CN111849846A - Gene modification method for high-yield monorhamnolipid and application thereof - Google Patents
Gene modification method for high-yield monorhamnolipid and application thereof Download PDFInfo
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Abstract
The invention discloses a gene modification method for high-yield monorhamnolipid, which is characterized by comprising the following steps of: according to Pseudomonas aeruginosarhlCDesigning PCR primer with gene sequence, purifying PCR product, cloning to pMD series T simple vector to construct pMD-rhlC,enzyme digestionrhlCInside the gene sequence, constructing a plasmid pMD-rhanlC with the deleted internal sequence of the target gene, inserting the rhalC fragment into the plasmid pK18mobSacB by double enzyme digestion to construct a targeting plasmid pK 18-rhalC, introducing into pseudomonas aeruginosa, performing secondary homologous recombination under the action of screening pressure, and obtaining the knockoutrhlCAnd (3) knocking out a pseudomonas aeruginosa strain of the gene, and fermenting and verifying the generated single rhamnolipid by the knocked-out strain. The invention has application value in the aspects of fermentation production, development and utilization of rhamnolipid.
Description
Technical Field
The invention relates to the technical field of biological engineering and the field of rhamnolipid production and application, in particular to a genetic modification method for high-yield single rhamnolipid of pseudomonas aeruginosa, which solves the problem that the existing method expresses the single rhamnolipid by heterologous expressionrhlABGeneThe method has the technical problems of low yield of the synthetic monorhamnolipid, complicated operation of extracting and separating the monorhamnolipid from the conventional rhamnolipid product and the like, and lays a foundation for the application and research of the monorhamnolipid.
Background
Rhamnolipid is a biosurfactant which is mainly metabolized and synthesized by pseudomonas aeruginosa, and is widely applied to the fields of oil exploitation, environmental pollution remediation, medicines, foods, agricultural biocontrol control, composting and the like due to the advantages of high surface/interface activity, safety, no toxicity, low critical micelle concentration, biodegradability, high stability and the like. Rhamnolipids are a series of homologues, consisting of 1 to 2 rhamnosyl groups and 1 to 2 saturated or unsaturated fatty acid chains of different carbon chain lengths. Nearly 60 rhamnolipid molecules with different structures are reported to be found.
According to the number of rhamnosyl groups in rhamnolipid molecules, the rhamnolipid is divided into mono-rhamnolipid and di-rhamnolipid. The physical and chemical activity of the rhamnolipid is necessarily influenced by the number of rhamnosyl groups contained in the rhamnolipid, namely the single rhamnolipid and the double rhamnolipid have different activity and different application potentials. Research shows that the monorhamnolipid has better oleophylic property and emulsifying activity compared with the dirhamnolipid. The rhamnolipid has great application advantages in the aspects of improving the bioavailability of hydrophobic organic pollutants, emulsifying, carrying, oil displacement, petroleum emulsifying, viscosity reduction and the like. Therefore, the production and preparation of the monorhamnolipid have important application values.
The prior art method is to synthesize the coding gene of rhamnolipid synthetase I (RhLAB) of monorhamnolipid in pseudomonas aeruginosarhlABHeterologous expression in hosts such as escherichia coli can realize the synthesis of the monorhamnolipid, but the yield of the monorhamnolipid is low, namely about hundreds of milligrams per liter. The extraction and separation of the monorhamnolipid from the rhamnolipid product of wild pseudomonas aeruginosa is complicated and time-consuming and labor-consuming. If the coding gene of rhamnolipid synthetase II (RhlC) catalyzing the synthesis of dual rhamnolipids from single rhamnolipid in pseudomonas aeruginosa is knocked outrhlCThe rhamnolipid synthesis pathway would be stopped at monorhamnolipids, which would be able to solve the above bottleneck problem. Book (I)Invention to knock outrhlCThe gene pseudomonas aeruginosa is a production bacterium, so that the synthetic rhamnolipid is completely the single rhamnolipid, the high yield of the single rhamnolipid is realized, and a foundation is laid for the application and research of the single rhamnolipid.
Disclosure of Invention
The invention aims to provide a genetic modification method for high yield of monorhamnolipid of pseudomonas aeruginosa, which is beneficial to solving the problem that the existing method expresses monorhamnolipid by heterologous expressionrhlABThe gene synthesis of the monorhamnolipid has low yield, and the extraction and separation of the monorhamnolipid from the conventional rhamnolipid product have the technical problems of complicated operation and the like, thereby laying a foundation for the application and research of the monorhamnolipid. The method can be used for directionally and efficiently constructing the pseudomonas aeruginosa engineering bacteria with high yield of the single rhamnolipid.
In order to achieve the purpose, the invention adopts the technical scheme that:
a genetic modification method for high yield of rhamnolipid is characterized in that pseudomonas aeruginosa is used as an original strain, and a coding gene of rhamnolipid synthetase II (RhlC) for catalyzing single rhamnolipid to synthesize dual rhamnolipidrhlCTo a retrofit target; the method comprises the following main steps: 1) according to Pseudomonas aeruginosarhlCDesigning a PCR primer by using a gene sequence; 2) using pseudomonas aeruginosa genome DNA as a template for PCR amplificationrhlCCloning the gene into pMD series carrier to construct recombinant plasmid pMD-rhlC; 3) according torhlCUnique cleavage site within the gene sequence, cleavagerhlCPurification and self-ligation of the interior of the gene sequencerhlCNew plasmid pMD-rhlC with deletion of internal gene sequence; 4) inserting the Δ rhlC fragment into the plasmid pK18mobSacB by a double-enzyme cutting method to construct a targeting plasmid pK18- Δ rhlC; 5) introducing the plasmid pK18- Δ rhlC into Pseudomonas aeruginosa, and performing secondary homologous recombination of the plasmid pK 18-rhlC and the chromosome gene of the Pseudomonas aeruginosa under the action of screening pressure to obtain knockoutrhlCA genetic pseudomonas aeruginosa knock-out strain; 6) and (3) performing fermentation production verification on the pseudomonas aeruginosa knockout strain, verifying a single rhamnolipid product by using an HPLC-MS method, and determining the yield of the single rhamnolipid by using an oil-discharge ring method.
Step (ii) of1) Designed for the pseudomonas aeruginosarhlCThe PCR primers of the gene are C-f and C-r, and the sequences of the primers are as follows
C-f：5'-CCGAAGCTTATGAGCGGCCTGTTCCACT-3'，SEQ ID NO:1
C-r: 5'-CTTGGAATTCCCGGAAGCTACGGACG-3' SEQ ID NO:2
The upstream and downstream primers are respectively introduced inrhlCRestriction sites (underlined in the primers), Hind III and EcoRI which are not present in the gene sequence and are contained in the Multiple Cloning Site (MCS) of pK18 mobSacB.
Step 2) PCR amplificationrhlCReaction system of the gene: 2.5 μ L of 10 XPCR Buffer, 2 μ L of 2.5mM dNTPMixture, 0.8 μ L of each of C-f and C-r primers with the size of 10 μ M, 0.125 μ L of Extaq Polymerase, 1 μ L of pseudomonas aeruginosa genomic DNA, and supplementing PCR water to 25 μ L of the total system; reaction conditions are as follows: pre-denaturation at 95 ℃ for 3min, 35 cycles: denaturation at 94 deg.C for 1min, annealing at 56 deg.C for 30s, extension at 72 deg.C for 1min30s, and extension at 72 deg.C for 10 min; after the PCR product is subjected to 1.2 percent agarose electrophoresis and gel cutting and purification by a kit,rhlCthe gene fragment is connected with a pMD series (18/19/20) T simple vector by AT to construct a recombinant plasmid pMD-rhlC.
Step 3) digesting plasmid pMD-rhlC by using restriction enzyme SalI kitrhlCThe internal sequence of the gene, the enzyme digestion product is subjected to 1.2 percent agarose electrophoresis and kit gel cutting purification, and then self-ligation is carried out to obtain the generhlCThe new plasmid pMD- Δ rhlC with 400-500 bp of sequence deleted inside the gene.
And step 4) utilizing restriction enzymes Hind III and EcoRI kit, carrying out double enzyme digestion on the plasmid pMD-rhlC and the suicide plasmid pK18mobSacB of pseudomonas aeruginosa, carrying out 1.2% agarose electrophoresis on double enzyme digestion products of the plasmid pK18mobSacB, carrying out gel cutting purification by the kit, and connecting to construct a targeting plasmid pK 18-rhlC.
Step 5) transforming the plasmid pK18- Δ rhlC into competent cells of E.coli S17-1 to construct recombinant E.coli S17-1 (pK18- Δ rhlC), and introducing the plasmid pK18- Δ rhlC into Pseudomonas aeruginosa by a conjugative transfer method; plasmid pK18- Δ rhlC and Pseudomonas aeruginosa chromosome under the screening pressure of ampicillin, kanamycin and sucroserhlCThe homologous sequence of the gene is subjected to secondary homologous recombination, and the rhlC gene fragment with the deletion of the internal sequence replaces the chromosomerhlCGene, screening to obtainrhlCA gene disrupted pseudomonas aeruginosa knock-out strain.
Step 6), fermenting the pseudomonas aeruginosa knockout strain for 4-10 days by shaking a flask; collecting 0.2-1L of the supernatant of the aseptic fermentation, extracting a rhamnolipid product in the fermentation liquor by using 0.6-3L of ethyl acetate, dissolving the extract in 10% acetonitrile water, wherein the concentration of the rhamnolipid is 300-1000 mg/L, and filtering a sample by using a 0.45-micron organic filter membrane; relevant parameters for HPLC-MS: the sample feeding amount is 5 mu L, the linear gradient elution is carried out on acetonitrile water solution (10% acetonitrile is added in water for 1min, the volume fraction of acetonitrile is increased from 10% to 60% within 30min, the volume fraction of acetonitrile is decreased from 60% to 10% within 5 min), the flow rate is 0.6mL/min, the effluent enters a mass spectrum system after being split by 1:1, the ion source temperature is 120 ℃, the detection is carried out in a negative ion mode, and the mass spectrum scanning range is 50-1000 m/z. And (3) dripping 10-100 times diluted fermentation liquid of 10 mu L on an oil film, and calculating the yield of the monorhamnolipid according to the linear relation between the diameter of the oil discharge ring and the concentration of the rhamnolipid and the dilution times of the fermentation liquid.
The fermentation medium formula comprises 30-60 g of glycerol and NaNO33-6 g, 0.5-1.5 g of yeast powder and KH2PO43~5g，K2HPO43~5g， MgSO40.4-1.0 g of sodium chloride and 0.5-2 g of deionized water 1L. The culture temperature is 30-40 ℃, and the rotation speed is 160-220 rpm.
The invention further discloses application of the genetic modification method for high yield of the rhamnolipid in the aspects of genetic modification of pseudomonas aeruginosa and production, development and utilization of the rhamnolipid, and experimental results show that: the method can directionally transform the pseudomonas aeruginosa strain, has high transformation efficiency and clear target; the rhamnolipid products synthesized by the pseudomonas aeruginosa knockout strain modified by the method are all single rhamnolipid and have high yield.
The invention mainly solves the problem of heterologous expression of pseudomonas aeruginosa by the existing methodrhlABThe yield of the gene synthesized single rhamnolipid is very low, and the operation of extracting and separating the single rhamnolipid from the conventional rhamnolipid product is complicated, time-consuming and labor-consuming and the like. Book (I)The invention takes pseudomonas aeruginosa as an original strain, and takes the coding gene of rhamnolipid synthetase II (RhlC) for catalyzing the synthesis of dirhamnolipid from monorhamnolipidrhlCIn order to achieve the aim of modification, the pseudomonas aeruginosa knockout strain with rhamnolipid products all being single rhamnolipid is obtained, and the main difficulty is that the pseudomonas aeruginosa knockout strain contains rhamnolipid rhlCConstructing the gene upstream and downstream homologous sequence targeting plasmid.
The invention discloses a gene modification method for high-yield monorhamnolipid, which has the beneficial effects that:
the pseudomonas aeruginosa is transformed by the method, the target property is strong, the transformation efficiency is high, and the stability of engineering bacteria is good; the pseudomonas aeruginosa knockout strain constructed by the method disclosed by the invention can ensure that all the synthesized rhamnolipid products are the mono-rhamnolipid while the rhamnolipid yield is not influenced, so that the high yield of the mono-rhamnolipid is realized, and a method foundation is laid for the research and application of the mono-rhamnolipid.
Drawings
FIG. 1 shows the HPLC-MS analysis results of rhamnolipid product of Pseudomonas aeruginosa knockout strain in example 2;
the chromatographic peak information of rhamnolipid product appearing in the figure is shown in the following table:
FIG. 2 shows the yield of monorhamnolipid of different genetically engineered bacteria in example 3.
Detailed Description
The present invention is described in detail with reference to the following specific examples to provide a more complete understanding of the present invention to those of ordinary skill in the art, but the present invention is not limited thereto in any way. In the following examples, materials and reagents used were all available from Biochemical materials, Inc., unless otherwise specified.
Example 1
Used in the present example to constructrhlCThe culture medium of the gene-knocked-out pseudomonas aeruginosa engineering bacteria is as follows:
liquid LB medium: 10g of peptone, 5g of yeast powder, 10g of NaCl, 1L of deionized water and pH 7.0.
LB agar medium: 10g of peptone, 5g of yeast powder, 10g of NaCl, 18 g of agar, 1L of deionized water and pH 7.0.
1) Of Pseudomonas aeruginosarhlCPCR primer design for genes
Download of Pseudomonas aeruginosa from NCBI databaserhlCGene sequence, Genbank sequence numbers are respectively: AE004091.2, CP000438.1, CP 002496.1. Pseudomonas aeruginosa according to downloadrhlCGene sequence, designed using Primer Premier 5 softwarerhlCPCR primer of gene, designed pseudomonas aeruginosarhlCThe PCR primer sequence of the gene is as follows: 5' -CCGAAGCTTATGAGCGGCCTGTTCCACT-3'，SEQ ID NO:1
C-r: 5'-CTTGGAATTCCCGGAAGCTACGGACG-3'， SEQ ID NO:2
The upstream and downstream primers are respectively introduced inrhlCRestriction sites (underlined in the primers), Hind III and EcoRI which are not present in the gene sequence and are contained in the Multiple Cloning Site (MCS) of pK18 mobSacB.
2) PCR amplificationrhlCGene
10mL of LB liquid culture medium is taken to culture the rhamnolipid high-producing strain pseudomonas aeruginosa SG at 37 ℃ and 180rpm, after 12h of culture, 2mL of culture solution is taken to centrifuge (10,000 rpm, 2 min) to collect thalli, and the bacterial genome extraction kit is used for extracting the genome DNA of the pseudomonas aeruginosa SG. Taking the genome DNA of pseudomonas aeruginosa SG as a template, and carrying out PCR amplification rhlCA gene. Reaction system: 2.5 μ L of 10 XPCR Buffer, 2 μ L of 2.5mM dNTP mix, 0.8 μ L of each of C-f and C-r primers of 10 μ M, 0.125 μ L of Extaq Polymerase, 1 μ L of Pseudomonas aeruginosa SG genomic DNA, and supplementing PCR water to 25 μ L of the total system; reaction conditions are as follows: pre-denaturation at 95 ℃ for 3min, 35 cycles: denaturation at 94 deg.C for 1min, annealing at 56 deg.C for 30s, extension at 72 deg.C for 1min30s, and extension at 72 deg.C for 10 min.
3) Construction of recombinant plasmid pMD-rhlC
After the PCR product is subjected to 1.2% agarose electrophoresis, gel cutting and purification are carried out by using a gel purification kitrhlCA gene fragment; after PCR amplification with Extaq polymeraserhlCGene fragmentThe terminal A base is connected with the T base AT the terminal of the pMD19-T simple vector by AT; the connecting liquid is heat-shocked for 60s at 42 ℃ to transform escherichia coli DH5 alpha competent cells, the transformation liquid is coated on an LB agar medium plate containing 100mg/L ampicillin and cultured for 18h at 37 ℃; the positive transformant was selected and inoculated into 10mL of LB liquid medium containing 100mg/L ampicillin, cultured at 37 ℃ and 180rpm for 12 hours, 4mL of the culture was centrifuged (10,000 rpm, 2 min) to collect the cells, and the recombinant plasmid pMD-rhlC was extracted using a plasmid extraction kit.
4) Construction ofrhlCNew plasmid pMD-rhlC with deletion of internal sequence of gene
By sequence analysis rhlC2 SalI restriction sites exist in the gene, and the plasmid pMD-rhlC is restricted by restriction enzyme SalI kit, so thatrhlCCutting 493bp sequence inside gene, subjecting the cut pMD-rhlC fragment to 1.2% agarose electrophoresis, cutting gel with gel purification kit to purify pMD-rhlC cut fragment, and cutting T4The pMD-rhlC enzyme digestion fragment is self-connected by DNA ligase to constructrhlCThe new plasmid pMD- Δ rhlC with 493bp sequence deleted inside the gene; the connecting liquid is heat-shocked for 60s at 42 ℃ to transform escherichia coli DH5 alpha competent cells, the transformation liquid is coated on an LB agar medium plate containing 100mg/L ampicillin and cultured for 18h at 37 ℃; the positive transformants were selected and inoculated into 10mL LB liquid medium containing 100mg/L ampicillin, cultured at 37 ℃ for 12h at 180rpm, 4mL of the culture was centrifuged (10,000 rpm, 2 min) to collect the cells, and the recombinant plasmid pMD- Δ rhlC was extracted using the plasmid extraction kit.
5) Construction of targeting plasmid pK18- Δ rhlC
Using restriction enzymes HindIII and EcoRI kit, double restriction enzyme plasmid pMD-rhanC and suicide plasmid pK18mobSacB of pseudomonas aeruginosa, double restriction enzyme products of rhanC fragment and pK18mobSacB plasmid are electrophoresed by 1.2% agarose, gel-cutting and purifying by using gel purification kit, using T-restriction enzyme 4Connecting the purified fragment with DNA ligase to construct a targeting plasmid pK18- Δ rhlC; the connecting liquid is heat-shocked for 60s at 42 ℃ to transform escherichia coli DH5 alpha competent cells, the transformation liquid is coated on an LB agar medium plate containing 100mg/L ampicillin and cultured for 18h at 37 ℃; picking positive transformant to inoculateCulturing in 10mL LB liquid medium containing 50mg/L kanamycin at 37 deg.C and 180rpm for 12h, centrifuging 4mL culture solution (10,000 rpm, 2 min), collecting thallus, and extracting recombinant plasmid pK18- Δ rhlC with plasmid extraction kit.
6) Introducing plasmid pK18- Δ rhlC into Pseudomonas aeruginosa, and screening and knocking outrhlCGenetic pseudomonas aeruginosa knock-out strains
The plasmid pK18- Δ rhlC is transformed into E.coli S17-1 competent cells by heat shock 60S at 42 ℃, the transformation fluid is coated on an LB agar medium plate containing 50mg/L kanamycin, the culture is carried out for 18h at 37 ℃, and the recombinant E.coli S17-1(pK18- Δ rhlC) is screened; mixing the recombinant Escherichia coli S17-1(pK18- Δ rhlC) and the Pseudomonas aeruginosa suspension in a ratio of 1:2, transferring the mixed solution into an LB agar culture medium, culturing at 37 ℃ for 12h, and introducing the plasmid pK18- Δ rhlC into the Pseudomonas aeruginosa by a conjugative transfer method; scraping lawn on the plate into 1mL LB liquid culture medium, diluting 5 times, taking 150 μ L of the diluted solution, coating double-resistant LB agar plate containing 100mg/L ampicillin and 350mg/L kanamycin, and culturing at 37 ℃ for 24 h; 10 positive clones were picked and used rhlCCarrying out colony PCR verification on the gene primers, and screening to obtain a single-exchanger strain, wherein the reaction system and the reaction conditions are the same as those in the ' 2 ' of the embodiment '; preparation of Single-exchanger bacterial suspension dilution 106Taking 150 mu L of the diluent, coating an LB agar plate containing 100mg/L ampicillin and 50g/L sucrose, culturing for 24h at 37 ℃, and inducing to generate secondary recombination by utilizing the screening pressure of sucrose SacB gene on plasmid pK18- Δ rhlC; 10 positive clones were picked and usedrhlCThe gene primer is used for carrying out colony PCR verification, screening and knocking offrhlCThe pseudomonas aeruginosa knockout strain SG Δ rhlC of the gene. The results fully show that the invention is used for transforming pseudomonas aeruginosarhlCHigh efficiency and feasibility of the genetic approach.
Example 2
The pseudomonas aeruginosa knock-out strain SG Δ rhlC in the embodiment 1 of the invention is used as a fermentation strain, and the culture medium used in the embodiment is as follows:
liquid LB medium: 10g of peptone, 5g of yeast powder, 10g of NaCl, 1L of deionized water and pH 7.0.
Fermentation medium: glycerol 45g, NaNO33g, 1g of yeast powder and KH2PO43g，K2HPO43 g，MgSO40.5 g, 1g of sodium chloride, 1L of deionized water and pH 6.8.
Inoculating pseudomonas aeruginosa knock-out strain SG Δ rhlC to 10mL of LB liquid culture medium, culturing at 37 ℃ and 180rpm for 12h to prepare a seed solution, inoculating 1mL of the seed solution to a triangular flask containing 120mL of fermentation medium, setting 5 parallel experiments, and culturing at 37 ℃ and 180rpm for 5 d.
Collecting 600mL fermentation liquid, centrifuging at 10,000rpm for 10min, removing thallus cells, and adjusting the pH of the supernatant to 2.0 by using 2mol/L HCl solution; extracting the supernatant with equal volume (600 mL) of ethyl acetate for 3 times, collecting the organic phase, and placing in a vacuum rotary evaporator (50 ℃,60 rpm) to obtain a light yellow viscous substance, namely the rhamnolipid extract in the fermentation broth.
The rhamnolipid extract is dissolved in 10% acetonitrile water, the concentration of rhamnolipid is 500mg/L, and the sample is filtered by a 0.45 mu m organic filter membrane and then subjected to HPLC-MS analysis. Relevant parameters for HPLC-MS: the sample feeding amount is 5 mu L, the linear gradient elution is carried out on acetonitrile water solution (10% acetonitrile is added in water for 1min, the volume fraction of acetonitrile is increased from 10% to 60% within 30min, the volume fraction of acetonitrile is decreased from 60% to 10% within 5 min), the flow rate is 0.6mL/min, the effluent enters a mass spectrum system after being split by 1:1, the ion source temperature is 120 ℃, the detection is carried out in a negative ion mode, and the mass spectrum scanning range is 50-1000 m/z. The results of HPLC-MS analysis are shown in FIG. 1.
The results of example 2 (FIG. 1) show that the rhamnolipid products produced by fermentation of Pseudomonas aeruginosa knock-out strain SG rhlC are all monorhamnolipids. The results fully show the reliability of the gene modification method for producing the monorhamnolipid by the pseudomonas aeruginosa.
Example 3
Comparing the yields of monorhamnolipid of different genetically engineered strains, the media used in this example are as follows:
liquid LB medium: 10g of peptone, 5g of yeast powder, 10g of NaCl, 1L of deionized water and pH 7.0.
Fermentation medium: glycerol 45g, NaNO33g, 1g of yeast powder and KH2PO43g，K2HPO43 g，MgSO40.5 g, 1g of sodium chloride, 1L of deionized water and pH 6.8.
Taking 3 genetic engineering bacteria for synthesizing the monorhamnolipid, glycerol tube strains of escherichia coli DH5 alpha (pBBR1-2rhl), pseudomonas stutzeri DNB (pBBR1-2rhl) and pseudomonas aeruginosa SG Δ rhlC, respectively inoculating into 10mL LB liquid culture medium, culturing at 37 ℃ and 180rpm for 12h to prepare seed liquid, respectively inoculating 1mL of the seed liquid into a triangular flask containing 120mL fermentation culture medium, setting 3 parallel fermentation experiments of each strain, and culturing at 37 ℃ and 180rpm for 5 d. Respectively taking 2mL of fermentation liquor from each triangular flask, centrifuging at 10,000rpm for 5min, and removing thallus cells; the supernatant of the fermentation broth of Pseudomonas aeruginosa (flam) rhalc was diluted 32 times, and the supernatant of the fermentation broth of Escherichia coli DH5 alpha (pBBR1-2rhl) and Pseudomonas stutzeri DNB (pBBR1-2rhl) was not diluted.
Placing a culture dish with the diameter of 90mm on millimeter coordinate paper, adding 30ml of distilled water, and dripping 20 mu l of crude oil with the viscosity of 20 mPa ∙ s at the center of the water surface in the culture dish to form an oil film on the water surface; and (3) dripping 10 mu l of the fermentation broth supernatant sample solution to the center of an oil film, and recording the diameter of an oil discharge ring. According to the linear relation of the oil discharge ring diameter (y, mm) and the concentration (x, mg/L) of the monorhamnolipid (y =0.0632x +4.6484, R) 2= 0.9939) and the dilution factor of the fermentation liquid, the result is shown in figure 2, the yield of the rhamnolipid of the pseudomonas aeruginosa SG Δ RhlC transformed by the method reaches 15g/L, which is far more than that of the pseudomonas aeruginosarhlAB2 genetically engineered bacteria with heterogeneously expressed genes.
The results of example 3 (see FIG. 2) show that the modification by the method of the inventionrhlCThe gene pseudomonas aeruginosa SG Δ rhlC can produce single rhamnolipid with high yield, effectively solving the problem of heterologous expression of pseudomonas aeruginosa by the existing methodrhlABThe gene synthesis technology has low yield of the monorhamnolipid. The results of example 3 demonstrate the reliability and utility of the present invention as a genetic modification method for high yield of monorhamnolipid from pseudomonas aeruginosa.
SEQUENCE LISTING
<110> university of Qufu Master
<120> gene modification method for high-yield monorhamnolipid and application thereof
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<170>PatentIn version 3.5
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<212>DNA
<213> Artificial sequence
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ccgaagctta tgagcggcct gttccact 28
<210>2
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cttggaattc ccggaagcta cggacg 26
Claims (7)
1. A genetic modification method for high yield of monorhamnolipid is characterized in that pseudomonas aeruginosa is used as an original strain, and a coding gene of rhamnolipid synthetase II (RhlC) for catalyzing monorhamnolipid to synthesize dirhamnolipid is usedrhlCThe method is carried out for the reconstruction target according to the following steps:
1) according to Pseudomonas aeruginosa rhlCDesigning a PCR primer by using a gene sequence; designed for the pseudomonas aeruginosarhlCThe PCR primers of the gene are C-f: 5' -CCGAAGCTTATGAGCGGCCTGTTCCACT-3'，C-r: 5'-CTTGGAATTCCCGGAAGCTACGGACG-3', respectively introduced into the upstream and downstream primersrhlCRestriction sites (underlined in the primers), Hind III and EcoRI which are not present in the gene sequence and are contained in the Multiple Cloning Site (MCS) of pK18 mobSacB;
2) using pseudomonas aeruginosa genome DNA as a template for PCR amplificationrhlCCloning the gene into pMD series carrier to construct recombinant plasmid pMD-rhlC;
3) according torhlCUnique cleavage site within the gene sequence, cleavagerhlCPurification and self-ligation of the interior of the gene sequencerhlCNew plasmid pMD-rhlC with deletion of internal gene sequence;
4) inserting the Δ rhlC fragment into the plasmid pK18mobSacB by a double-enzyme cutting method to construct a targeting plasmid pK18- Δ rhlC;
5) introducing the plasmid pK18- Δ rhlC into Pseudomonas aeruginosa, and performing secondary homologous recombination of the plasmid pK 18-rhlC and the chromosome gene of the Pseudomonas aeruginosa under the action of screening pressure to obtain knockoutrhlCA genetic pseudomonas aeruginosa knock-out strain;
6) and (3) performing fermentation production verification on the pseudomonas aeruginosa knockout strain, verifying a single rhamnolipid product by using an HPLC-MS method, and determining the yield of the single rhamnolipid by using an oil-discharge ring method.
2. The method of claim 1, wherein step 2): PCR amplificationrhlCReaction system of the gene: 10 XPCR buffer2.5 uL, 2.5mM dNTP mix 2 uL, 10 Mm C-f and C-r primers each 0.8 uL, Extaq polymerase0.125 uL, Pseudomonas aeruginosa genomic DNA 1 uL, make up PCR water to total system 25 uL; reaction conditions are as follows: pre-denaturation at 95 ℃ for 3min, 35 cycles: denaturation at 94 deg.C for 1min, annealing at 56 deg.C for 30s, extension at 72 deg.C for 1min30s, and extension at 72 deg.C for 10 min; after the PCR product is subjected to 1.2 percent agarose electrophoresis and gel cutting and purification by a kit,rhlCthe gene fragment is connected with a pMD series (18/19/20) T simple vector by AT to construct a recombinant plasmid pMD-rhlC.
3. The method of claim 1, wherein step 3): the plasmid pMD-rhlC is digested by using a restriction enzyme Sal I kitrhlCThe internal sequence of the gene, the enzyme digestion product is subjected to 1.2 percent agarose electrophoresis and kit gel cutting purification, and then self-ligation is carried out to obtain the generhlCThe new plasmid pMD- Δ rhlC with 400-500 bp of sequence deleted inside the gene.
4. The method of claim 1, wherein step 4): using restriction endonuclease HindIII and EcoRI kit, double restriction enzyme plasmid pMD- Δ rhlC and suicide plasmid pK18mobSacB of Pseudomonas aeruginosa, the double restriction enzyme products of the rhlC fragment and pK18mobSacB plasmid are subjected to 1.2% agarose electrophoresis, the kit is subjected to gel cutting and purification, and then are connected to construct targeting plasmid pK18- Δ rhlC.
5. The method of claim 1, wherein step 5): the plasmid pK18- Δ rhlC is transformed into competent cells of Escherichia coli S17-1 to construct recombinant Escherichia coli S17-1 (pK18- Δ rhlC), and the plasmid pK18- Δ rhlC is introduced into Pseudomonas aeruginosa by conjugative transfer method; plasmid pK18- Δ rhlC and Pseudomonas aeruginosa chromosome under the screening pressure of ampicillin, kanamycin and sucroserhlCThe homologous sequence of the gene is subjected to secondary homologous recombination, and the rhlC gene fragment with the deletion of the internal sequence replaces the chromosomerhlCGene, screening to obtainrhlCA gene disrupted pseudomonas aeruginosa knock-out strain.
6. The method of claim 1, wherein step 6): fermenting the pseudomonas aeruginosa knockout strain for 4-10 days by using a shake flask; collecting 0.2-1L of the supernatant of the aseptic fermentation, extracting a rhamnolipid product in the fermentation liquor by using 0.6-3L of ethyl acetate, dissolving the extract in 10% acetonitrile water, wherein the concentration of the rhamnolipid is 300-1000 mg/L, and filtering a sample by using a 0.45-micron organic filter membrane; relevant parameters for HPLC-MS: the sample feeding amount is 5 mu L, acetonitrile water solution is subjected to linear gradient elution (10% acetonitrile is added in water for 1min, the volume fraction of acetonitrile is increased from 10% to 60% within 30min, the volume fraction of acetonitrile is decreased from 60% to 10% within 5 min), the flow rate is 0.6mL/min, the effluent enters a mass spectrum system after being split by 1:1, the ion source temperature is 120 ℃, the detection is carried out in a negative ion mode, and the scanning range of mass spectrum is 50-1000 m/z; and (3) dripping 10-100 times diluted fermentation liquid of 10 mu L on an oil film, and calculating the yield of the monorhamnolipid according to the linear relation between the diameter of the oil discharge ring and the concentration of the rhamnolipid and the dilution times of the fermentation liquid.
7. The use of the genetic modification method for producing high-yield rhamnolipid as claimed in claim 1 in the genetic modification of pseudomonas aeruginosa and the production, development and utilization of rhamnolipid.
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Application publication date: 20201030 |
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