An engineered strain containing fatty acyl-coa dehydrogenase and its application method

Abstract

The present application relates to a kind of containing fatty acyl-coa dehydrogenase engineering bacteria and its application method, belong to microbial fermentation technical field.The engineering bacteria containing fatty acyl-coa dehydrogenase gene in the present application include fatty acyl-coa dehydrogenase gene and fatty acyl-coa thioesterase gene, the fatty acyl-coa dehydrogenase gene is derived from Candida tropicalis or Yarrowia lipolytica, its nucleotide sequence is SEQ ID NO.1 or SEQ ID NO.2;The nucleotide sequence of the fatty acyl-coa thioesterase gene is SEQ ID NO.3.Fatty acyl-coa dehydrogenase from Candida tropicalis and Yarrowia lipolytica, combined with existing fatty acyl-coa thioesterase, jointly convert 10-hydroxy decanoic acid, significantly improve the conversion rate of 10-hydroxy-2-decenoic acid, so that the industrialization production of 10-hydroxy-2-decenoic acid becomes possible.

Description

TECHNICAL FIELD

[0001] The present application relates to a kind of containing fatty acyl coenzyme A dehydrogenase engineering bacteria and its application method, specifically to a kind of containing fatty acyl coenzyme A dehydrogenase gene engineering bacteria and its application in the preparation of 10-hydroxy-2-decenoic acid, belong to microbial fermentation technical field. BACKGROUND

[0002] 10-hydroxy-2-decenoic acid (10-hydroxy-2-decenoic acid, 10-HDA for short) is the fatty acid unique to royal jelly, also known as royal jelly acid, its content in royal jelly is about 1.4% to 2.0%, and it is the highest content fatty acid in royal jelly, and it has antibacterial, anti-inflammatory, antitumor, immune enhancement, blood sugar reduction, blood lipid reduction, anti-radiation and nerve regulation and other functions. The compound structure is as follows:

[0003]

[0004] So far, the methods for obtaining 10-HDA mainly include extraction method, chemical synthesis method and microbial fermentation method. There are many extraction methods, including ether extraction method, ethanol extraction method, etc. However, the content of 10-HDA in royal jelly is relatively low, which is difficult to meet the extensive demand of the market, and the extraction cost is high. Since the 1960s of last century, various chemical synthesis methods of 10-HDA have been designed and applied to production, such as Doebmer condensation method, the substrate of which is 1,6-hexanediol, and the yield is 61%; condensation method, which uses γ-bromobutyric acid ethyl ester Grignard and organic magnesium compound as substrate, and generates 10-HDA after condensation, and the yield of this method reaches 70%. The above experimental methods can obtain 10-HDA, but chemical synthesis method has unavoidable shortcomings, such as harsh production conditions, easy pollution, etc., and the raw materials are not easy to obtain, the synthesis route is long, the yield is low, and there are cis-trans isomerism problems in chemical synthesis, and the product singleness is poor. Therefore, the production of 10-HDA based on biological synthesis method 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 E. coli engineering bacteria, the steps are as follows: (1) construct E. coli engineering bacteria containing recombinant plasmid pET-28a-ydII; (2) take E. coli engineering bacteria containing recombinant plasmid pET-28a-ydII to prepare induced cells; (3) culture the induced cells in a transformation medium to obtain resting cells, then add 10-hydroxy decanoic acid to the culture medium to prepare 10-hydroxy-2-decenoic acid. The synthesis pathway of 10-hydroxy-2-decenoic acid in the patent document: taking 10-hydroxy decanoic acid as the reaction substrate, relying on the first two steps of beta oxidation, after forming the trans double bond, hydrolyzing coenzyme A by E. coli ester acyl coenzyme A 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 E. coli engineering bacteria with decanoic acid as raw material, the steps are as follows: (1) construct recombinant plasmid pBbB5K-P450 fusion enzyme, recombinant plasmid pBbB5K-FadK, recombinant plasmid pBbB5K-MCAD, and recombinant plasmid pBbB5K-ydII; (2) construct pBbB5K-ydII-MCAD-FadK-P450 fusion enzyme combination plasmid; (3) transform E. coli with the fusion enzyme combination plasmid, screen, induce culture, and obtain induced cells; (4) culture the induced cells in a transformation medium to obtain resting cells, then add decanoic acid to the culture medium, culture, and obtain 10-hydroxy-2-decenoic acid. The synthesis pathway of 10-hydroxy-2-decenoic acid in the patent document: using substrate decanoic acid to produce 10-HDA by two-step method, the first step reaction is from decanoic acid to trans-2-decenoic acid, decanoic acid is added with coenzyme A to form decanoyl-CoA under the action of fatty acyl CoA synthetase FadK, then decanoyl-CoA is converted to trans-decenoic acid-CoA by removing two hydrogen atoms from the carboxyl group (beta position) of decanoyl-CoA under the action of fatty acyl CoA dehydrogenase MCAD, and finally trans-decenoic acid-CoA is removed from coenzyme A to form trans-2-decenoic acid under the action of fatty acyl CoA thioesterase YdII; the second step reaction 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 mutant enzyme CYP153A M228L is a mutant of the CYP153A enzyme at position 228, where the amino acid is mutated from M to L. The method for biosynthesizing 10-hydroxy-2-decenoic acid from decanoic acid in two steps mainly includes constructing the optimized recombinant plasmid pCDFDuet-1-MaMACS-PpFadE, the optimized recombinant plasmid pET21b-CYP153A M228L-CPR BM3 , and the optimized recombinant plasmid pET28a-SUMO-ctYdiI. The resting cells of the recombinant E. coli are prepared, and 10-hydroxy-2-decenoic acid is further prepared by culturing. The patent document is optimized based on patent document CN 109897870 A (application number 201910088897.0), and 10-HDA is generated by a two-step method. The fatty acyl-CoA dehydrogenase gene PpFadE, the fatty acyl-CoA synthetase gene MaMACS, and the acyl-CoA thioesterase gene ctydiI in the patent document are not the same as the fatty acyl-CoA dehydrogenase gene MCAD, the fatty acyl-CoA synthetase gene FadK, and the acyl-CoA thioesterase gene ydiI in Chinese patent document CN 109897870 A (application number: 201910088897.0). The alkane hydroxylase CYP153A is a mutant of the amino acid at position 228, where the amino acid is mutated from M to L. The conversion rate of 10-hydroxy-2-decenoic acid is increased to 54.6%.

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

[0009] The present application provides an engineered bacterium containing a fatty acyl-CoA dehydrogenase and its application method, specifically an engineered bacterium containing a fatty acyl-CoA dehydrogenase gene and its application in the preparation of 10-hydroxy-2-decenoic acid. The engineered bacterium can be used to prepare 10-hydroxy-2-decenoic acid from 10-hydroxy decanoic acid in one step.

[0010] Based on previous research, the present application further constructs expression elements, perfects key enzymes in the synthesis pathway of 10-hydroxy-2-decenoic acid, including fatty acyl-CoA dehydrogenase and fatty acyl-CoA thioesterase, and provides a method for preparing 10-hydroxy-2-decenoic acid from 10-hydroxy decanoic acid in one step using an engineered bacterium.

[0011] Term explanation:

[0012] acyl-CoA dehydrogenase, abbreviated as ACD.

[0013] acyl-CoA thioeaterase, abbreviated as ACOT.

[0014] The technical scheme of the present application is as follows:

[0015] An engineering bacterium containing an acyl-CoA dehydrogenase gene, wherein the engineering bacterium comprises an acyl-CoA dehydrogenase gene and an acyl-CoA thioeaterase gene.

[0016] According to the present application, preferably, the acyl-CoA dehydrogenase gene is derived from Candida tropicalis or Yarrowia lipolytica, and has a nucleotide sequence of SEQ ID NO. 1 or SEQ ID NO. 2; the acyl-CoA thioeaterase gene has a nucleotide sequence of SEQ ID NO. 3.

[0017] Further preferably, the acyl-CoA dehydrogenase has an amino acid sequence of SEQ ID NO. 4 or SEQ ID NO. 5, and the acyl-CoA thioeaterase has an amino acid sequence of SEQ ID NO. 6.

[0018] According to the present application, preferably, the host bacterium of the genetically engineered bacterium is Escherichia coli or Saccharomyces cerevisiae.

[0019] The construction method of the above engineering bacterium containing an acyl-CoA dehydrogenase gene is as follows:

[0020] (1) Constructing recombinant plasmids pETDuet-1-ACD, pET28a-SUMO-ACOT and pESC-URA-SUMO-ACOT;

[0021] The nucleotide sequence of the acyl-CoA dehydrogenase ACD gene is shown in SEQ ID NO. 1 or SEQ ID NO. 2, and the nucleotide sequence of the acyl-CoA thioeaterase ACOT gene is shown in SEQ ID NO. 3;

[0022] (2) Co-transforming the recombinant plasmids pETDuet-1-ACD and pET28a-SUMO-ACOT of step (1) into Escherichia coli, and obtaining an Escherichia coli genetically engineered bacterium after screening and identification; co-transforming the recombinant plasmids pETDuet-1-ACD and pESC-URA-SUMO-ACOT of step (1) into Saccharomyces cerevisiae, and obtaining a Saccharomyces cerevisiae genetically engineered bacterium after screening and identification.

[0023] The above engineering bacterium containing an acyl-CoA dehydrogenase gene is applied to the preparation of 10-hydroxy-2-decenoic acid by taking 10-hydroxydecanoic acid as a substrate.

[0024] According to the preferred technical solution of the present application, the method for preparing 10-hydroxy-2-decenoic acid by using the above-mentioned engineered bacteria containing the fatty acyl-CoA dehydrogenase gene and taking 10-hydroxy decanoic acid as the substrate has the following steps:

[0025] After the above-mentioned constructed Escherichia coli gene engineering bacteria are induced and cultured, induced cells are prepared, the induced cells are inoculated into a transformation culture medium, 10-hydroxy decanoic acid is added, and 10-hydroxy-2-decenoic acid is prepared by fermentation culture at 37℃.

[0026] According to the preferred technical solution of the present application, the induction culture is as follows: the Escherichia coli gene engineering bacteria are inoculated into a liquid LB culture medium containing kanamycin with a concentration of 50 μg / mL and streptomycin with a concentration of 40 μg / mL, and the bacteria liquid is shaken and cultured at 37℃ until the OD 600 of the bacteria liquid is 0.8-1.2; then the temperature is lowered to 16-20℃ for 1 hour of adaptation; then IPTG is added to a concentration of 0.5 mM; and then the induction culture is continued for 20-25 hours; and then the cells are separated to prepare the induced cells.

[0027] Further preferably, the separation of the cells is carried out by centrifugation at 5000 rpm for 15 min, and then the cell precipitate is collected and washed with 100 mM PBS buffer with a pH of 7.2-7.4.

[0028] According to the preferred technical solution of the present application, the components of the transformation culture medium are as follows, all in percentage by mass:

[0029] glycerol 1%, glucose 0.4%, kanamycin 50 μg / mL, streptomycin 40 μg / mL, and the rest is potassium phosphate buffer with a pH of 7.4 and a concentration of 100 mM.

[0030] According to the preferred technical solution of the present application, 10-hydroxy decanoic acid is added to a concentration of 0.1 g / L.

[0031] According to the preferred technical solution of the present application, 10-hydroxy decanoic acid is dissolved in dimethyl sulfoxide and then added to the transformation culture medium.

[0032] According to the preferred technical solution of the present application, the method for preparing 10-hydroxy-2-decenoic acid by using the above-mentioned engineered bacteria containing the fatty acyl-CoA dehydrogenase gene and taking 10-hydroxy decanoic acid as the substrate has the following steps:

[0033] After the above-mentioned constructed Saccharomyces cerevisiae gene engineering bacteria are induced and cultured, induced cells are prepared, the induced cells are inoculated into a transformation culture medium, 10-hydroxy decanoic acid is added, and 10-hydroxy-2-decenoic acid is prepared by fermentation culture at 30℃.

[0034] According to the application, preferably, the induction culture is as follows: the genetically engineered Saccharomyces cerevisiae is inoculated into liquid YPD medium containing 100 μg / mL ampicillin and 40 μg / mL streptomycin, and is cultured at 30°C with shaking until the OD 600 is 0.8-1.2, and then the temperature is lowered to 16-20°C for 1 hour of acclimation, 2% galactose is added, and the induction culture is continued for 48-55 hours, the cells are separated, and the induced cells are prepared.

[0035] Further preferably, the separation of the cells is by centrifugation at 5000 rpm for 15 min, the cell precipitate is collected, and the cell precipitate is washed with 100 mM PBS buffer at pH 7.2-7.4.

[0036] According to the application, preferably, the components of the transformation medium are as follows, all in mass percentage:

[0037] glycerol 1%, glucose 0.4%, ampicillin 100 μg / mL, streptomycin 40 μg / mL, and the rest is 100 mM potassium phosphate buffer at pH 7.4.

[0038] According to the application, preferably, 10-hydroxydecanoic acid is added to a concentration of 0.1 g / L.

[0039] According to the application, preferably, 10-hydroxydecanoic acid is dissolved in dimethyl sulfoxide and then added to the transformation medium.

[0040] The steps not described in detail in the application are performed according to conventional operations in the art.

[0041] Advantages:

[0042] 1. The application integrates the key enzymes for biosynthesis of 10-hydroxy-2-decenoic acid, co-transfers the fatty acyl-CoA dehydrogenase gene and the fatty acyl-CoA thioesterase gene into the host bacteria, realizes efficient expression of the 10-hydroxy-2-decenoic acid expression element, and after induction treatment, can produce 10-hydroxy-2-decenoic acid by fermentation using 10-hydroxydecanoic acid as raw material in the form of resting cells, realizing the process of producing high-value 10-hydroxy-2-decenoic acid from low-value 10-hydroxydecanoic acid.

[0043] 2、The present application optimizes the synthesis pathway of 10-hydroxy-2-decenoic acid, and optimizes the related key enzymes in the conversion process. Through pre-experiments, it is found that not all microorganisms contain the synthesis pathway from 10-hydroxy decanoic acid to 10-hydroxy-2-decenoic acid, and through further optimization, the fatty acyl coenzyme A dehydrogenase from Candida tropicalis and Yarrowia lipolytica is used, combined with the existing fatty acyl coenzyme A thioesterase, to co-convert 10-hydroxy decanoic acid, which significantly improves the conversion rate of 10-hydroxy-2-decenoic acid, thereby making it possible to industrialize the production of 10-hydroxy-2-decenoic acid. BRIEF DESCRIPTION OF DRAWINGS

[0044] Figure 1 It is the consumption amount change graph of different strains in example 1 to the substrate 10-hydroxy decanoic acid.

[0045] Figure 2 It is the recombinant plasmid pETDuet-1- Ct The structural diagram of ACD;

[0046] Figure 3 It is the agarose gel electrophoresis graph of PCR product of ACD gene on pETDuet-1 plasmid in recombinant E. coli in example 3, wherein, lane M is marker, lane 1 is Ct ACD, lane 2 is Yl ACD;

[0047] Figure 4 It is the SDS-PAGE graph of recombinant E. coli induced product, M is marker, 1 is blank control, 2 is recombinant bacteria (BL21) / pET28a-SUMO-YdII, pETDuet-1- Ct ACD, 3 is recombinant bacteria (BL21) / pET28a-SUMO-YdII, pETDuet-1- Yl ACD, wherein the size of the target protein fatty acyl coenzyme A thioesterase YdII is 15kDa, the size of the fatty acyl coenzyme A dehydrogenase Ct ACD is 49kDa, the size of the fatty acyl coenzyme A dehydrogenase Yl ACD is 59kDa;

[0048] Figure 5 It is the mass spectrum graph of fermentation product 10-hydroxy-2-decenoic acid

[0049] Figure 6 It is the yield change graph of fermentation product 10-hydroxy-2-decenoic acid of genetically engineered bacteria in example 5. DETAILED DESCRIPTION

[0050] The technical solutions of the present application are further described below in combination with examples, but the scope of protection of the present application is not limited thereto. The operation methods not described in detail in the examples are all conventional operation methods known to those skilled in the art.

[0051] Biological material sources:

[0052] Fatty acyl-CoA dehydrogenase Ct ACD gene: derived from Candida tropicalis MYA-3404, Genbank Accession No. XM_002546434.1; the nucleotide sequence is SEQ ID NO. 1, and the encoded amino acid sequence is SEQ ID NO. 4.

[0053] Fatty acyl-CoA dehydrogenase Yl ACD gene: derived from Yarrowia lipolytica CLIB122, Genbank Accession No. XM_501919.2; the nucleotide sequence is SEQ ID NO. 2, and the encoded amino acid sequence is SEQ ID NO. 5.

[0054] The fatty acyl-CoA thioesterase YdiI gene is described in Chinese patent document CN 113106109 A (application number 202110211118.9), and the nucleotide sequence thereof is SEQ ID NO. 3, and the encoded amino acid sequence is SEQ ID NO. 6.

[0055] The reagents and drugs used in the present application are all ordinary commercially available products.

[0056] Example 1: verification of metabolism of 10-hydroxydecanoic acid by different strains:

[0057] Through 10-hydroxydecanoic acid substrate fermentation detection of different strains such as Candida tropicalis, Escherichia coli, Staphylococcus aureus, Corynebacterium glutamicum, Bacillus subtilis, Yarrowia lipolytica, and Saccharomyces cerevisiae, it was found that Candida tropicalis and Yarrowia lipolytica had the ability to metabolize 10-hydroxydecanoic acid to synthesize 10-hydroxy-2-decenoic acid. The specific steps are as follows:

[0058] (1) Strain activation: different strains were inoculated into 50 mL of liquid medium at an inoculation amount of 1%, and the culture conditions were as follows:

[0059] Candida tropicalis: YPD medium, 30℃, 200rpm shaking culture for 48h,

[0060] Escherichia coli: LB medium, 37℃, 200rpm shaking culture for 48h,

[0061] Staphylococcus aureus: LB medium, 37°C, 200 rpm shaking culture for 48 h,

[0062] Corynebacterium glutamicum: LB medium, 37°C, 200 rpm shaking culture for 48 h,

[0063] Bacillus subtilis: LB medium, 37°C, 200 rpm shaking culture for 48 h,

[0064] Yarrowia lipolytica: YPD medium, 30°C, 200 rpm shaking culture for 48 h,

[0065] Saccharomyces cerevisiae: YPD medium, 30°C, 200 rpm shaking culture for 48 h,

[0066] The medium components include the following, all in mass percentage:

[0067] LB medium: yeast extract powder 0.5%, peptone 1%, NaCl 1%, pH adjusted to 7.0 with NaOH.

[0068] YPD medium: yeast extract powder 1%, peptone 2%, glucose 2%;

[0069] (2) Bacterial transfer: take 1 mL of the above activated bacterial strain and inoculate into 50 mL of liquid medium, the culture conditions are the same as when the strain is activated, and the culture is carried out until the OD 600 of the bacterial solution is 0.8, then the temperature is lowered to 16°C for 1 hour, IPTG is added to make the concentration of IPTG in the medium 0.5 mM, and the culture is carried out at the culture temperature as when activated, 200 rpm for 20 h;

[0070] (3) Collecting bacterial cells: take 50 mL of the above bacterial solution, centrifuge at 5000 rpm, 4°C for 15 min, and collect the bacterial cells;

[0071] (4) Wash the precipitate three times with 100 mM PBS buffer (pH 7.2-7.4), and resuspend the bacterial cells with a transformation medium containing 1% glycerol, 0.4% glucose, the rest being 100 mM potassium phosphate buffer (pH 7.4), and 10-hydroxydecanoic acid is added to make the concentration of 10-hydroxydecanoic acid in the medium 1 g / L, and the reaction is carried out at the culture temperature as when activated, 200 rpm for 48 h, to obtain the fermentation liquor.

[0072] Fermentation broth silanization treatment: take 1 mL of fermentation broth in a 2 mL centrifuge tube, add 1 mL of 4M HC1 solution, vortex centrifuge, heat at 100°C for 10 min, then refrigerate at -80°C for 15 min, after thawing at room temperature, add 1 mL of ethyl acetate, vortex centrifuge, collect the organic phase into a new tube, add 100 μL of N, O-bis (trimethylsilyl) trifluoroacetamide, shake well, and use after 40 min in a 70°C water bath.

[0073] Gas chromatography-mass spectrometry detection of product formation and substrate consumption: gas chromatography with nitrogen as carrier gas, constant flow mode, injection volume 1 μL, split injection, split ratio 1:50, injection temperature 250°C, 50°C for 1 min, then increase to 250°C at 15°C / min, hold for 10 min.

[0074] During fermentation, the content of 10-hydroxydecanoic acid in the fermentation broth of the strain changes as shown in Figure 1 The consumption rate of 10-hydroxydecanoic acid by different strains was calculated, and the results are as follows:

[0075] Table 1: Consumption rate of 10-hydroxydecanoic acid by different strains

[0076] Strains Consumption rate Candida tropicalis 100％ Escherichia coli 2％ Staphylococcus aureus 1％ Corynebacterium glutamicum 1％ Bacillus subtilis 1％ Yarrowia lipolytica 100％ Saccharomyces cerevisiae 7％

[0077] From the above table data, after 48 h of reaction, the consumption rate of 10-hydroxydecanoic acid by Candida tropicalis and Yarrowia lipolytica was 100%, while Escherichia coli, Staphylococcus aureus, Corynebacterium glutamicum, Bacillus subtilis and Saccharomyces cerevisiae could not consume 10-hydroxydecanoic acid.

[0078] The synthesis route of 10-hydroxy-2-decenoic acid disclosed in the prior art is: first react the substrate decanoic acid to form trans-2-decenoic acid, and then form 10-hydroxy-2-decenoic acid through terminal hydroxylation of trans-2-decenoic acid, as disclosed in Chinese Patent Document CN109897870A (application number 201910088897.0) and Chinese Patent Document CN113106109A (application number 202110211118.9). In the process of research, another synthesis route of 10-hydroxy-2-decenoic acid is constructed, that is, first hydroxylate the substrate decanoic acid to form 10-hydroxydecanoic acid, and then form 10-hydroxy-2-decenoic acid through beta oxidation of 10-hydroxydecanoic acid. However, in the previous research, it is found that in this synthesis route, after the formation of 10-hydroxydecanoic acid, the reaction activity of 10-hydroxydecanoic acid to form 10-hydroxy-2-decenoic acid is weak, resulting in the accumulation of a large amount of intermediate product 10-hydroxydecanoic acid, and the formation of 10-hydroxy-2-decenoic acid from 10-hydroxydecanoic acid is the rate-limiting step of this synthesis route. In the technical solution of Chinese Patent Document CN109402182A (application number 201811126048.1), 10-hydroxydecanoic acid is used as the substrate to produce 10-hydroxy-2-decenoic acid, and the unstable phenomenon of sometimes having product and sometimes not having product also occurs. The present application finds through the above experiment that not all strains can metabolize 10-hydroxydecanoic acid to form 10-hydroxy-2-decenoic acid, and the enzyme in the beta oxidation pathway may be a key factor affecting the formation of 10-hydroxy-2-decenoic acid from 10-hydroxydecanoic acid.

[0079] Example 2: Construction of recombinant plasmid

[0080] Recombinant plasmid pETDuet-1- Ct ACD, pETDuet-1- Yl Synthesis of ACD, pET28a-SUMO-Ydil, pESC-URA-SUMO-Ydil:

[0081] Among them, the fatty acyl-CoA dehydrogenase Ct ACD, Yl The nucleotide sequences of ACD genes are shown in SEQ ID NO. 1, SEQ ID NO. 2, respectively, and the nucleotide sequence of the fatty acyl-CoA thioesterase Ydil gene is shown in SEQ ID NO. 3; the amino acid sequences of the proteins encoded by the ACD genes are shown in SEQ ID NO. 4, SEQ ID NO. 5, respectively, and the amino acid sequence of the protein encoded by the fatty acyl-CoA thioesterase Ydil gene is shown in SEQ ID NO. 6; Ct ACD, Yl The nucleotide sequences of ACD genes are shown in SEQ ID NO. 1, SEQ ID NO. 2, respectively, and the nucleotide sequence of the fatty acyl-CoA thioesterase Ydil gene is shown in SEQ ID NO. 3; the amino acid sequences of the proteins encoded by the ACD genes are shown in SEQ ID NO. 4, SEQ ID NO. 5, respectively, and the amino acid sequence of the protein encoded by the fatty acyl-CoA thioesterase Ydil gene is shown in SEQ ID NO. 6;

[0082] The recombinant plasmid pETDuet-1- CtACD, pETDuet-1- Yl ACD, pET28a-SUMO-YdiI, pESC-URA-SUMO-YdiI were completed by Nanjing Kings River Biotechnology Co., Ltd. Among them, the recombinant plasmid pETDuet-1- Ct The structural diagram of ACD is shown in Figure 2 .

[0083] Example 3: Construction of Escherichia coli genetically engineered bacteria

[0084] (1) Preparation of Escherichia coli competent cells

[0085] ① Pick a single colony of Escherichia coli BL21 (or pick a preserved strain) and inoculate into 50 mL of liquid LB medium, and incubate at 37°C, 200 rpm overnight;

[0086] ② Take 1 mL of Escherichia coli bacterial solution and inoculate into 50 mL of LB medium, and incubate at 37°C, 200 rpm until the OD 600 of the bacterial solution is about 0.5-0.6;

[0087] ③ Place the bacterial solution in an ice-water mixture for 10 min, and pre-cool a 50 mL centrifuge tube at the same time;

[0088] ④ Transfer the bacterial solution to the centrifuge tube, centrifuge at 3700 rpm for 10 min at 4°C to collect the bacterial cells;

[0089] ⑤ Add 10 mL of pre-cooled 0.1M CaCl2 solution to each centrifuge tube, resuspend the bacterial cells, and then add 30 mL of pre-cooled 0.1M CaCl2 solution, mix well by inverting, and stand on ice for 20 min;

[0090] ⑥ Centrifuge the bacterial cells at 3700 rpm for 10 min at 4°C, and add pre-cooled 0.1M CaCl2 solution containing 15% glycerol in a ratio of 3:125 to the volume of the bacterial solution in step ④, resuspend the bacterial cells, and obtain competent cells;

[0091] ⑦ Aliquot the competent cells and store them at -80°C.

[0092] (2) Transformation of recombinant plasmids in Escherichia coli

[0093] ① The recombinant plasmids pETDuet-1- Ct ACD and pET28a-SUMO-YdiI are a group, pETDuet-1- Yl ACD and pET28a-SUMO-YdiI are a group, and the volume ratio between the two plasmids in each group is 1:1, a total of 10 μL, respectively, into 100 μL of freshly prepared competent cells, mix gently, and incubate on ice for 30 min;

[0094] ②42℃ heat shock 90s, then quickly placed in ice bath cooling 3 min;

[0095] ③ Add 900 μL LB medium, 37℃, 200 rpm shaking culture 60 min;

[0096] ④ 37 00 rpm centrifugation 10 min to collect bacteria, discard the supernatant, the precipitate was resuspended with 100 μL LB liquid medium;

[0097] ⑤ Take 100 μL of the above bacteria, E. coli bacteria were plated on LB solid medium with 50 μg / mL kanamycin and 40 μg / mL streptomycin;

[0098] ⑥ LB solid medium based on 37℃ incubator positive 30 min, after the bacteria were absorbed dry, inverted plate at 37℃ for 12-16 h.

[0099] (3) Identification of positive clones:

[0100] ① Colony PCR identification

[0101] Pick the above cultured single colony, E. coli was inoculated into 1 mL containing 50 μg / mL kanamycin and 40 μg / mL streptomycin liquid LB medium, 37℃, 200 rpm shaking culture 6-8 h; 2 μL of bacteria were taken, according to 50 μL PCR reaction system, colony PCR identification, the appearance of the band and single band showed that the colony was positive clone;

[0102] ACD gene on pETDuet-1 plasmid was verified by colony PCR, all the designed primer sequences in this experiment were shown in the following table, and the agarose gel electrophoresis map of the PCR product of ACD gene was shown in Figure 3

[0103] Table 2: Primer sequence

[0104]

[0105] ② Protein expression and solubility identification

[0106] Take 900 μL of the above bacteria, IPTG was added to the concentration of 0.5 mM in E. coli, induced for 20 hours; 12000 rpm centrifugation 1 min, collected bacteria, added 2 times loading buffer, resuspended bacteria, 100℃ water bath denaturation 10 min, protein expression was detected by SDS-PAGE, the results were shown in Figure 4

[0107] ③ Bacterial sequencing

[0108] ​​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.

[0109] The final construction obtained the E. coli genetically engineered bacteria:

[0110] (BL21) / pET28a-SUMO-YdiI, pETDuet-1- Ct ACD,

[0111] (BL21) / pET28a-SUMO-YdiI, pETDuet-1- Yl ACD.

[0112] Example 4: Construction of Saccharomyces cerevisiae genetically engineered bacteria

[0113] (1) Preparation of Saccharomyces cerevisiae competent cells

[0114] ① Pick a single colony of Saccharomyces cerevisiae (or pick a preserved strain) and inoculate it into 50 mL of liquid YPD medium, and incubate it at 30°C, 200 rpm overnight;

[0115] ② Take 1 mL of Saccharomyces cerevisiae bacterial solution and inoculate it into 50 mL of liquid YPD medium, and incubate it at 30°C, 200 rpm until the OD 600 of the bacterial solution is about 3.0-5.0;

[0116] ③ Dilute 10 mL of bacterial solution to an OD 600 of about 0.2-0.4;

[0117] ④ Continue to incubate the bacterial solution at 30°C in a constant temperature shaker for 3-6 h, so that the OD 600 reaches about 0.6-1.0;

[0118] ⑤ Transfer the bacterial solution to a centrifuge tube and centrifuge it at 4°C, 3700 rpm for 10 min to collect the bacterial cells;

[0119] ⑥ Resuspend the bacterial cells with 10 mL of pre-cooled PBS buffer (pH 7.4) with a concentration of 100 mM, and centrifuge it at 4°C, 3700 rpm for 10 min to collect the bacterial cells;

[0120] ⑦ Add 1 mL of TE / LiAc solution to each centrifuge tube to resuspend the bacterial cells and obtain competent cells;

[0121] ⑧ Divide the competent cells and store them at -80°C.

[0122] (2) Transformation of recombinant plasmid in Saccharomyces cerevisiae

[0123] ① Transform the recombinant plasmid pETDuet-1- CtACD and pESC-URA-SUMO-YdiI are a group, the volume ratio of two plasmids in each group is 1:1, 2 μL each into 50 μL freshly prepared competent cells, 500 μL transformation solution (PEG / LiAc, DMSO) is added, mixed gently, ice bath 30 min; Yl ACD and pESC-URA-SUMO-YdiI are a group, the volume ratio of two plasmids in each group is 1:1, 2 μL each into 50 μL freshly prepared competent cells, 500 μL transformation solution (PEG / LiAc, DMSO) is added, mixed gently, ice bath 30 min;

[0124] ② 30℃ constant temperature shaker culture 1h, every 15min pop the wall mixed;

[0125] ③ 900 μL of liquid YPD medium is added, 30℃, 200 rpm shaking culture 60 min;

[0126] ④ 3700 rpm centrifugal 10 min to collect the bacteria, discard the supernatant, the precipitate is resuspended with 100 μL TE;

[0127] ④ Take 100 μL of the above bacteria, spread on solid YPD medium with 100 μg / mL ampicillin and 40 μg / mL streptomycin;

[0128] ⑤ the solid YPD medium after coating is placed in 37℃ incubator for 30 min, after the bacteria are absorbed, inverted, cultured at 37℃ for 12-16h.

[0129] (3) identification of positive clones:

[0130] ① colony PCR identification

[0131] Pick the above cultured single colony, inoculated into 1 mL containing 100 μg / mL ampicillin and 40 μg / mL streptomycin liquid YPD medium, 30℃, 200 rpm shaking culture 6-8h; 2 μL of bacteria were taken, and the colony PCR identification was carried out according to the method of example 3, the ACD gene on the pETDuet-1 plasmid was verified, and the band appeared and the band was single, which showed that the colony was positive clone.

[0132] ② protein expression and solubility identification

[0133] Take 900 μL of the above bacteria, add 2% galactose, induce culture for 48 hours, centrifuge at 12000 rpm for 1 min, collect the bacteria, add 2 times loading buffer, resuspend the bacteria, denature at 100℃ water bath for 10 min, detect protein expression by SDS-PAGE according to the method of example 3, and screen for positive clones.

[0134] ③ bacteria sequencing

[0135] 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.

[0136] Finally, the genetically engineered Saccharomyces cerevisiae strain was constructed:

[0137] Sc. / pESC-URA-SUMO-YdiI, pETDuet-1- Ct ACD,

[0138] Sc. / pESC-URA-SUMO-YdiI, pETDuet-1- Yl ACD.

[0139] Example 5: Preparation of 10-hydroxy-2-decenoic acid using genetically engineered bacteria

[0140] (1) Activation of the strain:

[0141] The genetically engineered Escherichia coli strain constructed in Example 3 was inoculated into 50 mL of liquid LB medium containing 50 μg / mL kanamycin and 40 μg / mL streptomycin at an inoculation amount of 1%, and cultured at 37°C with 200 rpm shaking for 12 h;

[0142] The genetically engineered Saccharomyces cerevisiae strain constructed in Example 4 was inoculated into 50 mL of liquid YPD medium containing 100 μg / mL ampicillin and 40 μg / mL streptomycin at an inoculation amount of 1%, and cultured at 30°C with 200 rpm shaking for 12 h;

[0143] (2) Bacterial transfer:

[0144] 1 mL of the activated bacterial solution of the above genetically engineered Escherichia coli strain was inoculated into 50 mL of liquid LB medium containing 50 μg / mL kanamycin and 40 μg / mL streptomycin, and cultured at 37°C with 200 rpm shaking until the OD 600 of the bacterial solution was 0.8. After being cooled to 16°C for 1 h, IPTG was added to a concentration of 0.5 mM, and the induction culture was continued at 37°C with 200 rpm shaking for 20 h;

[0145] 1 mL of the activated bacterial solution of the above genetically engineered Saccharomyces cerevisiae strain was inoculated into 50 mL of liquid YPD medium containing 100 μg / mL ampicillin and 40 μg / mL streptomycin, and cultured at 30°C with 200 rpm shaking until the OD 600 of the bacterial solution was 0.8. After being cooled to 16°C for 1 h, 2% galactose was added, and the induction culture was continued at 30°C with 200 rpm shaking for 48 h;

[0146] (3) Collection of bacterial cells: 50 mL of the above cultured bacterial solution was centrifuged at 5000 rpm and 4°C for 15 min to collect the bacterial cells;

[0147] (4) Wash the bacteria three times with 100 mM PBS buffer (pH 7.2-7.4) and resuspend the bacteria in transformation medium; the E. coli transformation medium comprises 1% glycerol, 0.4% glucose, 50 μg / mL kanamycin and 40 μg / mL streptomycin, the balance being 100 mM potassium phosphate buffer at pH 7.4, 10-hydroxydecanoic acid is dissolved in dimethyl sulfoxide and then added to the transformation medium to give a 10-hydroxydecanoic acid concentration of 0.1 g / L in the medium, and the reaction is carried out at 37°C for 24 h to obtain a fermentation liquor; the S. cerevisiae transformation medium comprises 1% glycerol, 0.4% glucose, 100 μg / mL ampicillin and 40 μg / mL streptomycin, the balance being 100 mM potassium phosphate buffer at pH 7.4, 10-hydroxydecanoic acid is dissolved in dimethyl sulfoxide and then added to the transformation medium to give a 10-hydroxydecanoic acid concentration of 0.1 g / L in the medium, and the reaction is carried out at 30°C for 24 h to obtain a fermentation liquor.

[0148] Silanization treatment of the fermentation liquor: 1 mL of the fermentation liquor is taken in a 2 mL centrifuge tube, 1 mL of 4 M HCl solution is added, vortexed and centrifuged, heated at 100°C for 10 min, then stored at -80°C for 15 min, thawed at room temperature, 1 mL of ethyl acetate is added, vortexed and centrifuged, the organic phase is collected in a new tube, 100 μL of N,O-bis(trimethylsilyl)trifluoroacetamide is added, shaken and mixed, and then used after 40 min of 70°C water bath.

[0149] Gas chromatography-mass spectrometry detection of the generated product: gas chromatography-mass spectrometry uses helium as the carrier gas, constant flow mode, injection volume 1 μL, split injection, split ratio 1:50, injection temperature 250°C, 50°C for 1 min, then increased to 250°C at 15°C / min, and maintained for 10 min.

[0150] The mass spectrum of the fermentation product 10-hydroxy-2-decenoic acid is shown in Figure 5 .

[0151] The yield of 10-hydroxy-2-decenoic acid produced by the recombinant bacteria is shown in Figure 6 .

[0152] The yield of 10-hydroxy-2-decenoic acid detected and the calculated conversion rate are shown in Table 3:

[0153] Table 3: Yield of 10-hydroxy-2-decenoic acid and conversion rate of each genetically engineered bacterium

[0154] Strains Yield / (g / L) Conversion rate Initial host bacteria Escherichia coli 0 0 (BL21) / pET28a-SUMO-YdiI, pETDuet-1- Ct ACD]] 0.043 43％ (BL21) / pET28a-SUMO-YdiI, pETDuet-1- Yl ACD]] 0.035 35％ Initial host bacteria Saccharomyces cerevisiae 0 0 Sc. / pESC-URA-SUMO-YdiI, pETDuet-1- Ct ACD]]> 0.032 32％ Sc. / pESC-URA-SUMO-YdiI, pETDuet-1- Yl ACD]]> 0.028 28％

[0155] From the above data, it can be seen that the initial host bacteria cannot synthesize 10-hydroxy-2-decenoic acid due to the lack of key enzymes for synthesizing 10-hydroxy-2-decenoic acid. After transforming the fatty acyl-CoA dehydrogenase gene and the fatty acyl-CoA thioesterase gene into the host bacteria, the genetically engineered bacteria can synthesize 10-hydroxy-2-decenoic acid from 10-hydroxy decanoic acid in one step, and the synthesis pathway is as follows: fatty acyl-CoA dehydrogenase catalyzes 10-hydroxy decanoic acid to synthesize 10-hydroxy-2-decenoic acid, and fatty acyl-CoA thioesterase catalyzes 10-hydroxy-2-decenoic acid to synthesize 10-hydroxy-2-decenoic acid after removing coenzyme A, and the conversion rate is 28-43%, which is significantly improved.

Claims

1. An engineered bacterium containing a fatty acyl-CoA dehydrogenase gene, characterized in that, The engineering bacteria comprise a fatty acyl-CoA dehydrogenase ACD gene and a fatty acyl-CoA thioesterase ACOT gene; the nucleotide sequence of the fatty acyl-CoA dehydrogenase ACD gene is SEQ ID NO. 1 or SEQ ID NO. 2; the nucleotide sequence of the fatty acyl-CoA thioesterase ACOT gene is SEQ ID NO. 3; and the host bacteria of the engineering bacteria are Escherichia coli or Saccharomyces cerevisiae.

2. The engineered bacteria containing a fatty acyl-CoA dehydrogenase gene according to claim 1, wherein, The fatty acyl-CoA dehydrogenase ACD gene is derived from Candida tropicalis or Yarrowia lipolytica.

3. The engineered bacteria containing a fatty acyl-CoA dehydrogenase gene according to claim 1, characterized in that, The amino acid sequence of the fatty acyl-CoA dehydrogenase ACD is SEQ ID NO. 4 or SEQ ID NO. 5, and the amino acid sequence of the fatty acyl-CoA thioesterase ACOT is SEQ ID NO.

6.

4. The method for constructing an engineered bacterium containing a fatty acyl-CoA dehydrogenase gene according to claim 1, characterized by, The steps are as follows: (1) constructing recombinant plasmids pETDuet-1-ACD, pET28a-SUMO-ACOT and pESC-URA-SUMO-ACOT; The nucleotide sequence of the fatty acyl-CoA dehydrogenase ACD gene is shown in SEQ ID NO. 1 or SEQ ID NO. 2, and the nucleotide sequence of the fatty acyl-CoA thioesterase ACOT gene is shown in SEQ ID NO. 3; (2) co-transforming the recombinant plasmids pETDuet-1-ACD and pET28a-SUMO-ACOT of step (1) into Escherichia coli, and obtaining the Escherichia coli genetic engineering bacteria after screening and identification; Co-transforming the recombinant plasmids pETDuet-1-ACD and pESC-URA-SUMO-ACOT of step (1) into Saccharomyces cerevisiae, and obtaining the Saccharomyces cerevisiae genetic engineering bacteria after screening and identification.

5. The application of the engineering bacteria containing the fatty acyl-CoA dehydrogenase gene in claim 1 in the preparation of 10-hydroxy-2-decenoic acid by taking 10-hydroxy decanoic acid as a substrate.

6. A method for preparing 10-hydroxy-2-decenoic acid using an engineered bacterium containing a fatty acyl-CoA dehydrogenase gene as a substrate for 10-hydroxy decanoic acid, characterized in that, The steps are as follows: After the Escherichia coli genetic engineering bacteria constructed in claim 4 are induced and cultured, induced cells are obtained, the induced cells are inoculated into a transformation culture medium, 10-hydroxy decanoic acid is added, and 10-hydroxy-2-decenoic acid is prepared by fermentation culture at 37℃.

7. The method of claim 6, wherein, One or more of the following conditions are met: i. The induction culture is: the E. coli genetically engineered bacteria is inoculated into liquid LB medium containing 50 μg / mL kanamycin and 40 μg / mL streptomycin, and is cultured at 37°C with shaking until the OD 600 of the bacterial solution is 0.8-1.2; then the temperature is lowered to 16-20°C for 1 hour of adaptation, IPTG is added to a concentration of 0.5 mM, and the induction culture is continued for 20-25 hours; the cells are separated, and the induced cells are prepared. ii. The components of the transformation culture medium are as follows, all in percentage by mass: glycerol 1%, glucose 0.4%, kanamycin 50 μg / mL, streptomycin 40 μg / mL, and the rest is potassium phosphate buffer with a concentration of 100 mM and pH 7.4; iii. 10-hydroxy decanoic acid is added to a concentration of 0.1 g / L; iv. 10-hydroxy decanoic acid is dissolved in dimethyl sulfoxide and then added to the transformation culture medium.

8. The method of claim 7, wherein, In condition i, the separation of the cells is performed by centrifugation at 5000 rpm for 15 min, the cell precipitate is collected, and then the cell precipitate is washed with PBS buffer with a concentration of 100 mM and pH 7.2-7.

4.

9. A method for preparing 10-hydroxy-2-decenoic acid using an engineered bacterium containing a fatty acyl-CoA dehydrogenase gene as a substrate for 10-hydroxy decanoic acid, characterized in that, The steps are as follows: After the Saccharomyces cerevisiae genetic engineering bacteria constructed in claim 4 are induced and cultured, induced cells are obtained, the induced cells are inoculated into a transformation culture medium, 10-hydroxy decanoic acid is added, and 10-hydroxy-2-decenoic acid is prepared by fermentation culture at 30℃.

10. The method of claim 9, wherein, One or more of the following conditions are met: i. The induction culture is: the genetically engineered Saccharomyces cerevisiae is inoculated into liquid YPD medium containing 100 μg / mL ampicillin and 40 μg / mL streptomycin, and cultured at 30°C with shaking until the OD 600 is 0.8-1.2, and then the temperature is lowered to 16-20°C for 1 hour of adaptation, 2% galactose is added, and the induction culture is continued for 48-55 hours, the cells are separated, and the induced cells are prepared. ii. The transformation medium components are as follows, all in mass percentage: glycerol 1%, glucose 0.4%, ampicillin 100 μg / mL, streptomycin 40 μg / mL, the rest being potassium phosphate buffer at a concentration of 100 mM, pH 7.4; iii. 10-hydroxydecanoic acid is added to a concentration of 0.1 g / L; iv. 10-hydroxydecanoic acid is dissolved in dimethyl sulfoxide and added to the transformation medium.

11. The method of claim 10, wherein, The separation of the cells in condition i is by centrifugation at 5000 rpm for 15 min, the cell pellet is collected and the cell pellet is washed with PBS buffer at 100 mM, pH 7.2-7.4.

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