A method for constructing an engineered bacterium for preparing high-yield royal jelly acid precursor trans-2-decenoic acid

Abstract

The application provides a construction method of preparing high-yield royal jelly acid precursor trans-2-decenoic acid engineering bacteria, belongs to the technical field of biological fermentation, and specifically comprises the following steps: as a service Knocking out genes in the E. coli engineering bacteria BL21 Delta FadB, R and J to obtain the E. coli engineering bacteria BL21 Delta FadB, R and J Delta as a service ; genes in the E. coli engineering bacteria BL21 Delta FadB, R and J Delta ftsQ are continuously knocked out, plasmid elimination is carried out after the knocking out is completed, and the E. coli engineering bacteria BL21 Delta FadB, R and J Delta as a service Delta ftsQ are obtained; the transformation method is used to transform the recombinant plasmid pCDFDuet-1-MaMACS-PpFadE and the recombinant plasmid pET28a-SUMO-ctYdiI, and the E. coli engineering bacteria BL21 Delta FadB, R and J Delta as a service Delta ftsQ-MEI are obtained, and the engineering bacteria provided by the application effectively promote the synthesis of decanoic acid into trans-2-decenoic acid.

Description

Technical Field

[0001] This invention belongs to the field of bio-fermentation technology, specifically relating to a method for constructing an engineered bacterium that produces high-yield royal jelly acid precursor trans-2-decenoic acid. Background Technology

[0002] Royal jelly acid (10-HDA) is a medium-chain fatty acid derivative containing both double and unsaturated bonds, with the molecular formula C1. 10 H 18 O3. With a relative molecular mass of 186.25 and a melting point of 64℃, royal jelly acid has been found only in royal jelly in nature, at a concentration of only 1.4%-2.4%. Trans-2-decenoic acid is an important intermediate in the biosynthesis of royal jelly acid; therefore, its biosynthesis will lay the foundation for the future biosynthesis of 10-HDA. Besides its use in the synthesis of royal jelly acid, trans-2-decenoic acid is also a chemical intermediate in the synthesis of important drugs and can be used as a ligand to synthesize desired anisotropic nanocrystalline materials. In addition to these applications, trans-2-decenoic acid can also be used to produce fragrances and flavorings. Therefore, trans-2-decenoic acid, as both an intermediate and final product, has important functions and significant commercial value.

[0003] Currently, the chemical synthesis of trans-2-decenoic acid involves reacting octaldehyde and malonic acid, or octaldehyde and dibromoacetic acid, with extraction by organic solvents. However, chemical synthesis of this target substance using various raw materials generally suffers from drawbacks such as severe environmental pollution, low reaction controllability, and low safety. Therefore, compared to traditional methods, biocatalytic synthesis of the target substance has attracted widespread attention due to its advantages, including high safety, strong specificity, and low environmental pollution. The current bottleneck in the biosynthesis of royal jelly acid is that the substrate decanoic acid and fatty acid products have a certain inhibitory effect on the fermentation strain, making it difficult to increase yield.

[0004] Current gene editing technologies rely on the synthesis of DNA sequence-specific binding protein modules for targeted gene editing. This step is cumbersome, time-consuming, and has a long experimental cycle, resulting in low gene editing efficiency.

[0005] Chinese patent document CN114958700A (application number: 202210510742.3) discloses an engineered strain of *E. coli* and its applications. This patent document knocks out the FadB, FadR, and FadJ genes using the RED recombination method. Chinese patent document CN110684794A (application number: 201911038194.3) discloses a method for preparing α- and β-unsaturated fatty acids from fatty acids using engineered *E. coli* strains. This patent document's method involves overexpressing the FadE, FadD, and ydii genes while simultaneously knocking out the FadR and FadB genes. This invention blocks the effects of β-oxidation and the regulation of cellular regulators, optimizing the trans-2-decenoic acid expression pathway. Chinese patent document CN109402182A (application number: 201811126048.1) discloses a method for preparing 10-hydroxy-2-decenoic acid using engineered *E. coli* 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. Chinese patent document CN113106109B (application number: 202110211118.9) discloses a mutant enzyme CYP153A M228L and a two-step method for biosynthesizing 10-hydroxy-2-decenoic acid using decanoic acid as a raw material. This patent constructs a 10-hydroxy-2-decenoic acid synthesis pathway by modifying the CYP153A protein. The technical solutions and effects disclosed in existing patent documents are significantly different from those of this invention. Summary of the Invention

[0006] To address the shortcomings of existing technologies, this invention provides a method for constructing engineered bacteria that produce high-yield royal jelly acid precursor trans-2-decenoic acid.

[0007] The purpose of this invention is to provide an engineered strain capable of producing high levels of trans-2-decenoic acid and its applications.

[0008] The technical solution of the present invention is as follows: A method for constructing an engineered strain that produces high levels of trans-2-decenoic acid includes the following steps: (1) The engineered Escherichia coli BL21 ΔFadB, R, J aaS Gene knockout was performed using CRISPR / Cas9 technology. aaS The nucleotide sequence of the gene is shown in SEQ ID NO.15, yielding the engineered Escherichia coli BL21 ΔFadB, R, JΔ. aaS ; (2) The engineered Escherichia coli BL21 ΔFadB, R, JΔ from step (1) aaS As the starting strain, continue to use it. ftsQGene knockout was performed using CRISPR / Cas9 technology. ftsQ The nucleotide sequence of the gene is shown in SEQ ID NO.16. After knockout, plasmid elimination was performed to obtain engineered E. coli strains BL21ΔFadB, R, and JΔ. aaS Δ ftsQ ; (3) The engineered Escherichia coli BL21 ΔFadB, R, JΔ obtained in step (2) aaS Δ ftsQ The recombinant plasmids pCDFDuet-1-MaMACS-PpFadE and pET28a-SUMO-ctYdiI were transformed into Escherichia coli engineered strains BL21 ΔFadB, R, and JΔ. aaS Δ ftsQ-MEI .

[0009] According to a preferred embodiment of the present invention, the method for gene knockout using CRISPR / Cas9 technology in steps (1) and (2) includes the following: ① A gene containing 20 bases in sgRNA was designed using the website CRISPRRGEN Tools (rgenome.net); The nucleotide sequence of this gene segment is shown in SEQ ID NO.1; ②Design primers to amplify the target gene by PCR using the genome of primitive Escherichia coli BL21 as a template. aaS upstream and downstream Homologous arms; aaS The nucleic acid sequences of the upstream primer of the upstream homologous arm of the gene are shown in SEQ ID NO.2, the nucleic acid sequences of the downstream primers are shown in SEQ ID NO.3, the nucleic acid sequences of the upstream primers of the downstream homologous arm are shown in SEQ ID NO.4, and the nucleic acid sequences of the downstream primers are shown in SEQ ID NO.5. ③ Use overlap PCR to ligate the upstream and downstream homologous arms of the previously prepared homologous fragments to form DonorDNA- aaS Linear segments; ④ Construct plasmids expressing sgRNA The corresponding N20 sequence was determined. The nucleic acid sequence of the upstream primer is shown in SEQ ID NO.6, and the nucleic acid sequence of the downstream primer is shown in SEQ ID NO.7. Using pTargetF plasmid as a template, pTargetF- was amplified by reverse PCR. N20 / aaS The linear fragment was recovered and purified using a kit to obtain pTargetF- carrying the target gene N20 sequence. N20 / aaS Linear gene fragments; pTargetF- N20 / aaS Linear gene fragmentation was transfected into DH5α competent cells and circularized to produce pTargetF- N20 / aaS Plasmids were obtained and extracted using reagents; ⑤ The engineered Escherichia coli BL21 ΔFadB, R, J cells were made into competent cells and transformed with pCAS9 plasmid. Subsequently, the engineered Escherichia coli BL21 ΔFadB, R, J cells containing pCAS9 plasmid were prepared into electrotransfer competent cells. ⑥ Co-transformation of recombinant sgRNA plasmid with Donor DNA: The prepared pTargetF- N20 / aaS Plasmids and DonorDNA- aaS Transformed Escherichia coli engineered strain BL21 ΔFadB, R, J competent cells containing pCAS9 plasmid and screened for positive clones; ⑦ Eliminate pTargetF plasmid to obtain engineered E. coli strain BL21 ΔFadB, R, JΔ aaS The cells were then converted into electrocompetent cells, and the ftsQ gene was knocked out using the same method as described above. Finally, the Cas9 plasmid was eliminated to obtain engineered E. coli strain BL21 ΔFadB, R, JΔ. aaS Δ ftsQ ; ftsQ During the gene knockout process, the nucleotide sequence of the N20 gene in the designed sgRNA is shown in SEQ ID NO.8; the nucleic acid sequence of the upstream primer of the upstream homologous arm is shown in SEQ ID NO.9, and the nucleic acid sequence of the downstream primer is shown in SEQ ID NO.10; the nucleic acid sequence of the upstream primer of the downstream homologous arm is shown in SEQ ID NO.11, and the nucleic acid sequence of the downstream primer is shown in SEQ ID NO.12; in the plasmid expressing sgRNA, the nucleic acid sequence of the upstream primer is shown in SEQ ID NO.13, and the nucleic acid sequence of the downstream primer is shown in SEQ ID NO.14.

[0010] The above-mentioned engineered Escherichia coli strains BL21 ΔFadB, R, JΔ aaS Δ ftsQ-MEI Applications in the production of trans-2-decenoic acid.

[0011] A method for high-yield trans-2-decenoic acid includes the following steps: The above-mentioned engineered Escherichia coli strains BL21ΔFadB, R, JΔ aaS Δ ftsQ-MEIWhen the bacterial cell count reaches OD600 = 0.8-1.2, inoculate the culture medium at a volume fraction of 5-10% and culture until OD600 = 0.8-1.2. Then, add lactose to a final concentration of 0.5-5 g / L for induction. The induction conditions are 28-30 ℃, 150-500 r / min, and 2-2.5 h to obtain the fermentation culture. Then, add decanoic acid to the fermentation culture for whole-cell catalysis to obtain trans-2-decenoic acid.

[0012] According to a preferred embodiment of the present invention, in the method, the engineered Escherichia coli BL21ΔFadB, R, JΔ aaS Δ ftsQ- MEI Inoculate into LB liquid medium and incubate at 35-37℃ until the bacterial OD600 is 0.8-1.2.

[0013] According to a preferred embodiment of the present invention, the induction conditions are as follows: after cooling the cultured bacterial solution to 30°C, lactose with a final concentration of 2-5 g / L is added, and the culture is carried out at 150-500 r / min for 2-2.5 h.

[0014] According to a preferred embodiment of the present invention, the fermentation culture medium components in the method include the following: Sodium chloride 8-12 g / L, peptone 8-12 g / L, yeast extract 2-6 g / L, kanamycin 40-60 μg / mL, ampicillin 50-150 μg / mL and streptomycin 20-60 μg / mL, balance water, pH 7.0-7.2.

[0015] More preferably, in the method, the fermentation culture medium components include the following: Sodium chloride 10 g / L, peptone 10 g / L, yeast extract 5 g / L, kanamycin 50 μg / mL, ampicillin 100 μg / mL and streptomycin 40 μg / mL, balance water, pH 7.0-7.2.

[0016] According to a preferred embodiment of the present invention, in the method, decanoic acid is added to the fermentation culture and reacted at 28-37°C for 6-60 hours to obtain trans-2-decenoic acid.

[0017] More preferably, in the method, the final concentration of the added decanoic acid is 0.5-6 g / L.

[0018] The aforementioned engineered Escherichia coli strains BL21ΔFadB, R, and J, and the method for constructing these strains, are existing technologies and have been disclosed in patent document CN113106109A, specifically in paragraphs

[00197] -

[0221] . The patent title is: "A mutant enzyme CPY153M228L and its application in the synthesis of 10-hydroxy-2-decenoic acid," application number: 202110211118.9, publication date: July 13, 2021.

[0019] The construction and transformation of the above-mentioned recombinant plasmids pCDFDuet-1-MaMACS-PpFadE and pET28a-SUMO-ctYdiI are existing technologies and have been disclosed in Chinese patent document CN113106109A, specifically refer to

[0048] -

[0065] .

[0020] Beneficial effects 1. The Escherichia coli BL21 ΔFadB, R, JΔ provided by this invention aaS Δ ftsQ-MEI During fermentation, the engineered strains significantly improved the tolerance to the substrate decanoic acid, effectively promoting the synthesis of trans-2-decenoic acid from decanoic acid.

[0021] 2. The method for knocking out target genes in Escherichia coli provided by this invention has higher knockout efficiency and is more convenient to operate. Attached Figure Description

[0022] Figure 1 To knock out E. coli aaS The agarose gel electrophoresis image after gene knockout shows that the original gene length was 3008 bp, and the knockout length should be 856 bp. In the diagram, lane M is the marker; lanes 1-3 are... aaS Gene bands.

[0023] Figure 2 To knock out E. coli ftsQ The agarose gel electrophoresis image after gene knockout shows that the original gene length was 1716 bp, and the knockout length should be 885 bp. In the diagram, lane M is the marker; lane 1 is... ftsQ Gene bands.

[0024] Figure 3 For Escherichia coli BL21 ΔFadB, R, J- MEI With Escherichia coli BL21 ΔFadB, R, JΔ aaS Δ ftsQ - MEI Escherichia coli BL21 ΔFadB, R, JΔ aaS Δ paL - MEIEscherichia coli BL21 ΔFadB, R, JΔ ftsQ ΔpaL - MEI Comparison of growth under different concentrations of decanoic acid.

[0025] Figure 4 For Escherichia coli BL21 ΔFadB, R, J- MEI With Escherichia coli BL21 ΔFadB, R, JΔ aaS Δ ftsQ - MEI Escherichia coli BL21 ΔFadB, R, JΔ aaS Δ paL - MEI Escherichia coli BL21 ΔFadB, R, JΔ ftsQ Δ paL - MEI Comparison of yields of catalytic decanoic acid to trans-2-decenoic acid. Detailed Implementation

[0026] The present invention will be further described below with reference to embodiments, but the scope of protection of the present invention is not limited thereto.

[0027] All details not described in the embodiments are based on existing technology in the field.

[0028] The liquid fermentation medium consists of: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract, 50 μg / mL kanamycin, 100 μg / mL ampicillin and 40 μg / mL streptomycin, with the remainder being water, and a pH of 7.0-7.2.

[0029] The composition of LB liquid medium is: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract, and the balance is water, pH 7.0-7.2.

[0030] n-decanoic acid was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.

[0031] Example 1 A method for constructing an engineered strain that produces high levels of trans-2-decenoic acid includes the following steps: (1) Constructing engineered Escherichia coli strains BL21 ΔFadB, R, J; the engineered Escherichia coli strains BL21 ΔFadB, R, J and the method of constructing the strains are existing technologies and have been disclosed in patent document CN113106109A, specifically referring to paragraphs

[00197] -

[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; (2) The engineered Escherichia coli BL21 ΔFadB, R, J aaS Gene knockout was performed using CRISPR / Cas9 technology. aaS The nucleotide sequence of the gene is shown in SEQ ID NO.15, yielding the engineered Escherichia coli BL21 ΔFadB, R, JΔ. aaS ; (3) The engineered Escherichia coli BL21 ΔFadB, R, JΔ from step (2) aaS As the starting strain, the ftsQ gene was further knocked out using CRISPR / Cas9 technology. The nucleotide sequence of the ftsQ gene is shown in SEQ ID NO. 16. After knockout, plasmid elimination was performed to obtain engineered Escherichia coli strains BL21ΔFadB, R, JΔ. aaS Δ ftsQ .

[0032] The specific methods for gene knockout using CRISPR / Cas9 technology in steps (2) and (3) are as follows: ① A gene containing 20 bases in sgRNA was designed using the website CRISPRRGEN Tools (rgenome.net); The nucleotide sequence of this gene segment is shown in SEQ ID NO.1; ②Design primers to amplify the target gene by PCR using the genome of primitive Escherichia coli BL21 as a template. aaS upstream and downstream Homologous arms; aaS The nucleic acid sequences of the upstream primer for the upstream homologous arm of the gene are shown in SEQ ID NO.2, the nucleic acid sequences of the downstream primers are shown in SEQ ID NO.3, the nucleic acid sequences of the upstream primers for the downstream homologous arm are shown in SEQ ID NO.4, and the nucleic acid sequences of the downstream primers are shown in SEQ ID NO.5. The PCR amplification system is as follows, with a total volume of 50 μL: 2.0 μL of 100 μM upstream primer, 2.0 μL of 100 μM downstream primer, 2.0 μL of template, 25 μL of 5 U / μL phanta enzyme, and 19 μL of ddH2O; The PCR amplification conditions are as follows: Pre-denaturation at 98℃ for 3 min; denaturation at 95℃ for 10 s, annealing at 62℃ for 5 s, extension at 72℃ for 5 s, cycled 30 times, and finally extended at 72℃ for 1 min.

[0033] ③ Use overlap PCR to ligate the upstream and downstream homologous arms of the previously prepared homologous fragments to form DonorDNA- aaSLinear segments; The PCR amplification system is as follows, with a total volume of 50 μL: 100mM aaS 2.0 μL of upstream primer for the upstream homologous arm of the gene, 100 μM aaS 2.0 μL of the downstream primer of the gene's downstream homologous arm, 2.0 μL each of the upstream and downstream homologous arms as templates, 23 μL of 5 U / μL phanta enzyme, and 19 μL of ddH2O; The PCR amplification conditions are as follows: Pre-denaturation at 98℃ for 3 min; denaturation at 95℃ for 10 s, annealing at 62℃ for 5 s, extension at 72℃ for 5 s, cycled 30 times, and finally extended at 72℃ for 1 min.

[0034] ④ Construct plasmids expressing sgRNA The corresponding N20 sequence was determined. The nucleic acid sequence of the upstream primer is shown in SEQ ID NO.6, and the nucleic acid sequence of the downstream primer is shown in SEQ ID NO.7. Using pTargetF plasmid as a template, pTargetF- was amplified by reverse PCR. N20 / aaS The linear fragment was recovered and purified using a kit to obtain pTargetF- carrying the target gene N20 sequence. N20 / aaS Linear gene fragments.

[0035] The PCR amplification system is as follows, with a total volume of 50 μL: 2.0 μL of 100 μM upstream primer, 2.0 μL of 100 μM downstream primer, 2.0 μL each of pTargetF plasmid as template, 25 μL of 5 U / μL phanta enzyme, and 19 μL of ddH2O; The PCR amplification conditions are as follows: Pre-denaturation at 98℃ for 3 min; denaturation at 95℃ for 10 s, annealing at 62℃ for 5 s, extension at 72℃ for 15 s, cycled 30 times, followed by complete extension at 72℃ for 1 min.

[0036] pTargetF- N20 / aaS Linear gene fragmentation was transfected into DH5α competent cells and circularized to produce pTargetF- N20 / aaS Plasmids were extracted using reagents.

[0037] ⑤ The engineered Escherichia coli strains BL21 ΔFadB, R, and J were made into competent cells and transformed with the pCAS9 plasmid (this plasmid is temperature-sensitive and the culture temperature is 30℃). Subsequently, the strains containing the pCAS9 plasmid were prepared into electrotransfer competent cells.

[0038] Single colonies were inoculated into LB+Kan (kanamycin 50 μg / mL) liquid medium and incubated overnight at 30 °C for 200 rpm. The cultured BL21ΔFadB, R, and J bacterial cultures carrying the pCas plasmid were then inoculated into 40 mL of LB+Kan (kanamycin 50 μg / mL) medium, and arabinose was added to a final concentration of 60 mM. The amount of bacterial culture added was adjusted to achieve an OD value between 0.04 and 0.1. The cultures were incubated at 30 °C for 200 rpm until the logarithmic phase was reached, at which point the culture was stopped, indicating the induction was complete. Subsequently, the induced *E. coli* engineered bacteria BL21ΔFadB, R, and J carrying the pCas plasmid were used to prepare competent cells.

[0039] ⑥ Co-transformation of recombinant sgRNA plasmid with Donor DNA The previously prepared pTargetF- N20 / aaS Plasmids and Donor DNA- aaS Thaw on ice, and add approximately 100 ng of pTargetF- to the electroshock cup in the following order: N20 / aaS Plasmid, 400 ng Donor DNA aaS 50 μL of prepared *E. coli* BL21 ΔFadB, R, J competent cells carrying pCas plasmid and induced were used for electroporation. After transformation, the cells were revived and plated.

[0040] ⑦ Screening for positive clones by colony PCR.

[0041] The original gene length was 3008 bp, and the knockout length should be 856 bp. Electrophoresis results are shown below. Figure 1 .

[0042] ⑧ Eliminate pTargetF plasmid to obtain engineered E. coli strain BL21 ΔFadB, R, JΔ aaS The cells were then converted into electrocompetent cells, and the ftsQ gene was knocked out using the same method as described above. Finally, the Cas9 plasmid was eliminated to obtain the desired engineered E. coli strain BL21ΔFadB, R, JΔ. aaS Δ ftsQ .

[0043] ftsQDuring the gene knockout process, the nucleotide sequence of the N20 gene in the designed sgRNA is shown in SEQ ID NO. 8; the nucleic acid sequence of the upstream primer for the upstream homologous arm is shown in SEQ ID NO. 9, and the nucleic acid sequence of the downstream primer is shown in SEQ ID NO. 10; the nucleic acid sequence of the upstream primer for the downstream homologous arm is shown in SEQ ID NO. 11, and the nucleic acid sequence of the downstream primer is shown in SEQ ID NO. 12; in the plasmid expressing the sgRNA, the nucleic acid sequence of the upstream primer is shown in SEQ ID NO. 13, and the nucleic acid sequence of the downstream primer is shown in SEQ ID NO. 14. The original length of the designed gene was 1716 bp, and the knockout length should be 885 bp. The electrophoresis results are shown in […]. Figure 2 .

[0044] Comparative Example 1 The engineered Escherichia coli strains BL21ΔFadB, R, JΔ obtained in Example 1 aaS Using the starting strain, another cell division-controlling strain was knocked out using the same method as in Example 1. paL Genes were obtained to produce engineered E. coli strains BL21 ΔFadB, R, JΔ aaS Δ paL The paL The nucleotide sequence of the gene is shown in SEQ ID NO.17.

[0045] Comparative Example 2 Using the engineered Escherichia coli strains BL21 ΔFadB, R, and J mentioned in Example 1 as the starting strains, the ΔFadB, R, and J strains were first knocked out using the same method as in Example 1. ftsQ, We obtain BL21 ΔFadB, R, JΔ ftsQ, Then, using the same method as in Example 1, knock out another cell division control factor. paL Genes were obtained from E. coli BL21 ΔFadB, R, JΔ ftsQ Δ paL The paL The nucleotide sequence of the gene is shown in SEQ ID NO.17.

[0046] Example 2 The construction and transformation of the recombinant plasmids pCDFDuet-1-MaMACS-PpFadE and pET28a-SUMO-ctYdiI are existing technologies, disclosed in Chinese patent document CN113106109A. Specifically, referring to sections

[0048] -

[0064] , the recombinant plasmids pCDFDuet-1-MaMACS-PpFadE and pET28a-SUMO-ctYdiI were transformed into the engineered Escherichia coli strains BL21 ΔFadB, R, J, Example 1, Comparative Example 1, and Comparative Example 2, respectively, according to existing methods. The transformed strains were then inoculated into LB liquid medium containing 50 μg / mL kanamycin, 100 μg / mL ampicillin, and 40 μg / mL streptomycin, respectively, to obtain engineered Escherichia coli strains BL21 ΔFadB, R, J, and [other strains]. -MEI、 Engineered Escherichia coli BL21 ΔFadB, R, JΔ aaS Δ ftsQ-MEI BL21 ΔFadB、R、JΔ aaS Δ paL-MEI and BL21 ΔFadB, R, JΔ ftsQ Δ paL-MEI .

[0047] Example 1 Example 2 constructed engineered Escherichia coli strains BL21ΔFadB, R, JΔ aaS Δ ftsQ-MEI The growth under different concentrations of decanoic acid was investigated using the following steps: Take five 50ml bottles of LB medium and add decanoic acid to final concentrations of 0.5g / L, 1g / L, 2.5g / L, 3g / L, and 5g / L, respectively. Then add 800μL of engineered Escherichia coli BL21 ΔFadB, R, and JΔ, respectively. aaS Δ ftsQ-MEI The bacterial culture was incubated at 37℃ and 200 r / min for 12 h, and then the OD value was measured using a spectrophotometer. The results are shown in Table 1.

[0048] Table 1 substrate concentration g / L OD value 0.5 12.3 1 11.5 2.5 10.7 3 10.2 5 9.6 Comparative Example 3 Measurement of engineered Escherichia coli BL21ΔFadB, R, J -MEI The growth under different concentrations of decanoic acid followed the same experimental procedure as in Example 1, and the test results are shown in Table 2.

[0049] Table 2 substrate concentration g / L OD value 0.5 11.7 1 7.9 2.5 6.3 3 5.8 5 3.1 Comparative Example 4 Measurement of engineered Escherichia coli BL21ΔFadB, R, JΔ ftsQ Δ paL-MEI The growth under different concentrations of decanoic acid followed the same experimental procedure as in Example 1, and the test results are shown in Table 3.

[0050] Table 3 substrate concentration g / L OD value 0.5 11.82 1 6.6 2.5 4.1 3 3.5 5 2.8 Comparative Example 5 Measurement of engineered Escherichia coli BL21ΔFadB, R, JΔ aaS Δ paL-MEI The growth under different concentrations of decanoic acid followed the same experimental procedure as in Example 1, and the test results are shown in Table 4.

[0051] Table 4 substrate concentration g / L OD value 0.5 10.12 1 7.35 2.5 6.5 3 4.8 5 4.2 Example 2 In a 1L fermenter, engineered Escherichia coli BL21 ΔFadB, R, JΔ was used. aaS Δ ftsQ-MEI The fermentation process to produce trans-2-decenoic acid follows these steps: Inoculate 50 μl of bacterial culture into 50 ml of LB medium and incubate at 37°C until the OD600 of the bacterial culture is 0.8-1.2 to obtain the seed culture required for fermentation.

[0052] 50 ml of seed culture was inoculated into a fermenter containing 800 mL of fermentation medium and cultured at 37 °C and 500 r / min for 2 h. The temperature was lowered by 30 °C beforehand and cultured until OD600 = 0.8-1.2.

[0053] The fermentation medium consists of: 10 g / L sodium chloride, 10 g / L peptone, 5 g / L yeast extract, 50 μg / mL kanamycin, 100 μg / mL ampicillin and 40 μg / mL streptomycin, with the remainder being water, pH 7.0-7.2.

[0054] 40 ml of lactose (100 g / L) was added as an inducer (final concentration 5 g / L), and the mixture was induced for 2 h at 30 °C and 500 rpm. Then, 701 μl of decanoic acid (600 g / L) was added as a substrate (final concentration 1.5 g / L), and the mixture was cultured at 37 °C and 500 rpm. The substrate was fed at a rate of 0.5 g / L every 6 h. A 10 ml sample was taken every 6 h, based on the time of decanoic acid addition. The results are shown in Table 5.

[0055] Table 5 Time h 6 12 18 24 30 36 42 48 54 60 trans-2-decenoic acid yield (g / L) 0.47 0.80 1.35 1.88 2.32 2.86 3.63 3.76 4.32 4.86 Comparative Example 6 In a 1L fermenter, engineered Escherichia coli BL21 ΔFadB, R, J was used. -MEIFermentation was carried out to produce trans-2-decenoic acid. The experimental procedure was the same as in Example 2. The test results are shown in Table 6.

[0056] Table 6 Time h 6 12 18 24 30 36 42 48 54 60 trans-2-decenoic acid yield (g / L) 0.31 0.60 1.28 1.78 2.16 2.45 2.82 3.15 3.24 3.59 Comparative Example 7 In a 1L fermenter, engineered Escherichia coli BL21 ΔFadB, R, JΔ was used. ftsQ Δ paL-MEI Fermentation was carried out to produce trans-2-decenoic acid. The experimental procedure was the same as in Example 2. The test results are shown in Table 7.

[0057] Table 7 Time h 6 12 18 24 30 36 42 48 54 60 trans-2-decenoic acid yield (g / L) 0.29 0.69 1.33 1.66 1.79 1.97 2.23 2.56 2.89 3.43 Comparative Example 8 In a 1L fermenter, engineered Escherichia coli BL21 ΔFadB, R, JΔ was used. aaS Δ paL-MEI Fermentation was carried out to produce trans-2-decenoic acid. The experimental procedure was the same as in Example 2. The test results are shown in Table 8.

[0058] Table 8 Time h 6 12 18 24 30 36 42 48 54 60 trans-2-decenoic acid yield (g / L) 0.42 0.75 1.35 1.72 1.87 1.96 2.78 2.96 3.7 3.76 As can be seen from the experimental results of Example 1 and Comparative Examples 3, 4, and 5, see Figure 3 Escherichia coli BL21 ΔFadB, R, JΔ aaS Δ ftsQ-MEI The growth was best under different concentrations of decanoic acid, as can be seen from the experimental results of Example 2 and Comparative Examples 6, 7, and 8. Figure 4 After 60 hours of fermentation, E. coli BL21 ΔFadB, R, JΔ aaS Δ ftsQ-MEI The yield of trans-2-decenoic acid produced by consuming decanoic acid can reach 4.86 g / L, with a conversion rate of 97.2%. Therefore, it can be concluded that knocking out... aaS Genes and ftsQ The gene significantly improved the resistance of engineered E. coli to decanoic acid and significantly increased the yield of trans-2-decenoic acid.

[0059] The Escherichia coli BL21ΔFadB, R, JΔ provided by this invention aaS Δ ftsQ-MEI During fermentation, the engineered strains significantly improved the tolerance to the substrate decanoic acid, effectively enhancing the synthesis of trans-2-decenoic acid from decanoic acid.

Claims

1. A method for constructing an engineered strain that produces high levels of trans-2-decenoic acid, characterized in that, Includes the following steps: (1) The engineered Escherichia coli BL21 ΔFadB, R, J aaS Gene knockout was performed using CRISPR / Cas9 technology. aaS The nucleotide sequence of the gene is shown in SEQ ID NO.15, yielding the engineered Escherichia coli BL21 ΔFadB, R, JΔ. aaS ; (2) The engineered Escherichia coli BL21 ΔFadB, R, JΔ from step (1) aaS As the starting strain, continue to use it. ftsQ Gene knockout was performed using CRISPR / Cas9 technology. ftsQ The nucleotide sequence of the gene is shown in SEQ ID NO.

16. After knockout, plasmid elimination was performed to obtain engineered E. coli strain BL21 ΔFadB, R, JΔ. aaS Δ ftsQ ; (3) The engineered Escherichia coli BL21 ΔFadB, R, JΔ obtained in step (2) aaS Δ ftsQ The recombinant plasmids pCDFDuet-1-MaMACS-PpFadE and pET28a-SUMO-ctYdiI were transformed into Escherichia coli engineered strains BL21 ΔFadB, R, and JΔ. aaS Δ ftsQ-MEI .

2. The method as described in claim 1, characterized in that, In steps (1) and (2), the methods for gene knockout using CRISPR / Cas9 technology include the following: ① The N20 sequence in sgRNA was designed using CRISPR RGEN Tools; the nucleotide sequence of the N20 sequence is shown in SEQ ID NO.1; ②Design primers to amplify the target gene by PCR using the genome of primitive Escherichia coli BL21 as a template. aaS Upstream and downstream homologous arms; aaS The nucleic acid sequence of the upstream primer of the upstream homologous arm of the gene is shown in SEQ ID NO.2, the nucleic acid sequence of the downstream primer is shown in SEQ ID NO.3, the nucleic acid sequence of the upstream primer of the downstream homologous arm is shown in SEQ ID NO.4, and the nucleic acid sequence of the downstream primer is shown in SEQ ID NO.5; ③ Use overlap PCR to ligate the upstream and downstream homologous arms of the previously prepared homologous fragments to form DonorDNA- aaS Linear segments; ④ Construct plasmids expressing sgRNA The corresponding N20 sequence was determined. The nucleic acid sequence of the upstream primer is shown in SEQ ID NO.6, and the nucleic acid sequence of the downstream primer is shown in SEQ ID NO.

7. pTargetF plasmid was used as a template for reverse PCR amplification of pTargetF- N20 / aaS The linear fragment was recovered and purified using a kit to obtain pTargetF- carrying the target gene N20 sequence. N20 / aaS Linear gene fragments; pTargetF- N20 / aaS Linear gene fragmentation was transfected into DH5α competent cells and circularized to produce pTargetF- N20 / aaS Plasmids were obtained and extracted using reagents; ⑤ The engineered Escherichia coli BL21 ΔFadB, R, J cells were made into competent cells and transformed with pCAS9 plasmid. Subsequently, the engineered Escherichia coli BL21 ΔFadB, R, J cells containing pCAS9 plasmid were prepared into electrotransfer competent cells. ⑥ Co-transform the plasmid expressing sgRNA with Donor DNA: The prepared pTargetF- N20 / aaS Plasmids and DonorDNA- aaS Transformed Escherichia coli engineered strain BL21 ΔFadB, R, J competent cells containing pCAS9 plasmid and screened for positive clones; ⑦ Eliminate pTargetF plasmid to obtain engineered E. coli strain BL21 ΔFadB, R, JΔ aaS The cells were then converted into electrocompetent cells, followed by ftsQ gene knockout; finally, the Cas9 plasmid was eliminated to obtain engineered E. coli strains BL21ΔFadB, R, and JΔ. aaS Δ ftsQ ; ftsQ During the gene knockout process, the nucleotide sequence of the N20 sequence in the designed sgRNA is shown in SEQ ID NO.8; the nucleic acid sequence of the upstream primer of the upstream homologous arm is shown in SEQ ID NO.9, and the nucleic acid sequence of the downstream primer is shown in SEQ ID NO.10; the nucleic acid sequence of the upstream primer of the downstream homologous arm is shown in SEQ ID NO.11, and the nucleic acid sequence of the downstream primer is shown in SEQ ID NO.12; in the plasmid expressing sgRNA, the nucleic acid sequence of the upstream primer is shown in SEQ ID NO.13, and the nucleic acid sequence of the downstream primer is shown in SEQ ID NO.

14.

3. The engineered Escherichia coli BL21 ΔFadB, R, JΔ constructed according to any one of claims 1-2 aaS Δ ftsQ- MEI Applications in the production of trans-2-decenoic acid.

4. A method for high-yield trans-2-decenoic acid, characterized in that, Includes the following steps: The engineered Escherichia coli BL21 ΔFadB, R, JΔ constructed according to any one of claims 1-2 aaS Δ ftsQ-MEI Cultivate the cells until the OD600 reaches 0.8-1.

2. Inoculate the cells into the fermentation medium at a volume fraction of 5-10%. Cultivate until the OD600 reaches 0.8-1.

2. Add lactose to achieve a final concentration of 0.5-5 g / L in the fermentation medium. Induce lactose induction at 28-30 ℃, 150-500 r / min, for 2-2.5 h to obtain the fermentation culture. Then, add decanoic acid to the fermentation culture and perform whole-cell catalysis to obtain trans-2-decenoic acid.

5. The method as described in claim 4, characterized in that, In the method, the engineered Escherichia coli BL21 ΔFadB, R, JΔ aaS Δ ftsQ-MEI Inoculate the culture medium into LB liquid medium and culture at 35-37℃ until the bacterial OD600 is 0.8-1.

2. Then, inoculate the culture medium with a volume fraction of 5-10%.

6. The method as described in claim 4, characterized in that, In the method, after cooling the cultured bacterial solution to 30°C, lactose is added to make the final concentration of lactose in the fermentation medium reach 2-5 g / L. The induction conditions are 28-30°C and 150-500 r / min for 2-2.5 h.

7. The method as described in claim 4, characterized in that, In the method, the fermentation medium components include the following: Sodium chloride 8-12 g / L, peptone 8-12 g / L, yeast extract 2-6 g / L, kanamycin 40-60 μg / mL, ampicillin 50-150 μg / mL and streptomycin 20-60 μg / mL, balance water, pH 7.0-7.

2.

8. The method as described in claim 7, characterized in that, In the method, the fermentation medium components include the following: Sodium chloride 10 g / L, peptone 10 g / L, yeast extract 5 g / L, kanamycin 50 μg / mL, ampicillin 100 μg / mL and streptomycin 40 μg / mL, balance water, pH 7.0-7.

2.

9. The method as described in claim 4, characterized in that, In the method described, decanoic acid is added to the fermentation culture and reacted at 28-37℃ for 6-60 hours to obtain trans-2-decenoic acid.

10. The method as described in claim 9, characterized in that, In the method, decanoic acid is added to the fermentation culture to achieve a final concentration of decanoic acid of 0.5-6 g / L in the fermentation culture.

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