CN112795587A

CN112795587A - A surfactin-producing Escherichia coli engineering bacterium and its construction method and application

Info

Publication number: CN112795587A
Application number: CN202110137604.0A
Authority: CN
Inventors: 魏韬; 郭玉婧; 邹苑; 林俊芳; 郑倩望; 叶志伟
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2021-02-01
Filing date: 2021-02-01
Publication date: 2021-05-14
Anticipated expiration: 2041-02-01
Also published as: CN112795587B

Abstract

The invention discloses a surfactin-producing Escherichia coli engineering bacteria and its construction method and application. In the present invention, through the ExoCET recombination technology, the surfactin synthesis gene cluster and the carrier fragment with homologous arms obtained by double enzyme digestion are annealed by T4 DNA polymerase in vitro, and then transferred into a strain with a recombination system to complete the recombination. , the recombinant plasmid pWG‑SRF was obtained. The E. coli wild-type strain was transferred to obtain the engineered strain BW25113/pWG‑SRF. The engineered strain has a de novo construction of a more concise surfactin anabolic network, a smaller genome and a shorter fermentative growth cycle. In addition, the surfactin obtained from the fermentation broth of the Escherichia coli strain has a strong surfactant function, and is expected to be developed and utilized in the fields of petroleum extraction, daily chemicals, antibacterial and antiviral preparations, etc., and has a good development and utilization prospect. .

Description

Escherichia coli engineering bacterium for producing surfactin and construction method and application thereof

Technical Field

The invention belongs to the field of microbial metabolic engineering and synthetic biology, and particularly relates to an escherichia coli engineering bacterium for producing surfactin, a construction method and application thereof, in particular to an engineering bacterium BW25113/pWG-SRF capable of producing surfactin.

Background

The surfactant is formed by condensing 7 amino acids and a beta hydroxy fatty acid chain (C12-C16), and is a cyclic lipopeptide mainly synthesized by bacillus bacteria [1 ]. The surfactant has extremely strong surface activity efficiency, is considered to be the strongest known biosurfactant at present, and has great application value in the aspects of petroleum, daily chemicals, sterilization and the like.

A great deal of past researches adopt methods such as culture medium optimization, reactor redesign, industrial and agricultural waste fermentation and the like to improve the synthesis level of the surfactant and reduce the price of raw materials, but all methods fail to achieve sufficient results [2 ].

A great deal of research shows that surfactin anabolism related genes in most of bacillus bacteria are also related to macroscopic growth and reproduction traits such as biofilm and spore formation [3 ]. In genome sequencing studies of Siamese Bacillus JFL15, it was found that the synthesis rate of surfactin is regulated by global regulators such as AbrB 4, DegU 5, CodY 6, etc., and is generally controlled to a lower range, thereby coordinating with other growth conditions of bacteria [7 ]. Meanwhile, synthesis of surfactin is also regulated by the quorum sensing system associated with sporulation such as ComXQPA [8 ].

When bacillus bacteria are used for fermenting and synthesizing surfactin, various cyclic lipopeptides and linear lipopeptides such as iturin (iturin) and fengycin (fengycin) can be simultaneously synthesized. Wherein, the three cyclic lipopeptides have similar structures and larger separation difficulty, and the production cost of the single-component surfactant is increased. Coutte et al report that the cost of separation and purification of currently marketed lipopeptide antibacterial peptide compounds accounts for more than 60% of the total production cost [9 ]. The separation of antibacterial substances from Siamese bacillus identifies 3 kinds of antibacterial lipopeptide compounds, siderophin compounds, polyketide compounds, cyclic dipeptide compounds and other antibacterial substances, and the complexity of the antibacterial lipopeptide kinds produced by bacillus bacteria is consistent with that reported in the past [10 ]. On the other hand, 2 structural analogues of iturin and fengycin were knocked out from Bacillus amyloliquefaciens NK-delta LP strains by Zhouyu et al, but synthesis of all kinds of antibacterial lipopeptides except for surfactin could not be completely prevented [11], suggesting that it is difficult to construct engineering bacteria of Bacillus for producing single-component surfactin.

In Bacillus, surfactin is synthesized by a multifunctional enzyme system (NRPS). The gene cluster for the synthesis of surfactin is the srf operon, which is 26kb in length [1 ].

Zhang Yogming et al reported that ExoCET recombination technology combines in vitro DNA recombination with in vivo RecET homologous recombination for the direct cloning of large fragment biosynthetic gene clusters. DNA fragments of sizes within 106kb were directly captured into expression vectors [12 ]. By using the above recombinant system, Zyoming et al successfully expressed 10 NRPS pathways in E.coli, of which 3 successfully detected the product [12 ]. Therefore, the successful construction of engineering bacteria for producing the single-component surfactant is of great significance.

Reference documents:

1. extreme metabolism mechanism and function research of efficient synthesis of surfactin by Bacillus [ D ] Jiangnan university, 2017.

2.Hamoen LW，Venema G，Kuipers OP.Controlling competence in Bacillus subtilis:shared use of regulators[J].Microbiology,2003.149(1):9-17.

3.Xu BH,Ye ZW,Zheng QW,et al.Isolation and characterization of cyclic lipopeptides withbroad-spectrum antimicrobial activity from Bacillus siamensis JFL15[J].3Biotech,2018.8(10):p.444-453.

4.Strauch MA,Bobay BG,Cavanagh J,et al.Abh and AbrB control of Bacillus subtilis antimicrobial gene expression[J].Journal of Bacteriology,2007.189(21):7720-7732.

5.Mitsuo O,Hirotake Y,Ken-Ichi Y，et al.DNA microarray analysis of Bacillus subtilis DegU,ComA and PhoP regulons:an approach to comprehensive analysis of B.subtilis two-component regulatory systems[J].Nucleic Acids Research,2001.29(18):3804-3813.

6.Serror P，Sonenshein AL.CodY is required for nutritional repression of Bacillus subtilis genetic competence[J].Journal of Bacteriology,1996.178(20):5910-5915.

7.Shaligram NS，Singhal RS.Surfactin--a review on biosynthesis,fermentation,purification and applications[J].Food Technology&Biotechnology,2010.48(2):119-134.

8.Oslizlo A,Stefanic P,Vatovec S,et al.Exploring ComQXPA quorum-sensing diversity and biocontrol potential of Bacillus spp.isolates from tomato rhizoplane[J].Microbial Biotechnology,2015.8(3):527-540.

9.Coutte F,Lecouturier D,Dimitrov K,et al.Microbial lipopeptide production and purification bioprocesses,current progress and future challenges[J].Biotechnology Journal,2017.12(7):1600566.

10.Xu BH,Lu YQ,Ye ZW,et al.Genomics-guided discovery and structure identification of cyclic lipopeptides from the Bacillus siamensis JFL15[J].PloS one,2018.13(8):e0202893-e0202893.

11. Zhouyu, Zhang Wan Ru, Zhang Dang Xuan Hemerocallis, and the like, the metabolic engineering transformation of Bacillus amyloliquefaciens improves the yield of Surfactin [ J ]. the university report of south China: nature science edition 2018.51(5):18-26.

12.Fu J,Bian X,Hu S,et al.Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting[J].Nature Biotechnology,2012.30:440-446.

Disclosure of Invention

In order to overcome the defects of various structural analogs and complex molecular regulation mechanism of a surfactin fermenting and synthesizing strain in the prior art, the invention aims to provide a construction method of escherichia coli engineering bacteria for producing surfactin.

The invention also aims to provide the escherichia coli engineering bacteria for producing the surfactant, which are obtained by the construction method.

The invention also aims to provide application of the escherichia coli engineering bacteria.

According to the invention, by an ExoCET recombination technology, a surfactant synthetic gene cluster and a vector fragment with a homologous arm obtained by double enzyme digestion are annealed by T4DNA polymerase in vitro and then transferred into a strain with a recombination system to complete recombination, so that a recombinant plasmid pWG-SRF is obtained. Extracting expression vector DNA connected with surfactant synthetic gene cluster, transferring into Escherichia coli wild strain, and obtaining engineering bacteria BW 25113/pWG-SRF. And (3) shaking the flask to ferment the engineering bacteria, measuring the surfactant content of the engineering bacteria by using an HPLC method, and measuring the activity of a crude extract of fermentation liquor according to an oil discharge ring method.

The purpose of the invention is realized by the following technical scheme:

a construction method of colibacillus engineering bacteria for producing surfactin comprises the following steps:

(1) analyzing a Siamese bacillus JFL15 genome, and comparing the genome with an NCBI database to obtain a surfactant synthetic gene cluster sequence;

(2) carrying out double enzyme digestion on genome DNA of Siamese bacillus JFL15 by KpnI/BamHI to obtain surfactin synthetic gene cluster DNA;

(3) carrying out T4DNA polymerase annealing on surfactant synthetic gene cluster DNA obtained by double enzyme digestion and p15A-Cm-HA in vitro by using an ExoCET method, transferring the DNA into a GB05-dir strain with a recombination system for recombination to obtain an engineering bacterium, and marking the engineering bacterium as GB 05-dir/pWG-SRF; the sequence of the p15A-Cm-HA is shown in SEQ ID NO: 1 is shown in the specification;

(4) extracting plasmid pWG-SRF from GB05-dir/pWG-SRF, transforming plasmid pWG-SRF into Escherichia coli wild strain BW25113, and obtaining engineering bacteria BW 25113/pWG-SRF.

In the step (1), the genome of Siamese Bacillus JFL15 is analyzed by using anti SMASH.

In the step (2), the fragment size of the surfactant synthetic gene cluster DNA is 32539 bp.

In the step (3), preferably, the GB05-dir strain with the recombination system is a GB05-dir strain with pSC101-BAD-ETgA-tet, and is marked as GB05-dir/pSC 101-BAD-ETgA-tet;

in step (3), preferably, the p15A-Cm-HA is constructed by the following steps:

counting 80bp from restriction enzyme sites KpnI and BamHI of a Siamese bacillus JFL15 genome to the inside of a gene cluster to be used as homology arms, designing a primer pair p15ACm F/p15ACm R, and amplifying a fragment with the 80bp homology arms by using pBAD-p15A-Cm-ccdB as a template, wherein the fragment is marked as p 15A-Cm-HA;

p15ACm F：5'-tcataaaatttcattaattgtcattgtagcataaacgccgaaaaaaagaagaagcgaaatcagtgtttcgcttcttctccggatccATGCGAGAGTAGGGAACTGC-3'; the crosshatched portion is the BamHI cleavage site.

p15ACm R：5'-gaggctgacggagctgtttcatttgaagcaaggagaaatacgaaggcatttattgtcctcaggcttaatacaaatgaaccggtaccATCTGATTAATAAGATGATC-3'; the underlined part represents a KpnI cleavage site.

Wherein the lower case part is a homology arm.

Preferably, the pBAD-p15A-Cm-ccdB is constructed by the following steps:

the vector pBAD33 is used as a template, p15A F/p15A R and Cm F/Cm R are used as primers, and pBAD-p15A with the size of 2070bp and Cm with the size of 1215bp are amplified; amplifying a 371bp ccdB gene fragment by using a plasmid pMD-ccdBKanS as a template and ccdB F/ccdB R as a primer, and assembling the 371bp ccdB gene fragment by using a TEDA one-step method to obtain a pBAD-p15A-Cm-ccdB plasmid;

p15A F：5'-AGTGAGAGGGCCGCGGCAAAG-3'；

p15A R：5'-GGTACCGAGCTCGAATTCGC-3'；

Cm F：5'-gccgcggccctctcactGCGAAAATGAGACGTTGATC-3'；

Cm R：5'-AAGCTTGGCTGTTTTGGCGG-3'；

ccdB F：5'-gcgaattcgagctcggtaccTTTCGGAATTAAGGAGGTAA-3'；

ccdB R：5'-ccgccaaaacagccaagcttTTATATTCCCCAGAACATCA-3'；

wherein the lower case part is a homology arm.

An escherichia coli engineering bacterium for producing surfactin is constructed by the construction method.

Fermenting the engineering bacteria of escherichia coli, extracting active substances to obtain a crude extract of fermentation liquor, and detecting by using a high performance liquid chromatography and an oil discharge ring method.

A recombinant expression vector pWG-SRF is constructed by the construction method.

The colibacillus engineering bacteria for producing the surfactin is applied to fermentation production of the surfactin.

The crude extract of fermentation liquor of escherichia coli engineering bacteria for producing surfactin can be applied to the fields of petroleum extraction, daily chemical industry, antibacterial and antiviral preparations and the like.

Compared with the prior art, the invention has the following advantages and effects:

the invention provides an escherichia coli engineering bacterium BW25113/pWG-SRF for producing surfactin, which is obtained by heterologous synthesis through a genetic engineering means and has the characteristics of simple culture, high growth speed and the like. Compared with the starting bacterium Siamese bacillus JFL15, the strain has a more concise surfactin anabolism network constructed de novo, a smaller genome and a shorter fermentation growth period. In addition, Escherichia coli does not produce surfactin structural analogs, and thus separation and purification costs are expected to be reduced. The surfactant extracted from the escherichia coli strain fermentation liquor has a strong surfactant function, is expected to be developed and utilized in the fields of petroleum extraction, daily chemical industry, antibacterial and antiviral preparations and the like, and has a good development and utilization prospect.

Drawings

FIG. 1 shows the EcoRI cleavage identification result of the vector pBAD-p 15A-Cm-ccdB; wherein, M: 1kb ladder DNA Maker; 1. 2, 3, 4: and (4) enzyme digestion to identify a correct transformant.

FIG. 2 shows the BglII cleavage identification result of pWG-SRF; wherein, M: 1kb ladder DNA Maker; 1. 2: BglII cleavage identified the correct transformants.

FIG. 3 shows the results of the restriction enzyme digestion of pWG-SRF with EcoRI, wherein M: 1kb ladder DNA Maker; 1. 2: EcoRI digestion identified the correct transformants.

FIG. 4 shows the results of measuring the content of surfactin in the fermentation broth of engineering bacteria BW25113/pWG-SRF by high performance liquid chromatography; wherein, 1: a surfactant standard (sigma standard); siemens JFL 15; 3: BW25113/pBAD 33; 4: BW 25113/pWG-SRF; the ordinate of 2 was spaced at 100mV intervals, the remainder at 10 mV.

FIG. 5 shows the results of the oil drainage test for crude fermentation broth; siemensis JFL 15; 2: BW25113/pBAD 33; 3: BW 25113/pWG-SRF.

Detailed Description

The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.

The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents and the like used are, unless otherwise specified, reagents and materials obtained from commercial sources.

The biomaterial used in the examples of the invention is as follows:

siamese Bacillus (Bacillus siamensis) JFL15 is disclosed in 201811086453.5, a method for transforming Siamese Bacillus by electric shock.

Coli BW25113 strain, pBAD33 vector, lysozyme and proteinase K were all commercially available.

pMD-ccdBKanS is disclosed in "Wei T, et al," Genome engineering Escherichia coli for L-DOPA over production from glucose.2016.6: p.30080 ".

GB05-dir/pSC101-BAD-ETgA-tet is obtained by transforming pSC101-BAD-ETgA-tet into E.coli GB05-dir, wherein E.coli GB05-dir and pSC101-BAD-ETgA-tet are disclosed in the literature "Wang H, et al, Exocet: exogenous enzyme in vitro assembly with RecET combining for high efficiency functional direct DNA cloning from complex genes, 2017.46(5): p.e28.";

TEDA one-step, multi-fragment assembly is disclosed in "Xia Y, et al, T5 exonuclease-dependent assembly of reagents a low-cost method for influencing and site-directed mutagenesis, nucleic Acids Res,2019.47(3): p.e 15.";

ExoCET large fragment cloning techniques are disclosed in "Wang, H., et al," ExoCET: exoenzyme in vitro assembly with RecET assembly for high throughput direct DNA cloning from complex genes, nucleic Acids Research,2017.46(5): p.e28.

EXAMPLE 1 cloning of surfactant Synthesis Gene Cluster

The vector pBAD33 is used as a template, p15A F/p15A R and Cm F/Cm R are used as primers, PrimeSTAR Max DNA Polymerase (Takara) is used for amplifying pBAD-p15A with 2070bp and Cm (chloramphenicol) with 1215bp, plasmid pMD-ccdBKanS is used as a template, ccdB F/ccdB R is used as a primer for amplifying 371bp ccdB, TEDA one-step method is used for multi-fragment assembly, after a transformant is obtained, enzyme digestion identification is carried out, and the pBAD-p15A-Cm-ccdB plasmid with 3599bp is obtained. The enzyme digestion identification is shown in figure 1, the EcoRI is used for carrying out enzyme digestion verification on the pBAD-p15A-Cm-ccdB, and 1210bp and 2389bp bands are cut out by the EcoRI, which indicates that the plasmid pBAD-p15A-Cm-ccdB is successfully constructed.

p15A F：5'-AGTGAGAGGGCCGCGGCAAAG-3'；

p15A R：5'-GGTACCGAGCTCGAATTCGC-3'；

Cm F：5'-gccgcggccctctcactGCGAAAATGAGACGTTGATC-3'；

Cm R：5'-AAGCTTGGCTGTTTTGGCGG-3'；

ccdB F：5'-gcgaattcgagctcggtaccTTTCGGAATTAAGGAGGTAA-3'；

ccdB R：5'-ccgccaaaacagccaagcttTTATATTCCCCAGAACATCA-3'；

Wherein the lower case part is a homology arm.

Analyzing a Siamese bacillus JFL15 genome by using anti SMASH, and comparing the genome with an NCBI database to obtain a surfactant synthetic gene cluster sequence; among these, the whole genome sequence of the siamese bacillus JFL15 genome has been published at NCBI with GenBank accession number LFWQ 00000000.1. The surfactant synthesis gene cluster sequence has the position 1904569-1937107 in LFWQ 00000000.1.

A primer pair p15ACm F/p15ACm R is designed by taking 80bp from the restriction enzyme sites KpnI and BamHI of a Siamese Bacillus JFL15 genome to the inside of a gene cluster as homology arms, a fragment with 80bp homology arms is amplified by using PrimeSTAR Max DNA Polymerase (Takara) and pBAD-p15A-Cm-ccdB as a template, the size of the fragment is 1784bp and is marked as p15A-Cm-HA (the sequence of the fragment is shown as SEQ ID NO: 1), and the gel is cut and recovered according to the operation of a gel recovery kit.

Wherein the lower case part is a homology arm.

Overnight cultured Siamese bacillus JFL15 thalli were collected. Cells were washed once with ultrapure water. 8mL of SET (75mM NaCl, 25mM EDTA, 20mM Tris, pH 8.0), 40mg of lysozyme were added in a water bath at 37 ℃ for 2 hours. 20mg of proteinase K, 1mL of 10% SDS and water bath at 50 ℃ for 4-5 hours until the mixture is clear. 3.5mL of 5M NaCl was added. The genomic DNA was extracted by phenol-chloroform-isoamyl alcohol (25:24:1) extraction, absolute ethanol precipitation, and finally dissolved in 300. mu.L of 10mM Tris-HCl (pH 8.0).

50 mu g of genome DNA is cut by KpnI/BamHI enzyme, and 400 mu L of the system is cut by enzyme at 37 ℃ for 1h to obtain the surfactant synthetic gene cluster DNA, and the fragment size is 32539 bp.

Enzyme digestion system:

the genomic DNA double-digested with KpnI/BamHI was extracted by phenol-chloroform-isoamyl alcohol (25:24:1) extraction and precipitated with absolute ethanol, and finally dissolved in 12. mu.L of ddH₂O, taking 2 mu L of agarose gel with the concentration of 1 percentAnd (5) carrying out electrophoresis detection. The results indicate that the restriction enzyme will cut the genomic DNA apart, thereby successfully obtaining the surfactant synthesis gene cluster.

mu.L of 1. mu.g/. mu.L of genomic DNA double-digested with KpnI/BamHI, 1. mu.L of 200 ng/. mu. L p 15-15A-Cm-HA, 2. mu.L of 10 XNEB Buffer 2.1, 0.13. mu.L of 3U/. mu. L T4DNA polymerase and 6.87. mu.L of ddH₂O mixed into a 20. mu.L reaction system. Treating the reaction system according to the reaction steps of 25 ℃ for 60min, 75 ℃ for 25min and 50 ℃ for 30min, cooling to 4 ℃ after the reaction is finished, stopping the reaction, and desalting at room temperature for 30min to obtain desalted reaction liquid.

By using an ExoCET method, recombining the surfactant synthetic gene cluster DNA obtained by enzyme digestion and a p15A-Cm-HA carrier fragment in a GB05-dir strain (engineering bacteria GB05-dir/pSC101-BAD-ETgA-tet) with a recombination system to obtain the engineering bacteria GB 05-dir/pWG-SRF; the method comprises the following specific steps:

40 mu L of engineering bacteria GB05-dir/pSC101-BAD-ETgA-tet (OD)₆₀₀3-4) was inoculated into 1.4mL of LB liquid medium. After shaking culture at 30 ℃ and 200rpm for 2h, the OD is waited₆₀₀Adding 35 mu L of 10% L-arabinose when the value reaches 0.35-0.4, and continuing culturing at 37 ℃ for 40min to OD₆₀₀0.7 to 0.8. Transferring the bacterial liquid to ice bath treatment at 2 deg.c to terminate the growth of thallus, and centrifuging at 8000rpm to collect thallus. With ice-cold 1mL ddH₂The cells were O-washed 2 times. Resuspended in 20. mu.L of ice-cold ddH₂O, 8. mu.L of the desalted reaction solution was added thereto and mixed. After the transformation by electric shock in an electric shock cup with the width of a liquid adding gap of 1mm, 1mL of LB liquid culture medium is added, and the mixture is subjected to oscillatory recovery culture at 37 ℃ and 200rpm for 1-2 h. After the completion of the recovery culture, centrifugation is carried out for 2min at 8000rpm, after partial supernatant is removed, the heavy suspension liquid is blown out and coated on an LB (Cm +) plate, and inversion culture is carried out overnight at 37 ℃. The next day, colony PCR preliminary identification was performed. The bacterial strain correctly identified by colony PCR is marked as GB 05-dir/pWG-SRF; further extracting a plasmid, marked as pWG-SRF, and carrying out enzyme digestion identification.

Example 2 enzyme cleavage identification

Plasmid pWG-SRF, size 34151bp, including surfactant synthesis gene cluster DNA fragment (size 32539bp) and p15A-Cm-HA (size 1784bp) with 80bp homology arm.

The DNA was cleaved with BglII, which has 8 cleavage sites at pWG-SRF, and EcoRI, respectively. The 8739bp, 6997bp, 5549bp, 5158bp, 2803bp, 2040bp, 1950bp and 915bp 8 bands are cut out. EcoRI has 7 cleavage sites at pWG-SRF. 7 bands of 12805bp, 8357bp, 3650bp, 3289bp, 3263bp, 1992bp and 795bp are cut out. After the system is prepared, the enzyme is cut for 30min at 37 ℃, and 10 mu L of the enzyme is detected by 1 percent agarose gel electrophoresis. As shown in FIGS. 2 and 3, the band 8739bp, 6997bp, 5549bp, 5158bp, 2803bp, 2040bp, 1950bp, 915bp 8 was excised from Bgl II; the 2040bp and 1950bp bands are not separated during electrophoresis. Cutting 7 bands of 12805bp, 8357bp, 3650bp, 3289bp, 3263bp, 1992bp and 795bp by EcoRI; the two bands of 3289bp and 3263bp are not separated during electrophoresis. Thus, the construction of the plasmid pWG-SRF is successful.

Enzyme digestion system:

example 3 engineering bacteria obtention

Extracting plasmid pWG-SRF, and electrically transferring plasmid pWG-SRF into E.coli BW25113 competent cells to obtain engineering bacteria BW 25113/pWG-SRF.

Example 4 fermentation extraction of surfactin

The fermentation medium is low-salt LB: 1% peptone; 0.5% yeast extract; 0.1% NaCl, pH 7.0. The seed solution was transferred to the fermentation medium (50mL/250mL) at 1% inoculum size, Cm was added at a final concentration of 15 μ g/mL, 30 ℃, 200rpm, 3 bottles were inoculated with each bacterium (engineering bacteria BW25113/pWG-SRF, positive control Bacillus siamensis JFL15, negative control BW25113/pBAD33) as three replicates, and the compound was co-fermented for 72h, wherein 1mL XAD 16 macroporous adsorbent resin (commercially available as usual) was added at 48h of fermentation. The resin-added 50mL of the bacterial solution was transferred to a 50mL centrifuge tube, centrifuged at 8000rpm for 15min, and the supernatant was discarded. Adding 30mL of methanol into the precipitate, carrying out ultrasonic resuspension, pouring into the original triangular flask, and shaking at 30 ℃ and 180rpm for 3 hours. The cells and resin in the flask were poured into a new 50mL centrifuge tube and centrifuged at 8000rpm for 15 min. The supernatant was filtered through filter paper into a 100mL conical flask, which was evaporated to dryness by a rotary evaporator, and the crude extract was dissolved in 1mL of methanol. 12000rpm, centrifugation for 10 min. Filtering the supernatant with filter head, transferring into centrifuge tube to obtain crude extract of fermentation liquid, and storing at-20 deg.C.

Example 5 surfactant detection method

Performing high performance liquid chromatography detection by using Shimadzu LC 2030 CN; the detector is an ultraviolet detector; chromatographic column ODS-SP (5.0 μm, 4.6X 250mm, GL Sciences); the ultraviolet detection wavelength is 205 nm; the column temperature is 30 ℃; the mobile phase was methanol/water (containing 0.1% trifluoroacetic acid) 90: 10; the flow rate is 1 mL/min; the amount of the sample was 10. mu.L.

The content of the surfactant in the fermentation liquor of the engineering bacteria BW25113/pWG-SRF is detected by using high performance liquid chromatography, and the result is shown in figure 4, the engineering bacteria BW25113/pWG-SRF can generate a peak consistent with a surfactant standard and a positive control B.siemensis JFL15 at 8.3min, and the negative control BW25113/pBAD33 has no peak. The engineering bacteria BW25113/pWG-SRF can produce the surfactant with higher purity. Solves the problem of difficult separation and purification caused by a plurality of structural analogs when the surface active element is produced by fermentation of bacillus.

Example 6 Activity test method of crude extract of fermentation broth of engineering bacteria

The oil discharge performance of the crude extract of the fermentation liquor is measured by adopting an oil discharge ring method, and the specific method comprises the following steps: taking 100 mu L of the crude extract of the fermentation liquid obtained in the example 4, naturally volatilizing, and redissolving in 100 mu L of ddH₂And O. Add 50mL ddH to a 9cm diameter Petri dish₂And O, dripping 200 mu L of Sudan III dyed olive oil on the water surface, after an oil layer is spread, slowly dripping 10 mu L of water-soluble crude extract of fermentation liquor into the center of the oil layer, and recording the result after an oil discharge ring is stable. The results are shown in FIG. 5, compared with positive control B.siemensis JFL15 and negative control BW25113/pBAD33, the crude extract of fermentation liquor of engineering bacteria BW25113/pWG-SRF can generate obvious oil drain circle. The engineering bacteria BW25113/pWG-SRF can produce the surfactant with higher purity.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> southern China university of agriculture

<120> Escherichia coli engineering bacterium for producing surfactin, construction method and application thereof

<160> 9

<170> SIPOSequenceListing 1.0

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<211> 1784

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> p15A-Cm-HA

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tcataaaatt tcattaattg tcattgtagc ataaacgccg aaaaaaagaa gaagcgaaat 60

cagtgtttcg cttcttctcc ggatccatgc gagagtaggg aactgccagg catcaaataa 120

aacgaaaggc tcagtcgaaa gactgggcct ttcgttttat ctgttgtttg tcggtgaacg 180

ctctcctgag taggacaaat ccgccgggag cggatttgaa cgttgcgaag caacggcccg 240

gagggtggcg ggcaggacgc ccgccataaa ctgccaggca tcaaattaag cagaaggcca 300

tcctgacgga tggccttttt ctagattacg ccccgccctg ccactcatcg cagtactgtt 360

gtaattcatt aagcattctg ccgacatgga agccatcaca gacggcatga tgaacctgaa 420

tcgccagcgg catcagcacc ttgtcgcctt gcgtataata tttgcccatg gtgaaaacgg 480

gggcgaagaa gttgtccata ttggccacgt ttaaatcaaa actggtgaaa ctcacccagg 540

gattggctga gacgaaaaac atattctcaa taaacccttt agggaaatag gccaggtttt 600

caccgtaaca cgccacatct tgcgaatata tgtgtagaaa ctgccggaaa tcgtcgtggt 660

attcactcca gagcgatgaa aacgtttcag tttgctcatg gaaaacggtg taacaagggt 720

gaacactatc ccatatcacc agctcaccgt ctttcattgc catacggaat tccggatgag 780

cattcatcag gcgggcaaga atgtgaataa aggccggata aaacttgtgc ttatttttct 840

ttacggtctt taaaaaggcc gtaatatcca gctgaacggt ctggttatag gtacattgag 900

caactgactg aaatgcctca aaatgttctt tacgatgcca ttgggatata tcaacggtgg 960

tatatccagt gatttttttc tccattttag cttccttagc tcctgaaaat ctcgataact 1020

caaaaaatac gcccggtagt gatcttattt cattatggtg aaagttggaa cctcttacgt 1080

gccgatcaac gtctcatttt cgcagtgaga gggccgcggc aaagccgttt ttccataggc 1140

tccgcccccc tgacaagcat cacgaaatct gacgctcaaa tcagtggtgg cgaaacccga 1200

caggactata aagataccag gcgtttcccc ctggcggctc cctcgtgcgc tctcctgttc 1260

ctgcctttcg gtttaccggt gtcattccgc tgttatggcc gcgtttgtct cattccacgc 1320

ctgacactca gttccgggta ggcagttcgc tccaagctgg actgtatgca cgaacccccc 1380

gttcagtccg accgctgcgc cttatccggt aactatcgtc ttgagtccaa cccggaaaga 1440

catgcaaaag caccactggc agcagccact ggtaattgat ttagaggagt tagtcttgaa 1500

gtcatgcgcc ggttaaggct aaactgaaag gacaagtttt ggtgactgcg ctcctccaag 1560

ccagttacct cggttcaaag agttggtagc tcagagaacc ttcgaaaaac cgccctgcaa 1620

ggcggttttt tcgttttcag agcaagagat tacgcgcaga ccaaaacgat ctcaagaaga 1680

tcatcttatt aatcagatgg taccggttca tttgtattaa gcctgaggac aataaatgcc 1740

ttcgtatttc tccttgcttc aaatgaaaca gctccgtcag cctc 1784

<210> 2

<211> 106

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> p15ACm F

<400> 2

tcataaaatt tcattaattg tcattgtagc ataaacgccg aaaaaaagaa gaagcgaaat 60

cagtgtttcg cttcttctcc ggatccatgc gagagtaggg aactgc 106

<210> 3

<211> 106

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> p15ACm R

<400> 3

gaggctgacg gagctgtttc atttgaagca aggagaaata cgaaggcatt tattgtcctc 60

aggcttaata caaatgaacc ggtaccatct gattaataag atgatc 106

<210> 4

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> p15A F

<400> 4

agtgagaggg ccgcggcaaa g 21

<210> 5

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> p15A R

<400> 5

ggtaccgagc tcgaattcgc 20

<210> 6

<211> 37

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Cm F

<400> 6

gccgcggccc tctcactgcg aaaatgagac gttgatc 37

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> Cm R

<400> 7

aagcttggct gttttggcgg 20

<210> 8

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> ccdB F

<400> 8

gcgaattcga gctcggtacc tttcggaatt aaggaggtaa 40

<210> 9

<211> 40

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> ccdB R

<400> 9

ccgccaaaac agccaagctt ttatattccc cagaacatca 40

Claims

1. a construction method of the Escherichia coli engineering bacteria producing surfactin, is characterized in that comprising the steps:

(1) Analyze the Bacillus siamese JFL15 genome and compare it with the NCBI database to obtain the surfactin synthesis gene cluster sequence;

(2) The genomic DNA of Bacillus siamese JFL15 was digested with KpnI/BamHI double enzyme to obtain surfactin synthesis gene cluster DNA;

(3) Using the method of ExoCET, the surfactin synthesis gene cluster DNA obtained by double enzyme digestion and p15A-Cm-HA were annealed by T4 DNA polymerase in vitro, and then transferred into GB05-dir strain with recombination system. Recombination to obtain engineering bacteria, which is recorded as GB05-dir/pWG-SRF; the sequence of described p15A-Cm-HA is shown in SEQ ID NO: 1;

(4) The plasmid pWG-SRF was extracted from GB05-dir/pWG-SRF, and the plasmid pWG-SRF was transformed into E. coli wild-type strain BW25113 to obtain engineering strain BW25113/pWG-SRF.

2. the construction method of the Escherichia coli engineering bacteria producing surfactin according to claim 1, is characterized in that:

In step (1), the genome of Bacillus siamese JFL15 was analyzed by antiSMASH.

3. the construction method of the Escherichia coli engineering bacteria producing surfactin according to claim 1, is characterized in that:

In step (2), the fragment size of the surfactin synthesis gene cluster DNA is 32539bp.

4. the construction method of the Escherichia coli engineering bacteria producing surfactin according to claim 1, is characterized in that:

In step (3), the GB05-dir strain with the recombination system is the GB05-dir strain with pSC101-BAD-ETgA-tet, which is denoted as GB05-dir/pSC101-BAD-ETgA-tet.

5. the construction method of the Escherichia coli engineering bacteria producing surfactin according to claim 1, is characterized in that:

In step (3), the construction steps of p15A-Cm-HA are as follows:

From Bacillus siamese JFL15 genome restriction sites KpnI and BamHI to the interior of the gene cluster with a number of 80bp as homology arms, the primer pair p15ACm F/p15ACm R was designed, and pBAD-p15A-Cm-ccdB was used as the template to amplify with 80bp The fragment of the homology arm, denoted as p15A-Cm-HA;

p15ACm F: 5'-tcataaaatttcattaattgtcattgtagcataaacgccgaaaaaaagaagaagcgaaatcagtgtttcgcttcttctcc ggatcc ATGCGAGAGTAGGGAACTGC-3'; the underlined part is the BamHI restriction site;

p15ACm R: 5'-gaggctgacggagctgtttcatttgaagcaaggagaaatacgaaggcatttattgtcctcaggcttaatacaaatgaacc ggtacc ATCTGATTAATAAGATGATC-3'; the underlined part is the KpnI restriction site;

Among them, the lowercase part is the homology arm;

The construction steps of pBAD-p15A-Cm-ccdB are as follows:

Using the vector pBAD33 as a template, p15A F/p15A R and Cm F/Cm R as primers, amplify pBAD-p15A with a size of 2070 bp and Cm with a size of 1215 bp; with plasmid pMD-ccdBKanS as a template, ccdB F/ccdB R is Primers to amplify the ccdB gene fragment with a size of 371 bp, and use TEDA one-step multi-fragment assembly to obtain the pBAD-p15A-Cm-ccdB plasmid;

p15A F:5'-AGTGAGAGGGCCGCCGGCAAAG-3';

p15A R: 5'-GGTACCGAGCTCGAATTCGC-3';

Cm F: 5'-gccgcggccctctcactGCGAAAATGAGACGTTGATC-3';

CmR: 5'-AAGCTTGGCTGTTTTGGCGG-3';

ccdB F: 5'-gcgaattcgagctcggtaccTTTCGGAATTAAGGAGGTAA-3';

ccdB R: 5'-ccgccaaaacagccaagcttTTATATTCCCCAGAACATCA-3';

Among them, the lowercase part is the homology arm.

6 . A surfactin-producing Escherichia coli engineering bacterium, characterized in that it is constructed by the construction method described in any one of claims 1 to 5 .

7. A recombinant expression vector pWG-SRF, characterized in that: the pWG-SRF is the pWG-SRF described in any one of the construction methods of claims 1-5.

8. The application of the surfactin-producing Escherichia coli engineering bacteria of claim 6 in the fermentation production of surfactin.

9. the application of the fermentation broth crude extract of the surfactin-producing Escherichia coli engineering bacteria of claim 6 in petroleum extraction, daily chemical industry, antibacterial and antiviral preparations.

10. The application according to claim 9, wherein:

The crude extract of the fermentation broth is obtained by fermenting the Escherichia coli engineering bacteria described in claim 6 and extracting active substances.