Genetically engineered bacterium with high capric acid tolerance as well as construction method and application of genetically engineered bacterium

Abstract

The invention relates to a genetically engineered bacterium with high capric acid tolerance as well as a construction method and application of the genetically engineered bacterium. The genetically engineered bacterium takes escherichia coli as a starting strain, an aaS gene is knocked out, and meanwhile, a mla Z gene and a mla Y gene are overexpressed; wherein the Gene ID (Identity) of the nucleotide sequence of the aaS gene is 947315; the Gene ID (Identity) of the nucleotide sequence of the mla Z gene is 882402; and the Gene ID (Identity) of the nucleotide sequence of the mla Y gene is 882401. The aaS gene in the escherichia coli is knocked out, and the mla Z and mla Y genes are overexpressed at the same time, so that the membrane lipid strength of the escherichia coli engineering bacteria is effectively enhanced, and the genetically engineered bacteria show better robustness and higher tolerance in the fermentation process of a high-concentration substrate capric acid. When the capric acid is fed to the maximum concentration of 3.0 g / L, the yield of the trans-2-decenoic acid can reach 1.47 g / L, the conversion rate is 49.13%, and the yield is remarkably improved.

Description

Technical Field

[0001] The invention relates to a genetically engineered bacterium with high decanoic acid tolerance and a construction method and application thereof, belonging to the technical field of microorganisms. Background Art

[0002] Trans-2-decenoic acid is an α, β-medium chain unsaturated fatty acid with extremely high industrial and biopharmaceutical value. As a key intermediate for the synthesis of the bioactive substance 10-hydroxy-2-decenoic acid (10-HDA) and other drugs, biosynthesis of trans-2-decenoic acid has become a hot research area today. Compared with chemical synthesis, the production of trans-2-decenoic acid through biosynthesis can achieve a more environmentally friendly production process, expand its application in various fields, and promote the green development of biopharmaceuticals.

[0003] In the biosynthetic pathway, Escherichia coli is widely used as a host bacterium because of its clear genetic background, easy genetic manipulation and relatively simple fermentation conditions. However, the existing Escherichia coli trans-2-decenoic acid production system has some limitations, such as low substrate utilization, product inhibition, poor cell tolerance, etc., which makes it difficult for production efficiency and output to meet industrial needs. Especially under high substrate concentration conditions, the growth of engineered strains is inhibited, resulting in a decrease in substrate conversion rate and product yield.

[0004] In terms of existing technologies, Chinese patent document CN117402797A discloses an engineered strain capable of producing high trans-2-decenoic acid and its application. The patent describes the use of CRISPR / Cas9 technology to knock out specific genes (aaS and ftsQ) in Escherichia coli BL21 to improve tolerance to the substrate decanoic acid and the yield of trans-2-decenoic acid, and the use of transformation to transfer the Escherichia coli engineered bacteria BL21ΔFadB, R, JΔaaSΔftsQ after the specific genes are knocked out into the recombinant plasmids pCDFDuet-1-MaMACS-PpFadE and pET28a-SUMO-ctYdiI to obtain the final engineered strains BL21ΔFadB, R, JΔaaSΔftsQ-MEI, etc. Chinese patent document CN114958700A discloses an Escherichia coli engineered bacteria and its application. The genes knocked out in the patent document are FadB gene, FadR gene, and FadJ gene, and the RED recombination method is used to knock out the genes. Chinese patent document CN109402182A discloses a method for preparing 10-hydroxy-2-decenoic acid using resting cells of Escherichia coli engineered bacteria, which constructs Escherichia coli containing a recombinant plasmid pET-28a-ydiI and prepares resting cells to achieve a large amount of biosynthesis of 10-hydroxy-2-decenoic acid. The technical solutions and technical effects disclosed in the existing patent documents are significantly different from those of the present invention. Summary of the invention

[0005] In view of the deficiencies in the prior art, the present invention provides a genetically engineered bacterium with high capric acid tolerance and a construction method and application thereof.

[0006] The technical solution of the present invention is as follows:

[0007] A genetically engineered bacterium with high decanoic acid tolerance, wherein the genetically engineered bacterium is based on Escherichia coli as a starting strain, the aaS gene is knocked out, and the mlaZ gene and the mlaY gene are overexpressed;

[0008] Among them, the Gene ID of the aaS gene nucleotide sequence is 947315; the Gene ID of the mlaZ gene nucleotide sequence is 882402; and the Gene ID of the mlaY gene nucleotide sequence is 882401.

[0009] Preferably according to the present invention, the aaS gene encodes a bifunctional protein including 2-acylglycerophospholipid ethanolamine (2-acyl-GPE) acyltransferase and acyl-acyl carrier protein (acyl-ACP) synthetase.

[0010] Preferably according to the present invention, the mlaZ gene and mlaY gene are derived from Pseudomonas aeruginosa and are key genes for maintaining the outer membrane lipid composition stability (MLA) pathway. The MLA pathway is particularly related to the process of lipid transport from the inner membrane to the outer membrane, and reverse lipid transport. The mlaZ gene and mlaY gene are used to maintain the inner membrane integrity and outer membrane stability of Pseudomonas aeruginosa.

[0011] Preferably, according to the present invention, the starting strain is the Escherichia coli engineered bacteria BL21 (DE3)-△fadB-△fadR-△fadJ. The construction method of the strain is prior art and has been disclosed in patent document CN113106109A, with specific reference to paragraphs

[0197] -

[0221] . The patent name is: A mutant enzyme CPY153M228L and its application in the synthesis of 10-hydroxy-decenoic acid, application number: 202110211118.9, and the publication date is 2021.07.13.

[0012] The method for constructing the above-mentioned genetically engineered bacteria with high decanoic acid tolerance comprises the following steps:

[0013] (1) Using CRISPR / Cas9 technology, the aaS gene in the Escherichia coli engineered bacteria BL21(DE3)-△fadB-△fadR-△fadJ was knocked out, and the plasmid was eliminated after the knockout, thereby obtaining the Escherichia coli engineered bacteria BL21(DE3)-△fadB-△fadR-△fadJ-△aaS;

[0014] (2) Artificially synthesizing the nucleotide sequence of the mlaY gene, the nucleotide sequence of the mlaZ gene, and the nucleotide sequence of the pelb signal peptide; then using the mixture of the pelb signal peptide and the mlaY gene as a template and pelb-mlaY-F / R as primers to perform PCR amplification to obtain the pelb-mlaY sequence; using the mixture of the pelb signal peptide and the mlaZ gene as a template and pelb-mlaZ-F / R as primers to perform PCR amplification to obtain the pelb-mlaZ sequence; after double restriction digestion of the plasmid vector pET22b, the pelb-mlaY sequence, and the pelb-mlaZ sequence, ligating them together with T4 DNA ligase to obtain the plasmid pET22b-pelb-mlaY-pelb-mlaZ;

[0015] (3) Construction of plasmid pET28a-sumo- Ct ydiII and plasmid pCDFDuet-1- Ma MACS- Pp fadE;

[0016] (4) Plasmid pET28a-sumo- CtydiII, plasmid pCDFDuet-1- Ma MACS- Pp fadE and plasmid pET22b-pelb-mlaY-pelb-mlaZ were co-transformed into the competent Escherichia coli engineered bacteria BL21 (DE3)-△fadB-△fadR-△fadJ-ΔaaS, and positive recombinants were screened using a culture medium containing kanamycin, ampicillin and streptomycin to obtain the highly decanoic acid-tolerant genetically engineered bacteria E. coli-△BRJA-SKA.

[0017] According to the present invention, in step (1), the method of using CRISPR / Cas9 technology to knock out the aaS gene in the Escherichia coli engineered bacteria BL21 (DE3)-△fadB-△fadR-△fadJ, and the method of eliminating the plasmid after the knockout are both prior arts and have been disclosed in patent document CN117402797A, with specific reference to paragraphs

[0031] -

[0042] . Patent name: A method for constructing an engineered bacterium for preparing high-yield royal jelly acid precursor trans-2-decenoic acid, application number: 2023101245679, publication date 2024.01.16.

[0018] Preferably according to the present invention, in step (2), the primer sequence is as follows:

[0019] pelb-mlaY-F: 5′-GCGACTCCTGCATTAGGAAG-3′,

[0020] pelb-mlaY-R: 5′-GGATCCGAATTAATTCCGATATCCATG-3′;

[0021] pelb-mlaZ-F: 5′-CGGCAACtaaCCATGGATATCGGA-3′,

[0022] pelb-mlaZ-R: 5′-TGGTGGTGCTCGAGttaGCCC-3′.

[0023] Preferably according to the present invention, in step (2), the molar ratio of the pelb signal peptide to the mlaY gene is 1:1; the molar ratio of the pelb signal peptide to the mlaZ gene is 1:1.

[0024] According to the preferred embodiment of the present invention, in step (3), the plasmid pET28a-sumo- Ct ydiI is an existing conventional plasmid, which can be constructed according to the method described in the article "Screening of key catalytic elements for biosynthesis of 10-HDA and study on rational design of P450 enzymes"; the plasmid pCDFDuet-1-Ma MACS- Pp fadE is an existing conventional plasmid, which can be constructed according to the method disclosed in Chinese patent document CN113106109A, a mutant enzyme CYP153A M228L and its application in the synthesis of 10-hydroxy-2-decenoic acid.

[0025] Preferably according to the present invention, in step (4), the formula of the culture medium containing kanamycin, ampicillin and streptomycin is: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract powder, 100 μg / mL kanamycin, 100 μg / mL ampicillin and 50 μg / mL streptomycin.

[0026] The application of the above-mentioned genetically engineered bacteria with high decanoic acid tolerance in the production of trans-2-decenoic acid.

[0027] A method for preparing trans-2-decenoic acid comprises the following steps:

[0028] The highly decanoic acid-tolerant genetically engineered bacteria E. coli-△BRJA-SKA were inoculated into 50 mL of LB liquid culture medium containing 100 μg / mL kanamycin, 100 μg / mL ampicillin, and 50 μg / mL streptomycin, and cultured overnight at 37° C. and 200 r / min to obtain activated E. coli-△BRJA-SKA;

[0029] The activated E. coli-△BRJA-SKA bacterial liquid is inoculated into a fermentation medium at an inoculation rate of 1%, and cultured at 37°C and 200 r / min until the bacterial volume OD600 is 0.8-1.2, and α-lactose with a final concentration of 14 mM is added, and the culture is induced for 50-60 minutes to obtain a fermentation culture; then, decanoic acid is added to the fermentation culture for whole-cell catalysis to prepare trans-2-decenoic acid.

[0030] Preferably according to the present invention, the formula of the fermentation medium is: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract powder, 100 μg / mL kanamycin, 100 μg / mL ampicillin, and 50 μg / mL streptomycin.

[0031] Beneficial effects:

[0032] The present invention uses CRISPR-Cas9 gene editing technology to knock out the aaS gene in Escherichia coli, thereby reducing the level of medium-chain fatty acids incorporated into the cell membrane, thereby improving the integrity of cell membrane lipids. At the same time, the mlaZ and mlaY genes derived from Pseudomonas aeruginosa are overexpressed to strengthen the membrane lipid strength of the Escherichia coli engineered bacteria, so that the genetically engineered bacteria E.coli-△BRJA-SKA constructed by the present invention exhibits better robustness in the fermentation process of high-concentration substrate decanoic acid, and has higher tolerance to high-concentration substrate decanoic acid during the fermentation process.

[0033] In addition, the OD600 value of the genetically engineered bacteria E. coli-△BRJA-SKA constructed by the present invention can reach up to 4.66, showing a good growth state; when the decanoic acid is added to a maximum concentration of 3.0 g / L, the yield of trans-2-decenoic acid can reach 1.47 g / L, and the conversion rate is 49.13%, which significantly improves the yield. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 Schematic diagram of the construction of plasmid pET22b-pelb-mlaY-pelb-mlaZ;

[0035] Figure 2 is an agarose gel electrophoresis diagram of a partial fragment of the mlaY gene in plasmid pET22b-pelb-mlaY-pelb-mlaZ;

[0036] In the figure: Lane M is a marker; Lanes 1 to 4 are agarose gel electrophoresis images of a partial segment of the mlaY gene, with a length of 435 bp.

[0037] Figure 3 This is a graph showing the growth of genetically engineered bacteria E.coli-△BRJA-SKA, E.coli-△BRJ-SKA, E.coli-△BRJA-SK, and E.coli-△BRJ-SK.

[0038] Figure 4 The graph shows the yield of trans-2-decenoic acid and the decanoic acid conversion rate produced by the genetically engineered bacteria E. coli-△BRJA-SKA with high decanoic acid tolerance. DETAILED DESCRIPTION

[0039] The content of the present invention is further described below with reference to examples, but the protection content of the present invention is not limited thereto.

[0040] The experimental operations involved in the examples are all routine operations in the art unless otherwise specified. The materials, reagents, etc. used in the examples are all available from commercial sources unless otherwise specified.

[0041] The formula of the fermentation medium used in the example is: sodium chloride 10 g / L, peptone 10 g / L, yeast extract 5 g / L, kanamycin 100 μg / mL, ampicillin 100 μg / mL, and streptomycin 50 μg / mL.

[0042] Example 1

[0043] A method for constructing a genetically engineered bacterium with high decanoic acid tolerance comprises the following steps:

[0044] (1) knocking out the aaS gene in the Escherichia coli engineered bacteria BL21 (DE3)-△fadB-△fadR-△fadJ using CRISPR / Cas9 technology, and eliminating the plasmid after the knockout, thereby obtaining the Escherichia coli engineered bacteria BL21 (DE3)-△fadB-△fadR-△fadJ-△aaS;

[0045] Among them, the construction method of the starting strain Escherichia coli engineered bacteria BL21 (DE3)-△fadB-△fadR-△fadJ is a prior art, which has been disclosed in patent document CN113106109A, specifically referring to paragraphs

[0197] -

[0221] , patent name: A mutant enzyme CPY153M228L and its application in the synthesis of 10-hydroxy-2-decenoic acid, application number: 202110211118.9, publication date 2021.07.13;

[0046] The method of knocking out the aaS gene in the Escherichia coli engineered bacteria BL21 (DE3)-△fadB-△fadR-△fadJ using CRISPR / Cas9 technology is a prior art, which has been disclosed in patent document CN117402797A, specifically referring to paragraphs

[0031] -

[0042] . Patent name: A method for constructing an engineered bacterium for preparing high-yield royal jelly acid precursor trans-2-decenoic acid, application number: 2023101245679, publication date 2024.01.16; the Gene ID of the aaS gene nucleotide sequence is 947315 (length 2159 bp);

[0047] (2) Genwi Company artificially synthesized the nucleotide sequence of the mlaY gene, the nucleotide sequence of the mlaZ gene, and the nucleotide sequence of the pelb signal peptide according to the sequence information; the Gene ID of the mlaZ gene nucleotide sequence is 882402 (length 803 bp), the Gene ID of the mlaY gene nucleotide sequence is 882401 (length 1361 bp), and the Gene ID of the pelb signal peptide nucleotide sequence is 878832 (length 3581 bp);

[0048] Using the mixture of pelb signal peptide and mlaY gene as template and pelb-mlaY-F / R as primers, PCR amplification was performed to obtain the pelb-mlaY sequence; the primer sequences are as follows:

[0049] pelb-mlaY-F: 5′-GCGACTCCTGCATTAGGAAG-3′,

[0050] pelb-mlaY-R: 5′-GGATCCGAATTAATTCCGATATCCATG-3′;

[0051] Among them, the molar ratio of pelb signal peptide and mlaY gene was 1:1;

[0052] The above PCR amplification system is as follows: total system 20 μL, 1 μL upstream primer, 1 μL downstream primer, 1 μL template, 2× phanta enzyme 10 μL, 7 μL ddH2O;

[0053] The above PCR amplification conditions are as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 58°C for 5 s, extension at 72°C for 5 s, 30 cycles, and complete extension at 72°C for 1 min;

[0054] Using the mixture of pelb signal peptide and mlaZ gene as template and pelb-mlaZ-F / R as primers, PCR amplification was performed to obtain the pelb-mlaZ sequence; the primer sequences are as follows:

[0055] pelb-mlaZ-F: 5′-CGGCAACtaaCCATGGATATCGGA-3′,

[0056] pelb-mlaZ-R: 5′-TGGTGGTGCTCGAGttaGCCC-3′;

[0057] Among them, the molar ratio of pelb signal peptide and mlaZ gene was 1:1;

[0058] The above PCR amplification system is as follows: total system 20 μL, 1 μL upstream primer, 1 μL downstream primer, 1 μL template, 2× phanta enzyme 10 μL, 7 μL ddH2O;

[0059] The above PCR amplification conditions are as follows: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10 s, annealing at 61°C for 5 s, extension at 72°C for 5 s, 30 cycles, and complete extension at 72°C for 1 min;

[0060] The plasmid vector pET22b, pelb-mlaY sequence and pelb-mlaZ sequence were double-digested, and the digested plasmid vector pET22b, pelb-mlaY and pelb-mlaZ were mixed together, and T4 DNA ligase was added for ligation to obtain the plasmid pET22b-pelb-mlaY-pelb-mlaZ. The specific construction diagram is shown in the figure. Figure 1 As shown;

[0061] (3) According to the method disclosed in the article "Screening of key catalytic elements for biosynthesis of 10-HDA and research on rational design of P450 enzymes", plasmid pET28a-sumo- Ct ydiI; construct plasmid pCDFDuet-1- according to the method disclosed in Chinese patent document CN113106109A a mutant enzyme CYP153A M228L and its application in the synthesis of 10-hydroxy-2-decenoic acid Ma MACS- Pp fadE;

[0062] (4) The E. coli engineered bacteria BL21 (DE3)-ΔfadB-ΔfadR-ΔfadJ-ΔaaS obtained in step (1) were made into competent cells, and then the plasmid pET28a-sumo- Ct ydiI, plasmid pCDFDuet-1- Ma MACS- Pp fadE and plasmid pET22b-pelb-mlaY-pelb-mlaZ were transformed into BL21(DE3)-△fadB-△fadR-△fadJ-ΔaaS competent cells, and positive recombinants were screened using a culture medium containing kanamycin, ampicillin, and streptomycin to obtain the genetically engineered bacteria E. coli-△BRJA-SKA with high decanoic acid tolerance.

[0063] The formula of the culture medium containing kanamycin, ampicillin and streptomycin is: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract powder, 100 μg / mL kanamycin, 100 μg / mL ampicillin and 50 μg / mL streptomycin.

[0064] The plasmid pET22b-pelb-mlaY-pelb-mlaZ constructed in step (2) was verified by electrophoresis. The results were as follows: Figure 2 shown.

[0065] The verification target is the upper fragment of the mlaY gene (435 bp), and the primer sequences used for electrophoresis verification are as follows:

[0066] pet22b-frw: 5′-GCGACTCCTGCATTAGGAAGCAGCCCAGTA-3′,

[0067] pet22b-rev: 5′-CATCGCCGGCTGGGCAGCGAGGAGCAGCAG-3′.

[0068] Depend on Figure 2 It can be seen that the partial fragment (435bp) of the mlaY gene was successfully amplified by PCR using primers pet22b-frw and pet22b-rev. The electrophoresis results showed that the length of the fragment was 435bp, which was consistent with the expected length of the mlaY gene fragment. This proves that the plasmid pET22b-pelb-mlaY-pelb-mlaZ was successfully transferred into the competent cell BL21(DE3)-△fadB-△fadR-△fadJ-△aaS.

[0069] Comparative Example 1

[0070] Escherichia coli engineered bacteria BL21 (DE3)-△fadB-△fadR-△fadJ were made into competent cells, and then the plasmid pET28a-sumo- Ct ydiI, plasmid pCDFDuet-1- Ma MACS- Pp fadE and plasmid pET22b-pelb-mlaY-pelb-mlaZ were co-transformed into BL21(DE3)-△fadB-△fadR-△fadJ competent cells, and positive recombinants were screened using a culture medium containing kanamycin, ampicillin, and streptomycin to obtain the genetically engineered bacteria E. coli-△BRJ-SKA.

[0071] The formula of the culture medium containing kanamycin, ampicillin and streptomycin is: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract powder, 100 μg / mL kanamycin, 100 μg / mL ampicillin and 50 μg / mL streptomycin.

[0072] Comparative Example 2

[0073] Escherichia coli engineered bacteria BL21 (DE3)-△fadB-△fadR-△fadJ-ΔaaS were made into competent cells, and then the plasmid pET28a-sumo- Ct ydiI, plasmid pCDFDuet-1- Ma MACS- PpfadE was co-transformed into BL21(DE3)-△fadB-△fadR-△fadJ-ΔaaS competent cells, and positive recombinants were screened using a culture medium containing kanamycin and streptomycin to obtain the genetically engineered bacteria E. coli-△BRJA-SK.

[0074] The formula of the culture medium containing kanamycin and streptomycin is: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract powder, 100 μg / mL kanamycin, and 50 μg / mL streptomycin.

[0075] Comparative Example 3

[0076] Escherichia coli engineered bacteria BL21 (DE3)-△fadB-△fadR-△fadJ were made into competent cells, and then the plasmid pET28a-sumo- Ct ydiI, plasmid pCDFDuet-1- Ma MACS- Pp fadE was co-transformed into BL21(DE3)-△fadB-△fadR-△fadJ competent cells, and positive recombinants were screened using a culture medium containing kanamycin and streptomycin to obtain the genetically engineered bacteria E. coli-△BRJ-SK.

[0077] The formula of the culture medium containing kanamycin and streptomycin is: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract powder, 100 μg / mL kanamycin, and 50 μg / mL streptomycin.

[0078] Example 2

[0079] The highly decanoic acid-tolerant genetically engineered bacteria E.coli-△BRJA-SKA constructed in Example 1 and the genetically engineered bacteria E.coli-△BRJ-SKA constructed in Comparative Example 1 were inoculated in 50 mL of LB liquid culture medium containing 100 μg / mL of kanamycin, 100 μg / mL of ampicillin, and 50 μg / mL of streptomycin; the genetically engineered bacteria E.coli-△BRJA-SK in Comparative Example 2 and the genetically engineered bacteria E.coli-△BRJ-SK in Comparative Example 3 were inoculated in 50 mL of LB liquid culture medium containing 100 μg / mL of kanamycin and 50 μg / mL of streptomycin. Then, they were cultured overnight at 37°C and 200 r / min to obtain activated E.coli-△BRJA-SKA, E.coli-△BRJ-SKA, E.coli-△BRJA-SK, and E.coli-△BRJ-SK.

[0080] The activated E. coli-△BRJA-SKA, E. coli-△BRJ-SKA, E. coli-△BRJA-SK and E. coli-△BRJ-SK bacterial liquid was inoculated into 100 ml of fermentation medium added with corresponding antibiotics at a 1% inoculum volume (1 mL), and cultured at 37°C, 200 r / min for 48 h. The OD value of each strain was measured at 8, 16, 24, 32, 40 and 48 h, respectively. The growth of each strain was judged according to the OD value. The results are shown in FIG. Figure 3 shown.

[0081] Depend on Figure 3 It can be seen that the growth of the engineered bacteria E.coli-△BRJA-SKA with aaS gene knocked out and mlaY and mlaZ genes overexpressed in the present invention at each time point is better than that of the genetically engineered bacteria E.coli-△BRJ-SKA, E.coli-△BRJA-SK, and E.coli-△BRJ-SK in comparative examples 1 to 3. Among them, the OD600 value of E.coli-△BRJA-SKA can reach up to 4.66, showing a good growth state.

[0082] Example 3

[0083] A method for preparing trans-2-decenoic acid comprises the following steps:

[0084] The highly decanoic acid-tolerant genetically engineered bacteria E. coli-△BRJA-SKA constructed in Example 1 and the genetically engineered bacteria E. coli-△BRJ-SKA, E. coli-△BRJA-SK, and E. coli-△BRJ-SK constructed in Comparative Examples 1 to 3 were respectively inoculated into 50 mL of LB liquid culture medium containing the corresponding antibiotics, and cultured overnight at 37° C. and 200 r / min to obtain activated E. coli-△BRJA-SKA, E. coli-△BRJ-SKA, E. coli-△BRJA-SK, and E. coli-△BRJ-SK.

[0085] The activated E. coli-△BRJA-SKA, E. coli-△BRJ-SKA, E. coli-△BRJA-SK and E. coli-△BRJ-SK bacterial liquids were inoculated into 100 ml of fermentation medium with corresponding antibiotics at an inoculum volume of 1% (1 mL), and cultured at 37°C and 200 r / min for 2 hours until the bacterial volume OD600 = 1; then α-lactose with a final concentration of 14 mM was added, and the induction culture was carried out for 55 minutes to obtain E. coli-△BRJA-SKA, E. coli-△BRJ-SKA, E. coli-△BRJA-SK and E. coli-△BRJ-SK fermentation cultures;

[0086] Decanoic acid was added to the fermentation cultures of E. coli-△BRJA-SKA, E. coli-△BRJ-SKA, E. coli-△BRJA-SK and E. coli-△BRJ-SK, with an initial concentration of 0.5 g / L. Subsequently, samples were taken from the fermentation cultures of each strain every 8 hours and 0.5 g / L of substrate decanoic acid was added thereto for a total whole-cell catalytic reaction of 48 hours to prepare trans-2-decenoic acid.

[0087] The substrates and products of the above whole-cell catalytic reaction were determined by Japanese Tsu GC-2030 gas chromatography. The results are as follows Figure 4 shown. Figure 4 The left vertical axis in the middle is the concentration of trans-2-decenoic acid, corresponding to the bar graph; the right vertical axis is the concentration percentage of the substrate, corresponding to the line graph.

[0088] Depend on Figure 4 It can be seen that the decanoic acid substrate conversion rate and trans-2-decenoic acid yield of the engineered bacteria E. coli-△BRJA-SKA in which the aaS gene is knocked out and the mlaY and mlaZ genes are overexpressed are significantly higher than those of the genetically engineered bacteria E. coli-△BRJ-SKA, E. coli-△BRJA-SK, and E. coli-△BRJ-SK in comparative examples 1 to 3.

[0089] Among them, when E.coli-△BRJA-SKA was fermented for 8 hours and the initial concentration of decanoic acid was 0.5 g / L, the trans-2-decenoic acid could reach 0.46 g / L, and the conversion rate was 91.39%; when the fermentation lasted for 48 hours and the final concentration of decanoic acid was 3 g / L, the trans-2-decenoic acid could reach 1.47 g / L, and the conversion rate was 49.13%.

[0090] In summary, the present invention knocks out the aaS gene in Escherichia coli through CRISPR-Cas9 gene editing technology, thereby reducing the level of medium-chain fatty acids incorporated into the cell membrane, thereby improving the integrity of the cell membrane lipids. At the same time, the mlaZ and mlaY genes derived from Pseudomonas aeruginosa are overexpressed, which strengthens the membrane lipid strength of the Escherichia coli engineered bacteria, so that the engineered bacteria show better robustness during the fermentation process of high-concentration substrate decanoic acid, and have higher tolerance to high-concentration substrate decanoic acid during the fermentation process. The OD600 value of E.coli-△BRJA-SKA can reach up to 4.66, showing a good growth state. When decanoic acid is added to the maximum concentration of 3.0g / L, the yield of trans-2-decenoic acid can reach 1.47g / L, and the conversion rate is 49.13%, which significantly increases the yield of trans-2-decenoic acid.

[0091] The above-described embodiments are only preferred specific implementation schemes of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A genetically engineered bacterium with high tolerance to decanoic acid, characterized in that: The genetically engineered bacteria uses Escherichia coli as a starting strain, knocks out the aaS gene, and simultaneously overexpresses the mlaZ gene and the mlaY gene; Among them, the Gene ID of the aaS gene nucleotide sequence is 947315; the Gene ID of the mlaZ gene nucleotide sequence is 882402; and the Gene ID of the mlaY gene nucleotide sequence is 882401.

2. The genetically engineered bacteria with high decanoic acid tolerance according to claim 1, characterized in that The starting strain is the engineered Escherichia coli BL21 (DE3)-△fadB-△fadR-△fadJ.

3. The method for constructing the genetically engineered bacteria with high decanoic acid tolerance according to claim 1, characterized in that: The steps include: (1) Using CRISPR / Cas9 technology, the aaS gene in the Escherichia coli engineered bacteria BL21(DE3)-△fadB-△fadR-△fadJ was knocked out, and the plasmid was eliminated after the knockout, thereby obtaining the Escherichia coli engineered bacteria BL21(DE3)-△fadB-△fadR-△fadJ-△aaS; (2) Artificially synthesizing the nucleotide sequence of the mlaY gene, the nucleotide sequence of the mlaZ gene, and the nucleotide sequence of the pelb signal peptide; then using the mixture of the pelb signal peptide and the mlaY gene as a template and pelb-mlaY-F / R as primers to perform PCR amplification to obtain the pelb-mlaY sequence; using the mixture of the pelb signal peptide and the mlaZ gene as a template and pelb-mlaZ-F / R as primers to perform PCR amplification to obtain the pelb-mlaZ sequence; double-enzyme-digesting the plasmid vector pET22b, the pelb-mlaY sequence, and the pelb-mlaZ sequence, and connecting them together to obtain the plasmid pET22b-pelb-mlaY-pelb-mlaZ; (3) Construction of plasmid pET28a-sumo- Ct ydiI and plasmid pCDFDuet-1- Ma MACS- Pp fadE; (4) Plasmid pET28a-sumo- Ct ydiI, plasmid pCDFDuet-1- Ma MACS- Pp fadE and plasmid pET22b-pelb-mlaY-pelb-mlaZ were co-transformed into the competent Escherichia coli engineered bacteria BL21 (DE3)-△fadB-△fadR-△fadJ-ΔaaS, and positive recombinants were screened using a culture medium containing kanamycin, ampicillin and streptomycin to obtain the highly decanoic acid-tolerant genetically engineered bacteria E. coli-△BRJA-SKA.

4. The construction method according to claim 3, characterized in that: In step (2), the primer sequence is as follows: pelb-mlaY-F: 5′-GCGACTCCTGCATTAGGAAG-3′, pelb-mlaY-R: 5′-GGATCCGAATTAATTCCGATATCCATG-3′; pelb-mlaZ-F: 5′-CGGCAACtaaCCATGGATATCGGA-3′, pelb-mlaZ-R: 5′-TGGTGGTGCTCGAGttaGCCC-3′.

5. The construction method according to claim 3, characterized in that: In step (2), the molar ratio of the pelb signal peptide to the mlaY gene is 1:1; the molar ratio of the pelb signal peptide to the mlaZ gene is 1:

1.

6. The construction method according to claim 3, characterized in that: In step (4), the formula of the culture medium containing kanamycin, ampicillin and streptomycin is: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract powder, 100 μg / mL kanamycin, 100 μg / mL ampicillin and 50 μg / mL streptomycin.

7. Use of the genetically engineered bacteria with high decanoic acid tolerance according to claim 1 in the production of trans-2-decenoic acid.

8. A method for preparing trans-2-decenoic acid, characterized in that: The steps include: The highly decanoic acid-tolerant genetically engineered bacteria E. coli-△BRJA-SKA of claim 1 was inoculated into 50 mL of LB liquid culture medium containing 100 μg / mL of kanamycin, 100 μg / mL of ampicillin, and 50 μg / mL of streptomycin, and cultured overnight at 37° C. and 200 r / min to obtain activated E. coli-△BRJA-SKA; The activated E. coli-△BRJA-SKA bacterial liquid is inoculated into a fermentation medium at an inoculation rate of 1%, and cultured at 37°C and 200 r / min until the bacterial volume OD600 is 0.8-1.2, and α-lactose with a final concentration of 14 mM is added, and the culture is induced for 50-60 minutes to obtain a fermentation culture; then, decanoic acid is added to the fermentation culture for whole-cell catalysis to prepare trans-2-decenoic acid.

9. The method for preparing trans-2-decenoic acid as claimed in claim 8, characterized in that: The formula of the fermentation medium is: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract powder, 100 μg / mL kanamycin, 100 μg / mL ampicillin, and 50 μg / mL streptomycin.

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