CN110331121B - A high-yielding lipopeptide recombinant bacteria and its application - Google Patents

Abstract

The invention discloses a high-yield lipopeptide-producing recombinant bacteria and its application, belonging to the technical field of genetic engineering. The high-lipopeptide-producing recombinant bacteria is 2-isopropylmalate synthase in the leucine synthesis pathway The gene was transformed into the original strain and constructed. The further overexpressed part of the leucine synthesis pathway (2-isopropylmalate synthase, 3-isopropylmalate dehydrogenase, 3-isopropylmalate dehydratase and branched-chain amino acid transaminase) of the present invention Compared with the starting strain, the recombinant strain has a maximum increase of 55.6% in surfactin production, which can be used for the production of lipopeptide biosurfactants and has a good industrial application prospect. The average yield of lipopeptide in the fermentation broth obtained by shaking flask fermentation is 13 ‑16g/L.

Description

Recombinant bacterium for high-yield lipopeptide and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a recombinant bacterium for high-yield lipopeptide and application thereof.

Background

Lipopeptide (lipopeptide) biosurfactant is an amphoteric substance formed by connecting hydrophilic cyclic oligopeptide and hydrophobic fatty acid chain by inner ester bonds, and is mainly synthesized by microorganisms such as bacillus, streptomyces and the like. Because the amino acid composition in the peptide ring of the lipopeptide is different from the ring forming mode, the bacillus lipopeptide can be divided into surfactant, muskonein, iturin and the like, wherein the surfactant forming a unique saddle conformation at an air/liquid interface has good surface activity, biodegradability and antibacterial activity, and has wide application prospect in the fields of oil exploitation, biological control, medicine, daily chemicals and the like. However, the low yield of surfactant produced by fermentation of bacillus limits the industrial production and application of surfactant.

At present, methods for improving the fermentation level of microbial lipopeptide comprise culture condition optimization, mutation breeding, surfactant transmembrane transport enhancement, surfactant synthetase expression enhancement, fatty acid precursor supply enhancement and the like. Patent literature (CN 101892176A, CN 101775427A, WO 2002026961a) and the like improve the yield of surfactant in fermentation liquid by optimizing the culture medium and culture conditions in the fermentation process of producing surfactant by fermenting bacillus subtilis. In mutagenic breeding, CN101928677A discloses the treatment of Streptomyces roseosporus by UV mutagenesis, and US05227294 discloses the treatment of Bacillus subtilis with nitroso-formazan, all of which show an increase in the amount of surfactant synthesis after mutagenesis. In the aspect of genetic engineering modification, chinese patent document CN103898038A discloses that the yield of surfactin produced by fermentation of bacillus subtilis is improved by 97% by strengthening transmembrane protein YcxA for lipopeptide transport from intracellular to extracellular. Chinese patent document CN1554747A discloses that the expression of comA gene in Bacillus subtilis can increase the lipopeptide yield by 50%. In addition, fatty acid and amino acid in bacillus subtilis catalyze and synthesize surfactin under the action of surfactin synthase, and Chinese patent document CN105400784A improves the yield of surfactin by 17.7 times by replacing a lipopeptide synthetase gene cluster promoter with an inducible strong promoter Pg 3. Chinese patent document CN109097315A shows that the yield of surfactin is increased by 47% by over-expressing biotin carboxylase YngH in the synthesis pathway of branched chain fatty acid. (Qun Wu, Yan Zhi, Yan Xu, systematic Engineering of the biosyntheses of green biosurfactant by Bacillus subtilis 168[ J ]. Metabolic Engineering,2019,52:87-97) discloses that enhancing the whole pathway of synthesis of branched fatty acids in cells increases the supply of precursor fatty acids, thereby increasing the yield of surfactant by 20.8 times. Another class of major precursor amino acids for surfactant synthesis (coupling F, Niehren J, Dhali D, et al, modeling leucoine's metabolic pathway and knock-out prediction of the production of surfactin, a biosurfactant from Bacillus subtilis [ J ]. Biotechnology Journal,2015,10(8SI):1216-1234) discloses that addition of leucine to the medium can increase surfactant production by 3-fold, suggesting that increasing leucine content is an effective strategy for increasing surfactant production. However, (Qun Wu, Yan Zhi, Yan Xu, systematic Engineering of the biosyntheses of green biosurfactant negative Bacillus subtilis 168[ J ]. Metabolic Engineering,2019,52:87-97) overexpresses acetolactate synthase AlsS, ketol acid reductoisomerase IlvC, dihydroxy acid dehydratase IlvD, 2-isopropylmalate synthase LeuA, 3-isopropylmalate dehydrogenase LeuB and 3-isopropylmalate dehydratase LeuCD in the leucine synthesis pathway in a surfactant-producing host bacterium, and the surfactant production is not improved. No report has been made on the studies for enhancing leucine synthesis in cells to effectively increase the production of surfactin.

Disclosure of Invention

In order to overcome the disadvantages of the prior art, the present invention aims to provide a recombinant bacterium for efficiently producing lipopeptides, which can improve the yield of lipopeptides.

The recombinant strain is constructed by transforming a 2-isopropylmalate synthase gene in a leucine synthesis pathway to an original strain.

In the recombinant bacteria, one or more than one of a 3-isopropylmalate dehydrogenase gene, a 3-isopropylmalate dehydratase gene and a branched-chain amino acid transaminase gene can be optionally transferred into the recombinant bacteria.

In the recombinant strain, the 3-isopropylmalate dehydrogenase, the 3-isopropylmalate dehydratase and the branched-chain amino acid transaminase biotin carboxylase are LeuB, LeuCD and IlvK proteins or mutants thereof with the same function respectively,

in the recombinant bacterium, preferably, the amino acid sequence of the LeuB protein is shown as SEQ ID No.2, the amino acid sequence of the LeuC protein is shown as SEQ ID No.3, the amino acid sequence of the LeuD protein is shown as SEQ ID No.4, and the amino acid sequence of the IlvK protein is shown as SEQ ID No. 5.

In the recombinant strain, the 2-isopropylmalate synthase is a LeuA protein or a mutant thereof with the same function, and preferably, the amino acid sequence of the LeuA protein is shown as SEQ ID No. 1.

In the recombinant strain, the original strain is a wild strain with lipopeptide production capacity and a mutant strain, a mutant strain or a genetically engineered strain thereof.

In the recombinant strain, the original strain is bacillus subtilis, bacillus cereus or pseudomonas.

In the recombinant bacteria, the original strain is Bacillus subtilis THY-7.

The Bacillus subtilis THY-7 is preserved in China general microbiological culture Collection center (CGMCC) at 3 and 11 months in 2014, and the preservation registration number is CGMCC No.8906 and is disclosed in China patent document CN 105400784A.

In the recombinant bacteria, the original strain is Bacillus subtilis THY-7/Pg 3-srfA.

The Bacillus subtilis THY-7/Pg3-srfA means that an inducible strong promoter Pg3 is added into the Bacillus subtilis THY-7, so that the strong promoter Pg3 controls the expression of surfactin synthetase srfA. An inducible strong promoter Pg3, a Bacillus subtilis THY-7/Pg3-srfA and a specific preparation method thereof are disclosed in Chinese patent document CN 105400784A.

In the recombinant strain, the LeuA, LeuB, LeuC and LeuD proteins are controlled by a strong promoter, and the IlvK protein is controlled by an original constitutive promoter.

The invention also aims to provide application of the recombinant bacterium in producing lipopeptide.

In the application, the lipopeptide biosurfactant is surfactant.

A preparation method of the recombinant bacterium comprises the following steps:

amplifying to obtain 2-isopropylmalate synthase, 3-isopropylmalate dehydrogenase, 3-isopropylmalate dehydratase gene cluster leuABCD and branched chain amino acid transaminase biotin carboxylase IlvK with self-constitutive promoter, inserting the gene sequences of leuABCD and IlvK into shuttle plasmid, and constructing expression plasmid;

and introducing the expression plasmid into an original strain to construct a recombinant strain.

Further, the specific method comprises the following steps:

1. and (3) performing polymerase chain reaction by using the upstream and downstream primers and using the bacillus subtilis genome as a template, amplifying and obtaining the leuABCD gene, wherein preferably, the nucleic acid sequence is shown as SEQ ID NO. 6. Similarly, the ilvK gene is amplified and obtained, preferably, the nucleic acid sequence thereof is shown in SEQ ID NO. 7.

2. And carrying out double enzyme digestion on the leuABCD gene, the ilvK gene and the shuttle plasmid respectively, and connecting the two enzyme digestion products by using ligase to obtain a connection product.

3. Coli TOP10 competent cells were transformed with the ligation products, and positive clones were screened for resistance to obtain expression plasmid pJMP-leuABCD-ilvK containing leuABCD and ilvK gene sequences.

4. Transferring the expression plasmid pJMP-leuABCD-ilvK into a starting strain to obtain the genetically engineered bacterium transformed with the pJMP-leuABCD-ilvK plasmid.

Among the above methods for preparing genetically engineered bacteria, the construction method of the shuttle plasmid pJMP is disclosed in Chinese patent document CN 109097315A.

A preparation method of the recombinant bacterium can be selected, and comprises the following specific steps:

1. using leuABCD-F and leuABCD-R as upstream and downstream primers, using a bacillus subtilis genome as a template to perform polymerase chain reaction, and amplifying to obtain a leuABCD gene sequence, wherein the nucleic acid sequence is shown as SEQ ID NO. 6; taking ilvK-F and ilvK-R as upstream and downstream primers, taking a bacillus subtilis genome as a template to carry out polymerase chain reaction, and amplifying to obtain an ilvK gene sequence, wherein the nucleic acid sequence of the ilvK gene sequence is shown as SEQ ID NO. 7;

2. carrying out double enzyme digestion on the leuABCD gene by Xba I and Mlu I, carrying out double enzyme digestion on the ilvK gene by Mlu I and Nco I, carrying out double enzyme digestion on the shuttle plasmid by Xba I and Nco I, purifying enzyme digestion products, and connecting the three enzyme digestion products by using T4 DNA ligase to obtain a connection product.

3. Coli TOP10 competent cells were transformed with the ligation products, plated on LB plates with kanamycin, and cultured overnight in an incubator at 37 ℃. And (3) selecting the resistant clone growing on the plate for culturing, extracting plasmids for enzyme digestion and sequencing verification to obtain the expression plasmid pJMP-leuABCD-ilvK containing the correct leuABCD-ilvK gene sequence.

4. The expression plasmid pJMP-leuABCD-ilvK is transferred into Bacillus subtilis THY-7/Pg3-srfA (CN 105400784A) by an electrotransformation method, spread on an LB plate containing chloramphenicol and kanamycin, resistant clones are picked up for culture, and PCR verification is carried out to obtain the genetically engineered bacterium B.subtilis THY-7/Pg3-srfA (leuABCD-ilvK) transformed with the pJMP-leuABCD-ilvK plasmid.

Preferably, the Bacillus subtilis genome described in step 1 may be selected from Bacillus subtilis 1012wt (MoBiTec), Bacillus subtilis THY-7(CN 105400784A), THY-8 (chemical evolution, 2013, 32: 2952-.

Preferably, the shuttle plasmid in step 2 is pJMP, and the construction method is disclosed in Chinese patent document CN 109097315A.

The invention also aims to provide the application of the genetically engineered bacteria in preparing lipopeptides.

Optionally, the recombinant bacterium is used for producing lipopeptide, and the steps are as follows:

inoculating the recombinant bacteria into a culture medium, and performing amplification culture to obtain a gene engineering bacteria liquid;

inoculating the engineering bacteria liquid obtained in the step (1) into a fermentation culture medium according to the volume percentage of 1-20%, and performing fermentation culture to obtain a fermentation liquid containing lipopeptide.

In the above method, the method of the expanded culture comprises: culturing for 10-20h at 35-40 deg.C and shaking table rotation speed of 150-.

In the method, the fermentation culture method is to add 0.5-1.5mM IPTG inducer to culture for 1.5-4h under the conditions of 35-40 ℃ and the rotation speed of a shaking table of 150-.

In the above method, the fermentation medium comprises: 30-100g/L of saccharide, 10-50g/L of inorganic nitrogen source, 0.5-3g/L of organic nitrogen source and KH₂PO₄ 0.1-1g/L,Na₂HPO₄·12H₂O 0.5-0.3g/L,CaCl₂ 0.002-0.01g/L,MnSO₄·H₂O 0.002-0.01g/L,FeSO₄·7H₂O 0.002-0.01g/L,pH 6.5-7.5。

The invention has the advantages and beneficial effects that:

the invention adopts the genetic engineering technology to construct recombinant bacteria, amplifies and expresses 2-isopropylmalate synthase LeuA, 3-isopropylmalate dehydrogenase LeuB, 3-isopropylmalate dehydratase LeuCD and branched chain amino acid transaminase IlvK in a leucine synthesis way in lipopeptide-producing bacillus subtilis cells, and strengthens the synthesis of leucine in a surfactant molecular structure, thereby obviously improving the yield of lipopeptide. Compared with the original strain, the recombinant strain of the over-expression partial leucine synthetic path has the advantages that the yield of the surfactant is improved by 55.6% to the maximum extent, the recombinant strain can be used for producing the lipopeptide biosurfactant, the industrial application prospect is good, and the average yield of the lipopeptide in the fermentation liquor obtained by the shake flask fermentation is 13-16 g/L.

Drawings

FIG. 1 is a schematic diagram of plasmid pJMP for gene overexpression.

FIG. 2 is a PCR validation chart of the over-expression plasmid pJMP-leuABCD-ilvK containing the leu ABCD-ilvK gene string: lane 1 shows DNA molecular weight standards, and lane 2 shows PCR amplification of the plasmid using primers ilvK-F and ilv-R, resulting in a 1.3kb band.

FIG. 3 is a schematic representation of the Bacillus subtilis-E.coli shuttle plasmid pJMP-leuABCD-ilvK carrying the 2-isopropylmalate synthase LeuA, 3-isopropylmalate dehydrogenase LeuB, 3-isopropylmalate dehydratase LeuCD and the branched chain amino acid transaminase IlvK genes.

FIG. 4 is a PCR verification diagram of genetically engineered bacteria B.subtilisTHY-7/Pg3-srfA (leuABCD-ilvK) overexpressing 2-isopropylmalate synthase LeuA, 3-isopropylmalate dehydrogenase LeuB, 3-isopropylmalate dehydratase LeuCD and branched-chain amino acid transaminase IlvK: lane 1 shows the result of PCR amplification of THY-7/Pg3-srfA (pJMP-yngH) with ilvK-F primer and pJMP-R universal primer, resulting in a 1.5kb band. Lane 2 is a DNA molecular weight standard.

FIG. 5 shows the concentration of surfactant in the fermentation product of original strain THY-7/Pg3-srfA and genetically engineered bacterium THY-7/Pg3-srfA (leuABCD-ilvK).

Detailed Description

The invention is further described with reference to the following figures and specific examples. The biochemical reagents used in the examples are all commercially available reagents, and the technical means used in the examples are conventional means in the books of those skilled in the art, unless otherwise specified.

In the leucine synthesis pathway: glucose produces pyruvate, which is a precursor for leucine synthesis, via the glycolytic pathway. In the process of producing alpha-ketoisovalerate from pyruvic acid, the synthesis of acetolactate from pyruvic acid is the rate-limiting step of the process. The enzyme catalyzing this step is an acetohydroxy acid synthetase, encoded by the gene ilvHB and the gene alsS. In the process of synthesizing L-leucine from alpha-ketoisovalerate and acetyl CoA, the rate-limiting step is to synthesize alpha-isopropylmalate catalyzed by alpha-isopropylmalate synthase, and the gene coding the synthase is leuA.

The inventor discovers that the 3 related genes are expressed respectively in previous experiments: the synthesis of the surfactant is obviously reduced when the alsS and ilvHB are over-expressed, the yield of the surfactant is improved when the leuA is over-expressed, the yield of the lipopeptide is 11.58g/L and is improved by 13.5 percent, and the yield of the lipopeptide is not further improved when the leuAB is over-expressed.

Example 1 construction of a plasmid carrying the genes leuABCD and ilvK in the leucine Synthesis pathway

A single colony of Bacillus subtilis THY-7 was selected, inoculated in LB liquid medium, cultured overnight in a shaker at 37 ℃ and 200rpm, collected at 12000rpm for 5min, and the THY-7 genome was extracted using a bacterial genome extraction kit from Omega. The obtained genome is used as a template, and upstream primer leuABCD-F (shown in SEQ ID NO. 8) and downstream primer leuABCD-R (shown in SEQ ID NO. 9) are used for PCR amplification to obtain leuABCD fragment. An ilvK fragment primer obtained by PCR amplification using an upstream primer ilvK-F (shown in SEQ ID NO. 10) and a downstream primer ilvK-R (shown in SEQ ID NO. 11) was synthesized by Shibata Biotechnology (Shanghai) Co., Ltd, dissolved in sterile water and diluted to 10. mu.M for use. Polymerase, buffer and restriction enzyme for PCR amplification were purchased from TaKaRa. The PCR amplification reaction system is as follows:

the thermal cycle conditions are

And amplifying and purifying to obtain a bacillus subtilis leuABCD gene segment, wherein the sequence of the gene segment is shown as SEQ ID NO.6, and amplifying and purifying to obtain a bacillus subtilis ilvK gene segment, wherein the sequence of the gene segment is shown as SEQ ID NO. 7. The leuABCD gene was digested simultaneously with Xba I and Mlu I, the ilvK gene was digested simultaneously with Mlu I and Nco I, the shuttle plasmid was digested simultaneously with Xba I and Nco I at 27-33 ℃ for 1-3 hours, and then purified using a DNA purification kit from Omega, followed by overnight ligation at 16-22 ℃ using T4 DNA ligase (NEB). Coli TOP10 competent cells (TiangGen Co.) were transformed with the ligation product, plated on LB plates containing kanamycin, and cultured overnight in an incubator at 37 ℃. Selecting resistant clones growing on the plate for colony PCR, taking the selected colonies as a template, taking ilvK-F and a plasmid primer ilvK-R as primers, amplifying a band (shown in figure 2) of about 1.3Kb to obtain positive clones, selecting the positive clones for culture, extracting plasmids and carrying out sequencing verification to obtain expression plasmids pJMP-leuABCD-ilvK containing leuABCD and ilvK genes, wherein figure 1 is a schematic diagram of the plasmid pJMP.

Example 2 construction of genetically engineered bacterium B.subtilis THY-7/Pg3-srfA (leuABCD-ilvK) overexpressing leucine synthetic pathway

The Bacillus subtilis-Escherichia coli shuttle plasmid pJMP-leuABCD-ilvK carrying 2-isopropylmalate synthase LeuA, 3-isopropylmalate dehydrogenase LeuB, 3-isopropylmalate dehydratase LeuCD and branched chain amino acid transaminase IlvK constructed in example 1 was electroporated into competent cells of Bacillus subtilis THY-7/Pg3-srfA to obtain genetically engineered bacteria THY-7/Pg3-srfA (leuABCD-ilvK) over-expressing part of the leucine synthesis pathway gene. Wherein, the preparation and the electric transformation of the bacillus subtilis THY-7/Pg3-srfA competent cell adopt the method in Chinese patent document CN 105400784A.

After recovery, 100uL of bacterial liquid is taken and coated on LB solid culture medium containing 10-30 mug/mL kanamycin, the bacterial liquid is inverted and placed in an incubator at 37 ℃ for overnight culture, a single colony is picked, PCR verification is carried out by using upstream and downstream primers ilvK-F and pJMP-R, a band of about 1.5kb can be amplified, and the verification result is shown in figure 4, namely the genetically engineered bacteria THY-7/Pg3-srfA (leuABCD-ilvK) which excessively express 2-isopropylmalate synthase LeuA, 3-isopropylmalate dehydrogenase LeuB, 3-isopropylmalate dehydratase LeuCD and branched chain amino acid transaminase IlvK are obtained. The sequence of the plasmid primer pJMP-R is shown in SEQ ID NO. 12.

EXAMPLE 3 production of lipopeptide surfactant-surfactant, surfactant-Lin, Using genetically engineered bacterium THY-7/Pg3-srfA (leuABCD-ilvK)

The genetically engineered bacteria THY-7/Pg3-srfA (leuABCD-ilvK) which are obtained in example 2 and overexpress 2-isopropylmalate synthase LeuA, 3-isopropylmalate dehydrogenase LeuB, 3-isopropylmalate dehydratase LeuCD and branched chain amino acid transaminase IlvK are inoculated into LB liquid culture medium (containing chloramphenicol and kanamycin), cultured for 16h at 37 ℃ and 200rpm, inoculated into a shake flask containing 100mL of fermentation culture medium in a proportion of 5 percent, added with IPTG when cultured for 2-6h at 37 ℃ and 200rpm, and continuously cultured for 2-3d to obtain the fermentation liquid containing lipopeptide.

The method for detecting the surface active element in the fermentation liquor is used for coma and the like (Chinese patent CN 105400784A). The statistical results of the concentrations of the surfactant in the fermentation broth of the genetically engineered bacterium THY-7/Pg3-srfA (leuABCD-ilvK) and the original strain THY-7/Pg3-srfA are shown in FIG. 5: the yield of the THY-7/Pg3-srfA (leuABCD-ilvK) surfactant can reach 15.8g/L at most, and is improved by 54.9 percent compared with the THY-7/Pg3-srfA spawn (10.2 g/L). THY-7/Pg3-srfA (leuABCD-ilvK) is cultured in a 5L fermentation tank, and the yield of the surfactant reaches 19g/L at most.

The above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

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<213> Bacillus subtilis

<400> 4

Met Glu Pro Leu Lys Ser His Thr Gly Lys Ala Ala Val Leu Asn Arg

1 5 10 15

Ile Asn Val Asp Thr Asp Gln Ile Ile Pro Lys Gln Phe Leu Lys Arg

20 25 30

Ile Glu Arg Thr Gly Tyr Gly Arg Phe Ala Phe Phe Asp Trp Arg Tyr

35 40 45

Asp Ala Asn Gly Glu Pro Asn Pro Glu Phe Glu Leu Asn Gln Pro Val

50 55 60

Tyr Gln Gly Ala Ser Ile Leu Ile Ala Gly Glu Asn Phe Gly Cys Gly

65 70 75 80

Ser Ser Arg Glu His Ala Pro Trp Ala Leu Asp Asp Tyr Gly Phe Lys

85 90 95

Ile Ile Ile Ala Pro Ser Phe Ala Asp Ile Phe His Gln Asn Cys Phe

100 105 110

Lys Asn Gly Met Leu Pro Ile Arg Met Pro Tyr Asp Asn Trp Lys Gln

115 120 125

Leu Val Gly Gln Tyr Glu Asn Lys Ser Leu Gln Met Thr Val Asp Leu

130 135 140

Glu Asn Gln Leu Ile His Asp Ser Glu Gly Asn Gln Ile Ser Phe Glu

145 150 155 160

Val Asp Pro His Trp Lys Glu Met Leu Ile Asn Gly Tyr Asp Glu Ile

165 170 175

Ser Leu Thr Leu Leu Leu Glu Asp Glu Ile Lys His Phe Glu Ser Gln

180 185 190

Arg Ser Ser Trp Leu Gln Ala

195

<210> 5

<211> 363

<212> PRT

<213> Bacillus subtilis

<400> 5

Met Thr Lys Gln Thr Ile Arg Val Glu Leu Thr Ser Thr Lys Lys Pro

1 5 10 15

Lys Pro Asp Pro Asn Gln Leu Ser Phe Gly Arg Val Phe Thr Asp His

20 25 30

Met Phe Val Met Asp Tyr Ala Ala Asp Lys Gly Trp Tyr Asp Pro Arg

35 40 45

Ile Ile Pro Tyr Gln Pro Leu Ser Met Asp Pro Ala Ala Met Val Tyr

50 55 60

His Tyr Gly Gln Thr Val Phe Glu Gly Leu Lys Ala Tyr Val Ser Glu

65 70 75 80

Asp Asp His Val Leu Leu Phe Arg Pro Glu Lys Asn Met Glu Arg Leu

85 90 95

Asn Gln Ser Asn Asp Arg Leu Cys Ile Pro Gln Ile Asp Glu Glu Gln

100 105 110

Val Leu Glu Gly Leu Lys Gln Leu Val Ala Ile Asp Lys Asp Trp Ile

115 120 125

Pro Asn Ala Glu Gly Thr Ser Leu Tyr Ile Arg Pro Phe Ile Ile Ala

130 135 140

Thr Glu Pro Phe Leu Gly Val Ala Ala Ser His Thr Tyr Lys Leu Leu

145 150 155 160

Ile Ile Leu Ser Pro Val Gly Ser Tyr Tyr Lys Glu Gly Ile Lys Pro

165 170 175

Val Lys Ile Ala Val Glu Ser Glu Phe Val Arg Ala Val Lys Gly Gly

180 185 190

Thr Gly Asn Ala Lys Thr Ala Gly Asn Tyr Ala Ser Ser Leu Lys Ala

195 200 205

Gln Gln Val Ala Glu Glu Lys Gly Phe Ser Gln Val Leu Trp Leu Asp

210 215 220

Gly Ile Glu Lys Lys Tyr Ile Glu Glu Val Gly Ser Met Asn Ile Phe

225 230 235 240

Phe Lys Ile Asn Gly Glu Ile Val Thr Pro Met Leu Asn Gly Ser Ile

245 250 255

Leu Glu Gly Ile Thr Arg Asn Ser Val Ile Ala Leu Leu Lys His Trp

260 265 270

Gly Leu Gln Val Ser Glu Arg Lys Ile Ala Ile Asp Glu Val Ile Gln

275 280 285

Ala His Lys Asp Gly Ile Leu Glu Glu Ala Phe Gly Thr Gly Thr Ala

290 295 300

Ala Val Ile Ser Pro Val Gly Glu Leu Ile Trp Gln Asp Glu Thr Leu

305 310 315 320

Ser Ile Asn Asn Gly Glu Thr Gly Glu Ile Ala Lys Lys Leu Tyr Asp

325 330 335

Thr Ile Thr Gly Ile Gln Lys Gly Ala Val Ala Asp Glu Phe Gly Trp

340 345 350

Thr Thr Glu Val Ala Ala Leu Thr Glu Ser Lys

355 360

<210> 6

<211> 4755

<212> DNA

<213> Bacillus subtilis

<400> 6

atgcgcaaaa ttaatttttt cgatacgacg cttcgtgatg gtgaacagtc ccctggagtg 60

aacttgaata cacaggagaa acttgccata gctaagcagc tcgaaagact cggggcagat 120

atcattgaag cgggatttcc cgcttcgtcc cgaggtgact ttttagctgt tcaggaaatc 180

gcaagaacca ttaaaaattg ttcagtaact ggtctggccc gttgtgtaaa aggtgatatt 240

gatgctgctt gggaagcgtt aaaagaaggt tctcacccga gaattcatgt ttttatcgcc 300

acatcggaca ttcatttgaa gcacaagctg aaaatgacac gtgagcaagt cattgaaaga 360

gcggttgaaa tggtgaaata cgcaaaagaa cgttttccga ttgtgcaatg gtcagctgaa 420

gatgcctgcc gcactgaact gccgtttcta gcagaaatcg tcgaaaaagt gattgacgca 480

ggcgccagtg ttatcaatct tccggacact gtcggctacc tggctccggc ggaatacgga 540

aatatcttta gatatatgaa ggaaaacgtt ccgaacattc acaaagcaaa gctttcagcc 600

cactgtcatg atgatttagg aatggcagtc gcaaactctc ttgctgcgat tgaaaatggc 660

gctgatcaaa tcgaatgcgc tgtgaacggg atcggtgaaa gagccggaaa cgcggcatta 720

gaggaaattg ccgtagccct ccataccaga aaagatttct accaagtcga aacaggcatt 780

acactgaacg agattaagag aacaagtgat ttagtaagca aactgacagg catggctgtc 840

ccgcgcaaca aagcggttgt tggagataat gcatttgctc atgaatcagg catccatcag 900

gacggctttt taaaggaaaa atcgacttat gaaattattt caccggagct tgtcggcgta 960

accgcagatg cgcttgtcct aggtaaacat tccggacgcc acgcatttaa agaccggctg 1020

actgctttag gattccaatt tgacagtgaa gagattaata aattctttac gatgttcaaa 1080

gagttgactg agaagaaaaa agaaatcact gatgaggatc ttgtttctct tattttagaa 1140

gaaaaagtaa cagatcgcaa gattgggtat gaatttcttt ctctgcaagt acattacgga 1200

acaagtcagg tccctacggc tactctttcg ttgaaaaatc aagaaaacgc agagcttatt 1260

caggaagctg caactggagc tggaagtgtg gaagcaatct acaatacgct tgagcgctgc 1320

atcgataagg acgtggagct cttagactac cgcattcagt ctaacagaaa aggcgaagat 1380

gcatttgccc aggtgtatgt aagagttttg gtgaacggaa aagaatcagc aggtcggggc 1440

atagcgcaag acgtattaga agcatcagcg aaagcctatt tgaacgcagt caaccgtcaa 1500

ttggttttcc agtcgaatat gagcggattg aaaaaccaca cagctgtcgg atcataaaag 1560

aaaggagaac ggttaacttg aagaaacgta ttgctctatt gcccggagac gggatcggcc 1620

ctgaagtatt agaatcagcg acagacgtac tgaaaagtgt tgccgaacgc tttaaccatg 1680

aatttgaatt tgaatatggc ctgattggag gggcggctat tgatgaacat cataaccccc 1740

tcccggagaa aaccgttgct gcttgtaaaa atgcagacgc gatattgctt ggcgctgtcg 1800

gcggaccgaa atgggatcaa aatccttcgg aactgagacc ggaaaaaggg ctgctcagca 1860

tcagaaaaca gcttgatttg tttgcgaatt tacggcctgt gaaggtattt gaaagccttt 1920

ctgacgcttc gcctttgaaa aaagaatata tagataatgt tgatttcgtt atcgttcgtg 1980

aactcacagg cggcttgtat ttcggccagc cgagcaaacg ctatgtaaac actgaaggtg 2040

agcaggaagc agtagataca ctgttttata agcgaacgga aattgaacga gtgatcagag 2100

agggcttcaa aatggcggca gccagaaaag gcaaagtgac ctctgtagat aaagcgaatg 2160

ttcttgaatc aagccggctg tggcgtgaag tggctgagga cgttgcgaaa gaatttcctg 2220

atgtgaagct tgagcacatg cttgtggata acgcggccat gcagctaatc tatgcaccga 2280

atcaatttga tgtcgttgtg actgaaaata tgttcggtga tattttaagc gatgaagcgt 2340

ccatgcttac aggctcgctc ggaatgctcc cgtcagccag cctatcaagc tctggtcttc 2400

atctgtttga acctgttcat ggctccgcgc ctgatattgc tggtaaaggg atggcaaatc 2460

cgttcgcagc catattgtca gcggcaatgc ttttgaggac atctttcggg cttgaagagg 2520

aagcgaaagc tgtagaagat gcggtaaaca aagtcttggc ttccggaaaa agaacaaaag 2580

acttggcacg gggtgaagag ttcagcagca ctcaggccat tacagaggaa gttaaggcag 2640

caatcatgag tgaaaataca atgtctaatg tgtgacagct tacgttaagc ggtcttagct 2700

ctaggtagag ggaggaaata aaagatgatg cctcgaacaa tcatcgaaaa gatttgggat 2760

cagcatattg taaaacatgg tgagggaaag ccggatcttc tctatattga tttgcacctc 2820

attcatgagg tgacgtctcc tcaggcattt gaaggcttga gacaaaaggg aagaaaggtc 2880

agaagacccc aaaacacatt tgcgacaatg gaccacaaca tcccgactgt caatcgtttt 2940

gagataaagg atgaagttgc gaaacgccag gtaacggcgc ttgaaagaaa ctgtgaggaa 3000

tttggcgtgc gccttgccga tcttcacagc gtggatcaag ggattgtcca tgtcgtcggc 3060

cctgaactgg gcttaacgct tccagggaaa acgattgtgt gcggggacag tcatacatca 3120

acacatggcg ctttcggcgc tcttgcattt ggaatcggga cgagtgaagt cgaacatgtt 3180

ctttccacac agacactttg gcagcagcgt ccaaaaacac ttgaagtgcg cgtagatgga 3240

acgcttcaaa aaggggtaac ggcaaaggat gtcatccttg ctgtcatcgg caaatacggt 3300

gtgaaattcg gcacaggcta cgtcattgaa tacactgggg aagtattcag aaatatgacg 3360

atggatgaac gaatgactgt ttgtaacatg tcaattgaag caggagcaag agcaggtttg 3420

attgcacctg acgaggtgac gtttgaatat tgcaaaaatc gcaagtacac gccaaaaggc 3480

gaagaatttg acaaggccgt agaggaatgg aaggcgctgc gcacagaccc gggcgctgtt 3540

tacgataaat ctatcgtcct tgacggcaac aaaatttccc ctatggtgac atggggcatt 3600

aacccgggaa tggttcttcc tgtcgattct gaagttcctg cgccggaaag cttttctgca 3660

gaagatgata aaaaagaagc gattcgcgct tatgaatata tgggactgac tcctcatcag 3720

aaaattgaag acattaaagt ggagcacgta tttatcggtt cctgcacaaa ttcccgcatg 3780

actgaccttc gccaggctgc tgacatgatc aaggggaaga aggtagctga cagcgtaagg 3840

gccatcgtcg tgcccggatc ccaaagagtg aagcttcagg ctgaaaaaga agggcttgac 3900

caaattttct tggaagctgg atttgaatgg agagagtcag gctgcagcat gtgtttgagt 3960

atgaataatg atgttgttcc tgagggagag cgctgtgcat caacctctaa ccgcaacttc 4020

gagggcagac aaggaaaagg tgcaagaaca catctcgtca gcccggcaat ggctgcgatg 4080

gctgccattc acggacactt cgttgatgtc agaaagtttt atcaggaaaa aacagttgtg 4140

taaggagtgc gcgagatgga acctttgaaa tcacatacgg ggaaagcagc cgtattaaat 4200

cggatcaatg tggatacaga ccagattatt cctaagcaat ttttgaagag gattgaaaga 4260

acaggctacg ggcgttttgc attctttgac tggagatatg atgcgaatgg tgaaccgaac 4320

cctgaatttg aattaaacca gcctgtttat caaggagctt ccattttaat agcaggagaa 4380

aacttcggct gcgggtcatc gcgtgaacat gctccgtggg cacttgatga ttatgggttt 4440

aaaattatca ttgcgccgtc attcgctgat attttccatc agaactgctt taaaaacggc 4500

atgcttccga tccgcatgcc atatgacaat tggaaacagc ttgtcggcca gtatgaaaac 4560

aagtcattgc aaatgactgt tgaccttgaa aatcagctga ttcatgacag tgaaggcaat 4620

caaatttcat ttgaagttga cccgcattgg aaagagatgc tgatcaacgg atatgatgaa 4680

atttcattaa cgctgctgct ggaagatgaa atcaagcatt ttgaatcaca aagaagctct 4740

tggcttcaag cctga 4755

<210> 7

<211> 1247

<212> DNA

<213> Bacillus subtilis

<400> 7

agcgcttata cgcgttttct cctgcttttt tcatatgaat ttcttacaaa tttgagcaaa 60

cctattgcga ttatttgttg aaggtataca atagaatata attattttca aataagtttg 120

ataatataaa caatttaaca gcagggagat tgaccatgac taaacaaaca attcgcgttg 180

aattgacatc aacaaaaaaa ccgaaaccag acccaaatca gctttcgttc ggaagagtgt 240

ttacagacca catgtttgta atggactatg ccgcagataa aggttggtac gatccaagaa 300

tcattcctta tcagccctta tcaatggatc cggccgcaat ggtctatcac tacggccaaa 360

ccgtgtttga agggttaaag gcttacgtgt cagaggatga ccatgttctg cttttcagac 420

cggaaaaaaa tatggaacgc ctgaatcaat caaacgaccg cctctgcatc ccgcaaattg 480

atgaagaaca ggttcttgaa ggcttaaagc agcttgtcgc aattgataaa gactggattc 540

caaatgcgga gggcacgtcc ctatacatcc gtccgttcat catcgcaacc gagcctttcc 600

ttggtgttgc ggcatctcat acgtataagc tcttgatcat tctttctccg gtcggctctt 660

attacaaaga aggcattaag ccggtcaaaa tcgctgttga aagtgaattt gtccgtgcgg 720

taaaaggcgg aacaggaaat gccaaaaccg cagggaacta cgcttcaagc ttaaaagcgc 780

agcaggtcgc cgaagagaaa ggattttccc aagtgctttg gctggacggc attgagaaga 840

aatacatcga agaagttgga agcatgaaca tcttcttcaa aatcaacggt gaaatcgtaa 900

cgccgatgct gaacggaagc atcctggaag gcattacgcg caattcagtc atcgccttgc 960

ttaagcattg gggccttcaa gtttcagaac ggaaaattgc gatcgatgag gtcatccaag 1020

cccataaaga cggcatcctg gaagaagcct tcggaacagg tacagcagct gttatttccc 1080

cagtcggcga gctgatctgg caggatgaaa cactttcgat caacaacggt gaaacaggag 1140

aaatcgcaaa aaaactatat gacacgatta caggcattca aaaaggcgct gtcgcagacg 1200

aattcggatg gacgaccgaa gttgcagcgc tgactgaaag caagtaa 1247

<210> 8

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cactctagaa tgcgcaaaat taattttttc gatac 35

<210> 9

<211> 35

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

taacgcgttc aggcttgaag ccaagagctt ctttg 35

<210> 10

<211> 34

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

acaacgcgtt ttctcctgct tttttcatat gaat 34

<210> 11

<211> 29

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cacccatggt tacttgcttt cagtcagcg 29

<210> 12

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cagcctttgt tggttctttt 20

Claims (6)

1. a high-yielding lipopeptide recombinant bacterium, which is to transform the 2-isopropylmalate synthase gene in the leucine synthesis pathway into the original strain and build up; the recombinant bacterium has also transferred into 3-isopropylmalate synthase gene. propylmalate dehydrogenase gene, 3-isopropylmalate dehydratase gene and branched-chain amino acid transaminase gene; the original strain is Bacillus subtilis; the 2-isopropylmalate synthase, 3-isopropylmalate synthase, The propylmalate dehydrogenase, 3-isopropylmalate dehydratase and branched-chain amino acid transaminase are LeuA, LeuB, LeuCD and IlvK proteins respectively; the lipopeptide is surfactin. 2. The recombinant bacteria according to claim 1, wherein the amino acid sequence of the LeuA protein is shown in SEQ ID NO.1, the amino acid sequence of the LeuB protein is shown in SEQ ID NO.2, and the LeuC The amino acid sequence of the protein is shown in SEQ ID NO.3, the amino acid sequence of the LeuD protein is shown in SEQ ID NO.4, and the amino acid sequence of the IlvK protein is shown in SEQ ID NO.5. 3 . The recombinant bacteria according to claim 1 , wherein the original strain is Bacillus subtilis THY-7, and the deposit number is CGMCC No.8906. 4 . 4. The recombinant bacteria according to claim 3, wherein the original strain is Bacillus subtilis THY-7/Pg3-srfA, and the Bacillus subtilis THY-7/Pg3-srfA refers to An inducible strong promoter Pg3 was added to Bacillus subtilis THY-7, so that the strong promoter Pg3 could control the expression of surfactin synthase srfA. 5 . The recombinant bacteria according to claim 4 , wherein the LeuA, LeuB, LeuC and LeuD proteins are controlled by a strong promoter, and the IlvK protein is controlled by a constitutive promoter. 6 . 6. Application of the recombinant bacteria according to any one of claims 1-5 in the production of surfactin.

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