A genetically engineered bacterium and its application in preparation of 10-hydroxy-2-decenoic acid

Abstract

The present application relates to a kind of genetically engineered bacteria and its application in preparation 10-hydroxy-2-decenoic acid, belong to biological fermentation technical field.The genetically engineered bacteria of the present application includes fatty acyl-coa oxidase gene, fatty acyl-coa thioesterase gene and CYP153AM228L-CPR BM3 Fusion enzyme gene, the fatty acyl-coa oxidase gene is derived from Candida tropicalis, its nucleotide sequence is SEQ ID NO.3.The key enzyme of biosynthesis 10-hydroxy-2-decenoic acid is supplemented, integrated in the present application, fatty acid hydroxylase, fatty acyl-coa oxidase, fatty acyl-coa thioesterase is jointly transferred into host bacteria, so that each enzyme system in engineering bacteria synergistic effect, to achieve the purpose of decanoic acid as raw material one-step synthesis 10-hydroxy-2-decenoic acid.

Description

Technical Field

[0001] The invention relates to a genetically engineered bacterium and application thereof in preparing 10-hydroxy-2-decenoic acid, belonging to the technical field of biological fermentation. Background Art

[0002] 10-Hydroxy-2-decenoic acid (10-HDA) is a medium-chain fatty acid derivative containing both double bonds and hydroxyl groups, found only in royal jelly. 10-Hydroxy-2-decenoic acid (10-HDA) possesses numerous important physiological functions, including antibacterial, anti-inflammatory, immunomodulatory, anticancer, antitumor, and antioxidant properties. Therefore, it is widely used in the food, pharmaceutical, and cosmetic industries, boasting broad application prospects and extremely high economic value. The structure of this compound is as follows:

[0003]

[0004] To date, the main methods for obtaining 10-HDA include extraction, chemical synthesis, and microbial fermentation. A wide range of extraction methods exist, including ether extraction and ethanol extraction. However, the low content of 10-HDA in royal jelly makes it difficult to meet widespread market demand, and the extraction cost is high. Since the 1960s, various chemical synthesis pathways for 10-HDA have been developed and applied to production. These include the Doebmer condensation method, which uses 1,6-hexanediol as a substrate and achieves a 61% yield; and the Grignard condensation method, which uses γ-bromocrotonate ethyl ester and an organomagnesium compound as substrates to produce 10-HDA in a 70% yield. While all of these methods can produce 10-HDA, chemical synthesis has inherent drawbacks, such as demanding production conditions and the potential for contamination. Furthermore, raw materials are difficult to obtain, the synthetic route is long, and the yield is low. Furthermore, chemical synthesis can be subject to cis-trans isomerization issues, resulting in poor product homogeneity. Therefore, biosynthetic production of 10-HDA has become a new research direction.

[0005] Chinese patent document CN 109402182 A (application number 201811126048.1) discloses a method for preparing 10-hydroxy-2-decenoic acid using resting cells of Escherichia coli, which comprises the following steps: (1) constructing an Escherichia coli engineered bacterium containing the recombinant plasmid pET-28a-ydiI; (2) preparing induced cells from the Escherichia coli engineered bacterium containing the recombinant plasmid pET-28a-ydiI; (3) culturing the induced cells in a transformation medium to obtain resting cells, and then adding 10-hydroxydecanoic acid to the medium to prepare 10-hydroxy-2-decenoic acid. The synthesis pathway of 10-hydroxy-2-decenoic acid in this patent document is as follows: using 10-hydroxydecanoic acid as the reaction substrate, relying on the first two steps of β-oxidation to form a trans double bond, the coenzyme A is hydrolyzed by the Escherichia coli acyl-CoA thioesterase ydiI to release 10-hydroxy-2-decenoic acid.

[0006] Chinese patent document CN 109897870 A (application number 201910088897.0) discloses a method for preparing 10-hydroxy-2-decenoic acid using decanoic acid as a raw material using Escherichia coli engineered bacteria, the steps of which are as follows: (1) constructing a recombinant plasmid pBbB5K-P450 fusion enzyme, a recombinant plasmid pBbB5K-FadK, a recombinant plasmid pBbB5K-MCAD, and a recombinant plasmid pBbB5K-YdiI; (2) constructing a pBbB5K-ydiI-MCAD-FadK-P450 fusion enzyme combination plasmid; (3) transforming the fusion enzyme combination plasmid into Escherichia coli, screening, inducing and culturing to obtain induced cells; (4) culturing the induced cells in a transformation medium to obtain resting cells, and then adding decanoic acid to the culture medium and culturing to obtain 10-hydroxy-2-decenoic acid. The synthesis pathway of 10-hydroxy-2-decenoic acid in this patent document is as follows: 10-HDA is produced by a two-step process using the substrate decanoic acid. The first step is to convert decanoic acid to trans-2-decenoic acid. Decanoic acid is added with coenzyme A under the action of acyl-CoA synthetase FadK to form decanoyl-CoA. Then, decanoyl-CoA removes two hydrogen atoms at the carboxyl position (β-position) of decanoyl-CoA under the action of acyl-CoA dehydrogenase MCAD to convert it into trans-decenoyl-CoA. Finally, trans-decenoyl-CoA removes coenzyme A under the action of acyl-CoA thioesterase YdiI to form trans-2-decenoic acid. The second step is the terminal hydroxylation of trans-2-decenoic acid under the action of P450 fusion enzyme to form 10-hydroxy-2-decenoic acid.

[0007] Chinese patent document CN 113106109 A (application number 202110211118.9) discloses a mutant enzyme CYP153A M228L and its application in the synthesis of 10-hydroxy-2-decenoic acid. The specific mutant enzyme CYP153A M228L is a method in which the amino acid at position 228 of the CYP153A enzyme is mutated from M to L; a two-step biosynthesis method of 10-hydroxy-2-decenoic acid using decanoic acid as a raw material mainly includes constructing an optimized recombinant plasmid pCDFDuet-1-MaMACS-PpFadE and an optimized recombinant plasmid pET21b-CYP153A M228L-CPR BM3 , optimized recombinant plasmid pET28a-SUMO-ctYdiI; constructed recombinant Escherichia coli bacteria to produce resting cells, which were further cultured to produce 10-hydroxy-2-decenoic acid. This patent document is optimized based on patent document CN109897870A (application number 201910088897.0), and uses a two-step method to generate 10-HDA. The acyl-CoA dehydrogenase gene PpFadE, acyl-CoA synthetase gene MaMACS, and acyl-CoA thioesterase gene ctydiI are not the same genes as the acyl-CoA dehydrogenase gene MCAD, acyl-CoA synthetase gene FadK, and acyl-CoA thioesterase gene ydiI in Chinese patent document CN109897870A (application number: 201910088897.0). The alkane hydroxylase CYP153A changes the amino acid at position 228 from M to L, and the conversion rate of 10-hydroxy-2-decenoic acid is increased to 54.6%.

[0008] It is still necessary to develop more diverse synthetic pathways for 10-hydroxy-2-decenoic acid for the industrial production of 10-hydroxy-2-decenoic acid. Summary of the Invention

[0009] In view of the deficiencies in the prior art, the present invention provides a genetically engineered bacterium and application thereof in the preparation of 10-hydroxy-2-decenoic acid.

[0010] Based on previous research, the present invention supplements and integrates key enzymes in the synthesis process of 10-hydroxy-2-decenoic acid, including fatty acid hydroxylase, acyl-CoA oxidase, and acyl-CoA thioesterase, so that the various enzyme systems in the engineered bacteria work synergistically to synthesize 10-hydroxy-2-decenoic acid in a one-step method using decanoic acid as a raw material.

[0011] The technical solutions of the present invention are as follows:

[0012] A genetically engineered bacterium comprising an acyl-CoA oxidase gene, an acyl-CoA thioesterase gene, and a CYP153A M228L-CPR BM3 Fusion enzyme gene.

[0013] According to the present invention, the acyl-CoA oxidase gene is derived from Candida tropicalis, and its nucleotide sequence is SEQ ID NO.3; the nucleotide sequence of the acyl-CoA thioesterase gene is SEQ ID NO.5; the CYP153AM228L-CPR BM3 The nucleotide sequence of the fusion enzyme gene is SEQ ID NO.6.

[0014] More preferably, the amino acid sequence of the acyl-CoA oxidase is SEQ ID NO.9, the amino acid sequence of the acyl-CoA thioesterase is SEQ ID NO.11, and the CYP153A M228L-CPR BM3 The amino acid sequence of the fusion enzyme is SEQ ID NO.12.

[0015] According to the present invention, preferably, the host bacteria of the genetically engineered bacteria is Escherichia coli.

[0016] The construction method of the above-mentioned genetically engineered bacteria comprises the following steps:

[0017] (1) Construction of recombinant plasmid pETDuet-1- Ct ACO5, pET21b-CYP153A M228L-CPR BM3 , pET28a-SUMO-YdiI;

[0018] Acyl-CoA oxidase Ct The nucleotide sequence of the ACO5 gene is shown in SEQ ID NO.3, the nucleotide sequence of the acyl-CoA thioesterase YdiI gene is shown in SEQ ID NO.5, and the CYP153A M228L-CPR BM3 The nucleotide sequence of the fusion enzyme gene is shown in SEQ ID NO.6;

[0019] (2) The recombinant plasmid pETDuet-1- Ct ACO5, pET21b-CYP153A M228L-CPR BM3 , pET28a-SUMO-YdiI, and transformed into Escherichia coli. After screening and identification, the genetically engineered Escherichia coli strain (BL21) / pETDuet-1- Ct ACO5, pET21b-CYP153A M228L-CPR BM3 、pET28a-SUMO- Ct YdiI.

[0020] The invention relates to an application of the genetically engineered bacteria in the preparation of 10-hydroxy-2-decenoic acid using decanoic acid as a raw material.

[0021] A preferred technical solution of the present invention is a method for preparing 10-hydroxy-2-decenoic acid using decanoic acid as a raw material using the above-mentioned genetically engineered bacteria, and the steps are as follows:

[0022] The constructed Escherichia coli genetically engineered bacteria were induced to obtain induced cells, which were inoculated into a transformation medium, and decanoic acid was added, and the cells were fermented at 37° C. to obtain 10-hydroxy-2-decenoic acid.

[0023] According to the preferred embodiment of the present invention, the induction culture is as follows: the genetically engineered Escherichia coli bacteria are inoculated into LB liquid culture medium containing 50 μg / mL kanamycin, 100 μg / mL ampicillin and 40 μg / mL streptomycin, and cultured at 37°C with shaking until the bacterial solution OD 600 is 0.8-1.2; after cooling to 16-20°C and adapting for 1 hour, IPTG is added to a concentration of 0.5 mM, and the induction culture is continued for 20-25 hours, and the cells are separated to obtain induced cells.

[0024] More preferably, the cells are separated by centrifugation at 5000 rpm for 15 min, collecting the cell pellet, and then washing the cell pellet with 100 mM PBS buffer at pH 7.2-7.4.

[0025] According to the preferred embodiment of the present invention, the components of the transformation medium are as follows, all in mass percentage:

[0026] Glycerol 1%, glucose 0.4%, kanamycin 50 μg / mL, ampicillin 100 μg / mL, streptomycin 40 μg / mL, and the balance is 100 mM potassium phosphate buffer at pH 7.4.

[0027] Preferably, according to the present invention, capric acid is added to a concentration of 0.1 g / L.

[0028] Preferably according to the present invention, capric acid is dissolved in anhydrous ethanol and then added to the transformation medium.

[0029] The steps not described in detail in the present invention are all performed according to conventional operations in the art.

[0030] Beneficial effects:

[0031] 1. The present invention supplements and integrates the key enzymes for the biosynthesis of 10-hydroxy-2-decenoic acid, and co-transfers fatty acid hydroxylase, acyl-CoA oxidase, and acyl-CoA thioesterase into the host bacteria, so that the various enzyme systems in the engineered bacteria act synergistically to achieve the purpose of synthesizing 10-hydroxy-2-decenoic acid in one step using decanoic acid as a raw material. The synthetic pathway of 10-hydroxy-2-decenoic acid of the present invention is: CYP153A M228L-CPR BM3 The fusion enzyme catalyzes the synthesis of decanoic acid into 10-hydroxydecanoic acid, the acyl-CoA oxidase catalyzes the synthesis of 10-hydroxydecanoic acid into 10-hydroxy-2-decenoyl-CoA, and the acyl-CoA thioesterase catalyzes the removal of Coenzyme A from 10-hydroxy-2-decenoyl-CoA to synthesize 10-hydroxy-2-decenoic acid, thereby solving the problem of low conversion rate of the intermediate product 10-hydroxydecanoic acid in the prior art and opening up the synthesis pathway of 10-hydroxy-2-decenoic acid.

[0032] 2. The present invention significantly improves the conversion rate of 10-hydroxy-2-decenoic acid by optimizing the expression pathway of 10-hydroxy-2-decenoic acid and optimizing the relevant conditions and key enzymes in the conversion process. The genetically engineered Escherichia coli (BL21) / pETDuet-1- Ct ACO5, pET21b-CYP153A M228L-CPR BM3 , pET28a-SUMO-YdiI, and the conversion rate reached 75%, which was significantly improved, making it possible to industrially produce 10-hydroxy-2-decenoic acid using decanoic acid as a substrate. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 For the recombinant plasmid pETDuet-1- Ct Schematic diagram of the structure of ACO5;

[0034] Figure 2 The agarose gel electrophoresis diagram of the PCR product of the ACO gene on the pETDuet-1 plasmid in the recombinant bacteria, where lane M is the marker and lane 1 is Ct ACO2,2 is Ct ACO4,3 is Ct ACO5,4 is Yl ACO;

[0035] Figure 3 is the SDS-PAGE image of the recombinant bacteria induced product, M is a marker, 1 is a blank control, 2 is a recombinant bacteria (BL21) / pETDuet-1- Ct ACO2, pET21b-CYP153A M228L-CPR BM3, pET28a-SUMO-YdiI, 3 is the recombinant bacteria (BL21) / pETDuet-1- Ct ACO4, pET21b-CYP153A M228L-CPR BM3 , pET28a-SUMO-YdiI, 4 is the recombinant bacteria (BL21) / pETDuet-1- Ct ACO5, pET21b-CYP153A M228L-CPR BM3 , pET28a-SUMO-YdiI, 5 is the recombinant bacteria (BL21) / pETDuet-1- Yl ACO, pET21b-CYP153A M228L-CPR BM3 , pET28a-SUMO-YdiI, where the target protein acyl-CoA thioesterase YdiI is 15kDa, CYP153A M228L-CPR BM3 The fusion enzyme is 120 kDa, acyl-CoA oxidase Ct ACO2 is 78 kDa, acyl-CoA oxidase Ct ACO4 is 79 kDa, acyl-CoA oxidase Ct ACO5 is 74 kDa, acyl-CoA oxidase Yl The size of ACO is 78 kDa;

[0036] Figure 4 This is the mass spectrum of the fermentation intermediate 10-hydroxydecanoic acid.

[0037] Figure 5 This is the mass spectrum of the fermentation product 10-hydroxy-2-decenoic acid.

[0038] Figure 6 This is a graph showing the changes in the yield of 10-hydroxy-2-decenoic acid, a fermentation product of the recombinant bacteria. DETAILED DESCRIPTION

[0039] The technical solution of the present invention is further described below with reference to the examples, but the scope of protection of the present invention is not limited thereto. The operating methods not described in detail in the examples are all conventional operating methods well known to those skilled in the art.

[0040] Sources of biological materials:

[0041] Acyl-CoA oxidase CtACO2 gene: derived from Candida tropicalis MYA-3404, Genbank Accession No: XM_002548031.1, nucleotide sequence is SEQ ID NO.1, and the encoded amino acid sequence is SEQ ID NO.7.

[0042] Acyl-CoA oxidase Ct ACO4 gene: derived from Candida tropicalis strain MYA-3404, Genbank Accession No: CP047872.1; its nucleotide sequence is SEQ ID NO.2, and the encoded amino acid sequence is SEQ ID NO.8.

[0043] Acyl-CoA oxidase Ct ACO5 gene: derived from Candida tropicalis strain MYA-3404, Genbank Accession No: CP047872.1; its nucleotide sequence is SEQ ID NO. 3, and the encoded amino acid sequence is SEQ ID NO. 9.

[0044] Acyl-CoA oxidase Yl ACO gene: derived from Yarrowia lipolytica strain W29, Genbank Accession No: CP028451.1; its nucleotide sequence is SEQ ID NO. 4, and the encoded amino acid sequence is SEQ ID NO. 10.

[0045] The acyl-CoA thioesterase YdiI gene is described in Chinese patent document CN 113106109 A (application number 202110211118.9), its nucleotide sequence is SEQ ID NO.5, and the encoded amino acid sequence is SEQ ID NO.11.

[0046] CYP153A M228L-CPR BM3 The fusion enzyme gene is recorded in Chinese patent document CN 113106109A (application number 202110211118.9), its nucleotide sequence is SEQ ID NO.6, and the encoded amino acid sequence is SEQ ID NO.12.

[0047] The reagents and drugs used in the present invention are all common commercially available products.

[0048] Example 1: Construction of recombinant plasmid

[0049] Recombinant plasmid pETDuet-1- Ct ACO2, pETDuet-1- Ct ACO4, pETDuet-1- Ct ACO5、pETDuet-1- Yl ACO, pET21b-CYP153A M228L-CPR BM3 , Synthesis of pET28a-SUMO-YdiI:

[0050] Acyl-CoA oxidase Ct ACO2, Ct ACO4, Ct ACO5, Yl The nucleotide sequences of the ACO gene are shown in SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, and SEQ ID NO.4, respectively. The nucleotide sequence of the acyl-CoA thioesterase YdiI gene is shown in SEQ ID NO.5. BM3 The nucleotide sequence of the fusion enzyme gene is shown in SEQ ID NO.6; and acyl-CoA oxidase Ct ACO2, Ct ACO4, Ct ACO5, Yl The amino acid sequences of the proteins encoded by the ACO gene are shown in SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9, and SEQ ID NO.10, respectively. The amino acid sequence of the protein encoded by the acyl-CoA thioesterase YdiI gene is shown in SEQ ID NO.11. BM3 The amino acid sequence of the protein encoded by the fusion enzyme gene is shown in SEQ ID NO.12;

[0051] The recombinant plasmid pETDuet-1- Ct ACO2, pETDuet-1- Ct ACO4, pETDuet-1- Ct ACO5、pETDuet-1- Yl ACO, pET21b-CYP153A M228L-CPR BM3 , pET28a-SUMO-YdiI were constructed by Nanjing GenScript Biotechnology Co., Ltd. Ct The structural diagram of ACO5 is shown in Figure 1 shown.

[0052] Example 2: Construction of genetically engineered bacteria

[0053] (1) Preparation of competent cells

[0054] ① Pick a single colony of Escherichia coli (BL21) (or pick a preserved strain) and inoculate it into 50 mL of liquid LB medium. Incubate overnight at 37°C and 200 rpm.

[0055] ②Take 1mL of E. coli culture medium and inoculate it into 50mL of liquid LB medium. Cultivate at 37℃ and 200rpm until the OD value of the culture medium reaches 600 About 0.5-0.6;

[0056] ③ Place the bacterial solution on the ice-water mixture for 10 minutes, and pre-cool the 50mL centrifuge tube;

[0057] ④ Transfer the bacterial solution to a centrifuge tube and centrifuge at 3700 rpm for 10 min at 4°C to collect the bacteria;

[0058] ⑤ Add 10 mL of pre-cooled 0.1 M CaCl2 solution to each centrifuge tube, resuspend the cells, then add 30 mL of pre-cooled 0.1 M CaCl2 solution, mix thoroughly by inversion, and let stand on ice for 20 minutes;

[0059] ⑥ Collect the cells by centrifugation at 3700 rpm for 10 min at 4°C. Add pre-chilled 0.1 M CaCl2 solution containing 15% glycerol at a volume ratio of 3:125 to the bacterial solution in step ④, and resuspend the cells to obtain competent cells.

[0060] ⑦ Aliquot the competent cells and freeze at -80℃.

[0061] (2) Transformation of recombinant plasmid

[0062] ① Take the recombinant plasmid pETDuet-1- Ct ACO2, pET21b-CYP153A M228L-CPR BM3 , pET28a-SUMO-YdiI as a group, pETDuet-1- Ct ACO4, pET21b-CYP153A M228L-CPR BM3 , pET28a-SUMO-YdiI as a group, pETDuet-1- Ct ACO5, pET21b-CYP153A M228L-CPR BM3 , pET28a-SUMO-YdiI as a group, pETDuet-1- Yl ACO, pET21b-CYP153A M228L-CPR BM3, pET28a-SUMO-YdiI were grouped together, with the volume ratio of each plasmid in each group being 1:1:1, for a total of 10 μL, which were added to 100 μL of freshly prepared competent cells, mixed gently, and placed on ice for 30 min;

[0063] ②Heat shock at 42°C for 90 seconds, then quickly cool in an ice bath for 3 minutes;

[0064] ③ The competent cells were inoculated into 500 μL LB medium and cultured at 37°C with shaking at 200 rpm for 60 min.

[0065] ④ Take 200 μL of the above bacterial solution and spread the E. coli bacterial solution on LB solid medium supplemented with 50 μg / mL kanamycin, 100 μg / mL ampicillin, and 40 μg / mL streptomycin;

[0066] ⑤ Place the solid culture medium upright in a 37℃ incubator for 30 minutes. After the bacterial liquid is aspirated dry, invert the plate and incubate at 37℃ for 12-16 hours.

[0067] (3) Identification of positive clones:

[0068] ① Colony PCR identification

[0069] Pick a single colony cultured above and inoculate the E. coli into 1 mL of liquid LB medium containing 50 μg / mL kanamycin, 100 μg / mL ampicillin, and 40 μg / mL streptomycin. Incubate the culture at 37°C and 200 rpm with shaking for 6-8 h. Pipette 2 μL of the bacterial solution and perform colony PCR identification using a 50 μL PCR reaction system. The presence of a single target band indicates that the colony is a positive clone.

[0070] The ACO gene on the pETDuet-1 plasmid was verified by colony PCR. The sequences of all designed primers in this experiment are shown in Table 1. The agarose gel electrophoresis of the PCR amplified ACO gene product is shown in Figure 2 As shown:

[0071] Table 1: Primer sequences

[0072]

[0073] ②Protein expression and solubility identification

[0074] Take 900 μL of the above bacterial solution and add IPTG with a final concentration of 0.32 mM to induce expression for 4 hours. Centrifuge at 12000 rpm for 1 minute, collect the bacteria, add 2 times loading buffer, resuspend the bacteria, denature in a 100℃ water bath for 10 minutes, and detect protein expression by SDS-PAGE. The results are as follows: Figure 3 As shown, positive clones are shown.

[0075] ③ Bacteria sample sequencing

[0076] The positive clones identified by the above two methods were sent to a sequencing company for sequencing to further prove the correctness of the constructed positive clones.

[0077] Finally, the genetically engineered Escherichia coli was constructed:

[0078] (BL21) / pETDuet-1- Ct ACO2, pET21b-CYP153A M228L-CPR BM3 、pET28a-SUMO-YdiI,

[0079] (BL21) / pETDuet-1- Ct ACO4, pET21b-CYP153A M228L-CPR BM3 、pET28a-SUMO-YdiI,

[0080] (BL21) / pETDuet-1- Ct ACO5, pET21b-CYP153A M228L-CPR BM3 、pET28a-SUMO-YdiI,

[0081] (BL21) / pETDuet-1- Yl ACO, pET21b-CYP153A M228L-CPR BM3 , pET28a-SUMO-YdiI.

[0082] Example 3: Preparation of 10-hydroxy-2-decenoic acid using genetically engineered bacteria

[0083] (1) Activation of the strain: The genetically engineered Escherichia coli constructed in Example 2 was inoculated at a 1% inoculum into 50 mL of liquid LB medium containing 50 μg / mL kanamycin, 100 μg / mL ampicillin, and 40 μg / mL streptomycin, and cultured at 37°C and 200 rpm for 12 h;

[0084] (2) Bacterial transfer: 1 mL of the activated strain of the genetically engineered E. coli was inoculated into 50 mL of liquid LB medium containing 50 μg / mL kanamycin, 100 μg / mL ampicillin, and 40 μg / mL streptomycin. The culture was shaken at 37°C and 200 rpm until the OD value of the bacterial solution reached 0. 600 The temperature was set to 0.8, and after acclimation at 16°C for 1 hour, IPTG was added to make the IPTG concentration in the culture medium 0.5 mM, and cultured at 37°C and 200 rpm for 20 hours;

[0085] (3) Collecting bacteria: Take 50 mL of the above culture solution and centrifuge at 5000 rpm at 4°C for 15 min to collect the bacteria;

[0086] (4) Wash the cells three times with 100 mM PBS buffer (pH 7.2-7.4), resuspend the cells in transformation medium, and inoculate into transformation medium. The transformation medium contains 1% glycerol, 0.4% glucose, 50 μg / mL kanamycin, 100 μg / mL ampicillin, and 40 μg / mL streptomycin, and the remainder is 100 mM potassium phosphate buffer at a pH of 7.4. Dissolve capric acid in anhydrous ethanol and add it to the transformation medium to make the capric acid concentration in the medium 0.1 g / L. React at 37°C for 24 h to obtain a fermentation broth.

[0087] Silanization treatment of fermentation broth: take 1 mL of fermentation broth into a 2 mL centrifuge tube, add 1 mL of 4 M HCl solution, vortex centrifuge, heat at 100°C for 10 min, and then refrigerate at -80°C for 15 min. After melting at room temperature, add 1 mL of ethyl acetate and vortex centrifuge. Collect the organic phase into a new tube, add 100 μL of N, O-bis(trimethylsilyl)trifluoroacetamide and shake to mix. Incubate in a 70°C water bath for 40 min before use.

[0088] Detection of generated products by gas chromatography-mass spectrometry: Gas chromatography-mass spectrometry was performed with helium as carrier gas, constant flow mode, injection volume of 1 μL, split injection, split ratio of 1:50, injection temperature of 250 °C, maintained at 50 °C for 1 min, increased to 250 °C at 15 °C / min, and maintained for 10 min.

[0089] The mass spectrum of 10-hydroxydecanoic acid in the fermentation broth is as follows Figure 4 As shown, the mass spectrum of 10-hydroxy-2-decenoic acid is as follows Figure 5 shown.

[0090] The yield changes of 10-hydroxy-2-decenoic acid produced by recombinant bacteria fermentation are as follows Figure 6 shown.

[0091] The detected yield of 10-hydroxy-2-decenoic acid and the calculated conversion rate are shown in Table 2:

[0092] Table 2: Yield and conversion rate of 10-hydroxy-2-decenoic acid of each genetically engineered strain

[0093]

[0094] From the above data, it can be seen that the initial host bacteria lacked the key enzyme for the synthesis of 10-hydroxy-2-decenoic acid, so it could not synthesize 10-hydroxy-2-decenoic acid. After the acyl-CoA oxidase gene, acyl-CoA thioesterase gene and fatty acid hydroxylase gene were transferred into the host bacteria, the genetically engineered bacteria constructed could synthesize 10-hydroxy-2-decenoic acid in one step using decanoic acid as a substrate. Its synthesis pathway is roughly as follows: CYP153A M228L-CPR BM3 The fusion enzyme catalyzes the synthesis of 10-hydroxydecanoic acid from decanoic acid, the acyl-CoA oxidase catalyzes the synthesis of 10-hydroxydecanoic acid into 10-hydroxy-2-decenoyl-CoA, and the acyl-CoA thioesterase catalyzes the removal of CoA from 10-hydroxy-2-decenoyl-CoA into 10-hydroxy-2-decenoic acid. Ct ACO5, pET21b-CYP153A M228L-CPR BM3 , pET28a-SUMO-YdiI, and their conversion rates reached 75%, which was significantly improved.

[0095] Comparative Example: Preparation of 10-hydroxy-2-decenoic acid by two-step method

[0096] (1) Genetically engineered bacteria (BL21) / pET21b-CYP153A M228L-CPR BM3 Construction

[0097] According to the description of Example 2, 10 μL of recombinant plasmid pET21b-CYP153A M228L-CPR was taken. BM3 Transformed into E. coli, after screening and identification, the genetically engineered bacteria (BL21) / pET21b-CYP153A M228L-CPR was obtained. BM3 .

[0098] (2) Genetically engineered bacteria (BL21) / pETDuet-1- Ct Construction of ACO5 and pET28a-SUMO-YdiI

[0099] According to the description of Example 2, the recombinant plasmid ETDuet-1- Ct ACO5 and pET28a-SUMO-YdiI, with a volume ratio of 1:1, a total of 10 μL, were co-transformed into Escherichia coli. After screening and identification, the genetically engineered bacteria (BL21) / pETDuet-1- Ct ACO5, pET28a-SUMO-YdiI.

[0100] (3) Fermentation

[0101] i. Genetically engineered bacteria (BL21) / pET21b-CYP153A M228L-CPR BM3 Inoculate 50 mL of liquid LB medium containing 100 μg / mL ampicillin at a 1% inoculum size and culture at 37°C and 200 rpm for 12 h;

[0102] ii. Take 1 mL of the above culture solution and inoculate it into 50 mL of liquid LB medium containing 100 μg / mL ampicillin, and culture it at 37°C and 200 rpm until the OD value of the culture solution reaches 0. 600 0.8; after cooling to 16°C for 1 hour, IPTG was added to a concentration of 0.5 mM and the induction culture was continued at 37°C and 200 rpm for 20 hours;

[0103] iii. Take 50 mL of the above culture solution, centrifuge at 5000 rpm, 4°C for 15 min, and collect the bacteria;

[0104] iv. The cells were washed three times with 100 mM PBS buffer (pH 7.2-7.4) and resuspended in transformation medium. The transformation medium contained 1% glycerol, 0.4% glucose, 100 μg / mL ampicillin, and the remainder was 100 mM potassium phosphate buffer at pH 7.4. Capric acid was dissolved in anhydrous ethanol and added to the transformation medium to a concentration of 0.1 g / L capric acid in the medium. The reaction was carried out at 37°C for 24 h to obtain a fermentation broth.

[0105] v. Genetically engineered bacteria (BL21) / pETDuet-1- Ct ACO5 and pET28a-SUMO-YdiI were inoculated into 50 mL of liquid LB medium containing 50 μg / mL kanamycin and 40 μg / mL streptomycin at a 1% inoculum size and cultured at 37°C with shaking at 200 rpm for 12 h;

[0106] vi. Take 1 mL of the culture solution from step v and inoculate it into 50 mL of liquid LB medium containing 50 μg / mL kanamycin and 40 μg / mL streptomycin. Incubate at 37°C and 200 rpm with shaking until the OD value of the culture solution reaches 0. 600 The temperature was set to 0.8, and after acclimation at 16°C for 1 hour, IPTG was added to make the IPTG concentration in the culture medium 0.5 mM, and cultured at 37°C and 200 rpm for 20 hours;

[0107] vii. Take 50 mL of the cultured bacteria from step vi, centrifuge at 5000 rpm and 4 ° C for 15 min to collect the bacteria;

[0108] viii. Wash the cells in step vii three times with 100 mM PBS buffer (pH 7.2-7.4) and resuspend them in transformation medium. The volume of the cells after resuspension is 3 mL and added to the fermentation broth in step iv. React at 37 ° C for 24 h to obtain a fermentation broth.

[0109] The generated product was detected according to the method of Example 3. The results showed that the genetically engineered bacteria (BL21) / pET21b-CYP153A M228L-CPR BM3 The yield of 10-hydroxydecanoic acid was 0.087 g / L, the conversion rate was 87%, and the genetically engineered bacteria (BL21) / pETDuet-1- Ct The yield of 10-hydroxy-2-decenoic acid using ACO5 and pET28a-SUMO-YdiI was 0.065 g / L, with a conversion rate of 65% relative to the substrate decanoic acid. This indicates that the conversion rate of 10-hydroxy-2-decenoic acid produced by the two-step method is lower than that of the one-step method.

Claims

1. A genetically engineered bacterium, characterized in that: The genetically engineered bacteria include acyl-CoA oxidase gene, acyl-CoA thioesterase gene and CYP153A M228L-CPR BM3 fusion enzyme gene; The acyl-CoA oxidase gene is derived from Candida tropicalis, and its nucleotide sequence is SEQ ID NO.3; the nucleotide sequence of the acyl-CoA thioesterase gene is SEQ ID NO.5; the CYP153A M228L-CPR BM3 The nucleotide sequence of the fusion enzyme gene is SEQ ID NO.

6.

2. The genetically engineered bacterium according to claim 1, wherein The amino acid sequence of the acyl-CoA oxidase is SEQ ID NO.9, the amino acid sequence of the acyl-CoA thioesterase is SEQ ID NO.11, and the CYP153AM228L-CPR BM3 The amino acid sequence of the fusion enzyme is SEQ ID NO.

12.

3. The genetically engineered bacterium according to claim 1, wherein The host bacteria of the genetically engineered bacteria is Escherichia coli.

4. The method for constructing the genetically engineered bacteria according to claim 1, comprising the following steps: (1) Construction of recombinant plasmid pETDuet-1- Ct ACO5, pET21b-CYP153A M228L-CPR BM3 , pET28a-SUMO-YdiI; in, Acyl-CoA oxidase Ct The nucleotide sequence of the ACO5 gene is shown in SEQ ID NO.3, the nucleotide sequence of the acyl-CoA thioesterase YdiI gene is shown in SEQ ID NO.5, and the CYP153A M228L-CPR BM3 The nucleotide sequence of the fusion enzyme gene is shown in SEQ ID NO.6; (2) The recombinant plasmid pETDuet-1- Ct ACO5, pET21b-CYP153A M228L-CPR BM3 , pET28a-SUMO-YdiI, and transformed into Escherichia coli. After screening and identification, the genetically engineered Escherichia coli strain (BL21) / pETDuet-1- Ct ACO5, pET21b-CYP153A M228L-CPR BM3 、pET28a-SUMO- Ct YdiI.

5. Use of the genetically engineered bacteria according to claim 1 in the preparation of 10-hydroxy-2-decenoic acid using decanoic acid as a raw material.

6. A method for preparing 10-hydroxy-2-decenoic acid using decanoic acid as a raw material using genetically engineered bacteria, characterized in that: Here are the steps: The genetically engineered Escherichia coli constructed according to claim 4 is induced to obtain induced cells, the induced cells are inoculated into a transformation culture medium, and decanoic acid is added, and the 10-hydroxy-2-decenoic acid is obtained by fermentation culture at 37°C.

7. The method according to claim 6, wherein The induction culture is as follows: genetically engineered Escherichia coli bacteria are inoculated into LB liquid culture medium containing 50 μg / mL kanamycin, 100 μg / mL ampicillin and 40 μg / mL streptomycin, and cultured at 37°C with shaking until the bacterial solution OD 600 is 0.8-1.2; after cooling to 16-20℃ and adapting for 1 hour, add IPTG to a concentration of 0.5mM, continue induction culture for 20-25 hours, separate the cells, and prepare induced cells.

8. The method according to claim 7, wherein The cells were separated by centrifugation at 5000 rpm for 15 min, collecting the cell pellets, and then washing the cell pellets with 100 mM PBS buffer at pH 7.2-7.

4.

9. The method according to claim 6, wherein Also includes one or more of the following conditions: i. The transformation medium components are as follows, all in mass percentage: Glycerol 1%, glucose 0.4%, kanamycin 50 μg / mL, ampicillin 100 μg / mL, streptomycin 40 μg / mL, the balance being 100 mM potassium phosphate buffer at pH 7.4; ii. Add decanoic acid to a concentration of 0.1 g / L; iii. Dissolve decanoic acid in anhydrous ethanol and add to the transformation medium.

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