A genetically engineered bacterium and a method for producing poly 3-hydroxybutyrate using the same

Abstract

The present invention discloses a genetically engineered bacterium, characterized in that the starting strain of the genetically engineered bacterium is Corynebacterium glutamicum, and the genetically engineered bacterium comprises phaC gene, phaA gene and phaB gene, and the nucleotide sequences thereof are shown in SEQ ID NO:38, SEQ ID NO:37 and SEQ ID NO:36, respectively. The modified strain obtained by the present invention can produce PHB by using glucose and xylose in wheat straw hydrolyzate without adding antibiotics, thereby realizing that Corynebacterium glutamicum produces PHB by fermenting glucose and xylose in lignocellulose.

Description

Technical Field

[0001] The invention belongs to the field of synthetic biology and relates to a construction method of Corynebacterium glutamicum for producing poly 3-hydroxybutyrate by utilizing lignocellulose as a raw material and an application thereof. Background Art

[0002] Polyhydroxyalkanoates (PHAs) are a class of high molecular weight polyesters fully synthesized by microorganisms. This polymer material has thermochemical properties and physical strength comparable to petroleum-based polypropylene materials, and has good biocompatibility and biodegradability. Therefore, it has the potential to replace traditional petroleum-based plastics and reduce the "white pollution" problem that traditional petroleum-based plastics bring to the environment. PHA has different material properties depending on the type of constituent monomers and the degree of polymerization. The simplest PHA is poly 3-hydroxybutyrate, or PHB, which is polymerized from a single 3-hydroxybutyryl coenzyme A.

[0003] At present, the technology of producing PHB by microbial fermentation uses grain starch as the raw material source of fermentable monosaccharides, and its cost accounts for about half of the total cost of PHB. The production of PHB from grain starch not only greatly increases the production cost, but also has the problem of "competing with people for food", and cannot achieve the replacement of PHB for petroleum-based polymers. Among the non-grain carbohydrate sugar raw materials of non-grain fermentable monosaccharides, lignocellulosic biomass such as corn straw, wheat straw, rice straw, bagasse, and forestry waste are characterized by low price and wide sources. Fermentable monosaccharides such as glucose and xylose are obtained by advanced biorefining methods, and then fermented to produce PHB as sugar raw materials. It not only has the advantages of sufficient raw material sources and low costs, but also has great potential in reducing environmental pollution, carbon neutrality technology, and promoting agricultural industry upgrading.

[0004] The main technical obstacles to the production of PHB from lignocellulosic biomass raw materials include the fact that lignocellulose produces a large number of inhibitors during the pretreatment process. For example, furfural, 5-hydroxymethylfurfural (HMF), 4-hydroxybenzaldehyde (HBA), vanillin and syringaldehyde. These inhibitors will cause some strains to be unable to grow in the lignocellulosic system, so the fermentation strain must have strong tolerance to inhibitors. However, traditional PHB production strains, such as Burkholderia, Pseudomonas putida, halophilic bacteria, eutrophic alcaligenes and Escherichia coli modified by metabolic engineering, have weak tolerance to inhibitors. So far, there has been no research report on the application of high-solid content lignocellulose to high-yield PHB. Corynebacterium glutamicum has a strong tolerance to inhibitors in the lignocellulosic system, and can convert inhibitors such as furfural, 5-hydroxymethylfurfural (HMF), 4-hydroxybenzaldehyde (HBA), vanillin and syringaldehyde in the lignocellulosic system into alcohols and acids that are non-toxic to the bacteria. Therefore, Corynebacterium glutamicum can grow and ferment normally in a high-solid content lignocellulosic system. Corynebacterium glutamicum is also a GRAS strain certified by the U.S. Food Safety Administration. The products produced by it have excellent safety, do not contain endotoxins, and save downstream processing costs.

[0005] Corynebacterium glutamicum is not a natural PHB-producing strain. In recent years, some studies have expressed PHB synthesis-related genes in Corynebacterium glutamicum through plasmids to enable it to synthesize PHB using glucose or starch, but these studies have not achieved the production of PHB in lignocellulose systems by Corynebacterium glutamicum. This is because these studies have shortcomings:

[0006] The invention with application number 201410748914.6 is entitled "A method for increasing the L-arginine production of Corynebacterium scutellariae by introducing a poly-β-hydroxybutyrate metabolic pathway". The poly-β-hydroxybutyrate (PHB) synthesis operon gene phbCAB is transferred into Corynebacterium scutellariae Pdxw-10 by plasmid expression, so that it acquires the ability to accumulate PHB intracellularly. Corynebacterium scutellariae has no guarantee of survival in the biorefining fermentation system, has poor tolerance to inhibitors, cannot produce PHB in the lignocellulose system, and the expression of the phbCAB gene by plasmid leads to genetic instability, which also limits the subsequent transformation strategy for increasing PHB production.

[0007] The invention with application number 201910703084.8 is entitled "A strain of Escherichia coli with a streamlined chassis and its application in high-yield PHB", which discloses a recombinant strain of PHB that can synthesize 81.9% of the cell dry weight in a synthetic medium obtained by genetic engineering. However, the traditional PHB production strain has a weak tolerance to inhibitors in the lignocellulose system, making it unable to survive in the lignocellulose hydrolysate system. Therefore, there has been no actual research report on high-yield PHB from lignocellulose. Summary of the invention

[0008] The invention discloses an engineered bacterium of Corynebacterium glutamicum for producing poly 3-hydroxybutyrate, wherein three key genes for PHB synthesis are integrated step by step into the genome of Corynebacterium glutamicum by gene integration, so that the engineered bacterium of Corynebacterium glutamicum can stably produce PHB. The engineered bacterium of Corynebacterium glutamicum has strong tolerance to lignocellulose inhibitors, and can use lignocellulose hydrolysate with high solid content to ferment and produce PHB, thus realizing the application of real lignocellulose hydrolysate in PHB production.

[0009] In order to solve the above technical problems, the first aspect of the present invention provides a genetically engineered bacterium, the starting strain of which is Corynebacterium glutamicum, the genetically engineered bacterium comprises phaC gene, phaA gene and phaB gene, and the nucleotide sequences encoding the phaC gene, phaA gene and phaB gene are shown in SEQ ID NO:38, SEQ ID NO:37 and SEQ ID NO:36, respectively.

[0010] Preferably, the upstream of the phaC gene, phaA gene and phaB gene also includes a Peftu promoter, and the nucleotide sequence of the Peftu promoter is shown in SEQ ID NO:39.

[0011] In a specific embodiment, it also includes a gene encoding a pentose transporter, such as the araE gene, whose nucleotide sequence is shown in SEQ ID NO:49.

[0012] Preferably, a gene encoding acetate kinase, such as the ack gene, is knocked out from the genome of the genetically engineered bacteria, and its nucleotide sequence is shown in SEQ ID NO:62.

[0013] More preferably, the genetically engineered bacteria further knock out the ldhA gene, the pdh gene and the MscCG gene, and the nucleotide sequences encoding the ldhA gene, the pdh gene and the MscCG gene are shown in SEQ ID NO:63, SEQ ID NO:64 and SEQ ID NO:65, respectively.

[0014] In a specific embodiment, the integration sites of the phaC gene, phaA gene and phaB gene are ldhA gene, pdh gene and MscCG gene respectively; and / or, the starting strain is C. glutamicum XyltoGA01; and / or, the phaC gene also includes a signal peptide upstream, such as a porB signal peptide or a CGR signal peptide, whose nucleotide sequences are shown in SEQ ID NO:57 and SEQ ID NO:70 respectively.

[0015] In order to solve the above technical problems, the second aspect of the present invention provides a method for constructing the genetically engineered bacteria as described in the first aspect of the present invention, the method comprising:

[0016] (1) integrating the phaC gene, phaA gene and phaB gene into Corynebacterium glutamicum;

[0017] (2) Knock out the gene encoding acetate kinase and integrate the gene encoding pentose transporter.

[0018] Preferably, it also includes:

[0019] (3) The phaC gene, phaA gene and phaB gene are respectively integrated into the ldhA gene, the pdh gene and the MscCG gene, thereby knocking out the ldhA gene, the pdh gene and the MscCG gene.

[0020] More preferably, it also includes:

[0021] (4) Connecting a nucleotide encoding a surface signal peptide to the phaC gene, wherein the surface signal peptide is, for example, a porB signal peptide or a CGR signal peptide, and the nucleotide sequences encoding the porB signal peptide and the CGR signal peptide are shown in SEQ ID NO: 57 and SEQ ID NO: 70, respectively.

[0022] In order to solve the above technical problems, the third aspect of the present invention provides a fusion gene, which comprises a nucleotide encoding a surface signal peptide and a phaC gene, the nucleotide sequences encoding the surface signal peptide are shown in SEQ ID NO:57 and SEQ ID NO:70, respectively, and the sequence of the phaC gene is shown in SEQ ID NO:38.

[0023] In order to solve the above technical problems, the fourth aspect of the present invention provides an expression cassette, which includes the fusion gene as described in the third aspect of the present invention, and the promoter of the expression cassette is, for example, Peftu, and its nucleotide sequence is shown in SEQ ID NO:39.

[0024] In order to solve the above technical problems, the fifth aspect of the present invention provides a recombinant expression vector, which comprises a backbone plasmid and the expression cassette as described in the fourth aspect of the present invention. Preferably, the backbone plasmid is pK18mobsacB.

[0025] In order to solve the above technical problems, the sixth aspect of the present invention provides a method for producing poly 3-hydroxybutyrate, which comprises: fermenting the genetically engineered bacteria as described in the first aspect of the present invention using pentose and / or hexose as substrates.

[0026] Preferably, the method further comprises adding ammonium sulfate and magnesium sulfate to the fermentation medium; and / or the hexose is glucose and the pentose is xylose.

[0027] In a specific embodiment, the fermentation is carried out under the following conditions: temperature 28-32° C., rotation speed 500-700 rpm, pH 6.8-7.2; and / or,

[0028] The content of glucose is 90-120 g / L, and the content of xylose is 0-50 g / L; and / or,

[0029] The content of the ammonium sulfate is 1-20 g / L, and the content of the magnesium sulfate is 0.2-0.8 g / L.

[0030] Preferably, the temperature is 30° C., the rotation speed is 600 rpm, and the pH is 7.0; and / or,

[0031] The content of glucose is 117.2 g / L, and the content of xylose is 45.7 g / L.

[0032] More preferably, 20.0 g / L ammonium sulfate, 5.0 g / L urea, 1.0 g / L potassium dihydrogen phosphate, 1.0 g / L dimethyl hydrogen phosphate, 0.25 g / L magnesium sulfate, 42.0 g / L morphinepropanesulfonic acid, 0.01 g / L calcium chloride, 0.01 g / L ferrous sulfate heptahydrate, 0.01 g / L manganese sulfate monohydrate, 1.0 mg / L zinc sulfate heptahydrate, 0.2 mg / L copper sulfate, 0.02 mg / L nickel chloride hexahydrate and 0.1 mg / L biotin.

[0033] In a specific embodiment, the substrate is derived from the hydrolyzate of natural raw materials, and the natural raw materials are agricultural waste or forestry waste. Preferably, the agricultural waste includes wheat straw, corn straw, rice straw, and bagasse.

[0034] In a specific embodiment, the method for preparing the hydrolyzate of the natural raw material comprises the following steps:

[0035] The natural raw materials are subjected to pretreatment, detoxification, enzymatic hydrolysis, solid-liquid separation to remove solids, and clear liquid is collected to obtain a hydrolyzate of the natural raw materials. Toxicity inhibitors that have an adverse effect on the fermentation of PHB are removed by detoxification. The pretreated natural raw materials or the enzymatic hydrolyzate can be detoxified by a strain that can metabolize the toxic inhibitors, thereby removing the toxic inhibitors. That is, the detoxification method can be biological detoxification.

[0036] Conventional pretreatment methods include acid pretreatment, alkali pretreatment, steam explosion pretreatment, hot water pretreatment and ionic liquid pretreatment, but the present invention is not limited to the above-mentioned pretreatment methods.

[0037] Preferably, the solid-liquid separation method includes: centrifugation and filtration; the centrifugal speed is preferably 10000-15000 rpm, and the centrifugal time is preferably 8-15 minutes.

[0038] More preferably, the serum is sterilized and filtered.

[0039] In order to solve the above technical problems, the seventh aspect of the present invention provides an application of the genetically engineered bacteria described in the first aspect of the present invention, the fusion gene described in the third aspect of the present invention, the expression cassette described in the fourth aspect of the present invention or the recombinant expression vector described in the fifth aspect of the present invention in the production of poly 3-hydroxybutyrate.

[0040] The starting strain of the present invention is Corynebacterium glutamicum XyltoGA01. The specific construction method refers to patent number 202011331840.8, and the name of the invention is implementation case 1 of "A method for constructing a synthetic pathway for generating glutamate using xylose in Corynebacterium glutamicum".

[0041] On the basis of being in accordance with the common sense in the art, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0042] The reagents and raw materials used in the present invention are commercially available.

[0043] The positive and progressive effect of the present invention is that the modified strain obtained by the present invention can produce PHB by using glucose and xylose in wheat straw hydrolyzate without adding antibiotics, and realizes that Corynebacterium glutamicum uses glucose and xylose in lignocellulose to ferment and produce PHB, and the ability to produce PHB exceeds that of existing Corynebacterium glutamicum. DETAILED DESCRIPTION

[0044] The strains used in the present invention include Escherichia coli DH5α, which is used for the construction of knockout integration plasmids; the starting strain comes from Patent No. 202011331840.8, and the invention name is "A method for constructing a synthetic pathway for producing glutamate using xylose in Corynebacterium glutamicum", which is constructed in Example 1 of Implementation Case 1 of the invention.

[0045] Reagents include:

[0046] Restriction endonucleases were used to cut plasmids or gene fragments to produce sticky ends and were purchased from Thermo Scientific (Wilmington, DE, USA). DNA polymerase was used to amplify gene fragments, and DNA ligase was used to ligate gene fragments and plasmid vectors cut by enzymes. Both enzymes were purchased from Takara (Otsu, Japan). Plasmid extraction kits, PCR product purification and recovery kits, and gel recovery kits were purchased from Shanghai Jierui Biotechnology (Shanghai, China). Other chemical reagents were purchased from local suppliers. PHB standards were used to make liquid phase markers and were purchased from Sigma-Aldrich (Germany).

[0047] The culture medium involved in the embodiments and comparative examples includes:

[0048] LB medium: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract.

[0049] LK medium: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract, 50 μg / L kanamycin.

[0050] LBG competent medium: 6g / L peptone, 6g / L sodium chloride, 3g / L yeast powder, 30g / L glycine, 1g / L Tween-80, 20g / L glucose.

[0051] YP medium: 1.0 g / L potassium dihydrogen phosphate, 3.0 g / L urea, 0.6 g / L magnesium sulfate, 5.0 g / L yeast extract and 10.0 g / L peptone, 50 g / L glucose.

[0052] Example 1 Integration of the phaCAB gene cluster into the C. glutamicum genome

[0053] 1.1 Preparation of competent cells of Corynebacterium glutamicum

[0054] ① Take out the seed tube containing Corynebacterium glutamicum from the -80℃ refrigerator, use an inoculation loop to dip a small amount of bacterial solution and streak on the LB plate, and place the streaked plate in a 30℃ incubator for 2 days.

[0055] ② Pick a single colony of Corynebacterium glutamicum from the plate with good bacteria growth, inoculate it into a test tube containing 5 mL of LB medium, and place the test tube in a shaking incubator at 30°C for overnight culture.

[0056] ③ Add the cultured bacteria in the test tube to a 250mL shake flask containing 30mL LBG competent medium at a rate of 1%, and culture in a shaker at 30℃ for 6 hours.

[0057] ④Put the cultured bacteria into a 50mL centrifuge tube and precool it in ice water for 20 minutes.

[0058] ⑤ After precooling, centrifuge the centrifuge tube containing the bacteria at 4°C, 5000rpm for 10 minutes, discard the supernatant, add 30mL of precooled 10% glycerol to the centrifuge tube, and blow the bacteria to make them evenly suspended in the glycerol.

[0059] ⑥ Repeat step 5 twice. After discarding the supernatant for the last time, add 1 mL of pre-cooled 10% glycerol and mix the cells thoroughly. Dispense the mixed cells into 1.5 mL centrifuge tubes at a volume of 100 μL per tube and place in ice water for later use.

[0060] 1.2 Integration of the phaCAB gene cluster into the C. glutamicum genome

[0061] The PHB synthetic gene cluster phaCAB (H16_RS07135) used in this study, NCBI accession number WP_011615085.1, was amplified by PCR using the synthetic fragment as a template using primers phaCAB-F (as shown in SEQ ID NO:3) and phaCAB-R (as shown in SEQ ID NO:4) to obtain a phaCAB (as shown in SEQ ID NO:35) fragment with restriction sites at both ends. The gene fragment and the two expression plasmids were double-digested with EcoRI and SacI endonucleases, respectively, and then the two were connected together using T4 ligase to obtain the Peftu-phaCAB expression plasmid. Using the Peftu-phaCAB expression plasmid as a template, primers phaA-F (as shown in SEQ ID NO: 5), phaA-R (as shown in SEQ ID NO: 6), phaB-F (as shown in SEQ ID NO: 7), phaB-R (as shown in SEQ ID NO: 8), phaC-F (as shown in SEQ ID NO: 9), and phaC-R (as shown in SEQ ID NO: 10) were used to amplify phaA, phaB and phaC fragments by PCR. The knockout integration vector used was the pK18mobsacB plasmid, and the construction method of the knockout integration plasmid used was as follows.

[0062] For the pK18-Δpdh::phaA plasmid, its specific construction method is as follows: using the genome of C. glutamicum S9114 as a template, using primers pdh-up-F (as shown in SEQ ID NO: 17) and pdh-up-R (as shown in SEQ ID NO: 18) to amplify by PCR the upstream fragment of pdh (pdh-up, as shown in SEQ ID NO: 40), using primers pdh-down-F (as shown in SEQ ID NO: 21) and pdh-down-R (as shown in SEQ ID NO: 22) to amplify by PCR the downstream fragment (pdh-down, as shown in SEQ ID NO: 41); then using the Peftu-phaA expression plasmid as a template, using primers Peftu_phaA-F (as shown in SEQ ID NO: 19) and Peftu_phaA-R (as shown in SEQ ID NO: 20) to amplify by PCR the Peftu-phaA fragment (as shown in SEQ ID NO: 41). NO:42); then, by enzyme cutting and ligation, the three fragments were inserted into the pK18mobsacB plasmid in the order of pdh-up, Peftu-phaA and pdh-down to construct the pK18-Δpdh::phaA integration plasmid, and the Δpdh::phaA fragment was shown in SEQ ID NO:55.

[0063] For the pK18-ΔMscCG::phaB plasmid, its specific construction method is as follows: using the genome of C. glutamicum S9114 as a template, using primers MscCG-up-F (as shown in SEQ ID NO:23) and MscCG-up-R (as shown in SEQ ID NO:24) to amplify by PCR the upstream fragment of MscCG (MscCG-up, as shown in SEQ ID NO:43), using primers MscCG-down-F (as shown in SEQ ID NO:27) and MscCG-down-R (as shown in SEQ ID NO:28) to amplify by PCR the downstream fragment (MscCG-down, as shown in SEQ ID NO:44); then using the Peftu-phaB expression plasmid as a template, using primers Peftu_phaB-F (as shown in SEQ ID NO:25) and Peftu_phaB-R (as shown in SEQ ID NO:26) to amplify by PCR the Peftu-phaB fragment (as shown in SEQ ID NO:47). NO:45); then, by enzyme digestion and ligation, the three fragments were sequentially inserted into the pK18mobsacB plasmid in the order of MscCG-up, Peftu-phaB and MscCG-down to construct the pK18-ΔMscCG::phaB integration plasmid, and the ΔMscCG::phaB fragment was shown in SEQ ID NO:56.

[0064] For the pK18-ΔldhA2::phaC plasmid, its specific construction method is as follows: using the genome of C. glutamicum S9114 as a template, using primers ldhA2-up-F (as shown in SEQ ID NO: 11) and ldhA2-up-R (as shown in SEQ ID NO: 12) to obtain an upstream fragment (ldhA2-up, as shown in SEQ ID NO: 46) by PCR amplification, using primers ldhA2-down-F (as shown in SEQ ID NO: 15) and ldhA2-down-R (as shown in SEQ ID NO: 16) to obtain a downstream fragment (ldhA2-down, as shown in SEQ ID NO: 47) by PCR amplification; then using the Peftu-phaC expression plasmid as a template, using primers Peftu_phaC-F (as shown in SEQ ID NO: 13) and Peftu_phaC-R (as shown in SEQ ID NO: 14) to obtain a Peftu-phaC fragment (as shown in SEQ ID NO: 48) by PCR amplification. NO:48); then, by means of overlap extension PCR, the three fragments were fused together in the order of ldhA2-up, Peftu-phaC and ldhA2-down to obtain the fusion fragment ΔldhA2::phaC (as shown in SEQ ID NO:54); the fusion fragment ΔldhA2::phaC and the pK18mobsacB plasmid were double-digested with XbaI and HindIII endonucleases, and then the two were connected together with T4 ligase to construct the pK18-ΔldhA2::phaC integration plasmid.

[0065] The integration plasmids pK18-ΔldhA2::phaC, pK18-Δpdh::phaA, and pK18-ΔMscCG::phaB were sequentially transferred into C. glutamicum XyltoGA01 by electroporation, and the strain with correct homologous recombination was screened out by colony PCR verification and named C. glutamicum JH01.

[0066] The bacteria obtained after electroporation are evenly spread on the LK plate, and the plate is placed in a 30°C incubator for 2 days. The colonies grown on the plate are picked and placed in a PCR tube containing 20 μL LB medium. The universal primers on the plasmid and the primers on the genome of the target gene are used as a pair of verification primers. The strain with the correct band verified by the colony PCR method is considered to be a strain that has completed a homologous recombination. The successfully verified primary recombinant strain is inoculated into a test tube containing 5 mL LB medium and cultured overnight. The cultured bacteria are then diluted to an appropriate concentration and spread on the LB plate. Using the primers on the genome of the target gene as a pair of verification primers, the strain grown on the LB plate is used as a template, and the strain with the correct band verified by the colony PCR method is considered to be a strain that has completed a secondary homologous recombination.

[0067] Take out the preserved strain from the cryopreservation tube and streak it on the LB plate for 2-3 days. Pick a single colony from the cultured plate and inoculate it into a 100mL shake flask containing 10mL YP medium, culture it in a shaker at 30℃, 200rpm for 12 hours, then inoculate it into 30mL YP medium at a 10% inoculation rate for expansion, and continue to culture for 12 hours to complete the seed culture. Then inoculate the seed liquid into a 250mL shake flask containing 30mL YP medium at a 10% inoculation rate for fermentation. The fermentation conditions are 30℃, 200rpm, pH 7.0, and fermentation for 48h.

[0068] The results showed that the recombinant strain could stably produce PHB.

[0069] Example 2 Integration of heterologous transporter araE into the genome of C. glutamicum JH01

[0070] Using the genome of C. glutamicum S9114 as a template, primers were designed according to the upstream 1000 base sequence and the downstream 1000 base sequence of the ack gene, and the upstream fragment (ack-up, as shown in SEQ ID NO:50) was amplified by PCR using primers ack-up-F (as shown in SEQ ID NO:29) and ack-up-R (as shown in SEQ ID NO:30), and the downstream fragment (ack-down, as shown in SEQ ID NO:51) was amplified by PCR using primers ack-down-F (as shown in SEQ ID NO:33) and ack-down-R (as shown in SEQ ID NO:34). Then, using the pPH36-araE expression plasmid as a template, the PH36-araE fragment (as shown in SEQ ID NO:52) was amplified by PCR using primers PH36-araE-F (as shown in SEQ ID NO:31) and PH36-araE-R (as shown in SEQ ID NO:32). NO:52); then, by means of overlap extension PCR, the three fragments were fused together in the order of ack-up, PH36-araE and ack-down to obtain the fusion fragment Δack::araE (as shown in SEQ ID NO:53); the fusion fragment Δack::araE and the pK18mobsacB plasmid were double-digested with XbaI and SphI endonucleases, and then the two were connected together by enzyme cutting and ligation to construct the pK18-Δack::araE integration plasmid. Then, the integration plasmid pK18-Δack::araE was transferred into C. glutamicum JH01 by electroporation, and the strain with correct homologous recombination was screened out by colony PCR verification, and it was named C. glutamicum JH02.

[0071] Comparative Example 1 Construction of C. glutamicum JHB1 (PhaB integrated into gdh site) strain

[0072] For the pK18-Δgdh::phaB plasmid, its specific construction method is as follows: using the genome of C. glutamicum S9114 as a template, using primers gdh-up-F (as shown in SEQ ID NO:66) and gdh-up-R (as shown in SEQ ID NO:67) to amplify the upstream fragment (gdh-up) of gdh by PCR, and using primers gdh-down-F (as shown in SEQ ID NO:68) and gdh-down-R (as shown in SEQ ID NO:69) to amplify the downstream fragment (gdh-down) by PCR; then using the Peftu-phaB expression plasmid as a template, using primers Peftu_phaB-F (as shown in SEQ ID NO:25) and Peftu_phaB-R (as shown in SEQ ID NO:26) to amplify the downstream fragment (gdh-down). NO:26) was amplified by PCR to obtain the Peftu-phaB fragment; then, the three fragments were inserted into the pK18mobsacB plasmid in the order of gdh-up, Peftu-phaB and gdh-down by enzyme cutting and ligation to construct the pK18-Δgdh::phaB integration plasmid.

[0073] The integration plasmids pK18-ΔldhA2::phaC, pK18-Δpdh::phaA, and pK18-Δgdh::phaB were sequentially transferred into C. glutamicum XyltoGA01 by electroporation, and the strain with correct homologous recombination was screened out by colony PCR verification and named C. glutamicum JHB1.

[0074] C. glutamicum JHB1 and C. glutamicum JH01 were fermented using synthetic medium separately.

[0075] The modified strains C.glutamicum JHB1 and C.glutamicum JH01 were taken out from the cryopreservation tube and streaked on LB plates for 2-3 days. A single colony was picked from the cultured plate and inoculated into a 100mL shake flask containing 10mL YP medium, and cultured in a shaker at 30°C and 200rpm for 12 hours, and then inoculated into 30mL YP medium at a 10% inoculation amount for expansion, and continued to culture for 12 hours to complete the seed culture. The seed liquid was then inoculated into a 250mL shake flask containing 30mL YP medium at a 10% inoculation amount for fermentation. The fermentation conditions were 30°C, 200rpm, pH 7.0, and fermentation for 48h. The results are shown in Table 1. As can be seen from Table 1, C.glutamicum JHB1 cannot produce PHB, which means that integrating PhaB into the gdh site cannot produce PHB.

[0076] Table 1 Comparison of the yields of PHB producing strains

[0077] PHB yield (g / L) Intracellular PHB content (wt%) C.glutamicum JH01 (PhaB integrated into the MscCG site) 0.24 4.75 C.glutamicum JHB1 (PhaB integrated into the gdh locus) 0 0

[0078] Comparison of promoters in comparative example 2

[0079] The promoters used were PH36 promoter and Peftu promoter, respectively, and the remaining steps were the same as in Example 1 and Example 2. The comparison results are shown in Table 2. The modified strains were taken out from the cryopreservation tubes and streaked on LB plates for 2-3 days. A single colony was picked from the cultured plate and inoculated into a 100mL shake flask containing 10mL YP medium, cultured in a shaker at 30°C and 200rpm for 12 hours, and then inoculated into 30mL YP medium at a 10% inoculum for expansion, and continued to culture for 12 hours to complete the seed culture. The seed liquid was then inoculated into a fermenter containing 300mL YP medium at a 10% inoculum for fermentation. The fermentation conditions were PH 7.0, temperature 30°C, stirring speed 600rpm, ventilation volume 0.47~1.4VVM, fermentation for 48 hours, and the results are shown in Table 2. It can be seen from the results that the Peftu promoter is more suitable for expressing the phaCAB gene in C.glutamicum XyltoGA01.

[0080] Table 2 Comparison of expression promoters and yields of PHB production strains

[0081] Expression PHB yield (g / L) PHB content (wt%) C.glutamicum XyltoGA01 PH36-phaCAB 0.18 0.60 C.glutamicum XyltoGA01 Peftu-phaCAB 1.20 3.91

[0082] Example 3 Comparison of fermentation of modified strains C. glutamicum JH02 and C. glutamicum JH01 in synthetic medium

[0083] The modified strains C. glutamicum JH01 and C. glutamicum JH02 were taken out of the cryopreservation tube and streaked on LB plates for 2-3 days. A single colony was picked from the cultured plate and inoculated into a 100mL shake flask containing 10mL YP medium, cultured in a shaker at 30°C and 200rpm for 12 hours, and then inoculated into 30mL YP medium at a 10% inoculation rate for expansion, and continued to culture for 12 hours to complete the seed culture. The seed liquid was then inoculated into a 250mL shake flask containing 30mL YP medium at a 10% inoculation rate for fermentation. The fermentation conditions were 30°C, 200rpm, pH 7.0, and 48h. The yield and other fermentation results are shown in Table 3. It can be concluded from Table 3 that compared with JH01, the cell dry weight of JH02 increased by 18.8% and the PHB yield increased by 20.8%.

[0084] Table 3 Comparison of the yield of PHB producing strains

[0085]

[0086] Example 4 Overexpression of the phaC gene fused with the porB surface display signal peptide

[0087] First, an expression plasmid of (PorB)phaC was constructed. The specific construction method was as follows: using the genome of C. glutamicum S9114 as a template, using primers porB-F (as shown in SEQ ID NO:58) and porB-R (as shown in SEQ ID NO:59) to amplify the porB surface display signal peptide by PCR, using the Peftu-phaCAB expression plasmid as a template, using primers phaC-F (as shown in SEQ ID NO:60) and phaC-R (as shown in SEQ ID NO:61) to amplify the phaC gene by PCR, and then connecting the surface display signal peptide and the phaC gene by fusion PCR to obtain the porB_phaC fusion fragment, and then inserting the fusion fragment into the Peftumob expression vector by enzyme cutting and ligation (the same operation as in Example 1) to obtain Peftu-(porB)phaC. Then, the expression plasmid Peftu-(porB)phaC was transferred into C. glutamicum JH02 by electroporation, and the universal primers PH36-F (as shown in SEQ ID NO: 1) and PH36-R (as shown in SEQ ID NO: 2) on the expression plasmid were used as verification primers. The strain with the correct band was verified by colony PCR and was considered to be the recombinant Corynebacterium glutamicum containing the target expression plasmid, and was named C. glutamicum JH02: Peftu-(porB)phaC.

[0088] Example 5 Fermentation production of PHB using engineered strains in synthetic medium

[0089] Control strain C. glutamicum JH02: Construction of Peftu-phaC:

[0090] First, construct the expression plasmid of phaC, and the specific construction method is as follows: using the Peftu-phaCAB expression plasmid as a template, using primers phaC-F (as shown in SEQ ID NO: 60) and phaC-R (as shown in SEQ ID NO: 61) to amplify the phaC gene by PCR, and then insert the fusion fragment into the Peftumob expression vector by restriction ligation (the same operation as in Example 1) to obtain Peftu-phaC. Then, the expression plasmid Peftu-phaC is transferred into C. glutamicum JH02 by electroporation, and the universal primers PH36-F (as shown in SEQ ID NO: 1) and PH36-R (as shown in SEQ ID NO: 2) on the expression plasmid are used as verification primers. The strain with the correct band verified by the colony PCR method is considered to be a recombinant Corynebacterium glutamicum containing the target expression plasmid, and is named C. glutamicum JH02: Peftu-phaC.

[0091] Capability verification of PHB production by fermentation in synthetic medium:

[0092] The modified strains C.glutamicum JH02:Peftu-phaC and C.glutamicum JH02:Peftu-(proB)phaC were taken out from the cryopreservation tube and streaked on LB plates for 2-3 days. A single colony was picked from the cultured plate and inoculated into a 100mL shake flask containing 10mL YP medium, and cultured in a shaker at 30°C and 200rpm for 12 hours, and then inoculated into 30mL YP medium at a 10% inoculation rate for expansion, and continued to culture for 12 hours to complete the seed culture. The seed liquid was then inoculated into a 250mL shake flask containing 30mL YP medium at a 10% inoculation rate for fermentation. The fermentation conditions were 30°C, 200rpm, pH 7.0, 48h, and the results are shown in Table 4. As can be seen from the table, compared with the control, the intracellular PHB content of the C. glutamicum JH02:Peftu-(proB)phaC modified strain increased by 3.1 times and the PHB yield increased by 1.4 times.

[0093] Table 4 Comparison of yields of PHB producing strains

[0094]

[0095] Example 6 Construction of fusion gene using CGR_RS04950 signal peptide (SEQ ID NO: 70) and phaC gene

[0096] The CGR_RS04950 signal peptide (SEQ ID NO: 70) was used to construct a fusion protein with the phaC gene, and the other conditions were referred to Example 4. The effect of the obtained strain on the production of PHB by fermentation was tested under the same YP medium and fermentation conditions as Example 5, thereby verifying the effect of the signal peptide on the production of PHB. The results are shown in Table 5.

[0097] Table 5 PHB production of C. glutamicum JH02:Peftu-(CGR)phaC

[0098]

[0099] The comparative experiment in Table 5 was a batch fermentation carried out in a shake flask. As shown in Table 5, the yield of the genetically engineered bacteria using the CGR_RS04950 signal peptide was slightly higher than the yield of PHB produced by C. glutamicum JH02. Specifically, it can be seen from the table that the intracellular PHB content of C. glutamicum JH02 is 6.1% (w / w) of the cell dry weight, and the intracellular PHB content of C. glutamicum JH02: Peftu-(CGR)phaC using the CGR signal peptide is 8.1% (w / w) of the cell dry weight, which increases the yield by about 25%.

[0100] Example 7: Fermentation of PHB in wheat straw hydrolysate using the engineered strain obtained by transformation

[0101] Wheat straw was purchased from Nanyang, Henan in the spring of 2021. After two-step acid hydrolysis, it was determined to contain 30.5% cellulose, 24.9% hemicellulose, 21.6% lignin and 9.7% ash. The fermentation broth obtained after dry pretreatment, detoxification and enzymatic hydrolysis of the collected wheat straw was centrifuged at 12000rpm for 10 minutes using a high-speed centrifuge. The supernatant was collected and sterilized under high pressure and filtered to obtain a straw hydrolyzate containing glucose and xylose. The concentrations of glucose and xylose in the hydrolyzate were 117.2g / L and 45.7g / L, respectively. The modified strain C. glutamicum JH02 was fermented in a 3L tank using wheat straw hydrolyzate at a fermentation temperature of 30°C and a speed of 600rpm. The results showed that the modified strain can stably produce PHB using wheat straw hydrolyzate for 72h.

[0102] Table 6 PHB production by production strains in wheat straw hydrolysate

[0103]

[0104] As can be seen from Table 6, the fermentation results indicate that the modified strain obtained by the present invention has the ability to produce PHB by fermenting straw hydrolyzate.

[0105] The above specifically describes the operation examples of the technical solution of the present invention, which is not to be regarded as limiting the application of the present invention. Any equivalent replacement of the operating conditions is within the protection scope of the present invention. SEQUENCE LISTING <110> Shanghai Cathay Biotechnology Co., Ltd. CIBT USA <120> A genetically engineered bacterium and a method for producing poly 3-hydroxybutyrate using the same <130> P21016727C <160> 70 <170> PatentIn version 3.5 <210> 1 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PH36-F <400> 1 ttgacagctt atcatcaaaa gctgggtacc tctatctggt 40 <210> 2 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PH36-F <400> 2 tttgcgatca aataatgaca tggatcccat gctactccta 40 <210> 3 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> phaCAB-F <400> 3 ggaattcatg gcaaccggca agggcgca 28 <210> 4 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> phaCAB-R <400> 4 tccccgcggt tagcccatgt gcaggccgcc g 31 <210> 5 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> phaA-F <400> 5 gctctagaat gaccgatgtt gtcatcgttt ctg 33 <210> 6 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> phaA-R <400> 6 acgcgtcgac ttacttacgt tccactgcaa gtgc 34 <210> 7 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> phaB-F <400> 7 gctctagact cgaattccat ggtttacgcc attaatggca gaagttgc 48 <210> 8 <211> 43 <212> DNA <213> Artificial Sequence <220> <223> phaB -R <400> 8 acgcgtcgac ttgacagctt atcatagcgg taaccatcac ggg 43 <210> 9 <211> 46 <212> DNA <213> Artificial Sequence <220> <223> phaC-F <400> 9 gctctagatg cgatcaaata atgacatggg taaaaaatcc tttcgt 46 <210> 10 <211> 48 <212> DNA <213> Artificial Sequence <220> <223> phaC-R <400> 10 acgcgtcgac acgaaaggat tttttaccca tgtcattatt tgatcgca 48 <210> 11 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> ldhA2-up-F <400> 11 gctctagaga ctgctatcgg tgtggcttta cg 32 <210> 12 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> ldhA2-up-R <400> 12 accagataga ggtacccagc ttttggtagc cctttcagtt gttgg 45 <210> 13 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Peftu_phaC-F <400> 13 ccaacaactg aaagggctac caaaagctgg gtacctctat ctggt 45 <210> 14 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> Peftu_phaC-R <400> 14 agtgacaaca ttttttctcc ttaagcctta gccttgacgt aacgg 45 <210> 15 <211> 45 <212> DNA <213> Artificial Sequence <220> <223> ldhA2-down-F <400> 15 ccgttacgtc aaggctaagg cttaaggaga aaaaatgttg tcact 45 <210> 16 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> ldhA2-down-R <400> 16 cccaagctta gcactccttc aacgcctcca ccttcaccgc 40 <210> 17 <211> 31 <212> DNA <213> Artificial Sequence <220> <223> pdh-up-F <400> 17 ggaattcaat tgccacagaa ccccaaatac c 31 <210> 18 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> pdh-up-R <400> 18 cgggatccct gaactcctca acgttatggc tatt 34 <210> 19 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> Peftu_phaA-F <400> 19 cgggatcccg aaaagcaatt tgcttttcga cgcc 34 <210> 20 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Peftu_phaA-R <400> 20 acgcgtcgac ttacttacgt tccactgcaa gtgcc 35 <210> 21 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> pdh-down-F <400> 21 acgcgtcgac tgattgatac acctgctgtt ctc 33 <210> 22 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> pdh-down-R <400> 22 aactgcagcg attactggaa gtggcacttt at 32 <210> 23 <211> 28 <212> DNA <213> Artificial Sequence <220> <223> MscCG-up-F <400> 23 ggaattcgat ggcaaccatg ggaacgga 28 <210> 24 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> MscCG -up-R <400> 24 cgggatccga gccaagatta gcgctgaaaa gtagc 35 <210> 25 <211> 33 <212> DNA <213> Artificial Sequence <220> <223> Peftu_phaB-F <400> 25 cgggatcccg aaaagcaatt tgcttttcga cgc 33 <210> 26 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Peftu_phaB-R <400> 26 acgcgtcgac ttagcccatg tgcaggccgc cgtta 35 <210> 27 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> MscCG -down-F <400> 27 acgcgtcgac gacgctgatt acagacgtgt cccattt 37 <210> 28 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> MscCG -down-R <400> 28 aactgcagtg catctgccac aatatcgccg acgttc 36 <210> 29 <211> 36 <212> DNA <213> Artificial Sequence <220> <223> ack-up-F <400> 29 ctagtctaga atggctgccc accagctgct tgatca 36 <210> 30 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> ack-up-R <400> 30 atagaggtac ccagcttttg tagctgcgtc ctcct 35 <210> 31 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> PH36-araE-F <400> 31 aggaggacgc agctacaaaa gctgggtacc tctat 35 <210> 32 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> PH36-araE-R <400> 32 ccaagaggtc taggggcctc agacgccgat atttctcaac 40 <210> 33 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> ack-down-F <400> 33 gttgagaaat atcggcgtct gaggcccta gacctcttgg 40 <210> 34 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> ack-down-R <400> 34 acatgcatgc ttgtctgggc aaccaacccc 30 <210> 35 <211> 3851 <212> DNA <213> Artificial Sequence <220> <223> phaCAB <400> 35 atggcaaccg gcaagggcgc agctgcttct acccaggaag gtaagtctca gccattcaag 60 gttaccccag gcccattcga tccagcaacc tggcttgaat ggtcccgtca gtggcagggc 120 accgaaggta acggtcacgc agctgcatcc ggtatcccag gccttgatgc tcttgctggt 180 gttaagatcg ctccagcaca gctcggcgat atacagcagc gttacatgaa ggatttctct 240 gcactttggc aggcaatggc agaaggtaag gctgaagcta ccggcccact tcacgatcgc 300 cgtttcgcag gcgatgcatg gcgtaccaac ctgccatacc gtttcgctgc tgcattctac 360 ctcctcaacg cacgcgctct gaccgaactt gctgatgcag ttgaagcaga tgcaaagacc 420 cgtcagcgta tccgtttcgc aatctctcag tgggttgatg ctatgtcccc agcaaacttc 480 ctcgctacca acccagaagc acagcgcctt cttatcgaat ccggcggtga atccctgcgc 540 gctggtgtgc gtaacatgat ggaggacctg acccgcggca aaattctca gaccgatgaa 600 tccgcattcg aagtcggccg taacgtggct gtgaccgaag gcgcagttgt ttcgaaaac 660 gaatacttcc agcttcttca gtacaagcca ctgaccgata aggttcacgc acgcccactc 720 cttatggttc caccatgcat caacaagtac tacatccttg atcttcagcc agaatcttcc 780 cttgtgcgtc acgtcgtgga acagggtcac accgtgttcc tcgtgtcttg gcgtaaccca 840 gatgcatcta tggctggttc cacctgggat gattacatcg aacacgctgc tatccgcgca 900 atcgaagttg cacgcgacat atccggtcag gataagatca acgtcctggg tttctgcgtc 960 ggcggaacta tcgtgtctac cgcactcgct gtcctcgctg ctcgtggtga acacccagca 1020 gcatctgtga cccttctgac caccctcctt gatttcgctg atactggaat cctggatgtg 1080 ttcgtggatg aaggccacgt tcagcttcgt gaagcaaccc tgggcggcgg cgctggtgct 1140 ccatgcgcac tcctccgcgg tctggaactc gcaaacacct tctctttcct ccgcccaaac 1200 gatcttgttt ggaactacgt tgtcgataac taccttaagg gtaacacccc agtgccattc 1260 gatctccttt tctggaacgg tgatgcaacc aacctgccag gtccttggta ctgctggtat 1320 ctgcgtcaca cctaccttca gaacgaactt aaggttccag gtaaattgac cgtttgcggt 1380 gtgccagttg atcttgcttc tatcgatgtg ccaacctaca tctacggttc ccgcgaagat 1440 cacatcgtgc cttggaccgc tgcatacgca tccaccgcac tcctcgctaa caagctccgt 1500 ttcgtcctcg gcgcttccgg tcacatcgca ggtgttatca acccaccagc tagaacaag 1560 cgttctcact ggaccaacga tgcactgcca gaatccccac agcagtggct tgctggtgct 1620 atcgaacacc acggctcttg gtggccagat tggaccgcat ggcttgcagg tcaggcaggt 1680 gcaaagcgtg ctgctccagc taactacggt aacgctcgtt accgcgctat cgaaccagct 1740 ccaggccgtt acgtcaaggc taaggcttaa cgcttgcatg agtgccggcg tgcgtcatgc 1800 acggcgccgg caggcctgca ggttccctcc cgtttccatt gaaggacta caaatgacc 1860 gatgttgtca tcgtttctgc tgcacgtacc gctgttggta agttcggcgg ctctcttgct 1920 aagatcccag ctccagaact tggcgctggt gtcatcaagg cagcactcga acgcgctggc 1980 gtgaagccag aacaggtttc cgaagtgatc atgggtcagg ttctcaccgc tggctccggt 2040 cagaacccag cacgtcaggc tgcaatcaag gcaggtcttc cagcaatggt gccagctatg 2100 accatcaaca aggtttgcgg ttctggcctc aaggcagtta tgcttgcagc taacgctatc 2160 atggcaggtg atgctgaaat cgttgtggct ggtggtcagg aaaacatgtc tgctgctcca 2220 cacgtccttc caggttcccg cgatggtttc cgtatgggtg atgcaaagct cgtggatacc 2280 atgatcgtcg atggcctttg ggatgtgtac aaccagtacc acatgggtat caccgctgaa 2340 aacgtcgcaa aggaatacgg tatcacccgc gaagcacagg atgagtttgc agtcggctct 2400 cagaacaagg cagaagcagc tcagaaggct ggtaagttcg atgaagaaat cgtgccagtc 2460 cttatcccac agcgtaaggg cgatccagtc gcattcaaga ccgatgagtt cgtgcgtcag 2520 ggcgctaccc tggattctat gtccggcctt aagccagcat tcgataaggc tggcactgtg 2580 accgctgcaa acgcatccgg ccttaacgat ggcgcagctg cggtcgttgt tatgtctgct 2640 gcaaaggcaa aggaactcgg cctgacccca ctcgcaacca tcaagtccta cgctaacgct 2700 ggtgttgatc caaaggttat gggcatgggc ccagtgccag catctaagcg tgctctgtcc 2760 cgcgctgaat ggaccccaca ggatcttgat ctcatggaaa tcaacgaggc attcgcagca 2820 caggcactcg cagttcacca gcagatgggt tgggatacct ctaaggttaa cgttaacggc 2880 ggtgcaatcg ctatcggtca cccaatcggt gcttccggtt gccgtatcct cgttaccctc 2940 cttcacgaaa tgaagcgccg tgatgctaag aagggtcttg cttccctgtg catcggcggc 3000 ggtatgggtg tggcacttgc agtggaacgt aagtaaggaa ggggttttcc ggggccgcgc 3060 gcggttggcg cggacccggc gacgataacg aagccaatca aggagtggac atgacccagc 3120 gtatcgcata cgtgacgggg ggtatgggcg gcatcggtac ggcaatctgc cagcgtcttg 3180 caaaggatgg tttccgcgtc gttgcaggtt gcggtccaaa ctccccacgc cgcgaaaagt 3240 ggctcgaaca gcagaaggca ctgggcttcg atttcatcgc ttccgaaggt aacgttgctg 3300 attgggattc taccaagacc gcattcgata aggtcaagtc cgaagtcggc gaagtggatg 3360 tccttatcaa caacgctggt atcacccgcg atgttgtgtt ccgtaagatg acccgtgctg 3420 attgggatgc agttatcgat accaacctca cctctctgtt caacgttacc aagcaggtga 3480 tcgatggtat ggcagatcgt ggctggggtc gtatcgttaa catctcctct gtgaacggtc 3540 agaagggtca gttcggccag accaactact ctaccgctaa ggcaggcctg cacggcttca 3600 caatggcact tgctcaggaa gtcgcaacca agggtgtgac cgtgaacacc gtgtctccag 3660 gttacatcgc taccgatatg gttaaggcaa tccgtcagga tgtgctcgat aagatcgtcg 3720 caaccatccc agtcaagcgc cttggcctgc cagaagaaat cgcttctatc tgcgcttggc 3780 tgtcctctga agaatccggt ttctccacgg gcgctgattt ctcccttaac ggcggcctgc 3840 acatgggcta at 3851 <210> 36 <211> 1194 <212> DNA <213> Artificial Sequence <220> <223> phaA <400> 36 ggaggacata caatgaccga tgttgtcatc gtttctgctg cacgtaccgc tgttggtaag 60 ttcggcggct ctcttgctaa gatcccagct ccagaacttg gcgctgttgt catcaaggca 120 gcactcgaac gcgctggcgt gaagccagaa caggttttccg aagtgatcat gggtcaggtt 180 ctcaccgctg gctccggtca gaacccagca cgtcaggctg caatcaaggc aggtcttcca 240 gcaatggtgc cagctatgac catcaacaag gtttgcggtt ctggcctcaa ggcagttatg 300 cttgcagcta acgctatcat ggcaggtgat gctgaaatcg ttgtggctgg tggtcaggaa 360 aacatgtctg ctgctccaca cgtccttcca ggttcccgg atggtttccg tatgggtgat 420 gcaaagctcg tggataccat gatcgtcgat ggcctttggg atgtgtacaa ccagtaccac 480 atgggtatca ccgctgaaaa cgtcgcaaag gaatacggta tcacccgcga agcacaggat 540 gagtttgcag tcggctctca gaacaaggca gaagcagctc agaaggctgg taagttcgat 600 gaagaaatcg tgccagtcct tatcccacag cgtaagggcg atccagtcgc attcaagacc 660 gatgagtcg tgcgtcaggg cgctaccctg gattctatgt ccggccttaa gccagcattc 720 gataaggctg gcactgtgac cgctgcaaac gcatccggcc ttaacgatgg cgcagctgcg 780 gtcgttgtta tgtctgctgc aaaggcaaag gaactcggcc tgaccccact cgcaaccatc 840 aagtcctacg ctaacgctgg tgttgatcca aaggttatgg gcatgggccc agtgccagca 900 tctaagcgtg ctctgtcccg cgctgaatgg accccacagg atcttgatct catggaaatc 960 aacgaggcat tcgcagcaca ggcactcgca gttcaccagc agatgggttg ggatacctct 1020 aaggttaacg ttaacggcgg tgcaatcgct atcggtcacc caatcggtgc ttccggttgc 1080 cgtatcctcg ttaccctcct tcacgaaatg aagcgccgtg atgctaagaa gggtcttgct 1140 tccctgtgca tcggcggcgg tatgggtgtg gcacttgcag tggaacgtaa gtaa 1194 <210> 37 <211> 752 <212> DNA <213> Artificial Sequence <220> <223> phaB <400> 37 gaggacatac aatgacccag cgtatcgcat acgtgacggg gggtatgggc ggcatcggta 60 cggcaatctg ccagcgtctt gcaaaggatg gtttccgcgt cgttgcaggt tgcggtccaa 120 actccccacg ccgcgaaaag tggctcgaac agcagaaggc actgggcttc gatttcatcg 180 cttccgaagg taacgttgct gattgggatt ctaccaagac cgcattcgat aaggtcaagt 240 ccgaagtcgg cgaagtggat gtccttatca acaacgctgg tatcacccgc gatgttgtgt 300 tccgtaagat gacccgtgct gattgggatg cagttatcga taccaacctc acctctctgt 360 tcaacgttac caagcaggtg atcgatggta tggcagatcg tggctggggt cgtatcgtta 420 acatctcctc tgtgaacggt cagaagggtc agttcggcca gaccaactac tctaccgcta 480 aggcaggcct gcacggcttc acaatggcac ttgctcagga agtcgcaacc aagggtgtga 540 ccgtgaacac cgtgtctcca ggttacatcg ctaccgatat ggttaaggca atccgtcagg 600 atgtgctcga taagatcgtc gcaaccatcc cagtcaagcg ccttggcctg ccagaagaaa 660 tcgcttctat ctgcgcttg ctgtcctctg aagaatccgg tttctccacg ggcgctgatt 720 tctcccttaa cggcggcctg cacatgggct aa 752 <210> 38 <211> 1780 <212> DNA <213> Artificial Sequence <220> <223> phaC <400> 38 aggacataca atggcaaccg gcaagggcgc agctgcttct acccaggaag gtaagtctca 60 gccattcaag gttaccccag gcccattcga tccagcaacc tggcttgaat ggtcccgtca 120 gtggcagggc accgaaggta acggtcacgc agctgcatcc ggtatcccag gccttgatgc 180 tcttgctggt gttaagatcg ctccagcaca gctcggcgat atacagcagc gttacatgaa 240 ggatttctct gcactttggc aggcaatggc agaaggtaag gctgaagcta ccggcccact 300 tcacgatcgc cgtttcgcag gcgatgcatg gcgtaccaac ctgccatacc gtttcgctgc 360 tgcattctac ctcctcaacg cacgcgctct gaccgaactt gctgatgcag ttgaagcaga 420 tgcaaagacc cgtcagcgta tccgtttcgc aatctctcag tgggttgatg ctatgtcccc 480 agcaaacttc ctcgctacca acccagaagc acagcgcctt cttatcgaat ccggcggtga 540 atccctgcgc gctggtgtgc gtaacatgat ggaggacctg acccgcggca aaatatctca 600 gaccgatgaa tccgcattcg aagtcggccg taacgtggct gtgaccgaag gcgcagttgt 660 tttcgaaaac gaatacttcc agcttcttca gtacaagcca ctgaccgata aggttcacgc 720 acgcccactc cttatggttc caccatgcat caacaagtac tacatccttg atcttcagcc 780 agaatcttcc cttgtgcgtc acgtcgtgga acagggtcac accgtgttcc tcgtgtcttg 840 gcgtaaccca gatgcatcta tggctggttc cacctgggat gattacatcg aacacgctgc 900 tatccgcgca atcgaagttg cacgcgacat atccggtcag gataagatca acgtcctggg 960 tttctgcgtc ggcggaacta tcgtgtctac cgcactcgct gtcctcgctg ctcgtggtga 1020 acacccagca gcatctgtga cccttctgac caccctcctt gatttcgctg atactggaat 1080 cctggatgtg ttcgtggatg aaggccacgt tcagcttcgt gaagcaaccc tgggcggcgg 1140 cgctggtgct ccatgcgcac tctccgcgg tctggaactc gcaaacacct tctctttcct 1200 ccgcccaaac gatcttgttt ggaactacgt tgtcgataac taccttaagg gtaacacccc 1260 agtgccattc gatctccttt tctggaacgg tgatgcaacc aacctgccag gtccttggta 1320 ctgctggtat ctgcgtcaca cctaccttca gaacgaactt aaggttccag gtaaattgac 1380 cgtttgcggt gtgccagttg atcttgcttc tatcgatgtg ccaacctaca tctacggttc 1440 ccgcgaagat cacatcgtgc cttggaccgc tgcatacgca tccaccgcac tcctcgctaa 1500 caagctccgt ttcgtcctcg gcgcttccgg tcacatcgca ggtgttatca acccaccagc 1560 taagaacaag cgttctcact ggaccaacga tgcactgcca gaatccccac agcagtggct 1620 tgctggtgct atcgaacacc acggctcttg gtggccagat tggaccgcat ggcttgcagg 1680 tcaggcaggt gcaaagcgtg ctgctccagc taactacggt aacgctcgtt accgcgctat 1740 cgaaccagct ccaggccgtt acgtcaaggc taaggcttaa 1780 <210> 39 <211> 371 <212> DNA <213> Artificial Sequence <220> <223> Peftu <400> 39 atccaccatg gaattccgaa aagcaatttg cttttcgacg ccccaccccg cgcgttttag 60 cgtgtcagta ggcgcgtagg gtaagtgggg tagcggcttg ttagatatct tgaaatcggc 120 tttcaacagc attgatttcg atgtatttag ctggccgtta ccctgcgaat gtccacaggg 180 tagctggtag tttgaaaatc aacgccgttg cccttaggat tcagtaactg gcacattttg 240 taatgcgcta gatctgtgtg ctcagtcttc caggctgctt atcacagtga aagcaaaacc 300 aattcgtggc tgcgaaagtc gtagccacca cgaagtccag gaggacatac agagctcggt 360 acccgggggat c 371 <210> 40 <211> 919 <212> DNA <213> Artificial Sequence <220> <223> pdh-up <400> 40 aattgccaca gaaccccaaa taccaattgc agtgttgcgc tcacgctcat cctcaaacgt 60 aatgcggatc agagccaaag ttgcgggcat catcgtcgcc gcaccgatgc caaggaaagc 120 tctcgcagca acaagagccc acgcagttgg agcaaacgca gcaccaagtg aagcgattcc 180 gaaaatgctc aagcccatga ggaacatccg gcggtggccg attttgtcac ccaaagtgcc 240 ggtacccaaa agaagacccg ccatgagcag gggatatgcg ttgatgatcc aaacgcttg 300 ggtttcagtg gctgcgagct gttcacgcag cagagggagt gcggtgtaga gaatcgagtt 360 gtctacaccg atcagaaaga gaccaccgct gataacggcg aggaaagccc aacgttgggt 420 tttcgtaggc gcttgcgcct gtaaagtttc tgaagtcatg gatcgtaact gtaacgaatg 480 gtcggtacag ttacaactct tttgttggtg ttttaggcca cggcgctgtg tggcgattta 540 aggcgtcgga aatcgtaggg gactgtcagt gtgggtcggg ttctttgagg cgcttagagg 600 cgattctgtg aggtcacttt ttatggggtc ggggtctaaa tttggccagt tttcgaggcg 660 accagacagg cgtgcccacg atgtttaaat aggcgttccg tgggcatctg tgtttggttt 720 cgacgggctg aaaccaaacc agactgccca gcaacgacgg aaatcccaaa agtgggcatc 780 cctgtttggt accgagtacc cacccgggcc tgaaactccc tggcaggcgg gcgaagcgtg 840 gcaacaactc gaattgaaga gcacaattga agtcgcacca agttaggcaa cacaatagcc 900 ataacgttga ggagttcag 919 <210> 41 <211> 900 <212> DNA <213> Artificial Sequence <220> <223> pdh-down <400> 41 tgattgatac acctgctgtt ctcattgacc gcgagcgctt gaccgccaac attttcaaaa 60 tggcggccca cgccgcagcc aatgggattg ctctgcgtcc gcatgtgaaa acacacaaaa 120 tcattgagat tgcgcagatg caggtcgatg ccggtgcccg agggattacc tgcgcaacca 180 ttggcgaggc agaaattttt gccggcgcag gttttacgga catctttatt gcatatccgc 240 tgtatctaac cgatcatgca gtgcaacgcc taaacgcgat ccctggagca atttccatcg 300 gcgtggattc ggtagagatg gcacaggcga cggcgggttt gcgggaagat atcaaggctc 360 tgattgaagt tgattcggga catcgtagaa gtggagtcac ggcgactgcg ttagaactga 420 gcgagatccg tcaggcgctg ggcagcaggt atacaggaggt gttcactttt cctggacatt 480 catatggtcc gggaaatggt gagcaggcag cagctgatga gcttcaggct ttaaacaaca 540 gcatccagcg acttggtggc ggcctgactt ctggcggttc ctcgccgtct gcgcagttca 600 caggcgcaat cgatgagatg cgaccaggtg tgtatgtgtt taacgattcc caacaaatca 660 cctcgggagc ctgcactgaa aagcaggtgg caatgacggt gctgtctact gtggtcagcc 720 gaaatgtgtc agatcgtcgg attattttgg atgcaggatc caaaatcctc agcactgata 780 aaccagcatg gattgatggc aatggtttg ttctggggaa tcctgaggcc cgaatctctg 840 ctttgtcgga gcatcacgca accattttct ggccagataa agtgccactt ccagtaatcg 900 <210> 42 <211> 1517 <212> DNA <213> Artificial Sequence <220> <223> Peftu-phaA <400> 42 cgaaaagcaa tttgcttttc gacgccccac cccgcgcgtt ttagcgtgtc agtaggcgcg 60 tagggtaagt ggggtagcgg cttgttagat atcttgaaat cggctttcaa cagcattgat 120 ttcgatgtat ttagctggcc gttaccctgc gaatgtccac agggtagctg gtagtttgaa 180 aatcaacgcc gttgccctta ggattcagta actggcacat tttgtaatgc gctagatctg 240 tgtgctcagt cttccaggct gcttatcaca gtgaaagcaa aaccaattcg tggctgcgaa 300 agtcgtagcc accacgaagt ccaggaggac atacaatgac cgatgttgtc atcgtttctg 360 ctgcacgtac cgctgttggt aagttcggcg gctctcttgc taagatccca gctccagaac 420 ttggcgctgt tgtcatcaag gcagcactcg aacgcgctgg cgtgaagcca gaacaggttt 480 ccgaagtgat catgggtcag gttctcaccg ctggctccgg tcagaaccca gcacgtcagg 540 ctgcaatcaa ggcaggtctt ccagcaatgg tgccagctat gaccatcaac aaggtttgcg 600 gttctggcct caaggcagtt atgcttgcag ctaacgctat catggcaggt gatgctgaaa 660 tcgttgtggc tggtggtcag gaaaacatgt ctgctgctcc acacgtcctt ccaggttccc 720 gcgatggttt ccgtatgggt gatgcaaagc tcgtggatac catgatcgtc gatggccttt 780 gggatgtgta caaccagtac cacatgggta tcaccgctga aaacgtcgca aaaggaatacg 840 gtatcacccg cgaagcacag gatgagtttg cagtcggctc tcagaacaag cgagaagcag 900 ctcagaaggc tggtaagttc gatgaagaaa tcgtgccagt ccttatccca cagcgtaagg 960 gcgatccagt cgcattcaag accgatgagt tcgtgcgtca gggcgctacc ctggattcta 1020 tgtccggcct taagccagca ttcgataagg ctggcactgt gaccgctgca aacgcatccg 1080 gccttaacga tggcgcagct gcggtcgttg ttatgtctgc tgcaaaggca areaactcg 1140 gcctgacccc actcgcaacc atcaagtcct acgctaacgc tggtgttgat ccaaaggtta 1200 tgggcatggg cccagtgcca gcatctaagc gtgctctgtc ccgcgctgaa tggaccccac 1260 aggatcttga tctcatggaa atcaacgagg cattcgcagc acaggcactc gcagttcacc 1320 agcagatggg ttgggatacc tctaaggtta acgttaacgg cggtgcaatc gctatcggtc 1380 acccaatcgg tgcttccggt tgccgtatcc tcgttaccct ccttcacagaa atgaagcgcc 1440 gtgatgctaa gaagggtctt gcttccctgt gcatcggcgg cggtatgggt gtggcacttg 1500 cagtggaacg stands 1517 <210> 43 <211> 800 <212> DNA <213> Artificial Sequence <220> <223> MscCG-up <400> 43 gatggcaacc atgggaacgg agtccaagtt tgcatcagcg attggggtaa ccaagttggt 60 tgctcctggg ccagaggttg caatgcagac gccaacgcgt ccagtaacct gcgcgtagcc 120 ggttgctgcg tggcctgcgc cctgctcgtg gcgcaccaag acgtggcgca cctttgtgga 180 ggaatagc gggtcataca ccggtagcac cgcaccacca ggataccga acacgatgtc 240 ggcgttaagc tcctcgagcg atcgaacaat tgcctgtgca cctgtcatcc gctcaggggc 300 ggcggatcga ccacggcttg caaccgtggc gggagtgggc tgttgagaag ctgccacatt 360 420. 420. 420. 420. 420. 420. 420. 420. 420. 420 gatttcaact cagcctaaag acaaagccct catttaaaat tgttccgacg cggatgcgtg tgcacgcagt gcgacagatg tctgttgcaa agttggctac ttgggtcata accaacaaga 540 aagccctcgt tccaacactg tggtgagtgt tgtcgagggc gcttgacgag acgacttgga 600 aggccgttac ggcaggcgcc gcgcggttac tactacaagt cgaataatgg tcatggtgtg 720. tcatgctaca cacatcgagt ttccaattcc acaacgcacg aaagttccca cccccaaaac tcccccactt cagttaagga atcaggattc tcacaaagtt cgggcaggct cccgctactt ttcagcgcta atcttggctc 800 <210> 44 <211> 1000 <212> DNA <213> Artificial Sequence <220> <223> MscCG-down <400> 44 gacgctgatt acagacgtgt cccatttctt tactactatt ggaaattatg agttcagacg cagaaaaggc atccgtggag ctttccgaaa aatttcaccc agaacgcacc catattttgg 120 gcgccgttgt ttttggcctg atctcattat tagtcatcgg cgcagcccct footcctgt 180 tttggctgct cgcactccct gtcatcttcg gttactgggt tctaaaatca tccacgatcg 240 ttgatgaaca gggcatcacc gcaaactacg ccttcaaggg caaaaaggtt gtggcctggg 300 aaaacctcgc aggaatcgga ttcaagggtg cccgcacttt cgctcgcacc acctctgatg 360 cagaagtcac cctccccggc gtcaccttca actcccttcc ccgccttgaa gctgcttccc 420 acggccgcat ccccgatgcg atcaccgcaa gcaaggaagc agccgacggc aaggttgtag 480 tcgttcaaga agacggctac tccgtgatga tgtccaagga agagtacttg gagcgccaaa 540 aggcactggg caagccagtt cagttgagct tcgatgacga caccgatggg aataacac 600 aaacagaaag cgttgaatcc caagagaccg gacaagccgc gtctgaaacc tcacatcgtg 660 ataaccctgc gtcacagcac tagaggttaa taagccgtcc gaaccaaagg tccacacctc 720 tgcacgagta gaagctcacc caagttttca aagtgccgtt gattcttgac acccccccgc 780 cgctccttag agcagatttg aaaagcgcat catgatccca cttcgttcaa aagtcaccac 840 cgtcggtcgc aatgcagctg gcgctcgcgc cctttggcgt gccaccggca ccaaggaaaa 900 tgagttcggc aagccaattg ttgccatcgt gaactcctac acccagttcg tgcccggaca 960 cgttcacctt aagaacgtcg gcgatattgt ggcagatgca 1000 <210> 45 <211> 1076 <212> DNA <213> Artificial Sequence <220> <223> Peftu-phaB <400> 45 cgaaaagcaa tttgcttttc gacgccccac cccgcgcgtt ttagcgtgtc agtaggcgcg 60 tagggtaagt ggggtagcgg cttgttagat atcttgaaat cggctttcaa cagcattgat 120 ttcgatgtat ttagctggcc gttaccctgc gaatgtccac agggtagctg gtagtttgaa 180 aatcaacgcc gttgccctta ggattcagta actggcacat tttgtaatgc gctagatctg 240 tgtgctcagt cttccaggct gcttatcaca gtgaaagcaa aaccaattcg tggctgcgaa 300 agtcgtagcc accacgaagt ccaggaggac atacaatgac ccagcgtatc gcatacgtga 360 cggggggtat gggcggcatc ggtacggcaa tctgccagcg tcttgcaaag gatggtttcc 420 gcgtcgttgc aggttgcggt ccaaactccc cacgccgcga aaagtggctc gaacagcaga 480 aggcactggg cttcgatttc atcgcttccg aaggtaacgt tgctgattgg gattctacca 540 agaccgcatt cgataaggtc aagtccgaag tcggcgaagt ggatgtcctt atcaacaacg 600 ctggtatcac ccgcgatgtt gtgttccgta agatgacccg tgctgattgg gatgcagtta 660 tcgataccaa cctcacctct ctgttcaacg ttaccaagca ggtgatcgat ggtatggcag 720 atcgtggctg gggtcgtatc gttaacatct cctctgtgaa cggtcagaag ggtcagttcg 780 gccagaccaa ctactctacc gctaaggcag gcctgcacgg cttcacaatg gcacttgctc 840 aggaagtcgc aaccaagggt gtgaccgtga acaccgtgtc tccaggttac atcgctaccg 900 atatggttaa ggcaatccgt caggatgtgc tcgataagat cgtcgcaacc atcccagtca 960 agcgccttgg cctgccagaa gaaatcgctt ctatctgcgc ttggctgtcc tctgaagaat 1020 ccggtttctc cacgggcgct gatttctccc ttaacggcgg cctgcacatg ggctaa 1076 <210> 46 <211> 1005 <212> DNA <213> Artificial Sequence <220> <223> ldhA2-up <400> 46 ctagagactg ctatcggtgt ggctttacga gccataacga ggtacatgaa taccgcgatc 60 atggcgaagc cgaggaagac aagcatagga acctcgagat gtaggaaaca agtgatctga 120 actatgactg cactcacatg cagtcccatt aggtataccg aagtgtattc ctgattgcaa 180 taggttgggt gtactaagat gatgcaatgt caataaaccc gcatgccaaa gtgataaaac 240 ctgatccccg caagactgat caggtatacg ctcaaattcg acatgagatt gaaaatggca 300 cactcccccc agggcaacgc atgtcagagg tgtggctcgt tgagcacact ggagcctcaa 360 ggacgccggt cagggatgct ttacgacgtt tagccgccga tgagttagtg attttggaac 420 cacgacaagc cccaatggtt acccccttat cactccagac aatcagggat cttttcgagt 480 tccgtcgcgt tgtcgaagtt gcggctttag aaaatatttc gtcgcgagtc tccaactccc 540 cagaaatcca ggaacagttt ggccaactct ctacaaaatt caataaactt ttagacagct 600 660. accgattttc gggacttc accgattttc gatgaacttg tctcaagata ttgcaacaac cagttcctag aaaagagcat tagcagcctc aaaccccata caacccgact gcgcctaatt gcccatgcag accatgatcg actcaggcaa tccatcactg agcacatcga tatgtgcaac gcagttactg caggcgacct ccgtacggca ggcgcagcgt gtcggcaaca cctcattcac gtggagcaa gcatcctaaa cgcactgatc acaggcaact caccgttcct caacagtatt gatctgcgga attach tgaagccttg cacaacctta ggccgttggt atacatttcg gtataccaac aactgaaagg gctac <210> 47 <211> 920 <212> DNA <213> Artificial Sequence <220> <223> ldhA2-down <400> 47 ggagaaaaaa tgttgtcact caccgttgat ctgcgcgtta aacctggcca tctggaagaa ttccttaacg ccatttcggt taatgcgcag cgcagctttc aagatgaaga aggttgtgtg tatttcgacg tttcacaaga tgttgatgat gagcttcatt tcagtttctt tgagctctac gcggatcaag cggctcttga tgctcaccgc tgcgcgccgc atttttacgt gtggcgcgaa 240 gcggtggcga aacatgttgt tcccgggagt cagaagaata tggtggggac gagaatgttg 300 caccatggct gaaatgagtg ggagggggcg tcgaaaagcg aaaatctttt aacgcttttc 360 aatgggcgcg acctcgcgct gctcctcgcc gttgtactcg gacagtgggc ggatgagcga 420 gttgttttcg tactgctcca cgatgtgtgc ggtccagccg gcgacgcgcg cgatgacgaa 480 cagcggggtg aagaaatcga ccgggaagcc gagcaggtgg taggcagggc cagcggggaa 540 atcgagattc ggcttgatgc cggtgcgggc gtccatggcg tcgcgcatgt tttcatacat 600 ggcaacccac ttttcgccat cgtggcgggc ggctagttcc cggaaggact tctccatgga 660 tggcacgcgg gaatcgccac gcttgtacac gcggtgacca aagcccatga ctacattctt 720 gttgtccaaa gcgttgttga tccagtcggc ggccttattt ggatcgtcga tctccaacat 780 ggtgtgcatg acaaactcgt tcgcgccgcc gtgcaatggg cccttgagag caccgatcgc 840 gccggtgatt gcggagtaca catccgattt agtggaggtg atcacgcggg cggtgaaggt 900 ggaggcgttg aaggagtgct 920 <210> 48 <211> 2216 <212> DNA <213> Artificial Sequence <220> <223> Peftu-phaC <400> 48 caaaagctgg gtacctctat ctggtgccct aaacggggga atattaacgg gcccagggtg 60 gtcgcacctt ggttggtagg agtagcatgg gatccaccat ggaattcgaa aagcaatttg 120 cttttcgacg ccccaccccg cgcgttttag cgtgtcagta ggcgcgtagg gtaagtgggg 180 tagcggcttg ttagatatct tgaaatcggc tttcaacagc attgatttcg atgtatttag 240 ctggccgtta ccctgcgaat gtccacaggg tagctggtag tttgaaaatc aacgccgttg 300 cccttaggat tcagtaactg gcacatttg taatgcgcta gatctgtgtg ctcagtcttc 360 caggctgctt atcacagtga aagcaaaacc aattcgtggc tgcgaaagtc gtagccacca 420 cgaagtccag gaggacatac aaattcatgg caaccggcaa gggcgcagct gcttctaccc 480 aggaaggtaa gtctcagcca ttcaaggtta ccccaggccc attcgatcca gcaacctggc 540 ttgaatggtc ccgtcagtgg cagggcaccg aaggtaacgg tcacgcagct gcatccggta tcccaggcct tgatgctctt gctggtgtta agatcgctcc agcacagctc ggcgatatac 660 agcagcgtta catgaaggat ttctctgcac tttggcaggc aatggcagaa ggtaaggctg aagctaccgg cccacttcac gatcgccgtt tcgcaggcga tgcatggcgt accaacctgc 780 cataccgttt cgctgctgca ttctacctcc tcaacgcacg cgctctgacc gaacttgctg 840 atgcagttga bite aagacccgtc agcgtatccg tttcgcaatc tctcagtggg ttgatgctat gtccccagca aacttcctcg ctaccaaccc agaagcacag cgccttctta 960 tcgaatccgg cggtgaatcc ctgcgcgctg gtgtgcgtaa catgatggag gacctgaccc gcggcaaaat atctcagacc gatgaatccg cattcgaagt cggccgtaac gtggctgtga ccgaaggcgc agttgttttc gaaacgaat acttccagct tcttcagtac aagccactga ccgataaggt tcacgcacgc ccctcctta tggttccacc atgcatcaac aagtactaca tccttgatct tcagccagaa tcttcccttg tgcgtcacgt cgtggaacag ggtcacaccg 1260 tgttcctcgt gtcttggcgt aacccagatg catctatggc tggttccacc tgggatgatt 1320 acatcgaaca cgctgctatc cgcgcaatcg aagttgcacg cgacatatcc ggtcaggata 1380 agatcaacgt cctgggtttc tgcgtcggcg gaactatcgt gtctaccgca ctcgctgtcc 1440 tcgctgctcg tggtgaacac ccagcagcat ctgtgaccct tctgaccacc ctccttgatt 1500 tcgctgatac tggaatcctg gatgtgttcg tggatgaagg ccacgttcag cttcgtgaag 1560 caaccctggg cggcggcgct ggtgctccat gcgcactcct ccgcggtctg gaactcgcaa 1620 acaccttctc tttcctccgc ccaaacgatc ttgtttggaa ctacgttgtc gataactacc 1680 ttaagggtaa caccccagtg ccattcgatc tccttttctg gaacggtgat gcaaccaacc 1740 tgccaggtcc ttggtactgc tggtatctgc gtcacaccta ccttcagaac gaacttaagg 1800 ttccaggtaa attgaccgtt tgcggtgtgc cagttgatct tgcttctatc gatgtgccaa 1860 cctacatcta cggttcccgc gaagatcaca tcgtgccttg gaccgctgca tacgcatcca 1920 ccgcactcct cgctaacaag ctccgtttcg tcctcggcgc ttccggtcac atcgcaggtg 1980 ttatcaaccc accagctaag aacaagcgtt ctcactggac caacgatgca ctgccagaat 2040 ccccacagca gtggcttgct ggtgctatcg aacaccacgg ctcttggtgg ccagattgga 2100 ccgcatggct tgcaggtcag gcaggtgcaa agcgtgctgc tccagctaac tacggtaacg 2160 ctcgttaccg cgctatcgaa ccagctccag gccgttacgt caaggctaag gcttaa 2216 <210> 49 <211> 1419 <212> DNA <213> Artificial Sequence <220> <223> araE <400> 49 atggttacta tcaatacgga atctgcttta acgccacgtt ctttgcggga tacgcggcgt 60 atgaatatgt ttgtttcggt agctgctgcg gtcgcaggat tgttatttgg tcttgatatc 120 ggcgtaatcg ccggagcgtt gccgttcatt accgatcact ttgtgctgac cagtcgtttg 180 caggaatggg tggttagtag catgatgctc ggtgcagcaa ttggtgcgct gtttaatggt 240 tggctgtcgt tccgcctggg gcgtaaatac agcctgatgg cgggggccat cctgtttgta 300 ctcggttcta tagggtccgc ttttgcgacc agcgtagaga tgttaatcgc cgctcgtgtg 360 gtgctgggca ttgctgtcgg gatcgcgtct tacaccgctc ctctgtatct ttctgaaatg 420 gcaagtgaaa acgttcgcgg taagatgatc agtatgtacc agttgatggt cacactcggc 480 atcgtgctgg cgtttttatc cgatacagcg ttcagttata gcggtaactg gcgcgcaatg 540 ttgggggttc ttgctttacc agcagttctg ctgattattc tggtagtctt cctgccaaat 600 agcccgcgct ggctggcgga aaaggggcgt catattgagg cggagaagagt attgcgtatg 660 ctgcgcgata cgtcggaaaa agcgcgagaa gaactcaacg aaattcgtga aagcctgaag 720 ttaaaacagg gcggttgggc actgtttaag atcaaccgta acgtccgtcg tgctgtgttt 780 ctcggtatgt tgttgcaggc gatgcagcag tttaccggta tgaacatcat catgtactac 840 gcgccgcgta tcttcaaaat ggcgggcttt acgaccacag aacaacagat gattgcgact 900 ctggtcgtag ggctgacctt tatgttcgcc acctttattg cggtgtttac ggtagataaa 960 gcagggcgta aaccggctct gaaaattggt ttcagcgtga tggcgttagg cactctggtg 1020 ctgggctatt gcctgatgca gtttgataac ggtacggctt ccagtggctt gtcctggctc 1080 tctgttggca tgacgatgat gtgtattgcc ggttatgcga tgagcgccgc gccagtggtg tggatcctgt gctctgaaat tcagccgctg aaatgccgcg atttcggtat tacctgttcg accaccacga actgggtgtc gaattgatt atcggcgcga ccttcctgac actgcttgat agcattggcg ctgccggtac gttctggctc tacactgcgc tgaacattgc gtttgtgggc attactttct ggctcattcc aatgtcacgc tggacatat cgaacgcaaa ctgatggcag gcgagaagtt gagaaatatc ggcgtctga <210> 50 <211> 893 <212> DNA <213> Artificial Sequence <220> <223> ack-up <400> 50 atggctgccc accagctgct tgatcaagac atctgtgaca tcacgatcct gggcgatcca gtaaagatca aggagcgcgc taccgaactt ggcctgcacc ttaacactgc atacctggtc aatccgctga cagatcctcg cctggagga ttcgccgac aattcgcgga gctgcgcaag tcaaagggcg tcactatcga tgaagcccgc gaaatcatga aggatattc ctacttcggc accatgatgg tccacaacgg cgacgccgac ggaatggtat ccggtgcagc aaacaccacc 300 gcacacacca ttaagccaag cttccagatc atcaaaactg ttccagaagc atccgtcgtt 360 tcttccatct tcctcatggt gctgcgcggg cgactgtggg cattcggcga ctgtgctgtt 420 aacccgaacc caactgctga acagcttggt gaaatcgccg ttgtgtcagc aaaaactgca 480 gcacaatttg gcattgatcc tcgcgtagcc atcttgtcct actccactgg caactccggc 540 ggaggctcag atgtggatcg cgccattgac gctcttgcag aagcacgccg actcaaccca 600 gaactatgcg tcgatggacc acttcagttc gacgccgccg tcgacccggg tgtggcgcgc 660 aagaagatgc cagactctga cgtcgctggc caggcaaatg tgtttatctt ccctgacctg 720 gaggccggaa acatcggcta caaaactgca caacgcaccg gtcacgccct ggcagttggt 780 ccgattctgc agggcctgaa caaaccagtc aacgaccttt cccgtggtgc gacagtccct 840 gacatcgtca acactgtcgc catcaccgct attcaggcag gaggacgcag cta 893 <210> 51 <211> 793 <212> DNA <213> Artificial Sequence <220> <223> ack-down <400> 51 ggcccctaga cctcttgggg ttgcgaattt tcgtccccac cgaacattaa aaggccggtt 60 ttggtcgaaa atttgctcta acaccttgct attatgcaaa tcttcgttcg atttaagcaa 120 atcccggcaa tctgtaatga gaagttgaac gggaaccta cattacccc gcagaaatca 180 catcagcccc aattgtccca aaagtaacgc ccccggaatc gcttctaagg gcctaactcg 240 cccaaagcca aactagttgg acactggagc caaaaggcc cttaaatcgc cacctaccaa 300 atagccccaa gccaaaacag ctagaaccaa ctcagtggcc gcaccgcatt cgccatatcc 360 actagtgcgt aacggtggtg ggggaaag gcggaacggg cctgaatccg gagtgcctcg 420 gaaatgccgg tgcgcaggcc tttttgggag aacgggtatt caaacaaagg gtttgcggag 480 gcagaagctt tgagttttcg ttctcgaagc cagctgaggc ctgctgacat gatggcaatt 540 ttgatttggt tgaagcgggg ttcgttggtg gggatttcgc tgagtcggcg ggcggctcgg 600 cggatgcggg attcactcaa attggagctg accagcaaca agatggtggt gagggtggcc 660 attcggtagt gggtggatga ttgggggagt ttatctaggg cttggactgc gagttcgatt 720 tggttttcgg ccatgagttg gcgggcgagc ccgaacgcgg aggacacagt ggtggggttg 780 gttgcccaga page 793 <210> 52 <211> 1514 <212> DNA <213> Artificial Sequence <220> <223> PH36_araE <400> 52 caaaagctgg gtacctctat ctggtgccct aaacggggga atattaacgg gcccagggtg 60 gtcgcacctt ggttggtagg agtagcatgg gatccatggt tactatcaat acggaatctg 120 cttaacgcc acgttctttg cgggatacgc ggcgtatgaa tatgtttgtt tcggtagctg 180 ctgcggtcgc aggattgtta tttggtcttg atacggcgt aatcgccgga gcgttgccgt 240 tcattaccga tcactttgtg ctgaccagtc gtttgcagga atgggtggtt agtagcatga 300 tgctcggtgc agcaattggt gcgctgttta atggttggct gtcgttccgc ctggggcgta 360 aatacagcct gatggcgggg gccatcctgt ttgtactcgg ttctataggg tccgcttttg 420 cgaccagcgt agagatgtta atcgccgctc gtgtggtgct gggcattgct gtcgggatcg 480 cgtcttacac cgctcctctg tatctttctg aaatggcaag tgaaaacgtt cgcggtaaga 540 tgatcagtat gtaccagttg atggtcacac tcggcatcgt gctggcgttt ttatccgata 600 cagcgttcag ttatagcggt aactggcgcg caatgttggg ggttcttgct ttaccagcag 660 ttctgctgat tattctggta gtcttcctgc caaatagccc gcgctggctg gcggaaaagg 720 ggcgtcatat tgaggcggaa gaagtattgc gtatgctgcg cgatacgtcg gaaaaagcgc 780 gagaagaact caacgaaatt cgtgaaagcc tgaagttaaa acagggcggt tgggcactgt 840 ttaagatcaa ccgtaacgtc cgtcgtgctg tgtttctcgg tatgttgttg caggcgatgc 900 agcagtttac cggtatgaac atcatcatgt actacgcgcc gcgtatcttc aaaatggcgg 960 gctttacgac cacagaacaa cagatgattg cgactctggt cgtagggctg acctttatgt 1020 tcgccacctt tattgcggtg tttacggtag ataaagcagg gcgtaaaccg gctctgaaaa 1080 ttggtttcag cgtgatggcg ttaggcactc tggtgctggg ctattgcctg atgcagtttg 1140 ataacggtac ggcttccagt ggcttgtcct ggctctctgt tggcatgacg atgatgtgta 1200 ttgccggtta tgcgatgagc gccgcgccag tggtgtggat cctgtgctct gaaattcagc 1260 cgctgaaatg ccgcgatttc ggtattacct gttcgaccac cacgaactgg gtgtcgaata 1320 tgattatcgg cgcgaccttc ctgacactgc ttgatagcat tggcgctgcc ggtacgttct 1380 ggctctacac tgcgctgaac attgcgtttg tgggcattac tttctggctc attccggaaa 1440 ccaaaaatgt cacgctggaa catatcgaac gcaaactgat ggcaggcgag aagttgagaa 1500 atatcggcgt ctga 1514 <210> 53 <211> 3200 <212> DNA <213> Artificial Sequence <220> <223> ack::araE <400> 53 atggctgcc accagctgct tgatcaagac atctgtgaca tcacgatcct gggcgatcca 60 gtaaagatca aggagcgcgc taccgaactt ggcctgcacc ttaacactgc atacctggtc 120 aatccgctga cagatcctcg cctggaggaa ttcgccgaac aattcgcgga gctgcgcaag 180 tcaaagggcg tcactatcga tgaagcccgc gaaatcatga aggatattc ctacttcggc accatgatgg tccacaacgg cgacgccgac ggaatggtat ccggtgcagc aaacaccacc 360. gcacacacca ttagccaag cttccagatc atcaaaactg ttccagaagc atccgtcgtt tcttccatct tcctcatggt gctgcgcggg cgactgtggg cattcggcga ctgtgctgtt 420 aacccgaacc caactgctga acagcttggt gaatcgccg ttgtgtcagc aaaaactgca gcacaatttg gcattgatcc tcgcgtagcc atcttgtcct actccactgg caactccggc 540 ggaggctcag atgtggatcg cgccattgac gctcttgcag aagcacgccg actcaaccca gaactatgcg tcgatggacc acttcagttc gacgccgccg tcgacccgg tgtggcgcgc 660 aagaagatgc cagactctga cgtcgctggc caggcaaatg tgtttatctt ccctgacctg gaggccgga acatcggcta caaaactgca caacgcaccg gtcacgccct ggcagttggt ccgattctgc agggcctgaa caaaccagtc aacgaccttt cccgtggtgc gacagtccct gacatcgtca acactgtcgc catcaccgct attcaggcag gaggacgcag ctacaaaagc tgggtacctc tatctggtgc cctaaacggg ggaatattaa cgggcccagg gtggtcgcac 960 cttggttggt aggagtagca tgggatccat ggttactatc aatacggaat ctgctttaac 1020 gccacgttct ttgcgggata cgcggcgtat gaatatgttt gtttcggtag ctgctgcggt 1080 cgcaggattg ttatttggtc ttgatatcgg cgtaatcgcc ggagcgttgc cgttcattac 1140 cgatcacttt gtgctgacca gtcgtttgca ggaatgggtg gttagtagca tgatgctcgg 1200 tgcagcaatt ggtgcgctgt ttaatggttg gctgtcgttc cgcctggggc gtaaatacag 1260 cctgatggcg ggggccatcc tgtttgtact cggttctata gggtccgctt ttgcgaccag 1320 cgtagagatg ttaatcgccg ctcgtgtggt gctgggcatt gctgtcggga tcgcgtctta 1380 caccgctcct ctgtatcttt ctgaaatggc aagtgaaaac gttcgcggta agatgatcag 1440 tatgtaccag ttgatggtca cactcggcat cgtgctggcg tttttatccg atacagcgtt 1500 cagttatagc ggtaactggc gcgcaatgtt gggggttctt gctttaccag cagttctgct 1560 gattattctg gtagtcttcc tgccaaatag cccgcgctgg ctggcggaaa aggggcgtca 1620 tattgaggcg gaagaagtat tgcgtatgct gcgcgatacg tcggaaaaag cgcgagaaga 1680 actcaacgaa attcgtgaaa gcctgaagtt aaaacagggc ggttgggcac tgtttaagat 1740 caaccgtaac gtccgtcgtg ctgtgtttct cggtatgttg ttgcaggcga tgcagcagtt 1800 taccggtatg aacatcatca tgtactacgc gccgcgtatc ttcaaaatgg cgggctttac 1860 gaccacagaa caacagatga ttgcgactct ggtcgtaggg ctgaccttta tgttcgccac 1920 cttattgcg gtgtttacgg tagataaagc agggcgtaaa ccggctctga aaattggtt 1980 cagcgtgatg gcgttaggca ctctggtgct gggctattgc ctgatgcagt ttgataacgg 2040 tacggcttcc agtggcttgt cctggctctc tgttggcatg acgatgatgt gtattgccgg 2100 ttatgcgatg agcgccgcgc cagtggtgtg gatcctgtgc tctgaaattc agccgctgaa 2160 atgccgcgat ttcggtatta cctgttcgac caccacgaac tgggtgtcga atatgattat 2220 cggcgcgacc ttcctgacac tgcttgatag cattggcgct gccggtacgt tctggctcta 2280 cactgcgctg aacattgcgt ttgtgggcat tactttctgg ctcattccgg aaaccaaaaa 2340 tgtcacgctg gaacatatcg aacgcaaact gatggcaggc gagaagttga gaaatatcgg 2400 cgtctgaggc ccctagacct cttggggttg cgaattttcg tccccaccga acattaaaag 2460 gccggttttg gtcgaaaatt tgctctaaca ccttgctatt atgcaaatct tcgttcgatt 2520 taagcaaatc ccggcaatct gtaatgagaa gttgaacggg aaacctacag taaccccgca 2580 gaaatcacat cagccccaat tgtcccaaaa gtaacgcccc cggaatcgct tctaagggcc 2640 taactcgccc aaagccaaac tagttggaca ctggagccac aaaggccctt aaatcgccac 2700 ctaccaaata gccccaagcc aaaacagcta gaaccaactc agtggccgca ccgcattcgc 2760 catatccact agtgcgtaac ggtggtgggg gaaaggagcg gaacgggcct gaatccggag 2820 tgcctcggaa atgccggtgc gcaggccttt ttgggagaac gggtattcaa acaaagggtt 2880 tgcggaggca gaagctttga gttttcgttc tcgaagccag ctgaggcctg ctgacatgat 2940 ggcaattttg atttggttga agcggggttc gttggtgggg atttcgctga gtcggcgggc 3000 ggctcggcgg atgcgggatt cactcaaatt ggagctgacc agcaacaaga tggtggtgag 3060 ggtggccatt cggtagtggg tggatgattg ggggagttta tctagggctt ggactgcgag 3120 ttcgatttgg ttttcggcca tgagttggcg ggcgagcccg aacgcggagg acacagtggt 3180 ggggttggtt gcccagacaa 3200 <210> 54 <211> 4141 <212> DNA <213> Artificial Sequence <220> <223> ldhA2::phaC <400> 54 ctagagactg ctatcggtgt ggctttacga gccataacga ggtacatgaa taccgcgatc 60 atggcgaagc cgaggaagac aagcatagga acctcgagat gtaggaaaca agtgatctga 120 actatgactg cactcacatg cagtcccatt aggtataccg aagtgtattc ctgattgcaa 180 taggttgggt gtactaagat gatgcaatgt caataaaccc gcatgccaaa gtgataaaac 240 ctgatccccg caagactgat caggtatacg ctcaaattcg acatgagatt gaaaatggca 300 cactcccccc agggcaacgc atgtcagagg tgtggctcgt tgagcacact ggagcctcaa 360 ggacgccggt cagggatgct ttacgacgtt tagccgccga tgagttagtg attttggaac 420 cacgacaagc cccaatggtt acccccttat cactccagac aatcagggat cttttcgagt tccgtcgcgt tgtcgaagtt gcggctttag aaaatattc gtcgcgagtc tccaactccc 540 cagaatcca ggacagttt ggccaactct ctacaaaatt caataactt ttagacagct 660. accgattttc gggacttc accgattttc gatgaacttg tctcaagata ttgcaacaac cagttcctag aaaagagcat tagcagcctc aaaccccata caacccgact gcgcctaatt gcccatgcag accatgatcg actcaggcaa tccatcactg agcacatcga tatgtgcaac gcagttactg caggcgacct ccgtacggca ggcgcagcgt gtcggcaaca cctcattcac gtggagcaa gcatcctaaa cgcactgatc acaggcaact caccgttcct caacagtatt gatctgcgga attach tgaagccttg cacaacctta ggccgttggt atacatttcg gtataccaac aactgaaagg gctaccaaaa gctgggtacc tctatctggt gccctaaacg ggggaatatt aacgggccca gggtggtcgc accttggttg gtaggagtag catgggatcc accatggat tcgaaaagca atttgctttt cgacgcccca ccccgcgcgt tttagcgtgt cagtaggcgc gtagggtaag tggggtagcg gcttgttaga 1200 tatcttgaaa tcggctttca acagcattga ttcgatgta tttagctggc cgttaccctg 1260 cgaatgtcca cagggtagct ggtagtttga aaatcaacgc cgttgccctt aggattcagt 1320 aactggcaca ttttgtaatg cgctagatct gtgtgctcag tcttccaggc tgcttatcac 1380 agtgaaagca aaaccaattc gtggctgcga aagtcgtagc caccacgaag tccaggagga 1440 catacaaatt catggcaacc ggcaagggcg cagctgcttc tacccaggaa ggtaagtctc 1500 agccattcaa ggttacccca ggcccattcg atccagcaac ctggcttgaa tggtcccgtc 1560 agtggcaggg caccgaaggt aacggtcacg cagctgcatc cggtatccca ggccttgatg 1620 ctcttgctgg tgttaagatc gctccagcac agctcggcga tatacagcag cgttacatga 1680 agatttctc tgcactttgg caggcaatgg cagaaggtaa ggctgaagct accggcccac 1740 ttcacgatcg ccgtttcgca ggcgatgcat ggcgtaccaa cctgccatac cgtttcgctg 1800 ctgcattcta cctcctcaac gcacgcgctc tgaccgaact tgctgatgca gttgaagcag 1860 atgcaaagac ccgtcagcgt atccgtttcg caatctctca gtgggttgat gctatgtccc 1920 cagcaaactt cctcgctacc aacccagaag cacagcgcct tcttatcgaa tccggcggtg 1980 aatccctgcg cgctggtgtg cgtaacatga tggaggacct gacccgcggc aaaatatctc 2040 agaccgatga atccgcattc gaagtcggcc gtaacgtggc tgtgaccgaa ggcgcagttg 2100 ttttcgaaaa cgaatacttc cagcttcttc agtacaagcc actgaccgat aaggttcacg 2160 cacgcccact ccttatggtt ccaccatgca tcaacaagta ctacatcctt gatcttcagc 2220 cagaatcttc ccttgtgcgt cacgtcgtgg aacagggtca caccgtgttc ctcgtgtctt 2280 ggcgtaaccc agatgcatct atggctggtt ccacctggga tgattacatc gaacacgctg 2340 ctatccgcgc aatcgaagtt gcacgcgaca tatccggtca ggataagatc aacgtcctgg 2400 gtttctgcgt cggcggaact atcgtgtcta ccgcactcgc tgtcctcgct gctcgtggtg 2460 aacacccagc agcatctgtg acccttctga ccaccctcct tgatttcgct gatactggaa 2520 tcctggatgt gttcgtggat gaaggccacg ttcagcttcg tgaagcaacc ctgggcggcg 2580 gcgctggtgc tccatgcgca ctcctccgcg gtctggaact cgcaaacacc ttctctttcc 2640 tccgcccaaa cgatcttgtt tggaactacg ttgtcgataa ctaccttaag ggtaacaccc cagtgccatt cgatctcctt ttctggaacg gtgatgcaac caacctgcca ggtccttggt actgctggta tctgcgtcac acctaccttc agaacgaact taaggttcca ggtaaattga ccgtttgcgg tgtgccagtt gatcttgctt ctatcgatgt gccaacctac atctacggtt 2880 cccgcgaaga tcacatcgtg ccttggaccg ctgcatacgc atccaccgca ctcctcgcta acaagctccg tttcgtcctc ggcgcttccg gtcacatcgc aggtgttatc aacccaccag ctaagaacaa gcgttctcac tggaccaacg atgcactgcc agaatcccca cagcagtggc ttgctggtgc tatcgaacac cacggctctt ggtggccaga ttggaccgca tggcttgcag gtcaggcagg tgcaaagcgt gctgctccag ctaactacgg taacgctcgt taccgcgcta 3180 3240. tcgaaccagc tccaggccgt tacgtcaagg ctaaggctta aggagaaaaa atgttgtcac tcaccgttga tctgcgcgtt aaacctggcc atctggaaga attccttaac gccatttcgg ttaatgcgca gcgcagcttt caagatgaag aaggttgtgt gtatttcgac gtttcacaag 3360 atgttgatga tgagcttcat ttcagtttct ttgagctcta cgcggatcaa gcggctcttg 3420 atgctcaccg ctgcgcgccg cattttacg tgtggcgcga agcggtggcg aaacatgttg 3480 ttcccgggag tcagaagaat atggtggggga cgagaatgtt gcaccatggc tgaaatgagt 3540 gggaggggc gtcgaaaagc gaaaatcttt taacgctttt caatgggcgc gacctcgcgc 3600 tgctcctcgc cgttgtactc ggacagtggg cggatgagcg agttgttttc gtactgctcc 3660 acgatgtgtg cggtccagcc ggcgacgcgc gcgatgacga acagcgggt gaagaaatcg 3720 accgggaagc cgagcaggtg gtaggcaggg ccagcggggga aatcgagatt cggcttgatg 3780 ccggtgcggg cgtccatggc gtcgcgcatg ttttcataca tggcaaccca cttttcgcca 3840 tcgtggcggg cggctagttc ccggaaggac ttctccatgg atggcacgcg ggaatcgcca 3900 cgcttgtaca cgcggtgacc aaagcccatg actacattct tgttgtccaa agcgttgttg 3960 atccagtcgg cggccttatt tggatcgtcg atctccaaca tggtgtgcat gacaaactcg 4020 ttcgcgccgc cgtgcaatgg gcccttgaga gcaccgatcg cgccggtgat tgcggagtac 4080 acatccgatt tagtggaggt gatcacgcgg gcggtgaagg tggaggcgtt gaaggagtgc 4140 t 4141 <210> 55 <211> 3348 <212> DNA <213> Artificial Sequence <220> <223> pdh::phaA <400> 55 aattgccaca gaaccccaaa taccaattgc agtgttgcgc tcacgctcat cctcaaacgt 60 aatgcggatc agagccaaag ttgcgggcat catcgtcgcc gcaccgatgc caaggaaagc 120 tctcgcagca acaagagccc acgcagttgg agcaaacgca gcaccaagtg aagcgattcc 180 gaaaatgctc aagcccatga ggaacatccg gcggtggccg atttgtcac ccaaagtgcc 240 ggtacccaaa agaagacccg ccatgagcag gggatatgcg ttgatgatcc acaacgcttg 300 ggtttcagtg gctgcgagct gttcacgcag cagaggggagt gcggtgtaga gaatcgagtt 360 gtctacaccg atcagaaaga gaccaccgct gataacggcg aggaaagccc aacgttgggt 420 ttcgtaggc gcttgcgcct gtaaagtttc tgaagtcatg gatcgtaact gtaacgaatg 480 gtcggtacag ttacaactct tttgttggtg ttttaggcca cggcgctgtg tggcgattta 540 aggcgtcgga aatcgtaggg gactgtcagt gtgggtcggg ttctttgagg cgcttagagg 600 cgattctgtg aggtcacttt ttatggggtc ggggtctaaa tttggccagt ttcgaggcg 660 accagacagg cgtgcccacg atgtttaaat aggcgttccg tgggcatctg tgtttggtttt 720 cgacgggctg aaaccaaacc agactgccca gcaacgacgg aaatcccaaa agtgggcatc 780 cctgtttggt accgagtacc cacccgggcc tgaaactccc tggcaggcgg gcgaagcgtg 840 gcaacaactc gaattgaaga gcacaattga agtcgcacca agttaggcaa cacaatagcc 900 ataacgttga ggagttcagg gatcccgaaa agcaatttgc tttcgacgc cccaccccgc 960 gcgttttagc gtgtcagtag gcgcgtaggg taagtggggt agcggcttgt tagatatctt 1020 gaaatcggct ttcaacagca ttgatttcga tgtatttagc tggccgttac cctgcgaatg 1080 tccacagggt agctggtagt ttgaaaatca acgccgttgc ccttaggatt cagtaactgg 1140 cacattttgt aatgcgctag atctgtgtgc tcagtcttcc aggctgctta tcacagtgaa 1200 agcaaaacca attcgtggct gcgaaagtcg tagccaccac gaagtccagg aggacataca 1260 atgaccgatg ttgtcatcgt ttctgctgca cgtaccgctg ttggtaagtt cggcggctct 1320 cttgctaaga tcccagctcc agaacttggc gctgttgtca tcaaggcagc actcgaacgc 1380 gctggcgtga agccagaaca ggtttccgaa gtgatcatgg gtcaggttct caccgctggc 1440 tccggtcaga acccagcacg tcaggctgca atcaaggcag gtcttccagc aatggtgcca 1500 gctatgacca tcaacaaggt ttgcggttct ggcctcaagg cagttatgct tgcagctaac 1560 gctatcatgg caggtgatgc tgaaatcgtt gtggctggtg gtcaggaaaa catgtctgct 1620 gctccacacg tccttccagg ttcccgcgat ggtttccgta tgggtgatgc aaagctcgtg 1680 gataccatga tcgtcgatgg cctttgggat gtgtacaacc agtaccacat gggtatcacc 1740 gctgaaaacg tcgcaaagga atacggtatc acccgcgaag cacaggatga gtttgcagtc 1800 ggctctcaga acaaggcaga agcagctcag aaggctggta agttcgatga agaaatcgtg 1860 ccagtcctta tcccacagcg taagggcgat ccagtcgcat tcaagaccga tgagttcgtg 1920 cgtcagggcg ctaccctgga ttctatgtcc ggccttaagc cagcattcga taaggctggc 1980 actgtgaccg ctgcaaacgc atccggcctt aacgatggcg cagctgcggt cgttgttatg 2040 tctgctgcaa aggcaaagga actcggcctg accccactcg caaccatcaa gtcctacgct 2100 aacgctggtg ttgatccaaa ggttatgggc atgggcccag tgccagcatc taagcgtgct 2160 ctgtcccgcg ctgaatggac cccacaggat cttgatctca tggaaatcaa cgaggcattc 2220 gcagcacagg cactcgcagt tcaccagcag atgggttggg atacctctaa ggttaacgtt 2280 aacggcggtg caatcgctat cggtcaccca atcggtgctt ccggttgccg tatcctcgtt 2340 accctccttc acgaaatgaa gcgccgtgat gctaagaagg gtcttgcttc cctgtgcatc 2400 ggcggcggta tgggtgtggc acttgcagtg gaacgtaagt aagtcgactg attgatacac 2460 ctgctgttct cattgaccgc gagcgcttga ccgccaacat tttcaaaatg gcggcccacg 2520 ccgcagccaa tgggattgct ctgcgtccgc atgtgaaaac acacaaaatc attgagattg 2580 cgcagatgca ggtcgatgcc ggtgcccgag ggattacctg cgcaaccatt ggcgaggcag 2640 aaatttttgc cggcgcaggt tttacggaca tctttattgc atatccgctg tatctaaccg 2700 atcatgcagt gcaacgccta aacgcgatcc ctggagcaat ttccatcggc gtggattcgg 2760 tagagatggc acaggcgacg gcgggtttgc gggagaatat caaggctctg attgaagttg 2820 attcgggaca tcgtagaagt ggagtcacgg cgactgcgtt agaactgagc gagatccgtc 2880 aggcgctggg cagcaggtat acaggagtgt tcacttttcc tggacattca tatggtccgg 2940 gaaatggtga gcaggcagca gctgatgagc ttcaggcttt aaacaacagc atccagcgac 3000 ttggtggcgg cctgacttct ggcggttcct cgccgtctgc gcagttcaca ggcgcaatcg 3060 atgagatgcg accaggtgtg tatgtgttta acgattccca acaaatcacc tcgggagcct 3120 gcactgaaaa gcaggtggca atgacggtgc tgtctactgt ggtcagccga aatgtgtcag 3180 atcgtcggat tattttggat gcaggatcca aaatcctcag cactgataaa ccagcatgga 3240 ttgatggcaa tggtttgtt ctgggggaatc ctgaggcccg aatctctgct ttgtcggagc 3300 atcacgcaac cattttctgg ccagataaag tgccacttcc agtaatcg 3348 <210> 56 <211> 2888 <212> DNA <213> Artificial Sequence <220> <223> MscCG::phaB <400> 56 gatggcaacc atgggaacgg agtccaagtt tgcatcagcg attggggtaa ccaagttggt 60 tgctcctggg ccagaggttg caatgcagac gccaacgcgt ccagtaacct gcgcgtagcc 120 ggttgctgcg tggcctgcgc cctgctcgtg gcgcaccaag acgtggcgca cctttgtgga 180 ggaatagagc gggtcataca ccggtagcac cgcaccacca ggaataccga acacgatgtc 240 ggcgttaagc tcctcgagcg atcgaacaat tgcctgtgca cctgtcatcc gctcaggggc 300 ggcggatcga ccacggcttg caaccgtggc gggagtgggc tgttgagaag ctgccacatt 360 cacgactttc tggctccttt actaaataag gattttcaca ggacccgtcc aagccaagcc 420 gatttcaact cagcctaaag acaaagccct catttaaaat tgttccgacg cggatgcgtg 480 tgcacgcagt gcgacagatg tctgttgcaa agttggctac ttgggtcata accaacaaga 540 aagccctcgt tccaacactg tggtgagtgt tgtcgagggc gcttgacgag acgacttgga 600 aggccgttac ggcaggcgcc gcgcggttac tactacaagt cgaataatgg tcatggtgtg 720. tcatgctaca cacatcgagt ttccaattcc acaacgcacg aaagttccca cccccaaaac tcccccactt cagttaagga atcaggattc tcacaaagtt cgggcaggct cccgctactt ttcagcgcta atcttggctc ggatcccgaa aagcaatttg cttttcgacg ccccaccccg cgcgttttag cgtgtcagta ggcgcgtagg gtaagtgggg tagcggcttg ttagatatct 900 tgaaatcggc tttcaacagc attgatttcg atgtatttag ctggccgtta ccctgcgaat gtccacaggg tagctggtag tttgaaaatc aacgccgttg cccttaggat tcagtaactg gcacattttg taatgcgcta gatctgtgtg ctcagtcttc caggctgctt atcacagtga 1080 aagcaaaacc aattcgtggc tgcgaaagtc gtagccacca cgaagtccag gaggacatac aatgacccag cgtatcgcat acgtgacggg gggtatgggc ggcatcggta cggcaatctg ccagcgtctt gcaaaggatg gtttccgcgt cgttgcaggt tgcggtccaa actccccacg 1260 ccgcgaaaag tggctcgaac agcagaaggc actgggcttc gattcatcg cttccgaagg taacgttgct gattgggatt ctaccaagac cgcattcgat aaggtcaagt ccgaagtcgg 1380 cgaagtggat gtccttatca acaacgctgg tatcacccg gatgttgtgt tccgtaagat 1440 gacccgtgct gattgggatg cagttatcga taccaacctc acctctctgt tcaacgttac 1500 caagcaggtg atcgatggta tggcagatcg tggctggggt cgtatcgtta acatctcctc 1560 tgtgaacggt cagaagggtc agttcggcca gaccaactac tctaccgcta aggcaggcct 1620 gcacggcttc acaatggcac ttgctcagga agtcgcaacc aagggtgtga ccgtgaacac 1680 cgtgtctcca ggttacatcg ctaccgatat ggttaaggca atccgtcagg atgtgctcga 1740 taagatcgtc gcaacatcc cagtcaagcg ccttggcctg ccagaagaaa tcgcttctat 1800 ctgcgcttgg ctgtcctctg aagaatccgg tttctccacg ggcgctgatt tctcccttaa 1860 cggcggcctg cacatgggct aagtcgacga cgctgattac agacgtgtcc catttcttta 1920 ctactattgg aaattatgag ttcagacgca gaaaaggcat ccgtggagct ttccgaaaaa 1980 tttcacccag aacgcaccca tattttgggc gccgttgttt ttggcctgat ctcattatta 2040 gtcatcggcg cagcccctca gtacctgttt tggctgctcg cactccctgt catcttcggt 2100 tactgggttc taaaatcatc cacgatcgtt gatgaacagg gcatcaccgc aaactacgcc 2160 ttcaagggca aaaaggttgt ggcctgggaa aacctcgcag gaatcggatt caagggtgcc 2220 cgcactttcg ctcgcaccac ctctgatgca gaagtcaccc tccccggcgt caccttcaac 2280 tcccttcccc gccttgaagc tgcttcccac ggccgcatcc ccgatgcgat caccgcaagc 2340 aaagcag ccgacggcaa ggttgtagtc gttcaagaag acggctactc cgtgatgatg 2400 tccaaggaag agtacttgga gcgccaaaag gcactgggca agccagttca gttgagcttc 2460 gatgacgaca ccgatgggaa tacaacacaa acagaaagcg ttgaatccca agagaccgga 2520 2580 agccgtccga accaaaggtc cacacctctg cacgagtaga agctcaccca agttttcaaa 2640 gtgccgttga ttcttgacac ccccccgccg ctccttagag cagatttgaa aagcgcatca 2700 tgatcccact tcgttcaaaa gtcaccaccg tcggtcgcaa tgcagctggc gctcgcgccc 2760 tttggcgtgc caccggcacc aaggaaaatg agttcggcaa gccaattgtt gccatcgtga 2820 actcctacac ccagttcgtg cccggacacg ttcaccttaa gaacgtcggc gatattgtgg 2880 cagatgca 2888 <210> 57 <211> 378 <212> DNA <213> Artificial Sequence <220> <223> porB signal peptide <400> 57 atgaagcttt cacaccgcat cgcagcaatg gcagcaaccg caggcatcgc agtggcagca 60 tttgcagcac ctgcttccgc atccgacttc gcaaacctat cctccaccaa caaagaactg 120 tccactcagt acaactgggt tgcttgtggc atccttgagg gtggcctcaa agcagctggc 180 gtccttgaag aaggccagta caaccgcgag ctcgcagaag caatcgccgc aaagggtgaa 240 ggcttctgga ccactcagtt cccacaaatc ggcgattgga acgaagatca ggcagcagca 300 cttgctgacc gcgcacagac ctgtggctta gttaaggctg acacctacct ttctgagctg 360 tcctccaact tctcttcc 378 <210> 58 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> porB-F <400> 58 gctctagaat gaagctttca caccgcatcg 30 <210> 59 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> porB-R <400> 59 gcagctgcgc ccttgccggt tgccatggaa gagaagttgg aggacagctc 50 <210> 60 <211> 50 <212> DNA <213> Artificial Sequence <220> <223> phac-F <400> 60 gagctgtcct ccaacttctc ttccatggca accggcaagg gcgcagctgc 50 <210> 61 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> phaC-R <400> 61 acgcgtcgac ttaagcctta gccttgacgt aacgg 35 <210> 62 <211> 1194 <212> DNA <213> Artificial Sequence <220> <223> ack <400> 62 atggcattgg cacttgtttt gaactccggt tcatcttcca tcaaattcca gctggtcaac 60 cccgaaaact ctgccatcga cgagccatat gtttctggtc ttgtggagca gattggtgag 120 ccaaacggcc gcatcgtact caagtagag ggtgaaaaat acaccctaga gacacccatc 180 gcagatcact ccgaaggcct aaacctggcg ttcgatctca tggaccagca caactgtggt 240 ccttcccaac tggaaatcac cgcagtcgga caccgtgtgg tccacggcgg aatcttgttc 300 tccgcaccgg aacttatcac tgatgaaatc gttgaaatga tccgcgacct cattccactc 360 gcaccactgc acaaccctgc aaacgttgac ggcattgatg ttgctcgaaa aattctcccc 420 gatgtcccac acgtagctgt ctttgacacc ggtttcttcc actcacttcc accagcagct 480 540 cacggcacct cccatgaatt tgtgtccaag cgcgtggtgg aaattttgga aaagcccacc 600 gaagaaatca acaccatcac cttccacctg ggaaacggcg catccatggc tgctgttcaa 660 ggtggtcgtg cggtagatac ttccatgggt atgacacctc tcgcgggact tgtcatgggt 720 acccgaagcg gtgacattga tccaggtatc gtcttccacc tttcacgcac cgctggcatg 780 agcatcgatg agatcgataa tctgctgaac aaaaagtcgg gtgtaaaggg actttccggt 840 gttaatgatt tccgtgaact gcgggaaatg atcgacaaca atgatcaaga tgcctggtcc 900 gcctacaaca tttacatcca ccagcttcgc cgctacctcg gttcctacat ggtggcacta 960 ggacgtgtgg acaccatcgt gtttaccgct ggtgttggtg aaaatgccca gtttgtccgt 1020 gaggatgcct tggcaggttt ggaaatgtac ggaattgaga tcgatccaga gcgtaacgca 1080 ttgccaaacg atggtcctcg attgatttcc actgatgcct ccaaagtgaa ggtgtttgtc 1140 atcccaacta atgaagaatt agctatcgct aggtacgcgg tgaagttcgc ttag 1194 <210> 63 <211> 1071 <212> DNA <213> Artificial Sequence <220> <223> ldhA <400> 63 atgacactca tcgattcacc acactcaacc tcagacacag ccgatattct tcttcccgct 60 cagaagctca ttgaacttgc cactggcatc cttgtggatc tcggtgttcc ccaatccgat 120 gcttttctgg ttgcagacag tctggttcaa gccgatttat gggggcatcc ttcccatggt 180 gtccttcgcc ttccctggta cgtgcgccgg ctgcagactg gcgccatgag tagcaatg 240 aacgcaaaag you cttgggtgct gttctcacca tcgatggcaa tgatggcatt ggccagatca ttacagaaca tgctcgagtc gaagcagttg cccgcgccca aaaaccggc ataggggctg tcgcagtaaa aaactccaac cacttcggca cagccatgta tttcacccgg cgtgcagctg cggcaggatg tatttctatc ttgactacca acgcttctcc tgcgatggca 480 ccgtggggtg gccgagaaaa aaagatcggc actaatcctt ggtctatcgc cgcaccattt 540. ggcgatacca ctgttgtggt ggatatggcc aacaccgcgg tggcccgtgg caaaatatat caagctcgcc agatgggtat cgaaatccca gaaacatggg cgattaccag tgagggcgct cccaccactg atccgtcaga ggctatcaac ggtgtcatcc ttcccatggc tggtcacaaa 720 ggctatgcca tcagcttcat gatggacgtg ttttctggag ttctcaccgg atctcaacac 780 agcacaaaag ttcacggccc ctatgagccc actcccaaag gtggagctgg gcatctgttc atcgcactcg acgtcgctgc gttccgtgat ccagaagatt tcgacgaagc cctcacgggt 900 ctagtccacg aagtaaaaa tacgcctacc gcggtaggtt tcgatgaggt cttttatcct 960 ggtgagctcg aggatcgcgc gcatgctaag aacatctcac aaggtgtccc gctcccccaa 1020 aaaacttggt cagaattgca agaactcgct aatgccgcgg gtcttaaata a 1071 <210> 64 <211> 1740 <212> DNA <213> Artificial Sequence <220> <223> pdh <400> 64 atggcacgca gctacgcaga acaattaatt gacactttgg aagctcaagg tgtgaagcga 60 atttatggtt tggtgggtga cagtctcaat ccgatcgtgg atgctgtccg ccaatcaggt 120 attgagtggg tgcacgttcg aaatgaggaa gcggcggcgt ttgcggctgg cgcggaatcg 180 ttgatcactg gggagttggc ggtgtgtgct gcttcttgtg gtcctggaaa cactcacctt 240 attcagggtc tttatgattc gcatcgaaat ggtgcgaagg ttttggccat cgctagtcat 300 attccgagtg cgcagattgg ttcgacgttc tttcaggaaa cgcatccgga gattttgttt 360 aaaggaatgct ctggttactg cgagatggtg aatggtggtg agcagggtga acgcattttg 420 catcacgcga ttcagtccac catggcgggt aaaggtgtgt cggtggtggt gattcctggt 480 gatatcgcta aggaagacgc aggtgacggc acttattcca attccactat ttcttcgggt 540 actcctgtag tgttcccgga tcctactgag gctccagcgc tggtggaggc gattaacaac 600 gctaagactg tcactttgtt ctgcggcgcg ggtgtgaaga atgctcgcgc gcaggtgttg 660 aagttggcgg agaagaattaa atcaccaatc gggcatgcgc tgggtggtaa gcagtacatc 720 cagcatgaga atccgtttga ggtcggcatg tctggtctgc ttggttacgg tgcctgcgtg 780 gatgcgtcca atgaggcgga tctgctgatt ctgctgggca cggattttcc ttattccgat 840 ttccttccca aagacaacgt cgctcaggtg gatatcaacg gcgcacacat tggtcgacgc 900 accacggtga aatacccggt gaccggtgat gttgctgcaa cgatcgagaa tattttgcct 960 catgtgaagg aaaagacgga tcgttccttc ctggatcgca tgctcaaggc acacgagcgt 1020 aagttgagct cggtggtgga gacgtacaca cataacgtcg agaagcatgt gcctattcac 1080 cctgaatacg ttgcctctat tttgaatgag ctggcggata aggacgcggt gtttaccgtg 1140 gacaccgaa tgtgcaatgt gtggcatgcg aggtacatcg aaaatccgga aggaacgcgc 1200 gactttgtgg gttcattccg ccacggcacg atggctaatg cgttgcctca tgcgattggt 1260 gcgcaaagtg ccgatcggaa ccgccaggtg atcgcgatgt gtggcgatgg tggtttgggc 1320 atgctgctgg gtgagcttct gaccgtgaag ctgcaccaac ttccgctgaa ggctgtggtg 1380 tttaacaaca gttctttggg catggtgaag ttggagatgc tcgtggaggg gcagccggaa 1440 tttggcacgg atcatgagga agtgaatttc gcagagattg cggcggctgc gggtatcaag 1500 tcggtacgca tcaccgatcc gaaaagtt cgcgagcagc tagctgaggc attggcatat 1560 cctggacctg tactgatcga tatcgttacg gatcctaatg cgctgtcgat cccaccaact 1620 atcacgtggg aacaaatcat gggattcagc aaggcagcca cccgaaccgt ctttggtgga 1680 ggagtgggag cgatgatcga tctcgcccgt tcgaacaataa ggaatattcc tactccatga 1740 <210> 65 <211> 1602 <212> DNA <213> Artificial Sequence <220> <223> MscCG <400> 65 atgattttag gcgtacccat tcaatatttg ctctattcat tgtggaattg gattgtcgat 60 accggttttg atgtagcaat tatcctggtc ttggcgtttt tgattccacg tatcggccga 120 ctggtcatgc gtattatcaa gcgccgagtg gagtctgcag ccgatgcgga caccactaag 180 aaccagctcg cctttgctgg cgttggcgtt tatatcgcgc aaattgtggc gtttttcatg 240 cttgccgtct ccgcgatgca ggcttttggt ttctctctcg cgggcgctgc gattccggca 300 accattgcgt cagctgccat tggtcttggt gcgcagtcga ttgttgcgga cttcttggcc 360 ggatttttca tcctgacgga aaagcaattc ggcgtgggtg actgggtgcg ttttgagggc 420 aacggcatcg ttgttgaagg cacagtcatt gagatcacca tgcgcgcgac caaaattcgc 480 acgattgcac aagagaccgt gattatcccc aactccacgg cgaaagtgtg catcaacaat 540 tctaataact ggtcgcgtgc ggttgtcgtt attccgatcc ccatgttggg ttctgaaaac 600 atcacagatg tcatcgcgcg ctctgaagct gcgactcgtc gcgcacttgg ccaggagaaa 660 atcgcgccgg aaattctcgg tgaactcgat gtgcacccag ccacggaagt cacgccgcca 720 acggtggtcg gcatgccgtg gatggtaacc atgcgtttcc tcgtgcaagt caccgccggc 780 840 tacggcagcg caaccactac atcgggaacc ctcattgatt ccttacacgt tgcgcatgaa 900 gagccaaaga cctcgcttat cgacgcctcc ccccaggctc ttaaggaacc gaagccggag 960 gctgcggcga cggttgcatc gctagctgca tcgtctaacg acgatgcaga caatgcagac 1020 gactcggtga tcaatgcagg caatccaaag aaagacttg attccgatgt gctggaacaa 1080 gaactctcca gcgaagaacc ggaagaaaca gcaaaaccag atcactctct ccgaggcttc 1140 ttccgcactg attactaccc aaatcggtgg caaaagatcc tgtcgtttgg cggacgtgtc 1200 cgcatgagca cttccctgtt gttgggtgcg ctgctcttgc tgtcactatt taaggtcatg 1260 actgtggaac ttggcaaaac tccagtggat ggctgtcacc aagcactgcc 1320 acctcaactg cggtgaccac ctccgaaact tccgcgccag taagcacgcc ttcgatgaca 1380 gtgcccacta cggtggagga gaccccaacg atggaatcta gcgtcgaaac gcagcaggaa 1440 acctcaaccc gtgcaaccgc aacgccccag cgagccgaca ccatcgaacc gaccgaggaa 1500 gccacgtccc aggaggaaac gactgcgtcg cagacgcagt ctccagcagt ggaagcacca 1560 accgcggtcc aagagacagt tgcgccaacg tccacccctt ag 1602 <210> 66 <211> 27 <212> DNA <213> Artificial Sequence <220> <223> gdh-up-F <400> 66 gaattcattg cgcagcttct gatgcgg 27 <210> 67 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> gdh-up-R <400> 67 ggatccgatt tcctcgttcc catctcggct gc 32 <210> 68 <211> 34 <212> DNA <213> Artificial Sequence <220> <223> gdh-down-F <400> 68 gtcgacgacc cctgcacttt acttaaaccc ctga 34 <210> 69 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> gdh-down-R <400> 69 ctgcagcttg atcgtcggta tccgtcaggg <210> 70 <211> 99 <212> DNA <213> Artificial Sequence <220> <223> CGR discovery <400> 70 60. atgcaaataa accgccgagg cttcttaaaa gccaccgcag gacttgccac tatcggcgct gccagcatgt ttatgccaaa ggccaacgcc cagggagca

Claims

1. A genetically engineered bacterium, characterized in that: The starting strain of the genetically engineered bacteria is C. glutamicum XyltoGA01, the genetically engineered bacteria include phaC Gene, phaA Genes and phaB Gene, phaC Gene, phaA Genes and phaB The integration sites of the genes are oeLh Gene, pdh Genes and MscCG Gene, encoding the phaC Gene, phaA Genes and phaB The nucleotide sequences of the genes are shown in SEQ ID NO: 38, SEQ ID NO: 37, and SEQ ID NO: 36, respectively; The genetically engineered bacteria also include a araE Gene, araE The nucleotide sequence of the gene is shown in SEQ ID NO: 49; The gene encoding acetate kinase is knocked out in the genome of the genetically engineered bacteria. ack Gene, ack The nucleotide sequence of the gene is shown in SEQ ID NO: 62; The genetically engineered bacteria also knocked out oeLh Gene, pdh Genes and MscCG Gene, encoding the oeLh Gene, pdh Genes and MscCG The nucleotide sequences of the genes are shown in SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65, respectively.

2. The genetically engineered bacterium according to claim 1, characterized in that Said phaC Gene, phaA Genes and phaB The upstream of the gene also includes the Peftu promoter, and the nucleotide sequence of the Peftu promoter is shown in SEQ ID NO:

39.

3. The genetically engineered bacterium according to claim 1 or 2, characterized in that: Said phaC The upstream of the gene also includes a signal peptide.

4. The genetically engineered bacterium according to claim 3, characterized in that: Said phaC Genes upstream include porB Signal peptide or CGR Signal peptide, encoding the porB Signal peptide and the CGR The nucleotide sequences of the signal peptides are shown in SEQ ID NO: 57 and SEQ ID NO: 70, respectively.

5. A method for constructing the genetically engineered bacteria according to any one of claims 1 to 3, characterized in that: The method comprises: (1) phaC Gene, phaA Genes and phaB Gene integration into C. glutamicum XyltoGA01; phaC Gene, phaA Genes and phaB Genes were integrated into oeLh Gene, pdh Genes and MscCG Gene, thus knocked out oeLh Gene, pdh Genes and MscCG Gene; encoding phaC Gene, phaA Genes and phaB The nucleotide sequences of the genes are shown in SEQ ID NO: 38, SEQ ID NO: 37, and SEQ ID NO: 36, respectively; oeLh Gene, pdh Genes and MscCG The nucleotide sequences of the genes are shown in SEQ ID NO: 63, SEQ ID NO: 64 and SEQ ID NO: 65, respectively; (2) Knockout of the gene encoding acetate kinase ack The gene encoding the pentose transporter araE gene; araE The nucleotide sequence of the gene is shown in SEQ ID NO: 49, ack The nucleotide sequence of the gene is shown in SEQ ID NO:

62.

6. The method according to claim 5, characterized in that Said phaC Gene, phaA Genes and phaB A Peftu promoter is also integrated upstream of the gene, and the nucleotide sequence of the Peftu promoter is shown in SEQ ID NO:

39.

7. The method according to claim 5, characterized in that The nucleotides encoding the surface signal peptide were phaC Genes are connected.

8. The method according to claim 7, characterized in that The surface signal peptide is porB Signal peptide or CGR Signal peptide, encoding the porB Signal peptide and CGR The nucleotide sequences of the signal peptides are shown in SEQ ID NO: 57 and SEQ ID NO: 70, respectively.

9. A method for producing poly 3-hydroxybutyrate, characterized in that: The method comprises: fermenting the genetically engineered bacteria according to any one of claims 1 to 4 using pentose and / or hexose as substrates.

10. The method according to claim 9, characterized in that The method further comprises adding ammonium sulfate and magnesium sulfate to the fermentation medium; and / or the hexose is glucose and the pentose is xylose.

11. The method according to claim 10, wherein: The fermentation is carried out under the following conditions: temperature 28-32° C., rotation speed 500-700 rpm, pH 6.8-7.2; and / or, The content of glucose is 90-120 g / L, and the content of xylose is 0-50 g / L; and / or, The content of the ammonium sulfate is 1-20 g / L, and the content of the magnesium sulfate is 0.2-0.8 g / L.

12. The method according to claim 11, characterized in that The temperature is 30° C., the rotation speed is 600 rpm, and the pH is 7.0; and / or, The content of glucose is 117.2 g / L, and the content of xylose is 45.7 g / L.

13. The method according to any one of claims 9 to 12, characterized in that: The substrate is derived from the hydrolyzate of natural raw materials, and the natural raw materials are agricultural waste or forestry waste.

14. The method according to claim 13, characterized in that The agricultural waste is wheat straw, corn straw, rice straw or bagasse.

15. The method according to claim 14, characterized in that The method for preparing the hydrolyzate of the natural raw material comprises the following steps: The natural raw materials are pretreated, detoxified, and hydrolyzed by enzymes, and solid-liquid separation is performed to remove solids, and clear liquid is collected to obtain a hydrolyzate of the natural raw materials.

16. The method according to claim 15, characterized in that The solid-liquid separation method includes: centrifugation and filtration.

17. The method according to claim 16, wherein: The centrifugal speed is 10000-15000 rpm.

18. The method according to claim 16, characterized in that The centrifugation time is 8 to 15 minutes; and / or the clear liquid is sterilized and filtered.

19. Use of the genetically engineered bacteria according to any one of claims 1 to 4 in the production of poly 3-hydroxybutyrate.

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