgggccgggaatccgctttattttatttttacaaggctgcttaa tgcgttgtaaatactgccataatagagacacctgggatttgcacggc ggtaaagaaatttccgttgaagaattaatgaaagaagtggtg acctatcgccattttatgaacgcctcgggcggcggagttaccgcttc cggcggtgaagctattttacaggcggaatttgtacgggactgg ttcagagcctgccataaagaaggaattaatacttgcttggataccaa cggtttcgtccgtcatcatgatcatattattgatgaattgattgat gacacggatcttgtgttgcttgacctgaaagaaatgaatgaacgggt tcacgaaagcctgattggcgtgccgaataaaagagtgctcg aattcgcaaaatatttagcggatcgaaatcagcgtacctggatccgc catgttgtagtgccgggttatacagatagtgacgaagatttgc acatgctggggaatttcattaaagatatgaagaatatcgaaaaagtg gaattattaccttatcaccgtctaggcgcccataaatgggaa gtactcggcgataaatacgagcttgaagatgtaaaaccgccgacaaa agaattaatggagcatgttaaggggttgcttgcaggctac gggcttaatgtgacatattag SEQIDNO:6(amtnoacidsequenceofPflAfrom strainDD1) MSVLGRIHSFETCGTVDGPGIRFILFLQGCLMRCKYCHNRDTWDLH GGKEISVEELMKEVVTYRHFMNASGGGVTASGGEAILQAEFVRDWFR ACHKEGINTCLDTNGFVRHHDHIIDELIDDTDLVLLDLKEMNERVHE SLIGVPNKRVLEFAKYLADRNQRTWIRHVVVPGYTDSDEDLHMLGNFI KDMKNIEKVELLPYHRLGAHKWEVLGDKYELEDVKPPTKELMEHVKGL LAGYGLNVTY SEQIDNO:7(nucleotidesequenceofpflD-gene fromstrainDD1) atggctgaattaacagaagctcaaaaaaaagcatgggaaggatt cgttcccggtgaatggcaaaacggcgtaaatttacgtgacttt atccaaaaaaactatactccgtatgaaggtgacgaatcattctt agctgatgcgactcctgcaaccagcgagttgtggaacagcgtga tggaaggcatcaaaatcgaaaacaaaactcacgcacctttagat ttcgacgaacatactccgtcaactatcacttctcacaagcctgg ttatatcaataaagatttagaaaaaatcgttggtcttcaaacag acgctccgttaaaacgtgcaattatgccgtacggcggtatcaaaat gatcaaaggttcttgcgaagtttacggtcgtaaattagatccgc aagtagaatttattttcaccgaatatcgtaaaacccataaccaagg cgtattcgacgtttatacgccggatattttacgctgccgtaaat caggcgtgttaaccggtttaccggatgcttacggtcgtggtcgt attatcggtgactaccgtcgtttagcggtatacggtattgattac ctgatgaaagataaaaaagcccaattcgattcattacaaccgcgt ttggaagcgggcgaagacattcaggcaactatccaattacgtgaa gaaattgccgaacaacaccgcgctttaggcaaaatcaaagaaatgg cggcatcttacggttacgacatttccggccctgcgacaaacgcac aggaagcaatccaatggacatattttgcttatctggcagcggtt aaatcacaaaacggtgcggcaatgtcattcggtcgtacgtctacat tcttagatatctatatcgaacgtgacttaaaacgcggtttaatca ctgaacaacaggcgcaggaattaatggaccacttagtaatgaaatt acgtatggttcgtttcttacgtacgccggaatacgatcaattatt ctcaggcgacccgatgtgggcaaccgaaactatcgccggtatgggc ttagacggtcgtccgttggtaactaaaaacagcttccgcgt attacatactttatacactatgggtacttctccggaaccaaactta actattctttggtccgaacaattacctgaagcgttcaaacgtttctgt gcgaaagtatctattgatacttcctccgtacaatacgaaaatgatga cttaatgcgtcctgacttcaacaacgatgactatgcaatcgcat gctgcgtatcaccgatggtcgtaggtaaacaaatgcaattcttcggt gcgcgcgcaaacttagctaaaactatgttatacgcaattaac ggcggtatcgatgagaaaaatggtatgcaagtcggtcctaaaactgcg ccgattacagacgaagtattgaatttcgataccgtaatcg aacgtatggacagtttcatggactggttggcgactcaatatgtaaccg cattgaacatcatccacttcatgcacgataaatatgcatatg aagcggcattgatggcgttccacgatcgcgacgtattccgtacaatgg cttgcggtatcgcgggtctttccgtggctgcggactcattatc cgcaatcaaatatgcgaaagttaaaccgattcgcggcgacatcaaaga taaagacggtaatgtcgtggcctcgaatgttgctatcga cttcgaaattgaaggcgaatatccgcaattcggtaacaatgatccgcg tgttgatgatttagcggtagacttagttgaacgtttcatgaaa aaagttcaaaaacacaaaacttaccgcaacgcaactccgacacaatct atcctgactatcacttctaacgtggtatacggtaagaaa accggtaatactccggacggtcgtcgagcaggcgcgccattcggaccg ggtgcaaacccaatgcacggtcgtgaccaaaaaggt gcggttgcttcacttacttctgtggctaaacttccgttcgcttacgcg aaagacggtatttcatataccttctctatcgtaccgaacgcattag gtaaagatgacgaagcgcaaaaacgcaaccttgccggtttaatggacg gttatttccatcatgaagcgacagtggaaggcggtcaa cacttgaatgttaacgttcttaaccgtgaaatgttgttagacgcgatg gaaaatccggaaaaatacccgcaattaaccattcgtgtttcag gttacgcggttcgtttcaactcattaactaaagagcaacaacaagacg tcatcactcgtacgtttacacaatcaatgtaa SEQIDNO:8(amtnoacidofPflDfromstrainDD1) MAELTEAQKKAWEGFVPGEWQNGVNLRDFIQKNYTPYEGDESFLADAT PATSELWNSVMEGIKIENKTHAPLDFDEHTPSTITSHKPGYINKDLEK IVGLQTDAPLKRAIMPYGGIKMIKGSCEVYGRKLDPQVEFIFTEYRKT HNQGVFDVYTPDILRCRKSGVLTGLPDAYGRGRIIGDYRRLAVYGIDY LMKDKKAQFDSLQPRLEAGEDIQATIQLREEIAEQHRALGKIKEMAAS YGYDISGPATNAQEAIQWTYFAYLAAVKSQNGAAMSFGRTSTFLDIYI ERDLKRGLITEQQAQELMDHLVMKLRMVRFLRTPEYDQLFSGDPMWAT ETIAGMGLDGRPLVTKNSFRVLHTLYTMGTSPEPNLTILWSEQLPEAF KRFCAKVSIDTSSVQYENDDLMRPDFNNDDYAIACCVSPMVVGKQMQF FGARANLAKTMLYAINGGIDEKNGMQVGPKTAPITDEVLNFDTVIERM DSFMDWLATQYVTALNIIHFMHDKYAYEAALMAFHDRDVFRTMACGIA GLSVAADSLSAIKYAKVKPIRGDIKDKDGNVVASNVAIDFEIEGEYPQ FGNNDPRVDDLAVDLVERFMKKVQKHKTYRNATPTQSILTITSNVVYG KKTGNTPDGRRAGAPFGPGANPMHGRDQKGAVASLTSVAKLPFAYAKD GISYTFSIVPNALGKDDEAQKRNLAGLMDGYFHHEATVEGGQHLNVNV LNREMLLDAMENPEKYPQLTIRVSGYAVRFNSLTKEQQQDVITRT FTQSM SEQIDNO:9(completenucleotidesequenceof plasmidpSacB) tcgagaggcctgacgtcgggcccggtaccacgcgtcatatgacta gttcggacctagggatatcgtcgacatcgatgctcttctgcgtt aattaacaattgggatcctctagactccataggccgctttcctgg ctttgcttccagatgtatgctctcctccggagagtaccgtgactt tattttcggcacaaatacaggggtcgatggataaatacggcgatag tttcctgacggatgatccgtatgtaccggcggaagacaagctgca aacctgtcagatggagattgatttaatggcggatgtgctgagagca ccgccccgtgaatccgcagaactgatccgctatgtgtttgcgg atgattggccggaataaataaagccgggcttaatacagattaagcc cgtatagggtattattactgaataccaaacagcttacggagg acggaatgttacccattgagacaaccagactgccttctgattatta atatttttcactattaatcagaaggaataaccatgaattttacccg gattgacctgaatacctggaatcgcagggaacactttgccctttat cgtcagcagattaaatgcggattcagcctgaccaccaaactcg atattaccgctttgcgtaccgcactggcggagacaggttataagtt ttatccgctgatgatttacctgatctcccgggctgttaatcagttt ccggagttccggatggcactgaaagacaatgaacttatttactggg accagtcagacccggtctttactgtctttcataaagaaaccgaaa cattctctgcactgtcctgccgttattttccggatctcagtgagtt tatggcaggttataatgcggtaacggcagaatatcagcatgatacca gattgtttccgcagggaaatttaccggagaatcacctgaatatatca tcattaccgtgggtgagttttgacgggatttaacctgaacatca ccggaaatgatgattattttgccccggtttttacgatggcaaagtt tcagcaggaaggtgaccgcgtattattacctgtttctgtacaggttc atcatgcagtctgtgatggctttcatgcagcacggtttattaatac acttcagctgatgtgtgataacatactgaaataaattaattaattct gtatttaagccaccgtatccggcaggaatggtggctttttttttata ttttaaccgtaatctgtaatttcgtttcagactggttcaggatgagc tcgcttggactcctgttgatagatccagtaatgacctcagaactcca tctggatttgttcagaacgctcggttgccgccgggcgttttttattg gtgagaatccaagcactagcggcgcgccggccggcccggtgtgaaat accgcacagatgcgtaaggagaaaataccgcatcaggcgctcttccg cttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcga gcggtatcagctcactcaaaggcggtaatacggttatccacagaatc aggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaagg ccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctc cgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtg gcgaaacccgacaggactataaagataccaggcgtttccccctggaa gctccctcgtgcgctctcctgttccgaccctgccgcttaccggatac ctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctc acgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgg gctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatc cggtaactatcgtcttgagtccaacccggtaagacacgacttatcgc cactggcagcagccactggtaacaggattagcagagcgaggtatgtag gcggtgctacagagttcttgaagtggtggcctaactacggctaca ctagaaggacagtatttggtatctgcgctctgctgaagccagttacc ttcggaaaaagagttggtagctcttgatccggcaaacaaacc accgctggtagcggtggtttttttgtttgcaagcagcagattacgcg cagaaaaaaaggatctcaagaagatcctttgatcttttctacgg ggtctgacgctcagtggaacgaaaactcacgttaagggattttggtca tgagattatcaaaaaggatcttcacctagatccttttaaagg ccggccgcggccgccatcggcattttcttttgcgtttttatttgttaa ctgttaattgtccttgttcaaggatgctgtctttgacaacagatgttt tcttgcctttgatgttcagcaggaagctcggcgcaaacgttgattgtt tgtctgcgtagaatcctctgtttgtcatatagcttgtaatcacgacatt gtttcctttcgcttgaggtacagcgaagtgtgagtaagtaaaggttac atcgttaggatcaagatccatttttaacacaaggccagttttgtt cagcggcttgtatgggccagttaaagaattagaaacataaccaagcat gtaaatatcgttagacgtaatgccgtcaatcgtcatttttgat ccgcgggagtcagtgaacaggtaccatttgccgttcattttaaagacgt tcgcgcgttcaatttcatctgttactgtgttagatgcaatcagc ggtttcatcacttttttcagtgtgtaatcatcgtttagctcaatcata ccgagagcgccgtttgctaactcagccgtgcgttttttatcgctttgca gaagtttttgactttcttgacggaagaatgatgtgcttttgccatagta tgctttgttaaataaagattcttcgccttggtagccatcttcagttcc agtgtttgcttcaaatactaagtatttgtggcctttatcttctacgtag tgaggatctctcagcgtatggttgtcgcctgagctgtagttgccttc atcgatgaactgctgtacattttgatacgtttttccgtcaccgtcaaag attgatttataatcctctacaccgttgatgttcaaagagctgtctg atgctgatacgttaacttgtgcagttgtcagtgtttgtttgccgtaatg tttaccggagaaatcagtgtagaataaacggatttttccgtcaga tgtaaatgtggctgaacctgaccattcttgtgtttggtcttttaggata gaatcatttgcatcgaatttgtcgctgtctttaaagacgcggccag cgtttttccagctgtcaatagaagtttcgccgactttttgatagaacat gtaaatcgatgtgtcatccgcatttttaggatctccggctaatgc aaagacgatgtggtagccgtgatagtttgcgacagtgccgtcagcgttt tgtaatggccagctgtcccaaacgtccaggccttttgcaga agagatatttttaattgtggacgaatcaaattcagaaacttgatatttt tcatttttttgctgttcagggatttgcagcatatcatggcgtgtaata tgggaaatgccgtatgtttccttatatggcttttggttcgtttctttc gcaaacgcttgagttgcgcctcctgccagcagtgcggtagtaaagg ttaatactgttgcttgttttgcaaactttttgatgttcatcgttcatgt ctccttttttatgtactgtgttagcggtctgcttcttccagccctcctg tttgaagatggcaagttagttacgcacaataaaaaaagacctaaaatat gtaaggggtgacgccaaagtatacactttgccctttacacatttt aggtcttgcctgctttatcagtaacaaacccgcgcgatttacttttcga cctcattctattagactctcgtttggattgcaactggtctattttcct cttttgtttgatagaaaatcataaaaggatttgcagactacgggcctaa agaactaaaaaatctatctgtttcttttcattctctgtattttttata gtttctgttgcatgggcataaagttgcctttttaatcacaattcagaaa atatcataatatctcatttcactaaataatagtgaacggcaggt atatgtgatgggttaaaaaggatcggcggccgctcgatttaaatc SEQIDNO:10(completenucleotidesequenceof plasmidpSacB_delta_ldhA) tcgagaggcctgacgtcgggcccggtaccacgcgtcatatgactagtt cggacctagggatgggtcagcctgaacgaaccgcactt gtatgtaggtagttttgaccgcccgaatattcgttataccttggtgga aaaattcaaaccgatggagcaattatacaattttgtggcggcgc aaaaaggtaaaagcggtatcgtctattgcaacagccgtagcaaag tggagcgcattgcggaagccctgaagaaaagaggcatttc cgcagccgcttatcatgcgggcatggagccgtcgcagcgggaagc ggtgcaacaggcgtttcaacgggataatattcaagtggtgg tggcgaccattgcttttggtatggggatcaacaaatctaatgtgc gttttgtggcgcattttgatttatctcgcagcattgaggcgtattat caggaaaccgggcgcgcggggcgggacgacctgccggcggaagcggta ctgttttacgagccggcggattatgcctggttgcataaaat tttattggaagagccggaaagcccgcaacgggatattaaacggca taagctggaagccatcggcgaatttgccgaaagccagacc tgccgtcgtttagtgctgttaaattatttcggcgaaaaccgccaaac gccatgtaataactgtgatatctgcctcgatccgccgaaaaaat atgacggattattagacgcgcagaaaatcctttcgaccatttatcgca ccgggcaacgtttcggcacgcaatacgtaatcggcgtaatg cgcggtttgcagaatcagaaaataaaagaaaatcaacatgatgagttg aaagtctacggaattggcaaagataaaagcaaagaat actggcaatcggtaattcgtcagctgattcatttgggctttgtgcaac aaatcatcagcgatttcggcatggggaccagattacagctcac cgaaagcgcgcgtcccgtgctgcgcggcgaagtgtctttggaactggc catgccgagattatcttccattaccatggtacaggctccgc aacgcaatgcggtaaccaactacgacaaagatttatttgcccgcctgc gtttcctgcgcaaacagattgccgacaaagaaaacattc cgccttatattgtgttcagtgacgcgaccttgcaggaaatgtcgttgt atcagccgaccagcaaagtggaaatgctgcaaatcaacggt gtcggcgccatcaaatggcagcgcttcggacagccttttatggcgatt attaaagaacatcaggctttgcgtaaagcgggtaagaatc cgttggaattgcaatcttaaaatttttaactttttgaccgcactttta aggttagcaaattccaataaaaagtgcggtgggttttcgggaattttt aacgcgctgatttcctcgtcttttcaatttyttcgyctccatttgttc ggyggttgccggatcctttcttgactgagatccataagagagtagaa tagcgccgcttatatttttaatagcgtacctaatcgggtacgctttt tttatgcggaaaatccatatttttctaccgcactttttctttaaaga tttatacttaagtctgtttgattcaatttatttggaggttttatgcaa cacattcaactggctcccgatttaacattcagtcgcttaattcaagga ttctggcggttaaaaagctggcggaaatcgccgcaggaattgcttaca ttcgttaagcaaggattagaattaggcgttgatacgctggatcat gccgcttgttacggggcttttacttccgaggcggaattcggacgggcg ctggcgctggataaatccttgcgcgcacagcttactttggtg accaaatgcgggattttgtatcctaatgaagaattacccgatataaaat cccatcactatgacaacagctaccgccatattatgtggtcg gcgcaacgttccattgaaaaactgcaatgcgactatttagatgtattg ctgattcaccgwctttctccctgtgcggatcccgaacaaatcg cgcgggcttttgatgaactttatcaaaccggraaagtacgttatttc ggggtatctaactatacgccggctaagttcgccatgttgcaatctt atgtgaatcagccgttaatcactaatcaaattgagatttcgcctcttc atcgtcaggcttttgatgacggtaccctggattttttactggaaaa acgtattcaaccgatggcatggtcgccacttgccggcggtcgtttatt caatcaggatgagaacagtcgggcggtgcaaaaaacattactcgaaat cggtgaaacgaaaggagaaacccgtttagatacattggcttatgcctg gttattggcgcatccggcaaaaattatgccggttatggggtccggtaa aattgaacgggtaaaaagcgcggcggatgcgttacgaatttccttcac tgaggaagaatggattaaggtttatgttgccgcacagggacgggatatt ccgtaacatcatccgtctaatcctgcgtatctggggaaagatgcgtca tcgtaagaggtctataatattcgtcgttttgataagggtgccatatcc ggcacccgttaaaatcacattgcgttcgcaacaaaattattccttac gaatagcattcacctcttttaacagatgttgaatatccgtatcggca aaaatatcctctatatttgcggttaaacggcgccgccagttagcatat tgagtgctggttcccggaatattgacgggttcggtcataccgagccag tcttcaggttggaatccccatcgtcgacatcgatgctcttctgcgtta attaacaattgggatcctctagactccataggccgctttcctggctt tgcttccagatgtatgctctcctccggagagtaccgtgactttattt tcggcacaaatacaggggtcgatggataaatacggcgatagtttcctg acggatgatccgtatgtaccggcggaagacaagctgcaaacctgtca gatggagattgatttaatggcggatgtgctgagagcaccgccccgtg aatccgcagaactgatccgctatgtgtttgcggatgattggc cggaataaataaagccgggcttaatacagattaagcccgtatagggt attattactgaataccaaacagcttacggaggacggaatg ttacccattgagacaaccagactgccttctgattattaatatttttca ctattaatcagaaggaataaccatgaattttacccggattgacct gaatacctggaatcgcagggaacactttgccctttatcgtcagcagat taaatgcggattcagcctgaccaccaaactcgatattaccg ctttgcgtaccgcactggcggagacaggttataagttttatccgctg atgatttacctgatctcccgggctgttaatcagtttccggagttcc ggatggcactgaaagacaatgaacttatttactgggaccagtcagacc cggtctttactgtctttcataaagaaaccgaaacattctctg cactgtcctgccgttattttccggatctcagtgagtttatggcaggt tataatgcggtaacggcagaatatcagcatgataccagattgtttc cgcagggaaatttaccggagaatcacctgaatatatcatcattaccg tgggtgagttttgacgggatttaacctgaacatcaccggaaa tgatgattattttgccccggtttttacgatggcaaagtttcagcagga aggtgaccgcgtattattacctgtttctgtacaggttcatcatgca gtctgtgatggctttcatgcagcacggtttattaatacacttcagctg atgtgtgataacatactgaaataaattaattaattctgtatttaagc caccgtatccggcaggaatggtggctttttttttatattttaaccgt aatctgtaatttcgtttcagactggttcaggatgagctcgcttggac tcctgttgatagatccagtaatgacctcagaactccatctggatttg ttcagaacgctcggttgccgccgggcgttttttattggtgagaatcc aagcactagcggcgcgccggccggcccggtgtgaaataccgcacagat gcgtaaggagaaaataccgcatcaggcgctcttccgcttcctcgctc actgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagct cactcaaaggcggtaatacggttatccacagaatcaggggataacgc aggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgta aaaaggccgcgttgctggcgtttttccataggctccgcccccctgacg agcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgaca ggactataaagataccaggcgtttccccctggaagctccctcgtgcg ctctcctgttccgaccctgccgcttaccggatacctgtccgcctttc tcccttcgggaagcgtggcgctttctcatagctcacgctgtaggta tctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcac gaaccccccgttcagcccgaccgctgcgccttatccggtaactat cgtcttgagtccaacccggtaagacacgacttatcgccactggcag cagccactggtaacaggattagcagagcgaggtatgtaggcggtgct acagagttcttgaagtggtggcctaactacggctacactagaagg acagtatttggtatctgcgctctgctgaagccagttaccttcggaaa aagagttggtagctcttgatccggcaaacaaaccaccgctggt agcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaa aggatctcaagaagatcctttgatcttttctacggggtctgacg ctcagtggaacgaaaactcacgttaagggattttggtcatgagatta tcaaaaaggatcttcacctagatccttttaaaggccggccgc ggccgccatcggcattttcttttgcgtttttatttgttaactgttaa ttgtccttgttcaaggatgctgtctttgacaacagatgttttcttgc ctttgatgttcagcaggaagctcggcgcaaacgttgattgtttgtct gcgtagaatcctctgtttgtcatatagcttgtaatcacgacattgtt tcctttcgcttgaggtacagcgaagtgtgagtaagtaaaggttacat cgttaggatcaagatccatttttaacacaaggccagttttgttcagc ggcttgtatgggccagttaaagaattagaaacataaccaagcatgta aatatcgttagacgtaatgccgtcaatcgtcatttttgatccgcggga gtcagtgaacaggtaccatttgccgttcattttaaagacgttcgcgcgt tcaatttcatctgttactgtgttagatgcaatcagcggtttcatc acttttttcagtgtgtaatcatcgtttagctcaatcataccgagagc gccgtttgctaactcagccgtgcgttttttatcgctttgcagaagttttt gactttcttgacggaagaatgatgtgcttttgccatagtatgctttg ttaaataaagattcttcgccttggtagccatcttcagttccagtgtttg cttcaaatactaagtatttgtggcctttatcttctacgtagtgagga tctctcagcgtatggttgtcgcctgagctgtagttgccttcatcgatg aactgctgtacattttgatacgtttttccgtcaccgtcaaagattga tttataatcctctacaccgttgatgttcaaagagctgtctgatgctga tacgttaacttgtgcagttgtcagtgtttgtttgccgtaatgtttac cggagaaatcagtgtagaataaacggatttttccgtcagatgtaaat gtggctgaacctgaccattcttgtgtttggtcttttaggatagaatca tttgcatcgaatttgtcgctgtctttaaagacgcggccagcgtttttc cagctgtcaatagaagtttcgccgactttttgatagaacatgtaaatc gatgtgtcatccgcatttttaggatctccggctaatgcaaagacgatg tggtagccgtgatagtttgcgacagtgccgtcagcgttttgtaatggc cagctgtcccaaacgtccaggccttttgcagaagagatatttttaat tgtggacgaatcaaattcagaaacttgatatttttcatttttttgctg ttcagggatttgcagcatatcatggcgtgtaatatgggaaatgccgta tgtttccttatatggcttttggttcgtttctttcgcaaacgcttgag ttgcgcctcctgccagcagtgcggtagtaaaggttaatactgttgctt gttttgcaaactttttgatgttcatcgttcatgtctccttttttatg tactgtgttagcggtctgcttcttccagccctcctgtttgaagatgg caagttagttacgcacaataaaaaaagacctaaaatatgtaaggggtg acgccaaagtatacactttgccctttacacattttaggtcttgcctgc tttatcagtaacaaacccgcgcgatttacttttcgacctcattctatt agactctcgtttggattgcaactggtctattttcctcttttgttt gatagaaaatcataaaaggatttgcagactacgggcctaaagaactaa aaaatctatctgtttcttttcattctctgtattttttatagtttctgt tgcatgggcataaagttgcctttttaatcacaattcagaaaatatcat aatatctcatttcactaaataatagtgaacggcaggtatatgtg atgggttaaaaaggatcggcggccgctcgatttaaatc SEQIDNO:11(completenucleotidesequenceof plasmidpSacB_delta_pflA) tcgagtcaatgcggatttgacttatgatgtggcaaacaaccgatttcc gattattactacacgtaaaagttattggaaagcggcgattgcg gagtttctgggttatatccgcggctacgataatgcggcggatttc cgtaaattaggagcaaaaacctgggatgccaacgctaatgaaa atcaggtatggctgaataaccctcatcgcaaaggcaccgacgacat ggggcgcgtttacggcgtacagggcagagcctggcgtaa gcctaacggcgaaaccgttgatcaattacgcaaaattgtcaacaat ttaagtcgcggcattgatgatcgcggcgaaattctgaccttttt aaacccgggcgaattcgatctcggttgtctgcgcccttgtatgtac aatcacacgttttctttgctgggcgatacgctttatttaaccagtt atcaacgctcctgtgacgtacctttaggcttgaatttcaatcaaa ttcaagtatttacattcttagctttaatggcgcagattaccggtaa aaaagccggtcaggcatatcacaaaatcgtcaatgcgcatatttac gaagaccagctggaactaatgcgcgacgtgcagttaaaacgcga accgttcccgtcgccaaaactggaaattaatccggacattaaaacc cttgaagatttagaaacctgggtaaccatggatgatttcaacg tcgttggttaccaatgccacgaaccgataaaatatccgttctcgg tataaaccgacaaaagtgcggtcaaaaatttaatattttcatctgtt atagaaaatatttttcaacataaaatctagggatgcctgtttggcg tccgtaaatacgcagaaaaatattaaatttttgaccgcactttttt catctcaattaacagcctgataattcttatggatcaacaaattagct ttgacgaaaaaatgatgaatcgagctcttttccttgccgacaagg cggaagctttaggggaaattcccgtaggtgccgtattggtggatga acggggcaatatcattggtgaaggctggaacctctctattgtg aactcggatcccaccgcccatgccgaaattattgcgttgcgtaacg ccgcgcagaaaatccaaaattaccgcctgctcaataccactttata cgtgactttagaaccctgcaccatgtgcgccggcgcgattttacaca gccgaatcaaacgcttggtattcggggcgtccgattacaaaaccggtg cggtgggttccagatttcatttttttgaggattataaaatgaatcatg gggttgagatcacaagcggtgtcttacaggatcaatgcagtcagaagt taagccgctttttccaaaagcgcagggaacagaaaaaacaacaaaaag ctaccgcacttttacaacacccccggcttaactcctctgaaaaatagtg acaaaaaaaccgtcataatgtttacgacggtttttttatttcttaata tgcccttaaataatcaacaaaatatagcaagaagattatagcaaagaa tttcgtttttttcagagaatagtcaaatcttcgcaaaaaactaccgcac ttttatccgctttaatcaggggaattaaaacaaaaaaattccgcctat tgaggcggaatttattaagcaataagacaaactctcaattacattgat tgtgtaaacgtacgagtgatgacgtcttgttgttgctctttagttaa tgagttgaaacgaaccgcgtaacctgaaacacgaatggttaattgcgggt atttttccggattttccatcgcgtctaacaacatttcacggttaagaa cgttaacattcaagtgttgaccgccttccactgtcgcttcatgatg gaaataaccgtccattaaaccggcaaggttgcgtttttgcgcttcgtc atctttacctaatgcgttcggtacgatagagaaggtatatgaaatacc gtctttcgcgtaagcgaacggaagtttagccacagaagtaagtgaagc aaccgcacctttttggtcacgaccgtgcattgggtttgcacccggtcc gaatggcgcgcctgctcgacgaccgtccggagtattaccggttttctt accgtataccacgttagaagtgatagtcaggatagattgtgtcggag ttgcgttgcggtaagttttgtgtttttgaacttttttcatgaaacgt tcaactaagtctaccgctaaatcatcaacacgcggatcattgttacc gaattgcggatattcgccttcaatttcgaagtcgatagcaacattcg aggccacgacattaccgtctttatctttgatgtcgccgcgaatcggt ttaactttcgcatatttgattgcggataatgagtccgcagccacgga aagacccgcgataccgcaagccattgtacggaatacgtcgcgatcgtgg aacgccatcaatgccgcttcatatgcatatttatcgtgcatgaagtgg atgatgttcaatgcggttacatattgagtcgccaaccagtccatgaaac tgtccatacgttcgattacggtatcgaaattcaatacttcgtctgtaat cggcgcagttttaggaccgacttgcataccatttttctcatcgatacc gccgttaattgcgtataacatagttttagctaagtttgcgcgcgcacc gaagaattgcatttgtttacctacgaccatcggtgatacgcagcatgcg attgcatagtcatcgttgttgaagtcaggacgcattaagtcatcattttc gtattgtacggaggaagtatcaatagatactttcgcacagaaacgtttga acgcttcaggtaattgttcggaccaaagaatagttaagtttggt tccggagaagtacccatagtgtataaagtatgtaatacgcggaagctctt tagttaccaacggacgaccgtctaagcccataccggcgatagtttcggtt gccctctagactccataggccgctttcctggctttgcttccagatgta tgctctcctccggagagtaccgtgactttattttcggcacaaatacagg ggtcgatggataaatacggcgatagtttcctgacggatgatccgtatgt accggcggaagacaagctgcaaacctgtcagatggagattgatttaatg gcggatgtgctgagagcaccgccccgtgaatccgcagaactgatccgct atgtgtttgcggatgattggccggaataaataaagccgggcttaatac agattaagcccgtatagggtattattactgaataccaaacagcttac ggaggacggaatgttacccattgagacaaccagactgccttctgatt attaatatttttcactattaatcagaaggaataaccatgaattttacc cggattgacctgaatacctggaatcgcagggaacactttgcccttt atcgtcagcagattaaatgcggattcagcctgaccaccaaactcgata ttaccgctttgcgtaccgcactggcggagacaggttataagttttatc cgctgatgatttacctgatctcccgggctgttaatcagtttccgg agttccggatggcactgaaagacaatgaacttatttactgggaccag tcagacccggtctttactgtctttcataaagaaaccgaaacattctc tgcactgtcctgccgttattttccggatctcagtgagtttatggcag gttataatgcggtaacggcagaatatcagcatgataccagattgtttc cgcagggaaatttaccggagaatcacctgaatatatcatcattaccgt gggtgagttttgacgggatttaacctgaacatcaccggaaatgatgatt attttgccccgOtttacgatggcaaagtttcagcaggaaggtgaccgc gtattattacctgtttctgtacaggttcatcatgcagtctgtgatggc tttcatgcagcacggtttattaatacacttcagctgatgtgtgataac atactgaaataaattaattaattctgtatttaagccaccgtatccggc aggaatggtggctttttttttatattttaaccgtaatctgtaatttc gtttcagactggttcaggatgagctcgcttggactcctgttgataga tccagtaatgacctcagaactccatctggatttgttcagaacgctcg gttgccgccgggcgttttttattggtgagaatccaagcactagcggcg cgccggccggcccggtgtgaaataccgcacagatgcgtaaggagaaaa taccgcatcaggcgctcttccgcttcctcgctcactgactcgctgcgc tcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggt aatacggttatccacagaatcaggggataacgcaggaaagaacatgt gagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgtt gctggcgtttttccataggctccgcccccctgacgagcatcacaaaa atcgacgctcaagtcagaggtggcgaaacccgacaggactataaaga taccaggcgtttccccctggaagctccctcgtgcgctctcctgttcc gaccctgccgcttaccggatacctgtccgcctttctcccttcggga agcgtggcgctttctcatagctcacgctgtaggtatctcagttcggt gtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgtt cagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaa cccggtaagacacgacttatcgccactggcagcagccactggtaaca ggattagcagagcgaggtatgtaggcggtgctacagagttcttgaag tggtggcctaactacggctacactagaaggacagtatttggtatctg cgctctgctgaagccagttaccttcggaaaaagagttggtagctctt gatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgca agcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttg atcttttctacggggtctgacgctcagtggaacgaaaactcacgttaa gggattttggtcatgagattatcaaaaaggatcttcacctagatcct tttaaaggccggccgcggccgccatcggcattttcttttgcgttttt atttgttaactgttaattgtccttgttcaaggatgctgtctttgacaa cagatgttttcttgcctttgatgttcagcaggaagctcggcgcaaacg ttgattgtttgtctgcgtagaatcctctgtttgtcatatagcttgta atcacgacattgtttcctttcgcttgaggtacagcgaagtgtgagtaa gtaaaggttacatcgttaggatcaagatccatttttaacacaaggcca gttttgttcagcggcttgtatgggccagttaaagaattagaaacataa ccaagcatgtaaatatcgttagacgtaatgccgtcaatcgtcattttt gatccgcgggagtcagtgaacaggtaccatttgccgttcattttaaag acgttcgcgcgttcaatttcatctgttactgtgttagatgcaatcagc ggtttcatcacttttttcagtgtgtaatcatcgtttagctcaatcata ccgagagcgccgtttgctaactcagccgtgcgttttttatcgctttgca gaagtttttgactttcttgacggaagaatgatgtgcttttgccatagt atgctttgttaaataaagattcttcgccttggtagccatcttcagttcc agtgtttgcttcaaatactaagtatttgtggcctttatcttctacgta gtgaggatctctcagcgtatggttgtcgcctgagctgtagttgccttc atcgatgaactgctgtacattttgatacgtttttccgtcaccgtcaaag attgatttataatcctctacaccgttgatgttcaaagagctgtctg atgctgatacgttaacttgtgcagttgtcagtgtttgtttgccgtaat gtttaccggagaaatcagtgtagaataaacggatttttccgtcaga tgtaaatgtggctgaacctgaccattcttgtgtttggtcttttaggat agaatcatttgcatcgaatttgtcgctgtctttaaagacgcggccag cgtttttccagctgtcaatagaagtttcgccgactttttgatagaaca tgtaaatcgatgtgtcatccgcatttttaggatctccggctaatgc aaagacgatgtggtagccgtgatagtttgcgacagtgccgtcagcgtt ttgtaatggccagctgtcccaaacgtccaggccttttgcaga agagatatttttaattgtggacgaatcaaattcagaaacttgatatttt tcatttttttgctgttcagggatttgcagcatatcatggcgtgtaata tgggaaatgccgtatgtttccttatatggcttttggttcgtttctttc gcaaacgcttgagttgcgcctcctgccagcagtgcggtagtaaagg ttaatactgttgcttgttttgcaaactttttgatgttcatcgttcatg tctccttttttatgtactgtgttagcggtctgcttcttccagccctcc tgtttgaagatggcaagttagttacgcacaataaaaaaagacctaaaa tatgtaaggggtgacgccaaagtatacactttgccctttacacatttt aggtcttgcctgctttatcagtaacaaacccgcgcgatttacttttcg acctcattctattagactctcgtttggattgcaactggtctattttcct cttttgtttgatagaaaatcataaaaggatttgcagactacgggcct aaagaactaaaaaatctatctgtttcttttcattctctgtattttttata gtttctgttgcatgggcataaagttgcctttttaatcacaattcagaaa atatcataatatctcatttcactaaataatagtgaacggcaggt atatgtgatgggttaaaaaggatcggcggccgctcgatttaaatc SEQIDNO:12(completenucleotidesequenceof plasmidpSacB_delta_pflD) tcgagaggcctgacgtcgggcccggtaccacgcgtcatatgactagttc ggacctagggatgggatcgagctcttttccttgccgaca aggcggaagctttaggggaaattcccgtaggtgccgtattggtggat gaacggggcaatatcattggtgaaggctggaacctctctatt gtgaactcggatcccaccgcccatgccgaaattattgcgttgcgtaa cgccgcgcagaaaatccaaaattaccgcctgctcaatacc actttatacgtgactttagaaccctgcaccatgtgcgccggcgcgat tttacacagccgaatcaaacgcttggtattcggggcgtccgat tacaaaaccggtgcggtgggttccagatttcatttttttgaggatta taaaatgaatcatggggttgagatcacaagcggtgtcttatagga tcaatgcagtcagaagttaagccgctttttccaaaagcgcagggaac agaaaaaacaacaaaaagctaccgcacttttacaacacc cccggcttaactcctctgaaaaatagtgacaaaaaaaccgtcataat gtttacgacgctttttatttcttctaatatgtcacattaagcccg tagcctgcaagcaaccccttaacatgctccattaattcttttgtc ggcggttttacatcttcaagctcgtatttatcgccgagtacttcccat ttatgggcgcctagacggtgataaggtaataattccactttttcga tattcttcatatctttaatgaaattccccagcatgtgcaaatcttcg tcactatctgtataacccggcactacaacatggcggatccaggtacg ctgatttcgatccgctaaatattttgcgaattcgagcactcttttat tcggcacgccaatcaggctttcgtgaacccgttcattcatttctttc aggtcaagcaacacaagatccgtgtcatcaatcaattcatcaataat atgatcatgatgacggacgaaaccgttggtatccaagcaagtatt aattccttctttatggcaggctctgaaccagtcccgtacaaattcc gcctgtaaaatagcttcaccgccggaagcggtaactccgccgcccgag gcgttcataaaatggcgataggtcaccacttctttcattaa ttcttcaacggaaatttctttaccgccgtgcaaatcccaggtgtctc tgttatggcaatatttacaacgcattaagcagccttgtaaaaataa aataaagcggattcccggcccgtcaactgtcccgcaggtttcaaatg aatgaattcgtcctaaaaccgacataatatgcccttaaataa tcaacaaaatatagcaagaagattatagcaaagaatttcgttttttt cagagaatagtcaaatcttcgcaaaaaactaccgcacttttatc cgctttaatcaggggaattaaaacaaaaaaattccgcctattgaggc ggaatttattaagcaataagacaaactctcaattttaatacttc cttcttttctagtattgataagattgaaaccttgcaaggatgacgg cggatttgccgtcactctcacccaactaatgtggacgactggtaa accattgcattagaccaatgcaaacaccaccaccgacgatgttacct aaagtaacaggaattaaatttttaattactaaatggtacatat ctaaatttgcaaactgctcggcatttaaacccgttgcctgccagaat tccggcgatgcgaaatttgcaattaccatgcccatagggatca taaacatatttgctacgcagtgttcaaagcctgaagcgacaaayaac ccgatcggcaggatcataataaaagctttatccgttagagt yttgccggcataggccatccaaacggcaatacataccataatgttg caaagaatacctaaacagaaggcttcaayccaggtatgttct attttatgttgtgccgtatttaaaatggttaatccccactgaccgt ttgccgccatgatctgaccggaaaaccaaattaatgcaacaataa ataaaccgccgacaaaattaccgaartaaaccacaatccagttacg taacatctgaattgttgtaattttactctcaaagcgggcaata gtcgataaagttgatgaagtaaatagttcacagccgcaaaccgcca ccataattaccccgagagagaacaccaaaccgccgacc agtttagttaatccccaaggcgctcccgcagaggctgtttgagttgt tgtataaaaaacgaatgcaagagcaataaacataccggcag agatcgccgataaaaatgaataggcttgttttttcgtagctttat aaacgccgacgtctaacccggtttgagccatctcggttggcgaagc catccaagccaatttaaaatcttccgatttcattgagctttcctta gtaataaaactactcggaaatgagtagaactgccttaaagcataa atgatagattaaaaaatccaaaattgttgaatattatttaacgggg ggattataaaagattcataaattagataatagctaatttgagtgat ccatatcaccttttacagattttttgacctaaatcaaaattacccaa atagagtaataataccattataaagggtgtggatttattcctttggttt acgagataaattgctatttaagctgatttctgataaaaagtgcggt agatttttcccaaaaataaggaaacacaaaatggcagaagaa acaattttcagtaaaattattcgtaaagaaattcccgccgacattat atatcaagacgatcttgtcaccgcatttcgcgatattgcgccgca ggcaaaaactcatattttaattattccgaataaattgattccgacag taaacgacgtaaccgcccatcgtcgacatcgatgctcttctgcg ttaattaacaattgggatcctctagactttgcttccagatgtatgctc tcctccggagagtaccgtgactttattttcggcacaaatacaggg gtcgatggataaatacggcgatagtttcctgacggatgatccgtatgt accggcggaagacaagctgcaaacctgtcagatggagatt gatttaatggcggatgtgctgagagcaccgccccgtgaatccgcagaa ctgatccgctatgtgtttgcggatgattggccggaataaat aaagccgggcttaatacagattaagcccgtatagggtattattactga ataccaaacagcttacggaggacggaatgttacccattga gacaaccagactgccttctgattattaatatttttcactattaatcag aaggaataaccatgaattttacccggattgacctgaatacctgg aatcgcagggaacactttgccctttatcgtcagcagattaaatgcgga ttcagcctgaccaccaaactcgatattaccgctttgcgtacc gcactggcggagacaggttataagttttatccgctgatgatttacctg atctcccgggctgttaatcagtttccggagttccggatggcact gaaagacaatgaacttatttactgggaccagtcagacccggtctttac tgtctttcataaagaaaccgaaacattctctgcactgtcctgc cgttattttccggatctcagtgagtttatggcaggttataatgcggta acggcagaatatcagcatgataccagattgtttccgcagggaa atttaccggagaatcacctgaatatatcatcattaccgtgggtgagtt ttgacgggatttaacctgaacatcaccggaaatgatgattatttt gccccggtttttacgatggcaaagtttcagcaggaaggtgaccgcgta ttattacctgtttctgtacaggttcatcatgcagtctgtgatgg ctttcatgcagcacggtttattaatacacttcagctgatgtgtgataa catactgaaataaattaattaattctgtatttaagccaccgtatcc ggcaggaatggtggctttttttttatattttaaccgtaatctgtaatt tcgtttcagactggttcaggatgagctcgcttggactcctgttgatag atccagtaatgacctcagaactccatctggatttgttcagaacgctcg gttgccgccgggcgttttttattggtgagaatccaagcactag cggcgcgccggccggcccggtgtgaaataccgcacagatgcgtaagga gaaaataccgcatcaggcgctcttccgcttcctcgctc actgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagct cactcaaaggcggtaatacggttatccacagaatcaggg gataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccagg aaccgtaaaaaggccgcgttgctggcgtttttccat aggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtca gaggtggcgaaacccgacaggactataaagataccaggcgtttcccc ctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccg gatacctgtccgcctttctcccttcgggaagcgtggcgctttctcata gctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagc tgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttat ccggtaactatcgtcttgagtccaacccggtaagacacgacttatcg ccactggcagcagccactggtaacaggattagcagagcgaggtatgt aggcggtgctacagagttcttgaagtggtggcctaactacggctaca ctagaaggacagtatttggtatctgcgctctgctgaagccagttac cttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccg ctggtagcggtggtttttttgtttgcaagcagcagattacgcgcaga aaaaaaggatctcaagaagatcctttgatcttttctacggggtctga cgctcagtggaacgaaaactcacgttaagggattttggtcatgaga ttatcaaaaaggatcttcacctagatccttttaaaggccggccgcgg ccgccatcggcattttcttttgcgtttttatttgttaactgttaattg tccttgttcaaggatgctgtctttgacaacagatgttttcttgccttt gatgttcagcaggaagctcggcgcaaacgttgattgtttgtctgcgta gaatcctctgtttgtcatatagcttgtaatcacgacattgtttccttt cgcttgaggtacagcgaagtgtgagtaagtaaaggttacatcgttag gatcaagatccatttttaacacaaggccagttttgttcagcggcttgt atgggccagttaaagaattagaaacataaccaagcatgtaaatatcgtt agacgtaatgccgtcaatcgtcatttttgatccgcgggagtcagtg aacaggtaccatttgccgttcattttaaagacgttcgcgcgttcaat ttcatctgttactgtgttagatgcaatcagcggtttcatcacttttttc agtgtgtaatcatcgtttagctcaatcataccgagagcgccgtttgcta actcagccgtgcgttttttatcgctttgcagaagtttttgactttcttg acggaagaatgatgtgcttttgccatagtatgctttgttaaataaagat tcttcgccttggtagccatcttcagttccagtgtttgcttcaaata ctaagtatttgtggcctttatcttctacgtagtgaggatctctcagcgt atggttgtcgcctgagctgtagttgccttcatcgatgaactgctgt acattttgatacgtttttccgtcaccgtcaaagattgatttataatcct ctacaccgttgatgttcaaagagctgtctgatgctgatacgttaac ttgtgcagttgtcagtgtttgtttgccgtaatgtttaccggagaaatca gtgtagaataaacggatttttccgtcagatgtaaatgtggctgaa cctgaccattcttgtgtttggtcttttaggatagaatcatttgcatcga atttgtcgctgtctttaaagacgcggccagcgtttttccagctgtca atagaagtttcgccgactttttgatagaacatgtaaatcgatgtgtcat ccgcatttttaggatctccggctaatgcaaagacgatgtggta gccgtgatagtttgcgacagtgccgtcagcgttttgtaatggccagctgt cccaaacgtccaggccttttgcagaagagatatttttaattg tggacgaatcaaattcagaaacttgatatttttcatttttttgctgttc agggatttgcagcatatcatggcgtgtaatatgggaaatgccgtat gtttccttatatggcttttggttcgtttctttcgcaaacgcttgagt tgcgcctcctgccagcagtgcggtagtaaaggttaatactgttgcttgtt ttgcaaactttttgatgttcatcgttcatgtctccttttttatgtactg tgttagcggtctgcttcttccagccctcctgtttgaagatggcaagttagt tacgcacaataaaaaaagacctaaaatatgtaaggggtgacgccaaagt atacactttgccctttacacattttaggtcttgcctgcttta tcagtaacaaacccgcgcgatttacttttcgacctcattctattagact ctcgtttggattgcaactggtctattttcctcttttgtttgatagaaa atcataaaaggatttgcagactacgggcctaaagaactaaaaaatctatc tgtttcttttcattctctgtattttttatagtttctgttgcatgggc ataaagttgcctttttaatcacaattcagaaaatatcataatatctcat ttcactaaataatagtgaacggcaggtatatgtgatgggttaaa aaggatcggcggccgctcgatttaaatc