CN110331121A

CN110331121A - A kind of recombinant bacterium of high yield lipopeptid and its application

Info

Publication number: CN110331121A
Application number: CN201910549289.5A
Authority: CN
Inventors: 于慧敏; 王苗苗; 许春梦
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2019-06-24
Filing date: 2019-06-24
Publication date: 2019-10-15
Anticipated expiration: 2039-06-24
Also published as: CN110331121B

Abstract

The invention discloses a kind of recombinant bacterium for the high yield lipopeptid for belonging to gene engineering technology field and its application, the recombinant bacterium of the high yield lipopeptid is that the 2-Isopropylmalate synthase genetic transformation in leucine route of synthesis is built-up to original strain.The recombinant bacterium of further overexpression part leucine synthesis path (2-Isopropylmalate synthase, 3-Isopropylmalate dehydrogenase, 3-Isopropylmalate dehydratase and branched-chain amino acid transaminase) of the invention is compared with starting strain, Surfactin yield highest improves 55.6%, it can be used for the production of Lipopeptide Biosurfactants, with good prospects for commercial application, lipopeptid yield average out to 13-16g/L in fermentation liquid obtained by shake flask fermentation.

Description

A kind of recombinant bacterium of high yield lipopeptid and its application

Technical field

The invention belongs to gene engineering technology fields, and in particular to a kind of recombinant bacterium of high yield lipopeptid and its application.

Background technique

Lipopeptid (lipopeptide) type biological surfactant is a kind of by hydrophilic cyclic oligopeptides and hydrophobic fatty acid The amphiprotic substance that chain is formed by connecting with lactone bond, mainly by Microbe synthesis such as bacillus, streptomycetes.Due to composition lipopeptid Amino acid composition is different from cyclic mode in peptide ring, and bacillus lipopeptid can be divided into Surfactin, fragrant mustard element, iturin Deng wherein there is good surface-active, biological degradability in the Surfactin that air liquid interface forms unique " saddle " conformation And antibacterial activity, all have broad application prospects in fields such as oil exploitation, biological control, medicine and daily use chemicals.But due to gemma Bacillus fermentation production Surfactin low yield limits the industrialized production and application of Surfactin.

Currently, the method for improving microorganism lipopeptid fermentation level includes training systern, mutation breeding, strengthening surface work Property element transdermal delivery, strengthening surface active extract synthesis expression of enzymes, strengthen fatty acid precursor supply etc..Patent document (CN 101892176A, CN 101775427A, WO 2002026961A) etc. by fermentation of bacillus subtilis produce surface-active Culture medium and condition of culture optimize in plain fermentation process, improve the yield of Surfactin in fermentation liquid.Mutation breeding Aspect, CN101928677A, which is disclosed, handles Streptomyces roseosporus by ultraviolet mutagenesis, and US05227294, which is disclosed, uses nitroso First handles bacillus subtilis for urethane, and the microorganism in above-mentioned document is after mutagenic treatment, and Surfactin synthetic quantity is all There is increase.Genetic engineering transformation aspect, Chinese patent literature CN103898038A are disclosed by strengthening lipopeptid from intracellular to born of the same parents The transmembrane protein YcxA of outer transport, the Surfactin output increased 97% of fermentation of bacillus subtilis production.Chinese patent Document CN1554747A discloses in bacillus subtilis expression comA gene, lipopeptid output increased 50%.In addition, withered grass Fatty acid and amino acid catalyze and synthesize Surfactin, Chinese patent under the action of Surfactin synzyme in bacillus Document CN105400784A is by being substituted for inducible strong promoter Pg3, surface-active for lipopeptid synthase gene cluster promoter Plain output increased 17.7 times.Chinese patent literature CN109097315A is by being overexpressed biology in branched chain fatty acid route of synthesis Plain carboxylase YngH, Surfactin output increased 47%.(Qun Wu,Yan Zhi,Yan Xu,Systematically engineering the biosynthesis of a green biosurfactant surfactin by Bacillus [J] .Metabolic of subtilis 168 Engineering, 2019,52:87-97) it discloses and strengthens intracellular Branched fatty Acid synthesizes the supply that precursor fatty acid is improved in whole path, thus 20.8 times of Surfactin output increased.It is living about surface Property element synthesis another kind of main producers amino acid, (Coutte F, Niehren J, Dhali D, et al.Modeling leucine's metabolic pathway and knockout prediction improving the production of surfactin,a biosurfactant from Bacillus subtilis[J].Biotechnology Journal, 2015,10 (8SI): 1216-1234) disclose in culture medium add leucine can make 3 times of Surfactin output increased, explanation Improving leucine content is the available strategy for improving Surfactin yield.But (Qun Wu, Yan Zhi, Yan Xu, Systematically engineering the biosynthesis of a green biosurfactant Surfactin by Bacillus subtilis 168 [J] .Metabolic Engineering, 2019,52:87-97) Acetolactate synthase AlsS, the keto-alcohol acid being overexpressed in leucine route of synthesis in the host strain of production Surfactin restore different Structure enzyme IlvC, dihydroxylated acid dehydratase IlvD, 2-Isopropylmalate synthase LeuA, 3-Isopropylmalate dehydrogenase LeuB and 3-Isopropylmalate dehydratase LeuCD, Surfactin yield are not improved.Have about leucine synthesis in cell is strengthened Effect improves Surfactin production quantifier elimination and has not been reported.

Summary of the invention

The shortcomings that in order to overcome in the prior art, the purpose of the present invention is to provide a kind of recombinations of efficient production lipopeptid The yield of lipopeptid can be improved in bacterium.

The recombinant bacterium is by the 2-Isopropylmalate synthase genetic transformation in leucine route of synthesis to original bacteria Strain is built-up.

Selectable in above-mentioned recombinant bacterium, it is different that the recombinant bacterium has also been transferred to 3-Isopropylmalate dehydrogenase gene, 3- One or more in propyl malic acid dehydrase gene and branched-chain amino acid aminotransferase gene.

In above-mentioned recombinant bacterium, the 3-Isopropylmalate dehydrogenase, 3-Isopropylmalate dehydratase and branched-amino Sour transaminase biotin carboxylase is respectively LeuB, LeuCD and IlvK albumen or its mutant with the same function,

In above-mentioned recombinant bacterium, it is preferred that the amino acid sequence of the LeuB albumen is described as shown in SEQ ID NO.2 The amino acid sequence of LeuC albumen is as shown in SEQ ID NO.3, the amino acid sequence of the LeuD albumen such as SEQ ID NO.4 institute Show, the amino acid sequence of the IlvK albumen is as shown in SEQ ID NO.5.

In above-mentioned recombinant bacterium, the 2-Isopropylmalate synthase is LeuA albumen or its mutation with the same function Body, it is preferred that the amino acid sequence of the LeuA albumen is as shown in SEQ ID NO.1.

In above-mentioned recombinant bacterium, the original strain be with produce lipopeptid ability wild mushroom and its mutagenic fungi, mutant strain or Strain is transformed in genetic engineering.

In above-mentioned recombinant bacterium, the original strain is bacillus subtilis, Bacillus cercus or pseudomonad.

Selectable in above-mentioned recombinant bacterium, the original strain is bacillus subtilis Bacillus subtilis THY-7。

The bacillus subtilis Bacillus subtilis THY-7 is preserved in the micro- life of China on March 11st, 2014 Object culture presevation administration committee common micro-organisms center, preservation registration number are CGMCC No.8906, and in Chinese patent literature It is disclosed in CN 105400784A.

Selectable in above-mentioned recombinant bacterium, the original strain is bacillus subtilis Bacillus subtilis THY-7/Pg3-srfA。

The bacillus subtilis Bacillus subtilis THY-7/Pg3-srfA, refers in bacillus subtilis Inducible strong promoter Pg3 is added in Bacillus subtilis THY-7, closes strong promoter Pg3 control Surfactin At the expression of enzyme srfA.About inducible strong promoter Pg3, bacillus subtilis Bacillus subtilis THY-7/Pg3- SrfA and its specific preparation method have disclosure in Chinese patent literature CN 105400784A.

In above-mentioned recombinant bacterium, LeuA, LeuB, LeuC and LeuD albumen is controlled by strong promoter, the IlvK albumen It is controlled by former constitutive promoter.

The purpose of the present invention also provides application of the above-mentioned recombinant bacterium in production lipopeptid.

In above-mentioned application, the Lipopeptide Biosurfactants are Surfactin.

A kind of preparation method of above-mentioned recombinant bacterium, includes the following steps:

Amplification obtains 2-Isopropylmalate synthase, 3-Isopropylmalate dehydrogenase, 3-Isopropylmalate dehydratase Gene cluster leuABCD and branched-chain amino acid transaminase biotin carboxylase IlvK with itself constitutive promoter, will LeuABCD and IlvK gene order is inserted into shuttle plasmid, constructs expression plasmid；

Expression plasmid is imported into original strain, constructs recombinant bacterium.

Further, method particularly includes:

1, using upstream and downstream primer, polymerase chain reaction is carried out by template of Bacillus subtilis genes group, amplification is simultaneously Obtain leuABCD gene, it is preferred that its nucleic acid sequence is as shown in SEQ ID NO.6.Likewise, ilvK gene is expanded and obtains, Preferably, nucleic acid sequence is as shown in SEQ ID NO.7.

2, leuABCD and ilvK gene and shuttle plasmid are subjected to double digestion respectively, using ligase by two kinds of digestions Product is attached, and obtains connection product.

3, connection product is converted into E. coli TOP10 competent cell, carries out resistance screening positive colony, Obtain the expression plasmid pJMP-leuABCD-ilvK containing leuABCD and ilvK gene order.

4, expression plasmid pJMP-leuABCD-ilvK is transferred in starting strain, obtaining conversion has pJMP-leuABCD- The genetic engineering bacterium of ilvK plasmid.

In the preparation method of said gene engineering bacteria, the construction method of the shuttle plasmid pJMP is in Chinese patent literature It is had disclosed in CN109097315A.

A kind of preparation method of above-mentioned recombinant bacterium, optionally, specific method includes the following:

1, using leuABCD-F and leuABCD-R as upstream and downstream primer, gathered using Bacillus subtilis genes group as template Polymerase chain reaction, expands and obtains leuABCD gene order, nucleic acid sequence is as shown in SEQ ID NO.6；With ilvK-F and IlvK-R is upstream and downstream primer, carries out polymerase chain reaction by template of Bacillus subtilis genes group, expands and obtain IlvK gene order, nucleic acid sequence is as shown in SEQ ID NO.7；

2, leuABCD gene is subjected to Xba I and Mlu I double digestion, ilvK gene is subjected to the bis- enzymes of Mlu I and Nco I It cuts, shuttle plasmid is subjected to Xba I and Nco I double digestion, digestion products is purified, is produced three kinds of digestions using T4 DNA ligase Object is attached, and obtains connection product.

3, connection product is converted into E. coli TOP10 competent cell, the LB for being coated on kanamycins is flat Plate is inverted in 37 DEG C of incubators and is incubated overnight.The resistance clone to grow on picking plate is cultivated, and is extracted plasmid and is carried out Digestion and sequence verification obtain the expression plasmid pJMP-leuABCD-ilvK containing correct leuABCD-ilvK gene order.

4, expression plasmid pJMP-leuABCD-ilvK is transferred to bacillus subtilis B.subtilis using electrotransformation method In THY-7/Pg3-srfA (CN 105400784A), it is coated on the LB plate containing chloramphenicol and kanamycins, picking resistance Clone is cultivated, and PCR verifying is carried out, and obtains the genetic engineering bacterium that conversion has pJMP-leuABCD-ilvK plasmid B.subtilis THY-7/Pg3-srfA(leuABCD-ilvK)。

Preferably, Bacillus subtilis genes group described in step 1 can choose bacillus subtilis 1012wt (MoBiTec company), bacillus subtilis THY-7 (CN 105400784A), THY-8 (chemical industry progress, 2013,32:2952- 2956)、THY-15(Journal of industrial microbiology&biotechnology.2015,42(8): 1139-1147) or carry leuABCD and ilvK gene other Bacillus strains.

Preferably, the preferred pJMP of shuttle plasmid described in step 2, construction method is in Chinese patent literature CN109097315A In have disclosed.

The purpose of the present invention lies also in offer said gene engineering bacteria and is preparing the application in lipopeptid.

Optionally, lipopeptid is produced using above-mentioned recombinant bacterium, steps are as follows:

Recombinant bacterium is accessed in culture medium, expands culture, obtains genetic engineering bacterium bacterium solution；

In the engineering bacteria bacterium solution access fermentation medium for obtaining step (1) according to 1-20% percent by volume, sent out Ferment culture obtains the fermentation liquid containing lipopeptid.

In the above method, the method for expanding culture are as follows: in 35-40 DEG C, the condition that shaking speed is 150-200rpm Lower culture 10-20h.

In the above method, the method for the fermented and cultured be 35-40 DEG C, shaking speed be 150-200rpm under conditions of Continue to cultivate 40-60h after 0.5-1.5mM IPTG inducer is added when cultivating 1.5-4h.

In the above method, the composition of the fermentation medium are as follows: carbohydrate 30-100g/L, inorganic nitrogen-sourced 10-50g/L, it is organic Nitrogen source 0.5-3g/L, 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 advantages of the present invention:

The present invention constructs recombinant bacterium using technique for gene engineering, expands and expresses in producing lipopeptid B. subtilis cell 2-Isopropylmalate synthase LeuA, 3-Isopropylmalate dehydrogenase LeuB, 3- isopropyl apple in leucine route of synthesis Tartaric acid dehydratase LeuCD and branched-chain amino acid transaminase IlvK, enhances the synthesis of leucine in Surfactin molecular structure, To significantly improve the yield of lipopeptid.Gained of the invention is overexpressed the recombinant bacterium and starting strain of part leucine synthesis path It compares, Surfactin yield highest improves 55.6%, can be used for the production of Lipopeptide Biosurfactants, has good Prospects for commercial application, lipopeptid yield average out to 13-16g/L in fermentation liquid obtained by shake flask fermentation.

Detailed description of the invention

Fig. 1 is the schematic diagram for gene overexpression plasmid pJMP.

Fig. 2 is the PCR proof diagram of the overexpression plasmid pJMP-leuABCD-ilvK containing leu ABCD-ilvK gene string: Swimming lane 1 is DNA molecular amount standard, and swimming lane 2 is for plasmid using primer ilvK-F and ilv-R progress PCR amplification as a result, available The band of 1.3kb.

Fig. 3 is to carry 2-Isopropylmalate synthase LeuA, 3-Isopropylmalate dehydrogenase LeuB, 3- isopropylmalate The B. subtilis-E. coli shuttle plasmid pJMP- of sour dehydratase LeuCD and branched-chain amino acid transaminase IlvK gene The schematic diagram of leuABCD-ilvK.

Fig. 4 is to be overexpressed 2-Isopropylmalate synthase LeuA, 3-Isopropylmalate dehydrogenase LeuB, 3- isopropyl apple The genetic engineering bacterium B.subtilisTHY-7/Pg3-srfA of tartaric acid dehydratase LeuCD and branched-chain amino acid transaminase IlvK (leuABCD-ilvK) PCR proof diagram: swimming lane 1 is THY-7/Pg3-srfA (pJMP-yngH) with primer ilvK-F and general Primer pJMP-R carry out PCR amplification as a result, the band of 1.5kb can be obtained.Swimming lane 2 is DNA molecular amount standard.

Fig. 5 is original strain THY-7/Pg3-srfA and genetic engineering bacterium THY-7/Pg3-srfA (leuABCD-ilvK) The concentration of Surfactin in tunning.

Specific embodiment

The present invention will be further explained below with reference to the attached drawings and specific examples.Such as not specified, institute in embodiment Biochemical reagents are commercial reagent, and technological means used in embodiment is the conventional hand in those skilled in the art's book Section.

In leucine route of synthesis: glucose generates pyruvic acid by glycolytic pathway, and pyruvic acid is leucine synthesis Precursor.During pyruvic acid generates α -one base isovaleric acid, the rate-limiting step that acetolactic acid is the process is synthesized by pyruvic acid. The enzyme for being catalyzed the step is Acetohydroxyacid synthase, is encoded by gene ilvHB and gene alsS.In α -one base isovaleric acid and second Acyl CoA synthesize L-Leu process, rate-limiting step be by α-isopropylmalate synthetase catalyzed synthesizing alpha-isopropylmolic acid, The gene for encoding the enzyme is leuA.

This 3 related gene discoveries: surface-active when being overexpressed alsS and ilvHB are expressed in inventor's previous experiments respectively The synthesis of element significantly reduces, and Surfactin yield is promoted when being overexpressed leuA, and lipopeptid yield is 11.58g/L, improves 13.5%, when being overexpressed leuAB, lipopeptid yield is not promoted further.

Embodiment 1 carries the plasmid construction of gene leuABCD and ilvK in leucine route of synthesis

Picking bacillus subtilis B.subtilis THY-7 single colonie, is inoculated in LB liquid medium, is placed in 37 DEG C, be incubated overnight in the shaking table of 200rpm, collect thallus under 12000rpm, 5min centrifugal condition, use the thin of Omega company Bacterium genome extraction kit extracts THY-7 genome.Using obtained genome as template, upstream primer leuABCD-F is used (sequence is as shown in SEQ ID NO.8) and downstream primer leuABCD-R (sequence is as shown in SEQ ID NO.9) carry out PCR amplification Obtain leuABCD segment.Use upstream primer ilvK-F (sequence is as shown in SEQ ID NO.10) and downstream primer ilvK-R (sequence Column as shown in SEQ ID NO.11) progress PCR amplification obtain ilvK fragment primer by Bo Shang biotechnology (Shanghai) Co., Ltd. Synthesis, dissolved with sterile water and be diluted to 10 μM it is spare.Polymerase, buffer used in PCR amplification and restriction enzyme are purchased from TaKaRa company.Pcr amplification reaction system are as follows:

Thermal cycle conditions are

Amplification purification obtains bacillus subtilis leuABCD gene segment, and sequence is as shown in SEQ ID NO.6, amplification Purifying obtains bacillus subtilis ilvK gene segment, and sequence is as shown in SEQ ID NO.7.LeuABCD gene is subjected to Xba IlvK gene is carried out Mlu I and Nco I double digestion by I and Mlu I double digestion, and it is bis- that shuttle plasmid is carried out Xba I and Nco I Digestion is purified with the DNA purification kit of Omega company after 27-33 DEG C of digestion 1-3h, and T4 DNA ligase is then used (NEB company) connects overnight under the conditions of 16-22 DEG C.Connection product converts E. coli TOP10 competent cell (TianGen company) is coated on the LB plate containing kanamycins, is inverted in 37 DEG C of incubators and is incubated overnight.On picking plate Longer resistance clone carries out bacterium colony PCR, is to draw with ilvK-F and Plasmid Primer ilvK-R using the bacterium colony of picking as template Object, amplifying about 1.3Kb band (as shown in Figure 2) is positive colony, and picking positive colony culture is extracted plasmid and is sequenced and tests Card, obtains the expression plasmid pJMP-leuABCD-ilvK containing leuABCD and ilvK gene, and wherein Fig. 1 is showing for plasmid pJMP It is intended to.

The genetic engineering bacterium B.subtilis THY-7/Pg3-srfA of the overexpression leucine route of synthesis of embodiment 2 (leuABCD-ilvK) building

By the carrying 2-Isopropylmalate synthase LeuA constructed in embodiment 1,3-Isopropylmalate dehydrogenase LeuB, The B. subtilis-E. coli shuttle matter of 3-Isopropylmalate dehydratase LeuCD and branched-chain amino acid transaminase IlvK Grain pJMP-leuABCD-ilvK can be obtained with the competent cell of electroporation conversion bacillus subtilis THY-7/Pg3-srfA To the genetic engineering bacterium THY-7/Pg3-srfA (leuABCD-ilvK) for being overexpressed leucine synthesis path portion gene.Wherein, The preparation of bacillus subtilis THY-7/Pg3-srfA competent cell and electrotransformation use Chinese patent literature Method in CN105400784A.

It takes 100uL bacterium solution to be coated on the LB solid medium of the g/mL kanamycins of μ containing 10-30 after recovery, is inverted in 37 It is incubated overnight in DEG C incubator, picking single colonie, carries out PCR verifying using upstream and downstream primer ilvK-F and pJMP-R, it is amplifiable About 1.5kb band out, verification result are overexpressed 2-Isopropylmalate synthase LeuA, 3- isopropyl apple as shown in figure 4, obtaining The genetic engineering bacterium of tartaric acid dehydrogenase LeuB, 3-Isopropylmalate dehydratase LeuCD and branched-chain amino acid transaminase IlvK THY-7/Pg3-srfA(leuABCD-ilvK).The Plasmid Primer pJMP-R sequence is as shown in SEQ ID NO.12.

Embodiment 3 produces lipopeptid class surface-active using genetic engineering bacterium THY-7/Pg3-srfA (leuABCD-ilvK) Agent-Surfactin

By the obtained overexpression 2-Isopropylmalate synthase LeuA of embodiment 2,3-Isopropylmalate dehydrogenase The genetic engineering bacterium THY-7/Pg3- of LeuB, 3-Isopropylmalate dehydratase LeuCD and branched-chain amino acid transaminase IlvK SrfA (leuABCD-ilvK) is inoculated in LB liquid medium (containing chloramphenicol and kanamycins), and 37 DEG C, under the conditions of 200rpm 16h is cultivated, with 5% ratio access equipped in the shaking flask of 100mL fermentation medium, 37 DEG C, cultivate 2-6h under the conditions of 200rpm When IPTG is added, continue to cultivate to 2-3d to get to the fermentation liquid containing lipopeptid.

Surfactin detection is used in the intelligent quick method for waiting (Chinese patent CN 105400784A) in fermentation liquid.Gene Engineering bacteria THY-7/Pg3-srfA (leuABCD-ilvK) and Surfactin in starting strain THY-7/Pg3-srfA fermentation liquid The statistical result of concentration is as shown in Figure 5: THY-7/Pg3-srfA (leuABCD-ilvK) Surfactin yield reaches as high as 15.8g/L goes out bacterium germination (10.2g/L) than THY-7/Pg3-srfA and improves 54.9%.5L fermentation tank culture THY-7/Pg3- SrfA (leuABCD-ilvK), Surfactin yield are up to 19g/L.

The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；Although referring to aforementioned each reality Applying example, invention is explained in detail, those skilled in the art should understand that: it still can be to aforementioned each Technical solution documented by embodiment is modified, or equivalent substitution of some or all of the technical features；And These are modified or replaceed, the range for technical solution of various embodiments of the present invention that it does not separate the essence of the corresponding technical solution.

Sequence table

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260 265 270

Tyr Asp Lys Ser Ile Val Leu Asp Gly Asn Lys Ile Ser Pro Met Val

275 280 285

Thr Trp Gly Ile Asn Pro Gly Met Val Leu Pro Val Asp Ser Glu Val

290 295 300

Pro Ala Pro Glu Ser Phe Ser Ala Glu Asp Asp Lys Lys Glu Ala Ile

305 310 315 320

Arg Ala Tyr Glu Tyr Met Gly Leu Thr Pro His Gln Lys Ile Glu Asp

325 330 335

Ile Lys Val Glu His Val Phe Ile Gly Ser Cys Thr Asn Ser Arg Met

340 345 350

Thr Asp Leu Arg Gln Ala Ala Asp Met Ile Lys Gly Lys Lys Val Ala

355 360 365

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

370 375 380

Gln Ala Glu Lys Glu Gly Leu Asp Gln Ile Phe Leu Glu Ala Gly Phe

385 390 395 400

Glu Trp Arg Glu Ser Gly Cys Ser Met Cys Leu Ser Met Asn Asn Asp

405 410 415

Val Val Pro Glu Gly Glu Arg Cys Ala Ser Thr Ser Asn Arg Asn Phe

420 425 430

Glu Gly Arg Gln Gly Lys Gly Ala Arg Thr His Leu Val Ser Pro Ala

435 440 445

Met Ala Ala Met Ala Ala Ile His Gly His Phe Val Asp Val Arg Lys

450 455 460

Phe Tyr Gln Glu Lys Thr Val Val

465 470

<210> 4

<211> 199

<212> PRT

<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

1. a kind of recombinant bacterium of high yield lipopeptid is by the 2-Isopropylmalate synthase genetic transformation in leucine route of synthesis It is built-up to original strain.

2. recombinant bacterium according to claim 1, which is characterized in that it is de- that the recombinant bacterium has also been transferred to 3- isopropylmolic acid One or more in hydrogenase gene, 3-Isopropylmalate dehydratase gene and branched-chain amino acid aminotransferase gene.

3. recombinant bacterium according to claim 2, which is characterized in that the 3-Isopropylmalate dehydrogenase, 3- isopropyl Malic acid dehydratase and branched-chain amino acid transaminase biotin carboxylase is respectively LeuB, LeuCD and IlvK albumen or it has The mutant of identical function, it is preferred that the amino acid sequence of the LeuB albumen is as shown in SEQ ID NO.2, the LeuC egg White amino acid sequence is as shown in SEQ ID NO.3, and the amino acid sequence of the LeuD albumen is as shown in SEQ ID NO.4, institute The amino acid sequence of IlvK albumen is stated as shown in SEQ ID NO.5.

4. recombinant bacterium according to claim 1, which is characterized in that the 2-Isopropylmalate synthase be LeuA albumen or Its mutant with the same function, it is preferred that the amino acid sequence of the LeuA albumen is as shown in SEQ ID NO.1.

5. recombinant bacterium according to claim 1-4, which is characterized in that the original strain is with production lipopeptid energy Strain is transformed in the wild mushroom and its mutagenic fungi of power, mutant strain or genetic engineering.

6. recombinant bacterium according to claim 5, which is characterized in that the original strain is bacillus subtilis, wax-like bud Spore bacillus or pseudomonad.

7. recombinant bacterium according to claim 6, which is characterized in that the original strain is bacillus subtilis Bacillus subtilis THY-7。

8. recombinant bacterium according to claim 7, which is characterized in that the original strain is bacillus subtilis Bacillus subtilis THY-7/Pg3-srfA。

9. recombinant bacterium according to claim 8, which is characterized in that LeuA, LeuB, LeuC and LeuD albumen is opened by force Mover control, the IlvK albumen are controlled by former constitutive promoter.

10. application of the described in any item recombinant bacteriums of claim 1-9 in production lipopeptid.