Genetically engineered bacterium for producing abienol as well as construction method and application of genetically engineered bacterium

Abstract

The invention discloses a genetically engineered bacterium for producing abienol as well as a construction method and application thereof, and belongs to the technical field of environmental biology. In order to solve the technical problems of long fermentation period, low yield, high cost and incapability of meeting industrial production of engineering bacteria for producing abienol in the prior art, the invention provides a genetically engineered bacterium for producing abienol. A complete abienol anabolism pathway is constructed in escherichia coli, a functional membrane-free organelle system is constructed, and enzyme is collected into a membrane-free organelle through interaction of ankylopeptides, so that a metabolite channel is formed. According to the genetically engineered bacterium provided by the invention, high-yield abienol can be directly obtained from thalli, the yield of abienol is increased, the time for producing abienol by the genetically engineered bacterium is shortened, the production efficiency is improved, and the genetically engineered bacterium has a good industrial application prospect.

Description

Technical Field

[0001] The invention belongs to the field of environmental biotechnology, and specifically relates to a genetic engineering bacterium for producing afir alcohol and a construction method and application thereof. Background Art

[0002] Abietin is a natural diterpene monool found mainly in Abies balsamina ( Abies balsamea ) and is also found in plants such as tobacco. As an important raw material for the spice industry, fir alcohol has important applications in sustainable alternatives to ambergris. However, traditional fir alcohol extraction methods rely on plant sources. Due to the low content of fir alcohol in plants and the presence of a large number of functionalized derivatives, large-scale purification and production of fir alcohol have technical problems such as high difficulty, high cost, low efficiency and easy environmental pollution. Therefore, the development of alternative fir alcohol production pathways is an urgent problem to be solved. Compared with traditional plant extraction methods, the use of microorganisms to synthesize fir alcohol not only has the advantages of low cost, high yield, simple process, and easy scale-up, but also avoids the limitations of seasonality and climate change. As a model microorganism, Escherichia coli has become an ideal host strain for the synthesis of fir alcohol due to its fast growth rate, clear genetic background and easy molecular manipulation.

[0003] The synthesis pathway of abirol depends on the mevalonic acid (MVA) pathway and the pyruvate / 3-phosphoglyceraldehyde (MEP) metabolic pathway, both of which synthesize terpenoid metabolic precursors - isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP); in the synthesis of abirol, IPP and DMAPP are first synthesized into geranyl pyrophosphate (GGPP) through the catalysis of geranyl pyrophosphate synthase (GGPPS), and then GGPP is cyclized by lysine pyrophosphate synthase (Lpps) to produce abirol under the catalysis of abirol synthase (Cas). Studies in recent years have shown that due to the low flux of the MEP pathway of Escherichia coli itself, the production of terpenoid compounds can be significantly increased by introducing a heterologous MVA pathway to enhance the supply of IPP and DMAPP. For example, Li et al. introduced Lpps and Cas gene and overexpressed the MVA pathway to successfully construct an Escherichia coli capable of producing abestrol (L. Li, X. Wang, X. Li, H. Shi, F. Wang, Y. Zhang, and X. Li, Journal of Agricultural and Food Chemistry, 2019, 67, 6523-6531).

[0004] Although there are engineered bacteria that can produce fir alcohol in the prior art, the engineered strains are still a certain distance away from the requirements of industrial production in terms of fermentation cycle and fir alcohol yield. Therefore, those skilled in the art are eager to further optimize the synthesis pathway of fir alcohol, improve metabolic flux and yield, and achieve high-yield, high-efficiency, and low-cost synthesis of fir alcohol. Summary of the invention

[0005] The present invention aims to solve the technical problems in the prior art of producing fir alcohol, such as long fermentation cycle, low yield and high cost, which cannot meet the requirements of industrial production, and provides a genetically engineered bacterium for producing fir alcohol, a construction method and application thereof.

[0006] One of the purposes of the present invention is to provide a genetically engineered bacterium for producing firwood alcohol, wherein the genetically engineered bacterium expresses acetyl-CoA acyltransferase / hydroxymethylglutaryl-CoA reductase, 3-hydroxy-3-methylglutaryl-CoA synthase, mevalonate kinase, mevalonate-5-phosphate kinase, mevalonate-5-diphosphate decarboxylase, isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthetase, geranyl pyrophosphate synthetase, pyrophosphate lisdendronol synthetase, firwood alcohol synthetase, RGG domain in LAF-1 protein, short peptide RIDD of kinase anchor protein and short peptide RIAD of cAMP-dependent protein kinase A, and the starting host bacterium of the genetically engineered bacterium is Escherichia coli.

[0007] In a preferred embodiment of the present invention, the acetyl-CoA acyltransferase / hydroxymethylglutaryl-CoA reductase is a gene mvaE Encoding, GenBank accession number is AAG02439.1; the 3-hydroxy-3-methylglutaryl-CoA synthase is gene mvaS Encoding, GenBank accession number is AAG02438.1; the mevalonate kinase is gene ERG12 Encoding, GenBank accession number is NM_001182715.1; the mevalonate-5-phosphate kinase is a gene ERG8 Encoding, GenBank accession number is NM_001182727.1; the mevalonate-5-diphosphate decarboxylase is gene ERG19 Encoding, GenBank accession number is X97557.1; the isopentenyl pyrophosphate isomerase is gene IDI1 Encoding, GenBank accession number is NM_001183931.1; the farnesyl pyrophosphate synthase is gene Erg20 Encoding, GenBank accession number is NM_001181600.1; the geranyl pyrophosphate synthase is gene CrtE Encoding, GenBank accession number is M87280.1; the pyrophosphate lysine ester synthase is geneLpps Encoding, GenBank accession number is JQ478434.1; the abies alcohol synthase is gene Cas The nucleotide sequence of the RGG domain in the LAF-1 protein is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; the nucleotide sequence of the short peptide RIAD of the cAMP-dependent protein kinase A is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4; the nucleotide sequence of the short peptide RIDD of the kinase anchor protein is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6.

[0008] A second object of the present invention is to provide a microbial preparation containing the above-mentioned genetically engineered bacteria.

[0009] The third object of the present invention is to provide a method for constructing the above-mentioned genetically engineered bacteria, which specifically comprises the following steps: S1: Construction of expression vector: acetyl-CoA acyltransferase / hydroxymethylglutaryl-CoA reductase gene mvaE , 3-hydroxy-3-methylglutaryl-CoA synthase gene mvaS Ligated into plasmid pCDFDuet-1 to obtain recombinant plasmid I; farnesyl pyrophosphate synthase gene Erg20 geranyl pyrophosphate synthase gene CrtE , pyrophosphate lysine synthase gene with a short peptide tag RIDD RIDD-Lpps , abirol synthase gene with a short peptide tag RIDD RIDD-Cas cloned into plasmid pACYCDuet-1 to obtain recombinant plasmid II; The RGG domain nucleotide sequence in the LAF-1 protein was cloned into the expression vector pET-28a(+) to obtain recombinant plasmid III; S2: Construction of mutant strains: mevalonate kinase gene ERG12 , mevalonate-5-phosphate kinase gene ERG8 , mevalonate-5-diphosphate decarboxylase gene ERG19 and isopentenyl pyrophosphate isomerase gene IDI1 The mutant strain I containing the above four genes was obtained by integrating into the genome of Escherichia coli BL21 (DE3); S3: transformation of recombinant bacteria: the recombinant plasmid I, recombinant plasmid II and recombinant plasmid III obtained in step S1 are transformed into the competent cells prepared by the mutant strain I obtained in step S2 to obtain genetically engineered bacteria for producing afel alcohol.

[0010] In a preferred embodiment of the present invention, the acetyl-CoA acyltransferase / hydroxymethylglutaryl-CoA reductase is a gene mvaE Encoding, GenBank accession number is AAG02439.1; the 3-hydroxy-3-methylglutaryl-CoA synthase is gene mvaS Encoding, GenBank accession number is AAG02438.1; the mevalonate kinase is gene ERG12 Encoding, GenBank accession number is NM_001182715.1; the mevalonate-5-phosphate kinase is a gene ERG8 Encoding, GenBank accession number is NM_001182727.1; the mevalonate-5-diphosphate decarboxylase is gene ERG19 Encoding, GenBank accession number is X97557.1; the isopentenyl pyrophosphate isomerase is gene IDI Encoding, GenBank accession number is NM_001183931.1; the farnesyl pyrophosphate synthase is gene Erg20 Encoding, GenBank accession number is NM_001181600.1; the geranyl pyrophosphate synthase is gene CrtE Encoding, GenBank accession number is M87280.1; the pyrophosphate lysine ester synthase is gene Lpps Encoding, GenBank accession number is JQ478434.1; the abies alcohol synthase is gene Cas The encoding number is JN254808.1 in GenBank; the nucleotide sequence of the RGG domain in the LAF-1 protein is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; the nucleotide sequence of the short peptide RIAD of the cAMP-dependent protein kinase A is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4; the nucleotide sequence of the short peptide RIDD of the kinase anchor protein is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6.

[0011] A fourth object of the present invention is to provide a method for producing abirol, wherein the method comprises fermenting the above-mentioned genetically engineered bacteria at 37° C. for at least 46 hours.

[0012] In a preferred embodiment of the present invention, the culture medium used for fermentation in the method uses glucose as the carbon source.

[0013] In a preferred embodiment of the present invention, the culture medium used for fermentation in the method uses yeast powder as a nitrogen source.

[0014] In a preferred embodiment of the present invention, the fermentation medium used for fermentation in the method contains yeast powder, K 2 HPO 4 ·3H 2 O, citric acid, ammonium ferric citrate, glucose, MgSO 4 7H 2 O and trace elements.

[0015] A fifth object of the present invention is to provide the use of the above-mentioned genetically engineered bacteria or microbial preparation in the production of abirol.

[0016] Beneficial effects of the present invention: The present invention provides a genetically engineered bacterium for producing fir alcohol, and a construction method and application thereof. Based on the fir alcohol synthase gene in fir, the present invention constructs a complete fir alcohol synthesis metabolic pathway in Escherichia coli, thereby achieving efficient fir alcohol biosynthesis.

[0017] Furthermore, the present invention constructs a functional membrane-less organelle system, wherein pyrophosphate lysine synthase and abestrol synthase are fused with the anchor peptide RIAD, and the membrane-less organelle protein RGG fused with the anchor peptide RIDD is overexpressed. While forming membrane-less organelles, the enzyme is recruited into the membrane-less organelles through the interaction of the anchor peptides to form a metabolite channel; thereby, the ability of the genetically engineered bacteria provided by the present invention to synthesize abestrol is increased by 37%, and the yield of abestrol can be as high as 1473 mg / L under batch fed fermentation conditions.

[0018] The genetically engineered bacteria provided by the present invention can directly obtain high-yield alfalfa alcohol from the bacteria. Compared with the genetically engineered bacteria for producing alfalfa alcohol in the prior art, not only the yield of alfalfa alcohol is increased, but also the time for producing alfalfa alcohol by the genetically engineered bacteria is shortened, the production efficiency is improved, and the production efficiency is improved, which has good industrial application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 GC spectra of the fir alcohol standard and the fir alcohol synthesized by genetically engineered bacteria in Example 2; a is the GC spectrum of the fir alcohol standard, the abscissa is the retention time, and the ordinate is the signal intensity; b is the GC spectrum of the fir alcohol sample synthesized by genetically engineered bacteria, the abscissa is the retention time, and the ordinate is the signal intensity; Figure 2 The graph is a graph showing the growth trend of the genetically engineered bacteria in shake flasks and the synthesis trend of abienol in Example 2; the horizontal axis is time, the left vertical axis Cell Density is biomass, and the right vertical axis Abienol is abienol content; Figure 3The graph is a graph showing the growth trend of the abietol high-density fermentation bacteria and the abietol synthesis trend in Example 3; the horizontal axis represents time, the left vertical axis Cell Density represents biomass, and the right vertical axis Abienol represents abietol content. DETAILED DESCRIPTION

[0020] Those skilled in the art can refer to the content of this article and appropriately improve the process parameters. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0021] In order to make the purpose, technical scheme and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments and the accompanying drawings of the specification. The test methods used in the following examples are conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained by those skilled in the art through commercial channels.

[0022] Extraction and detection method of afel alcohol: Take 0.5 mL of fermentation broth, add an equal volume of ethyl acetate, mix on a vortex oscillator for 2 min, centrifuge at 13000×g for 20 min, take the upper organic phase for filtration, and place it in a liquid phase vial for testing. Detection conditions: GC-MS instrument model: Shimadzu TQ8050; ion source: EI; detector: quadrupole. Detection sample: Take the liquid in the gas phase vial for detection. GC instrument model: Shimadzu GC 2010Pro; separation column model: HP-5; injection volume: 1 µL; detector: hydrogen flame detector (FID); column temperature: 50℃ for 2.5 min, then heat to 320℃ at a rate of 10℃ / min and keep for 5 min.

[0023] The primary seed culture medium is LB medium: 10 g / L peptone, 5 g / L yeast powder, 10 g / L NaCl, the final concentrations of kanamycin, chloramphenicol and streptomycin in the fermentation broth are 50 μg / mL, 34 μg / mL and 50 μg / mL, respectively, pH=7.0, and high-pressure steam sterilization is performed at 121°C for 20 min. The antibiotics are sterilized by filtration with a 0.22 μm filter membrane.

[0024] The secondary seed medium is M9 liquid medium (or M9 seed medium), including: 20 g / L glucose, 15.2 g / L Na 2 HPO 4•12H 2 O, 3 g / L KH 2 PO 4 , 1 g / L NH 4 Cl, 0.5 g / L NaCl, 0.4 g / L MgSO 4 The final concentrations of kanamycin, chloramphenicol and streptomycin in the fermentation broth were 50 μg / mL, 34 μg / mL and 50 μg / mL, respectively, and the pH was 7.0; among which: Na 2 HPO 4 •12H 2 O.KH 2 PO 4 NH 4 After mixing Cl and NaCl, the solution was adjusted to pH = 7.0 and sterilized by high pressure steam at 121 °C for 20 min; the glucose stock solution was 500 g / L and sterilized separately at 115 °C for 30 min; MgSO 4 •7H 2 O stock solution was 200 g / L and sterilized at 121 °C for 20 min. The antibiotics were sterilized by filtration with a 0.22 μm filter membrane. When the seed solution was transferred, the fermentation medium was supplemented with separately sterilized glucose and MgSO 4 •7H 2 O and antibiotics.

[0025] Fermentation medium: 0.5 g / L yeast powder, 9.8 g / L K 2 HPO 4 •3H 2 O, 2.1 g / L citric acid•H 2 O, 0.3 g / L ammonium ferric citrate, 20 g / L glucose, 0.4 g / L MgSO 4 •7H 2 O; 1 ml / L 1000× trace elements ((NH 4 ) 6 Mo 7 O 24 •4H 2 O 3.7 g / L, ZnSO 4 •7H 2 O 2.9 g / L, H 3 BO 3 24.7 g / L, CuSO 4 •5H 2 O 2.5 g / L, MnCl 2 •4H 2O 15.8 g / L), the final concentrations of kanamycin, chloramphenicol and streptomycin in the fermentation broth were 50 μg / mL, 34 μg / mL and 50 μg / mL, respectively, and pH = 7.0; where: K 2 HPO 4 •3H 2 O 9.8 g / L, Citric acid•H 2 O 2.1 g / L, ammonium ferric citrate 0.3 g / L, (NH 4 ) 2 SO 4 3.0 g / L, the solution was adjusted to pH = 7.0 after mixing, and sterilized by high pressure steam at 121 ° C for 20 min; 1000 × trace elements were sterilized by filtration using a 0.22 μm filter membrane; when transferring the seed solution, the fermentation medium was supplemented with separately sterilized glucose and MgSO 4 •7H 2 O, 1000× trace element stock solution and antibiotics.

[0026] Example 1: Construction of a genetically engineered bacterium for producing abirol The genetically engineered bacteria for producing abirol described in this embodiment uses Escherichia coli as a host bacterium, and the genetically engineered bacteria express acetyl-CoA acyltransferase / hydroxymethylglutaryl-CoA reductase, 3-hydroxy-3-methylglutaryl-CoA synthase, mevalonate kinase, mevalonate-5-phosphate kinase, mevalonate-5-diphosphate decarboxylase, isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthetase, geranyl pyrophosphate synthetase, pyrophosphate lisdendronol synthetase, abirol synthase, the RGG domain in LAF-1 protein, the short peptide RIDD of kinase anchor protein and the short peptide RIAD of cAMP-dependent protein kinase A; the acetyl-CoA acyltransferase / hydroxymethylglutaryl-CoA reductase is a gene mvaE Encoding, GenBank accession number is AAG02439.1; the 3-hydroxy-3-methylglutaryl-CoA synthase is gene mvaS Encoding, GenBank accession number is AAG02438.1; the mevalonate kinase is gene ERG12 Encoding, GenBank accession number is NM_001182715.1; the mevalonate-5-phosphate kinase is a gene ERG8 Encoding, GenBank accession number is NM_001182727.1; the mevalonate-5-diphosphate decarboxylase is gene ERG19 Encoding, GenBank accession number is X97557.1; the isopentenyl pyrophosphate isomerase is gene IDI1 Encoding, GenBank accession number is NM_001183931.1; the farnesyl pyrophosphate synthase is geneErg20 Encoding, GenBank accession number is NM_001181600.1; the geranyl pyrophosphate synthase is gene CrtE Encoding, GenBank accession number is M87280.1; the pyrophosphate lysine ester synthase is gene Lpps Encoding, GenBank accession number is JQ478434.1; the abies alcohol synthase is gene Cas The encoding number is JN254808.1 in GenBank; the nucleotide sequence of the RGG domain in the LAF-1 protein is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2; the nucleotide sequence of the short peptide RIAD of the cAMP-dependent protein kinase A is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4; the nucleotide sequence of the short peptide RIDD of the kinase anchor protein is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6.

[0027] The specific construction method of genetically engineered bacteria is as follows: S1: Construction of recombinant plasmid I The recombinant plasmid I (pCDF-ES), i.e., pCDF-mvaE-mvaS plasmid, contains the acetyl-CoA acyltransferase / hydroxymethylglutaryl-CoA reductase gene mvaE , 3-hydroxy-3-methylglutaryl-CoA synthase gene mvaS Ligated into plasmid pCDFDuet-1 to obtain recombinant plasmid I; For those carrying bacteria from Enterococcus faecalis ( Enterococcus faecalis ) acetyl-CoA acyltransferase gene / hydroxymethylglutaryl-CoA reductase gene mvaE (GenBank No. AAG02439), hydroxymethylglutaryl-CoA synthase gene mvaS The pET30a(+) plasmid (GenBank No. AAG02438) was constructed as follows: S1-1: The gene derived from Enterococcus faecalis mvaE and genes mvaS It was obtained by chemical synthesis by Shanghai Jierui Biotechnology Co., Ltd. and then connected with the vector pGH to obtain pGH / mvaE and pGH / mvaS respectively; the pGH / mvaE and pGH / mvaS plasmid vectors obtained in S1-1 were used as templates, and mvaE_F (nucleotide sequence is shown in SEQ ID NO.7) and mvaE_R (nucleotide sequence is shown in SEQ ID NO.8) were used as primers to amplify mvaEGene fragment; mvaS_F (nucleotide sequence is shown in SEQ ID NO.9) and mvaS_R (nucleotide sequence is shown in SEQ ID NO.10) were used as primers to amplify mvaS Gene fragment; PCR amplification conditions were: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 15 s, 56℃ annealing for 10 s, 72℃ extension for 60 s; after repeating the above three steps of denaturation, annealing and extension for 34 cycles, the target gene fragment was recovered using a gel recovery kit (purchased from Omega); S1-2: The amplified mvaE and mvaS Add both ends of the gene fragment Nco I / Bam HI or Sac I / Hind III restriction site, the PCR fragment was recovered by agarose gel electrophoresis, and the above mvaE The gene fragment was cleaved with the vector pCDFDuet-1 using endonuclease Nco I / Bam HI was digested at 37℃ for 1 h, and the digested products were recovered by gel chromatography and then purified by T 4 The cells were connected by DNA ligase. After overnight connection at 16°C, an appropriate amount of the connection solution was taken to transform into competent E. coli cells. After activation, 100 μL was coated on LB plates (Cm resistance) and cultured overnight at 37°C. 30 transformants were selected and cultured at 37°C and 180 rpm for 8 h. 2 μL of the bacterial solution was taken from each cell as a template for colony PCR amplification and identification, and positive clones were screened. The plasmids of the positive clones obtained from the above screening were extracted for restriction digestion verification. The plasmid that passed the restriction digestion verification was pCDF-mvaE. S1-3: The amplified mvaS The gene fragment was cleaved with the vector pCDF-mvaE plasmid obtained in S1-2 using endonuclease Sac I / Hind III was digested at 37°C for 1 h, and the digested products were recovered by gel electrophoresis and analyzed by T 4 DNA ligase was used for ligation. After ligation at 16°C overnight, an appropriate amount of ligation solution was taken to transform competent E. coli cells. After activation, 100 μL was taken to coat LB plates (Cm resistance) and cultured at 37°C overnight. 30 transformants were selected and cultured at 37°C and 180 rpm for 8 h. 2 μL of bacterial solution was taken from each plate as a template for colony PCR amplification and identification, and positive clones were screened. The plasmids of positive clones obtained from the above screening were extracted for restriction digestion verification. The plasmid that passed the restriction digestion verification was pCDF-mvaE-mvaS (abbreviated as pCDF-ES), which was the recombinant plasmid I. S2: Construction of recombinant plasmid II The recombinant plasmid II (pACY-ECRLRC), i.e., pACY-Erg20-CrtE-RIDD-Lpps-RIDD-Cas plasmid, contains the farnesyl pyrophosphate synthase gene Erg20 geranyl pyrophosphate synthase gene CrtE , pyrophosphate lysine synthase gene with a short peptide tag RIDD RIDD-Lpps , abirol synthase gene with a short peptide tag RIDD RIDD- Cas cloned into plasmid pACYCDuet-1 to obtain recombinant plasmid II; The farnesyl pyrophosphate synthase is a gene Erg20 Encoding, GenBank accession number is NM_001181600.1; the geranyl pyrophosphate synthase is gene CrtE Encoding, GenBank accession number is M87280.1; the pyrophosphate lysine ester synthase is gene Lpps Encoding, GenBank accession number is JQ478434.1; the abies alcohol synthase is gene Cas The encoding number of GenBank is JN254808.1; the nucleotide sequence of the short peptide RIDD of the kinase anchor protein is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.6; the specific construction steps are as follows: S2-1: Construction of Erg20-CrtE gene fragment The Saccharomyces cerevisiae genome was used as a template and Erg20_F_BglII (nucleotide sequence shown in SEQ ID NO.11) and Erg20_R (nucleotide sequence shown in SEQ ID NO.12) were used as primers to amplify Erg20 Gene fragment; amplified using primers Crte-F (nucleotide sequence shown in SEQ ID NO.13) and Crte-R_XhoI (nucleotide sequence shown in SEQ ID NO.14) CrtE The PCR amplification conditions were as follows: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 15 s, annealing at 56°C for 10 s, and extension at 72°C for 30 s. The above three steps of denaturation, annealing, and extension were repeated for 32 cycles, and then extended at 72°C for 5 min. The target gene fragment was recovered using a gel recovery kit (purchased from Omega); the target gene fragment obtained by the above PCR amplification was amplified using Overlap PCR. Erg20 and CrtEThe gene fragments were connected, and the PCR amplification conditions were as follows: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 15 s, annealing at 56°C for 10 s, and extension at 72°C for 30 s; after repeating the above three steps of denaturation, annealing, and extension for 32 cycles, the target gene fragment Erg20-CrtE was recovered using a gel recovery kit (purchased from Omega); S2-2: Construction of pACY-Erg20-CrtE plasmid use Bgl II and Xho I. The Erg20-CrtE gene fragment obtained in S2-1 was double-digested with the vector pACYDuet-1. The vector and the exogenous fragment Erg20-CrtE were ligated at a molar ratio of 1:2 using TaKaRa's DNA Ligation Kit Ver.2.1. The ligation was carried out at 16°C for 30 min. The recombinant product was transformed into E. coli DH5α, then coated on LB solid plates with 34 mg / L chloramphenicol (Cm), screened positive clones by PCR, extracted recombinant plasmids from positive clones, and then identified by restriction enzyme digestion and sequencing to obtain the pACYC-Erg20-CrtE (abbreviated as pACY-EC) recombinant plasmid; S2-3: Construction of pACY-Erg20-CrtE-Lpps-Cas plasmid The gene was chemically synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. RIDD-LPPS As a template, primers LPPS_F (nucleotide sequence is shown in SEQ ID NO.15) and LPPS_R (nucleotide sequence is shown in SEQ ID NO.16) were used to amplify LPPS Gene fragment; amplified using primers Cas_F (nucleotide sequence is shown in SEQ ID NO.17) and Cas_R (nucleotide sequence is shown in SEQ ID NO.18) Cas The PCR amplification conditions are as follows: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 15 s, annealing at 56°C for 10 s, and extension at 72°C for 60 s. The above three steps of denaturation, annealing, and extension are repeated for 32 cycles. The target gene fragment is recovered using a gel recovery kit (purchased from Omega); The vector pACY-EC obtained in S2-2 was digested with Ndi I and the vector pACY-EC was separated from the exogenous fragments. Lpps, CasThe TaKara seamless cloning kit was used for ligation at a molar ratio of 1:2, and the ligation was carried out at 50°C for 30 min. The recombinant product was transformed into E. coli DH5α, and then coated on LB solid plates with 34 mg / L chloramphenicol (Cm). Positive clones were screened by PCR, and recombinant plasmids were extracted from the positive clones. The recombinant plasmid pACY-Erg20-CrtE-Lpps-Cas (abbreviated as: pACY-ECLC) was obtained by restriction enzyme digestion and sequencing identification. S2-4: Construction of pACY-Erg20-CrtE-RIDD-Lpps-RIDD-Cas plasmid The gene was chemically synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. RIDD-LPPS As a template, primers RIDD-LPPS_F (nucleotide sequence is shown in SEQ ID NO.19) and RIDD-LPPS_R (nucleotide sequence is shown in SEQ ID NO.20) were used to amplify RIDD-LPPS Gene fragment; amplified using primers RIDD-Cas_F (nucleotide sequence is shown in SEQ ID NO.21) and RIDD-Cas_R (nucleotide sequence is shown in SEQ ID NO.22) RIDD-Cas The PCR amplification conditions are as follows: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 15 s, annealing at 56°C for 10 s, and extension at 72°C for 60 s. The above three steps of denaturation, annealing, and extension are repeated for 32 cycles. The target gene fragment is recovered using a gel recovery kit (purchased from Omega); The vector pACY-EC obtained in S2-2 was used Nde I was used for single enzyme digestion, and the vector pACY-EC was separated from the exogenous fragments RIDD-Lpps, RIDD-Cas The molar ratio was 1:2 using TaKara's seamless cloning kit for ligation at 50°C for 30 min. The recombinant product was transformed E. coli DH5α, then coated with LB solid plate with 34 mg / L chloramphenicol (Cm), PCR screened positive clones, extracted recombinant plasmids from positive clones, and then obtained pACY-Erg20-CrtE-RIDD-Lpps-RIDD-Cas (abbreviated as: pACY-ECRLRC) recombinant plasmid by restriction enzyme digestion and sequencing, which is recombinant plasmid II; S3: Construction of recombinant plasmid III The recombinant plasmid III (pET-RR) is the pET-RIAD-RGG plasmid. The RGG domain nucleotide sequence in the LAF-1 protein is cloned into the expression vector pET-28a(+). The RGG domain nucleotide sequence in the LAF-1 protein is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. The recombinant plasmid III is obtained. The specific construction steps are as follows: Gene synthesized by Suzhou Jinweizhi Biotechnology Co., Ltd. RIAD-RGG As a template, primers RIAD-RGG_F (nucleotide sequence is shown in SEQ ID NO.23) and RIAD-RGG_R (nucleotide sequence is shown in SEQ ID NO.24) were used to amplify RIAD-RGG The target gene fragment; the PCR amplification conditions are as follows: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 15 s, annealing at 56°C for 10 s, and extension at 72°C for 60 s, and the above three steps of denaturation, annealing, and extension are repeated for 32 cycles; the target gene fragment is recovered using a gel recovery kit (purchased from Omega); the PCR amplification conditions are as follows: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 15 s, annealing at 56°C for 10 s, and extension at 72°C for 30 s, and the above three steps of denaturation, annealing, and extension are repeated for 32 cycles; the target gene fragment is recovered using a gel recovery kit (purchased from Omega); Vector pET28a (+) used Nco R was used for enzyme digestion to separate the pET28a (+) vector and the exogenous fragment RIAD-RGG The molar ratio was 1:2 using TaKara's seamless cloning kit for ligation at 50°C for 30 min. The recombinant product was transformed E. coli DH5α, then coated with LB solid plate with 50 mg / L kanamycin (Kan), PCR screened positive clones, extracted recombinant plasmids from positive clones, and then obtained pET-RIAD-RGG (abbreviated as: pET-RR) recombinant plasmids by restriction enzyme digestion and sequencing, which is recombinant plasmid III; S4: Construction of mutant strain I-BL21(DE3)::Trc-low mevalonate kinase gene ERG12 , mevalonate-5-phosphate kinase gene ERG8 , mevalonate-5-diphosphate decarboxylase gene ERG19 and isopentenyl pyrophosphate isomerase gene IDI1 Integrate into the genome of Escherichia coli BL21 (DE3) to obtain a mutant strain I containing four genes; the specific construction steps are as follows: S4-1: Construction of suicide plasmid pRE-glmS-glmU Integrate the Low gene into the chromosome of Escherichia coli glmS and glmU In the middle of the two genes. When amplifying the downstream fragment, the Trc promoter should be amplified together to obtain the Trc-low fragment. First, the E. coli genome was used as a template and primers glms_F (nucleotide sequence shown in SEQ ID NO.25) and glmU_F (nucleotide sequence shown in SEQ ID NO.26) were used to amplify glmS Gene fragment; amplified using primers glmU_F (nucleotide sequence shown in SEQ ID NO.27) and glmU_R (nucleotide sequence shown in SEQ ID NO.28) glmU Gene fragment; the PCR amplification conditions: 98°C pre-denaturation for 2 min; 98°C denaturation for 15 s, 56°C annealing for 10 s, 72°C extension for 60 s; the above three steps of denaturation, annealing and extension were repeated for 32 cycles; the target gene fragment was recovered using a gel recovery kit (purchased from Omega); Overlap PCR amplification was used to glmS and glmU The two gene fragments were connected, and the PCR amplification conditions were as follows: pre-denaturation at 98°C for 2 min; denaturation at 98°C for 15 s, annealing at 56°C for 10 s, and extension at 72°C for 60 s; the above three steps of denaturation, annealing, and extension were repeated for 32 cycles; the target gene fragment glmS-glmU was recovered using a gel recovery kit (purchased from Omega); The vector pRE112 was used Sac I and Xba I was used for double restriction digestion, and the vector pRE112 and the exogenous fragment glmS-glmU were connected in a molar ratio of 1:2 using TaKara's DNA Ligation Kit Ver.2.1. The connection was carried out at 16°C for 30 min, and the recombinant product was transformed E. coli DH5α, then coated on LB solid plates with 34 mg / L chloramphenicol (Cm), PCR screened positive clones, extracted recombinant plasmids from positive clones, and obtained suicide plasmid pRE-glmS-glmU through restriction enzyme digestion and sequencing identification; S4-2: Construction of suicide plasmid pRE-glmS-Trc-low-glmU Using pYJM14 as a template, Trc-low_F (nucleotide sequence is shown in SEQ ID NO.29) and rc-low_R (nucleotide sequence is shown in SEQ ID NO.30) as primers to amplify the Trc-low fragment, and the PCR amplification conditions are as follows: 98°C pre-denaturation for 2 min; 98°C denaturation for 15 s, 56°C annealing for 10 s, and 72°C extension for 5 min; the above three steps of denaturation, annealing, and extension are repeated for 32 cycles; the target gene fragment Trc-low is recovered using a gel recovery kit (purchased from Omega); The suicide plasmid pRE-glmS-glmU obtained in S4-1 was used with the Trc-low fragment Not I and Xho I was used for double enzyme digestion, and the pRE-glmS-glmU vector and the exogenous fragment Trc-low were connected in a molar ratio of 1:1 using TaKara's DNA Ligation Kit Ver.2.1. The connection was carried out at 16°C for 30 min, and the recombinant product was transformed E. coli DH5α, then coated on LB solid plates with 34 mg / L chloramphenicol (Cm), PCR screened positive clones, extracted recombinant plasmids from positive clones, and obtained suicide plasmid pRE-glmS-Trc-low-glmU through restriction enzyme digestion and sequencing identification; S4-3: First recombination screening The suicide plasmid pRE-glmS-Trc-low-glmU obtained in S4-2 was transformed into E.coli In χ7213, the transformation method is as follows: 2 μL of suicide plasmid is added to the prepared competent cells, ice bathed for 30 min, heat shocked at 42°C for 90 s, 500 μL of LB medium and 3 μL of 10% DAP are added, and cultured at 37°C on a shaker for one hour, centrifuged at 6000 rpm for 2 min, resuspended with 100 μL of 10% DAP, 50 μL of the resuspended solution is spread on a plate containing chloramphenicol resistance, and cultured at 37°C for 15 h; Select the above E. coli Single colonies of χ7213 and BL21 (DE3) were inoculated into LB medium. E.coli 15 μL of 10% DAP was added to χ7213. After culturing at 37°C for 12 hours, 1 mL was taken for centrifugation, washed twice with water, and resuspended with 500 μL. 100 μL of the resuspended liquid was taken for mixing and spread on LB medium without resistance. After culturing at 37°C overnight, all the bacterial liquid was washed with 1 mL of water, and the bacterial cells were collected by centrifugation. After centrifugation, ddH2 Wash once more with ddHO to remove DAP. 2 O resuspended bacteria, and diluted the resuspended bacteria solution by 10 -1 -10 -4 , 30 μL of the resuspended bacterial solution was spread on a chloramphenicol plate at each multiple, cultured overnight at 37°C, and a single bacterial solution was selected for verification, and positive bacteria were marked; S4-4: Secondary recombination screening The positive bacteria screened in S4-3 were inoculated into non-resistant LB medium and cultured at 37°C for 15 h. -3 -10 -6 , take 30 μL of each multiple and spread it on a 10% sucrose plate, and culture it at 37℃ for 12 h; select ≥40 monoclones on the sucrose plate, streak them on a chloramphenicol plate and a sucrose plate respectively, and the two plates are streaked with the same number; culture it at 37℃ for 10 h, select the single colonies that grow on the sucrose plate but not on the chloramphenicol plate for PCR verification, and mark the positive bacteria; Single clones were separated twice on sucrose plates to ensure the purity of the secondary recombination. The bacteria that grew in LB but did not grow in the chloramphenicol LB test tube were the mutant strains I (BL21(DE3)::Trc-low) with successful recombination. The specific construction method of the mutant strain I-BL21(DE3)::Trc-low can also be found in the literature Cheng T, Zhao G, Xian M, et al . Improved cis -Abienol production through increasing precursor supply in Escherichia coli . Scientific Reports, 2020, 10:16791; S5: Construction of genetically engineered Escherichia coli The recombinant plasmid I (pCDF-ES) obtained in S1 and the recombinant plasmid pACY-ECLC obtained in S2-2 were heat-shock transformed into the mutant strain I ( E. coli , BL21(DE3)::Trc-low) competent cells were plated on LB solid plates containing streptomycin and chloramphenicol antibiotics to obtain genetically engineered Escherichia coli CAS06; The recombinant plasmid I (pCDF-ES) obtained in S1, the recombinant plasmid III (pET-RR) obtained in S3, and the recombinant plasmid II (pACY-ECRLRC) obtained in S2 were heat-shock transformed into the mutant strain I obtained in S4 ( E. coli, BL21(DE3)::Trc-low) competent cells were spread on LB solid plates containing streptomycin, kanamycin and chloramphenicol antibiotics to obtain genetically engineered Escherichia coli CAS08.

[0028] Example 2: A method for producing abirol S1: Pick the single clones (CAS06 and CAS08) obtained in Example 1 and inoculate them into 3 mL LB medium for cultivation. 600 The culture medium was inoculated at a ratio of 1% into 100 mL of M9 seed medium and cultured at 37°C and 180 rpm until the OD 600 The concentration of the induction agent was 0.6-1.0, and the final concentration of IPTG was 0.5 mmol. The induction agent was induced and cultured at 30°C and 180 rpm for 48 hours for shake flask fermentation; S2: After the shake flask fermentation in S1 was completed, 50 mL of the fermentation liquid in the shake flask was taken, and an equal volume of ethyl acetate was added for extraction. The mixture was vortexed for 3 min, centrifuged at 8000 rpm for 5 min, and the upper organic phase was filtered through a 0.22 μm organic phase filter to obtain the abies alcohol in the fermentation liquid.

[0029] In this example, the above-obtained abies alcohol was tested, and the results were as follows: Figure 1 As shown, a is the standard substance of abestrol, which has a peak at 21.48 min; b is the product synthesized by the engineered bacteria, which has the same peak time as the standard substance, and the proportions of each fragment peak of the MS of the two are consistent, indicating that the present example successfully biosynthesized abestrol.

[0030] This example compares the shaking fermentation data of CAS06 and CAS08 strains that use membraneless cells to fuse two pathway cascade enzymes (such as Figure 2 As shown in Figure 2, it was found that at the same time point, the OD of CAS06 cells was 600 Basically consistent with CAS08, by comparing the abirol production of engineered strains CAS06 and CAS08, the engineered strain CAS08 was 20-40% higher at the same time point, and the highest concentration of 112 mg / L was reached after 40 hours of shake flask fermentation.

[0031] It shows that the membraneless organelles formed by expressing RGG can successfully recruit pathway proteins LPPS and Cas to form metabolic channels, achieve rapid conversion of intermediates, and increase the metabolic flux from glucose to target products, thereby improving the growth of engineered bacteria and the synthesis of target products.

[0032] Example 3: Production of abestrol by high-density fermentation The monoclonal CAS08 obtained in Example 1 was inoculated into a primary seed culture medium to obtain a primary seed solution; the primary seed solution was inoculated into a secondary seed culture medium to obtain a secondary seed solution; the secondary seed solution was inoculated into a fermentation culture medium for fermentation culture, and the specific method is as follows: S1: The monoclonal CAS08 obtained in Example 1 was inoculated into a 50 ml sterile centrifuge tube containing 10 mL LB medium, and streptomycin with a final concentration of 50 μg / mL, 34 μg / mL chloramphenicol, and 50 μg / mL kanamycin were added, and cultured at 37°C with a shaker at 220 rpm until OD 600 When it grows to about 1 (the culture time is 8-12 h), the first-grade seed solution is obtained; S2: The first-level seed solution obtained in S1 was inoculated into a 500 ml Erlenmeyer flask containing 100 mL LB medium at a ratio of 1%, and streptomycin with a final concentration of 50 μg / mL, chloramphenicol at 34 μg / mL, and kanamycin at 50 μg / mL were added. The culture was shaken at 37°C and 220 rpm until the OD 600 When it grows to about 1 (the culture time is 8-12 h), the secondary seed solution is obtained; S3: The secondary seed solution obtained in S2 was inoculated into a 5L small fermentation tank containing 2L fermentation medium at a rate of 5% (mass fraction), with aeration of 1-2 vvm, dissolved oxygen controlled at about 20-40%, and a rotation speed of 400-1000 rpm. The culture was cultured at 37°C until OD 600 When the pH value was about 20, IPTG was added with a final concentration of 0.5 mM, and the expression was induced at 30°C. The fermentation was continued for 48 h, and the pH value was controlled at 7.0 with ammonia water to obtain the fir alcohol product. During the culture process, the residual glucose in the fermentation broth was detected, and a glucose solution with a concentration of 500 g / L was added by variable speed flow to maintain the glucose concentration in the fermentation broth below 0.5 g / L.

[0033] The qualitative and quantitative analysis of the abies alcohol product obtained in this example was performed by GC-MS. The results are as follows: Figure 3 As shown, the fermentation cycle is 60 hours. When the fermentation reaches 46 hours, the concentration of the genetically engineered bacteria (CAS08) can reach the highest OD 600 =160, at this time the yield of abietol can reach 1473 mg / L.

[0034] The genetically engineered bacteria (CAS08) provided by the present invention has greatly improved the production of firwood alcohol compared with the genetically engineered bacteria announced by Li et al. in 2019 in the prior art (L. Li, X. Wang, X. Li, H. Shi, F. Wang, Y. Zhang, and X. Li, Journal of Agricultural and Food Chemistry, 2019, 67, 6523-6531). The production of firwood alcohol by the genetically engineered bacteria provided by Li et al. after fermentation for 100 h was only 635 mg / L, which was much lower than the production of firwood alcohol by the genetically engineered bacteria provided by the present invention after fermentation for 44 h, which could reach 1473 mg / L.

[0035] It can be seen that the genetically engineered bacteria provided by the present invention, compared with the genetically engineered bacteria producing afir alcohol in the prior art, not only increase the yield of afir alcohol, but also shorten the time for the genetically engineered bacteria to produce afir alcohol, thereby improving the production efficiency.

[0036] The contents not described in detail in the specification of the present invention are well-known technologies for those skilled in the art. Although the present invention has been disclosed as above with preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be based on the definition of the claims.

Claims

1. A genetically engineered bacterium for producing abirol, characterized in that: The genetically engineered bacteria express acetyl-CoA acyltransferase / hydroxymethylglutaryl-CoA reductase, 3-hydroxy-3-methylglutaryl-CoA synthase, mevalonate kinase, mevalonate-5-phosphate kinase, mevalonate-5-diphosphate decarboxylase, isopentenyl pyrophosphate isomerase, farnesyl pyrophosphate synthetase, geranyl pyrophosphate synthetase, pyrophosphate lysine synthetase, abietol synthetase, the RGG domain in the LAF-1 protein, the short peptide RIDD of the kinase anchor protein and the short peptide RIAD of the cAMP-dependent protein kinase A, and the starting host bacteria of the genetically engineered bacteria are Escherichia coli.

2. The genetically engineered bacterium according to claim 1, characterized in that: The acetyl-CoA acyltransferase / hydroxymethylglutaryl-CoA reductase is encoded by the gene mvaE, with a GenBank accession number of AAG02439.1; the 3-hydroxy-3-methylglutaryl-CoA synthase is encoded by the gene mvaS, with a GenBank accession number of AAG02438.1; the mevalonate kinase is encoded by the gene ERG12, with a GenBank accession number of NM_001182715.1; the mevalonate-5-phosphate kinase is encoded by the gene ERG8, with a GenBank accession number of NM_001182727.1; the mevalonate-5-diphosphate decarboxylase is encoded by the gene ERG19, with a GenBank accession number of X97557.

1. ; The isopentenyl pyrophosphate isomerase is encoded by the gene IDI1, with a GenBank accession number of NM_001183931.1; the farnesyl pyrophosphate synthase is encoded by the gene Erg20, with a GenBank accession number of NM_001181600.1; the geranyl pyrophosphate synthase is encoded by the gene CrtE, with a GenBank accession number of M87280.1; the pyrophosphate lysine synthase is encoded by the gene Lpps, with a GenBank accession number of JQ478434.1; the abietol synthase is encoded by the gene Cas, with a GenBank accession number of JN254808.1; the nucleotide sequence of the RGG domain in the LAF-1 protein is SEQ The nucleotide sequence of the short peptide RIAD of the cAMP-dependent protein kinase A is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4; the nucleotide sequence of the short peptide RIDD of the kinase anchor protein is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.

6.

3. A microbial preparation containing the genetically engineered bacteria according to any one of claims 1 to 2.

4. The method for constructing the genetically engineered bacteria according to any one of claims 1 to 2, characterized in that: The construction method specifically comprises the following steps: S1: Construction of expression vector: The acetyl-CoA acyltransferase / hydroxymethylglutaryl-CoA reductase gene mvaE and the 3-hydroxy-3-methylglutaryl-CoA synthase gene mvaS were connected to the plasmid pCDFDuet-1 to obtain a recombinant plasmid I; The farnesyl pyrophosphate synthase gene Erg20, the geranyl pyrophosphate synthase gene CrtE, the pyrophosphate lisdendronol synthase gene RIDD-Lpps with a short peptide tag RIDD, and the abietol synthase gene RIDD-Cas with a short peptide tag RIDD were cloned into the plasmid pACYCDuet-1 to obtain the recombinant plasmid II; The RGG domain nucleotide sequence in the LAF-1 protein was cloned into the expression vector pET-28a(+) to obtain recombinant plasmid III; S2: Construction of mutant strain: The mevalonate kinase gene ERG12, the mevalonate-5-phosphate kinase gene ERG8, the mevalonate-5-diphosphate decarboxylase gene ERG19 and the isopentenyl pyrophosphate isomerase gene IDI1 were integrated into the genome of Escherichia coli BL21 (DE3) to obtain a mutant strain I containing the above four genes; S3: transformation of recombinant bacteria: the recombinant plasmid I, recombinant plasmid II and recombinant plasmid III obtained in step S1 are transformed into the competent cells prepared by the mutant strain I obtained in step S2 to obtain genetically engineered bacteria for producing afel alcohol.

5. The construction method according to claim 4, characterized in that: The acetyl-CoA acyltransferase / hydroxymethylglutaryl-CoA reductase is encoded by the gene mvaE, with a GenBank accession number of AAG02439.1; the 3-hydroxy-3-methylglutaryl-CoA synthase is encoded by the gene mvaS, with a GenBank accession number of AAG02438.1; the mevalonate kinase is encoded by the gene ERG12, with a GenBank accession number of NM_001182715.1; the mevalonate-5-phosphate kinase is encoded by the gene ERG8, with a GenBank accession number of NM_001182727.1; and the mevalonate-5-diphosphate decarboxylase is encoded by the gene ERG19, with a GenBank accession number of X97557. 1; the isopentenyl pyrophosphate isomerase is encoded by gene IDI, with GenBank accession number NM_001183931.1; the farnesyl pyrophosphate synthase is encoded by gene Erg20, with GenBank accession number NM_001181600.1; the geranyl pyrophosphate synthase is encoded by gene CrtE, with GenBank accession number M87280.1; the pyrophosphate lysine synthase is encoded by gene Lpps, with GenBank accession number JQ478434.1; the abietol synthase is encoded by gene Cas, with GenBank accession number JN254808.1; the nucleotide sequence of the RGG domain in the LAF-1 protein is SEQ The nucleotide sequence of the short peptide RIAD of the cAMP-dependent protein kinase A is shown in SEQ ID NO.3, and the amino acid sequence is shown in SEQ ID NO.4; the nucleotide sequence of the short peptide RIDD of the kinase anchor protein is shown in SEQ ID NO.5, and the amino acid sequence is shown in SEQ ID NO.

6.

6. A method for producing abirol, characterized in that: The method comprises fermenting the genetically engineered bacteria according to any one of claims 1 to 2 at 37° C. for at least 46 hours.

7. The method according to claim 6, characterized in that In the method, the culture medium used for fermentation uses glucose as the carbon source.

8. The method according to claim 6, characterized in that In the method, the culture medium used for fermentation uses yeast powder as a nitrogen source.

9. The method according to claim 6, characterized in that The fermentation medium used for fermentation in the method contains yeast powder, K2HPO4·3H2O, citric acid, ammonium ferric citrate, glucose, MgSO4·7H2O and trace elements.

10. Use of the genetically engineered bacteria according to any one of claims 1 to 2 or the microbial preparation according to claim 3 in the production of abirol.

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