A genetically engineered bacterium with high capric acid tolerance and a construction method and application thereof

Abstract

The present application relates to a kind of high capric acid resistant genetically engineered bacteria and its construction method and application.The genetically engineered bacteria take E. coli as starting strain, knock out aaaS gene, simultaneously overexpress mlaZ gene and mlaY gene;Wherein, the Gene ID of aaaS gene nucleotide sequence is 947315;The Gene ID of mlaZ gene nucleotide sequence is 882402;The Gene ID of mlaY gene nucleotide sequence is 882401.The present application effectively strengthens the membrane lipid intensity of E. coli engineering bacteria by knocking out aaaS gene in E. coli and simultaneously overexpressing mlaZ and mlaY gene, so that genetically engineered bacteria show better robustness and higher resistance in the fermentation process of high-concentration substrate capric acid.Trans-2-decenoic acid yield can reach 1.47g / L when capric acid is added to the highest concentration of 3.0g / L, and the conversion rate is 49.13%, which significantly improves the yield.

Description

Technical Field

[0001] This invention relates to a genetically engineered bacterium with high decanoic acid tolerance, its construction method and application, belonging to the field of microbial technology. Background Technology

[0002] Trans-2-decenoic acid (TDE) is an α,β-medium-chain unsaturated fatty acid with extremely high industrial and biopharmaceutical value. As a key intermediate in the synthesis of the bioactive substance 10-hydroxy-2-decenoic acid (10-HDA) and other drugs, the biosynthesis of TDE has become a hot research area. Compared to chemical synthesis, the biosynthetic production of TDE offers a more environmentally friendly process, expanding its applications across various fields and promoting the green development of biopharmaceuticals.

[0003] In biosynthetic pathways, *Escherichia coli* is widely used as a host bacterium due to its clear genetic background, ease of genetic manipulation, and relatively simple fermentation conditions. However, existing *E. coli* trans-2-decenoic acid production systems have limitations, such as low substrate utilization, product inhibition, and poor cell tolerance, making it difficult to meet industrial-scale production efficiency and yield requirements. Particularly under high substrate concentrations, the growth of engineered strains is inhibited, leading to 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 high production of trans-2-decenoic acid and its applications. This patent describes the use of CRISPR / Cas9 technology to knock out specific genes (aaS and ftsQ) in *E. coli* BL21 to improve tolerance to the substrate decanoic acid and the production of trans-2-decenoic acid. It also describes the transformation of the *E. coli* engineered strains BL21ΔFadB, R, JΔaaSΔftsQ, after the specific genes were knocked out, into recombinant plasmids pCDFDuet-1-MaMACS-PpFadE and pET28a-SUMO-ctYdiI, resulting in the final engineered strains BL21ΔFadB, R, JΔaaSΔftsQ-MEI, etc. Chinese patent document CN114958700A discloses an engineered *E. coli* strain and its applications. This patent document knocks out the FadB, FadR, and FadJ genes using the RED recombination method. Chinese patent document CN109402182A discloses a method for preparing 10-hydroxy-2-decenoic acid using engineered Escherichia coli resting cells. This patent document constructs E. coli containing the recombinant plasmid pET-28a-ydiI and prepares resting cells to achieve large-scale biosynthesis of 10-hydroxy-2-decenoic acid. The technical solutions and effects disclosed in existing patent documents are significantly different from those of this invention. Summary of the Invention

[0005] To address the shortcomings of existing technologies, this invention provides a genetically engineered bacterium with high decanoic acid tolerance, its construction method, and its application.

[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, the aaS gene is knocked out, and the mlaZ and mlaY genes are overexpressed.

[0008] 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] According to a preferred embodiment of the present invention, the aaS gene encodes a bifunctional protein comprising 2-acylglycerol phospholipid ethanolamine (2-acyl-GPE) acyltransferase and acyl-acyl carrier protein (acyl-ACP) synthase.

[0010] According to a preferred embodiment of the present invention, the mlaZ and mlaY genes are derived from *Pseudomonas aeruginosa* and are key genes in the lipid composition stability pathway (MLA). The MLA pathway specifically involves the transport of lipids from the inner membrane to the outer membrane, as well as the reverse lipid transport process. The mlaZ and mlaY genes are used to maintain the inner membrane integrity and outer membrane stability of *Pseudomonas aeruginosa*.

[0011] According to a preferred embodiment of the present invention, the starting strain is the engineered Escherichia coli BL21(DE3)-△fadB-△fadR-△fadJ. The construction method of this strain is prior art and has been disclosed in patent document CN113106109A, specifically referring to paragraphs

[0197] -

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

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

[0013] (1) The aaS gene in the engineered Escherichia coli BL21(DE3)-△fadB-△fadR-△fadJ was knocked out using CRISPR / Cas9 technology. After the knockout was completed, the plasmid was eliminated to obtain the engineered Escherichia coli BL21(DE3)-△fadB-△fadR-△fadJ-ΔaaS.

[0014] (2) The nucleotide sequences of the mlaY gene, mlaZ gene, and pelb signal peptide were artificially synthesized; then, using a mixture of pelb signal peptide and mlaY gene as a template, PCR amplification was performed with pelb-mlaY-F / R as primers to obtain the pelb-mlaY sequence; using a mixture of pelb signal peptide and mlaZ gene as a template, PCR amplification was performed with pelb-mlaZ-F / R as primers to obtain the pelb-mlaZ sequence; after double digestion of the plasmid vector pET22b, the pelb-mlaY sequence, and the pelb-mlaZ sequence, they were ligated together with T4 DNA ligase to obtain the plasmid pET22b-pelb-mlaY-pelb-mlaZ;

[0015] (3) Constructing 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 E. coli engineered strain BL21(DE3)-△fadB-△fadR-△fadJ-ΔaaS competent cells. Positive recombinants were screened using a medium containing kanamycin, ampicillin, and streptomycin to obtain the genetically engineered strain E. coli-△BRJA-SKA with high decanoic acid tolerance.

[0017] According to the present invention, in step (1), the method of knocking out the aaS gene in the engineered Escherichia coli BL21(DE3)-△fadB-△fadR-△fadJ using CRISPR / Cas9 technology, and the method of removing the plasmid after knockout, are both existing technologies and have been disclosed in patent document CN117402797A. Specifically, refer to paragraphs

[0031] -

[0042] , patent name: a method for constructing a high-yield royal jelly acid precursor trans-2-decenoic acid engineered bacteria, application number: 2023101245679, publication date 2024.01.16.

[0018] According to a preferred embodiment of 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] According to a preferred embodiment of 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 a preferred embodiment of the present invention, in step (3), the plasmid pET28a-sumo- Ct ydiI is an existing conventional plasmid that can be constructed according to the method described in the article "Screening of Key Catalytic Elements for Biosynthesis of 10-HDA and Rational Design of P450 Enzymes"; the plasmid pCDFDuet-1-Ma MACS- Pp fadE is an existing conventional plasmid that can be constructed according to the method disclosed in Chinese patent document CN113106109A, which describes a mutant enzyme CYP153A M228L and its application in the synthesis of 10-hydroxy-2-decenoic acid.

[0025] According to a preferred embodiment of the present invention, in step (4), the culture medium containing kanamycin, ampicillin and streptomycin is formulated as follows: 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.

[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 includes the following steps:

[0028] The above-mentioned genetically engineered E. coli-△BRJA-SKA with high decanoic acid tolerance was inoculated into 50 mL of LB liquid 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 culture was inoculated into the fermentation medium at an inoculum rate of 1% and cultured at 37℃ and 200 r / min until the bacterial count OD600 = 0.8-1.2. α-lactose with a final concentration of 14 mM was added and the culture was induced for 50-60 min to obtain the fermentation culture. Then, decanoic acid was added to the fermentation culture for whole-cell catalysis to prepare trans-2-decenoic acid.

[0030] According to a preferred embodiment of the present invention, the fermentation medium is formulated as follows: 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.

[0031] Beneficial effects:

[0032] This invention utilizes CRISPR-Cas9 gene editing technology to knock out the aaS gene in *E. coli*, thereby reducing the level of medium-chain fatty acids incorporated into the cell membrane and improving the integrity of cell membrane lipids. Simultaneously, overexpression of the mlaZ and mlaY genes derived from *Pseudomonas aeruginosa* strengthens the membrane lipid strength of the engineered *E. coli* strain. This results in the constructed genetically engineered strain *E. coli-△BRJA-SKA* exhibiting better robustness during fermentation with high concentrations of decanoic acid, demonstrating greater tolerance to high concentrations of decanoic acid during fermentation.

[0033] Furthermore, the genetically engineered E. coli-△BRJA-SKA constructed in this invention has an OD600 value as high as 4.66, indicating good growth. When decanoic acid is added to the highest concentration of 3.0 g / L, the yield of trans-2-decenoic acid can reach 1.47 g / L, with a conversion rate of 49.13%, which significantly improves the yield. Attached Figure Description

[0034] Figure 1 This is a schematic diagram illustrating the construction of plasmid pET22b-pelb-mlaY-pelb-mlaZ.

[0035] Figure 2 Agarose gel electrophoresis image of a partial fragment of the mlaY gene in plasmid pET22b-pelb-mlaY-pelb-mlaZ;

[0036] In the figure: lane M is the marker; lanes 1-4 are agarose gel electrophoresis images of partial fragments of the mlaY gene, with a length of 435 bp.

[0037] Figure 3 The diagram shows the growth of the genetically engineered bacteria E. coli-△BRJA-SKA, E. coli-△BRJ-SKA, E. coli-△BRJA-SK, and E. coli-△BRJ-SK.

[0038] Figure 4 The yield of trans-2-decenoic acid and the conversion rate of decanoic acid produced by the genetically engineered bacterium E. coli-△BRJA-SKA with high decanoic acid tolerance. Detailed Implementation

[0039] The invention will be further illustrated below with examples, but the scope of protection of the invention is not limited thereto.

[0040] Unless otherwise specified, the experimental procedures involved in the examples are standard practices in the art. Unless otherwise specified, all materials and reagents used in the examples are commercially available.

[0041] The fermentation medium used in the examples has the following formulation: 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 includes the following steps:

[0044] (1) The aaS gene in the engineered Escherichia coli BL21(DE3)-△fadB-△fadR-△fadJ was knocked out using CRISPR / Cas9 technology. After the knockout was completed, the plasmid was eliminated to obtain the engineered Escherichia coli BL21(DE3)-△fadB-△fadR-△fadJ-ΔaaS.

[0045] The construction method of the starting strain, Escherichia coli engineered strain BL21(DE3)-△fadB-△fadR-△fadJ, is existing technology and 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 engineered Escherichia coli BL21(DE3)-△fadB-△fadR-△fadJ using CRISPR / Cas9 technology is existing technology and has been disclosed in patent document CN117402797A (see paragraphs

[0031] -

[0042] for details). The patent title is: "A method for constructing a high-yield royal jelly acid precursor trans-2-decenoic acid engineered bacteria", application number: 2023101245679, publication date: 2024.01.16; the Gene ID of the aaS gene nucleotide sequence is 947315 (length is 2159bp).

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

[0048] Using a mixture of pelb signal peptide and mlaY gene as a template, PCR amplification was performed using pelb-mlaY-F / R primers 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] The molar ratio of pelb signal peptide to mlaY gene is 1:1.

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

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

[0054] Using a mixture of pelb signal peptide and mlaZ gene as a template, PCR amplification was performed using pelb-mlaZ-F / R primers 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] The molar ratio of pelb signal peptide to mlaZ gene is 1:1.

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

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

[0060] The plasmid vectors pET22b, pelb-mlaY, and pelb-mlaZ were double-digested with enzymes. The digested plasmid vectors pET22b, pelb-mlaY, and pelb-mlaZ were then mixed together and ligated with T4 DNA ligase to obtain the plasmid pET22b-pelb-mlaY-pelb-mlaZ. A schematic diagram of the construction process is shown below. Figure 1 As shown;

[0061] (3) The plasmid pET28a-sumo- was constructed according to the method disclosed in the article "Screening of Key Catalytic Elements for Biosynthesis of 10-HDA and Rational Design of P450 Enzymes". Ct ydiI; plasmid pCDFDuet-1- was 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". Ma MACS- Pp fadE;

[0062] (4) Prepare competent cells from the engineered E. coli strain BL21(DE3)-△fadB-△fadR-△fadJ-ΔaaS obtained in step (1), and then use 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. Positive recombinants were screened using a medium containing kanamycin, ampicillin, and streptomycin to obtain the genetically engineered E. coli-△BRJA-SKA with high decanoic acid tolerance.

[0063] The culture medium containing kanamycin, ampicillin, and streptomycin has the following formula: 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.

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

[0065] The target for validation was the upper part (435 bp) of the mlaY gene. The primer sequences used for electrophoresis validation are as follows:

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

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

[0068] Depend on Figure 2 It was found that a 435bp fragment of the mlaY gene was successfully amplified by PCR using primers pet22b-frw and pet22b-rev. Electrophoresis results showed that the fragment length was 435bp, consistent with the expected length of the selected mlaY gene fragment. This confirms that the plasmid pET22b-pelb-mlaY-pelb-mlaZ was successfully transformed into competent cells BL21(DE3)-△fadB-△fadR-△fadJ-ΔaaS.

[0069] Comparative Example 1

[0070] Competent cells were prepared from the engineered E. coli strain BL21(DE3)-△fadB-△fadR-△fadJ, 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. Positive recombinants were screened using a medium containing kanamycin, ampicillin, and streptomycin to obtain the genetically engineered E. coli-△BRJ-SKA.

[0071] The culture medium containing kanamycin, ampicillin, and streptomycin has the following formula: 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.

[0072] Comparative Example 2

[0073] Competent cells were prepared from the engineered E. coli strain BL21(DE3)-ΔfadB-ΔfadR-ΔfadJ-ΔaaS, 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 medium containing kanamycin and streptomycin to obtain the genetically engineered E. coli-△BRJA-SK.

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

[0075] Comparative Example 3

[0076] Competent cells were prepared from the engineered E. coli strain BL21(DE3)-△fadB-△fadR-△fadJ, 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 medium containing kanamycin and streptomycin to obtain the genetically engineered E. coli-△BRJ-SK.

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

[0078] Example 2

[0079] The genetically engineered strain E. coli-△BRJA-SKA with high decanoic acid tolerance constructed in Example 1 and the genetically engineered strain E. coli-△BRJ-SKA constructed in Comparative Example 1 were inoculated into 50 mL LB broth containing 100 μg / mL kanamycin, 100 μg / mL ampicillin, and 50 μg / mL streptomycin, respectively. The genetically engineered strains E. coli-△BRJA-SK (Comparative Example 2) and E. coli-△BRJ-SK (Comparative Example 3) were inoculated into 50 mL LB broth containing 100 μg / mL kanamycin and 50 μg / mL streptomycin, respectively. Then, they were cultured overnight at 37 °C and 200 rpm to obtain activated E. coli-△BRJA-SKA, E. coli-△BRJ-SKA, E. coli-△BRJA-SK, and E. coli-△BRJ-SK.

[0080] Activated E. coli-△BRJA-SKA, E. coli-△BRJ-SKA, E. coli-△BRJA-SK, and E. coli-△BRJ-SK bacterial cultures were inoculated at a rate of 1% (1 mL) into 100 mL of fermentation medium containing the corresponding antibiotics. The cultures were incubated at 37℃ and 200 rpm for 48 h. The OD values ​​of each strain were measured at 8, 16, 24, 32, 40, and 48 h. The growth status of each strain was determined based on the OD values. The results are as follows: Figure 3 As shown.

[0081] Depend on Figure 3 It can be seen that the engineered bacterium E. coli-△BRJA-SKA, which knocks out the aaS gene and overexpresses the mlaY and mlaZ genes, exhibits superior growth at all time points compared to the engineered bacteria E. coli-△BRJ-SKA, E. coli-△BRJA-SK, and E. coli-△BRJ-SK of comparative examples 1-3. Among them, E. coli-△BRJA-SKA has the highest OD600 value, reaching 4.66, indicating excellent growth.

[0082] Example 3

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

[0084] The genetically engineered bacteria E. coli-△BRJA-SKA with high decanoic acid tolerance 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 inoculated into 50 mL of LB liquid 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 cultures were inoculated at a rate of 1% (1 mL) into 100 mL of fermentation medium containing the corresponding antibiotics. The cultures were incubated at 37 °C and 200 rpm for 2 h until the bacterial count reached OD600 = 1. Then, α-lactose was added to a final concentration of 14 mM, and the cultures were induced for 55 min to obtain the fermentation cultures of E. coli-△BRJA-SKA, E. coli-△BRJ-SKA, E. coli-△BRJA-SK, and E. coli-△BRJ-SK.

[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. The initial concentration of decanoic acid was 0.5 g / L. Then, every 8 h, samples were taken from the fermentation cultures of each strain and 0.5 g / L of substrate decanoic acid was added. The whole-cell catalytic reaction was carried out for a total time of 48 h to prepare trans-2-decenoic acid.

[0087] The substrates and products of the above whole-cell catalytic reaction were determined using a GC-2030 gas chromatograph (Nippon Tsutomu). The results are as follows: Figure 4 As shown. Figure 4 The left vertical axis represents the concentration of trans-2-decenoic acid, corresponding to a bar chart; the right vertical axis represents the percentage concentration of the substrate, corresponding to a line chart.

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

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

[0090] In summary, this invention utilizes CRISPR-Cas9 gene editing technology to knock out the aaS gene in *E. coli*, thereby reducing the level of medium-chain fatty acids incorporated into the cell membrane and improving the integrity of cell membrane lipids. Simultaneously, overexpression of the *mlaZ* and *mlaY* genes derived from *Pseudomonas aeruginosa* strengthens the membrane lipid strength of the engineered *E. coli* strain, resulting in better robustness during fermentation with high concentrations of decanoic acid and greater tolerance to high concentrations of decanoic acid. The OD600 value of *E. coli-△BRJA-SKA* reached a maximum of 4.66, indicating good growth. When decanoic acid was fed to a maximum concentration of 3.0 g / L, the yield of trans-2-decenoic acid reached 1.47 g / L, with a conversion rate of 49.13%, significantly improving the yield of trans-2-decenoic acid.

[0091] The embodiments described above are merely preferred implementations 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 within the protection scope of the present invention.

Claims

1. A genetically engineered bacterium with high decanoic acid tolerance, E. coli-△BRJA-SKA, characterized in that, The genetically engineered bacterium E.coli-△BRJA-SKA was developed using Escherichia coli as the starting strain, with the aaS gene knocked out and the mlaZ and mlaY genes overexpressed. Wherein, 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. The starting strain was the engineered Escherichia coli BL21 (DE3)-△fadB-△fadR-△fadJ; The method for constructing the genetically engineered bacterium E. coli-△BRJA-SKA with high decanoic acid tolerance includes the following steps: (1) The aaS gene in the engineered Escherichia coli BL21 (DE3)-△fadB-△fadR-△fadJ was knocked out using CRISPR / Cas9 technology. After the knockout was completed, the plasmid was eliminated to obtain the engineered Escherichia coli BL21 (DE3)-△fadB-△fadR-△fadJ-ΔaaS. (2) The nucleotide sequences of the mlaY gene, mlaZ gene, and pelb signal peptide were artificially synthesized; then, using a mixture of pelb signal peptide and mlaY gene as a template, PCR amplification was performed using pelb-mlaY-F / R primers to obtain the pelb-mlaY sequence; using a mixture of pelb signal peptide and mlaZ gene as a template, PCR amplification was performed using pelb-mlaZ-F / R primers to obtain the pelb-mlaZ sequence; the plasmid vector pET22b, the pelb-mlaY sequence, and the pelb-mlaZ sequence were double-digested and ligated together to obtain the plasmid pET22b-pelb-mlaY-pelb-mlaZ; (3) Constructing plasmid pET28a-sumo- Ct ydiI and plasmid pCDFDuet-1- Ma MACS - Pp fadE ; (4) The plasmid pET28a-sumo- Ct ydiI plasmid pCDFDuet-1- Ma MACS - Pp fadE The plasmid pET22b-pelb-mlaY-pelb-mlaZ was co-transformed into the engineered E. coli strain BL21 (DE3)-△fadB-△fadR-△fadJ-ΔaaS competent cells. Positive recombinants were screened using a medium containing kanamycin, ampicillin, and streptomycin to obtain the genetically engineered strain E. coli-△BRJA-SKA with high decanoic acid tolerance.

2. The genetically engineered bacterium with high decanoic acid tolerance as described in claim 1, wherein 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′.

3. The genetically engineered bacterium with high decanoic acid tolerance as described in claim 1, 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.

4. The genetically engineered bacterium with high decanoic acid tolerance as described in claim 1, characterized in that, In step (4), the culture medium containing kanamycin, ampicillin and streptomycin is formulated as follows: 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.

5. The application of the genetically engineered bacteria with high decanoic acid tolerance as described in claim 1 in the production of trans-2-decenoic acid.

6. A method for preparing trans-2-decenoic acid, characterized in that, Includes the following steps: The genetically engineered E. coli-△BRJA-SKA with high decanoic acid tolerance as described in claim 1 was inoculated into 50 mL of LB liquid 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; The activated E. coli-△BRJA-SKA bacterial culture was inoculated into the fermentation medium at an inoculum rate of 1% and cultured at 37℃ and 200 r / min until the bacterial count OD600 = 0.8~1.

2. α-lactose with a final concentration of 14 mM was added and the culture was induced for 50~60 min to obtain the fermentation culture. Then, decanoic acid was added to the fermentation culture for whole-cell catalysis to prepare trans-2-decenoic acid.

7. The method for preparing trans-2-decenoic acid as described in claim 6, characterized in that, The fermentation medium is formulated as follows: 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.

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