A recombinant bacterium for producing (-)-α-bisabolol and its preparation method and use

Abstract

The present invention provides a recombinant bacterium for producing (‑)‑α‑bisabolol, which is deposited in China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC M 20221785. The present invention also provides a method for obtaining the aforementioned recombinant bacterium by utilizing a (‑)‑α‑bisabolol synthase high-activity mutant mEeBBS L530P / S547V / E558V, by weakening squalene synthesis, enhancing the expression of farnesyl diphosphate synthase ERG20 gene and EeBBS gene, strengthening the MVA pathway, enhancing the acetic acid utilization ability and regulating the glycolysis pathway. The method has a high (‑)‑α‑bisabolol yield, a shake flask stage yield of more than 2 g / L in the production process, and a fed-batch fermentation stage yield of more than 30 g / L, and is suitable for practical promotion and application.

Description

Technical Field

[0001] The invention belongs to the technical field of gene recombination fermentation, and specifically relates to a recombinant bacterium for producing (-)-alpha-bisabolol, and a preparation method and application thereof. Background Art

[0002] (-)-α-Bisabolol, also known as bisabolol and sweet bisabolol, is a natural monocyclic unsaturated sesquiterpene alcohol with the chemical formula C 15 H 26 O, CAS registration number 23089-26-1, relative molecular mass 222.37, density 0.9211g / mL. Due to its stability, good skin compatibility and skin healing properties, it is used as an active ingredient in cosmetics. (-)-α-Bisabolol also has antiseptic, anti-inflammatory, ulcer healing and gallstone dissolving effects, which attracts attention in the pharmaceutical industry.

[0003] At present, the methods for producing α-bisabolol include plant extraction, chemical synthesis and microbial synthesis. Commercial α-bisabolol is mainly produced by steam distillation from the essential oil extracted from the Brazilian Candia tree (Eremanthus erytropappus). High-purity α-bisabolol can also be obtained by separating and extracting other plants rich in α-bisabolol, such as chamomile and matricaria. However, the extraction and purification of α-bisabolol from plant sources are limited by environmental seasonal fluctuations, slow production cycles, high purification costs and low extraction rates. Therefore, it is difficult to achieve large-scale industrial production by plant extraction. The complex chiral chemical structure of (-)-α-bisabolol makes direct chemical synthesis difficult, and the synthetic products have low biological activity and low purity, which is not suitable for large-scale production. With the development of synthetic biology, heterologous expression of α-bisabolol synthase in microorganisms and obtaining α-bisabolol by microbial fermentation have become competitive methods. Compared with plant extraction and chemical synthesis, microbial synthesis has the advantages of low cost, easy availability of raw materials and no drug residues, making it the most promising method.

[0004] According to current literature reports, in 2020, Ji Xiaojun's team at Nanjing University of Science and Technology achieved a production of 364.23 mg / L of α-bisabolol by batch fermentation of Yarrowia lipolytica. In 2020, the brewer's yeast cell factory created by Jiangsu Ruiting Biotechnology Co., Ltd. can produce 10.26 g / L α-bisabolol in 100 hours at the fermentation tank level, with a yield of 0.1026 g / L / h. In 2021, the engineered Escherichia coli strain created by the Korea Institute of Life Science and Technology can produce 23.4 g / L (-)-α-bisabolol in 96 hours at the fermentation tank level, with a yield of 0.2438 g / L / h. Although certain results have been achieved in the heterologous production of (-)-α-bisabolol using microorganisms, the production capacity of recombinant strains needs to be further improved to meet industrial production applications. Summary of the invention

[0005] The object of the present invention is to provide a novel recombinant strain for producing (-)-α-bisabolol.

[0006] The invention provides a recombinant bacterium for producing (-)-α-bisabolol, which is deposited in China Center for Type Culture Collection (CCTCC) with a deposit number of CCTCC M 20221785.

[0007] Furthermore, compared with wild yeast, the recombinant bacteria expressed increased (-)-α-bisabolol synthase EeBBS activity, weakened squalene synthesis, enhanced expression of farnesyl diphosphate synthase ERG20 gene and (-)-α-bisabolol synthase mEeBBS gene, strengthened MVA pathway, enhanced acetic acid utilization ability, and enhanced glycolysis pathway.

[0008] Furthermore, the activity of the expressed (-)-α-bisabolol synthase EeBBS is improved because the expressed (-)-α-bisabolol synthase EeBBS is mutated into (-)-α-bisabolol synthase mEeBBS, and the mutations are: L530P, S547V and E558V mutations.

[0009] Furthermore, the above recombinant bacteria contains a high-activity mutant mEeBBS gene of the (-)-α-bisabolol synthase EeBBS gene, the sequence of which is shown in SEQ ID NO.4.

[0010] Furthermore, the weakening of squalene synthesis mentioned above is to down-regulate the transcription level of squalene synthesis gene ERG9 in wild yeast.

[0011] Furthermore, the promoter of the squalene biosynthesis gene ERG9 in the above recombinant bacteria is P HXT1 Preferably, the P HXT1 The nucleotide sequence is shown in SEQ ID NO.2.

[0012] Furthermore, the above-mentioned recombinant bacteria contain no less than 5 copies of the farnesyl diphosphate synthase ERG20 gene and the (-)-α-bisabolol synthase mEeBBS gene.

[0013] Furthermore, the above-mentioned MVA pathway enhancement is the overexpression of genes in the MVA pathway, and the genes in the MVA pathway are ERG10, ERG13, tHMG1, ERG12, ERG8, ERG19 and IDI1.

[0014] Furthermore, the above-mentioned overexpression of genes of the MVA pathway is the transfer of genes of the MVA pathway into wild yeast.

[0015] Furthermore, the enhancement of the acetic acid utilization ability is due to overexpression of acetyl-CoA synthetase synthesis genes; the acetyl-CoA synthetase synthesis genes are ACS1 and ACS2.

[0016] Furthermore, the above-mentioned acetyl-CoA synthetase synthesis gene overexpression is to transfer the acetyl-CoA synthetase synthesis gene into wild yeast.

[0017] Furthermore, the enhancement of the glycolytic pathway is to enhance the expression of the phosphofructokinase PFK gene.

[0018] Furthermore, the promoter for the phosphofructokinase PFK gene in the above recombinant bacteria is P TEF1 Preferably, the P TEF1 The nucleotide sequence is shown in SEQ ID NO.3.

[0019] Furthermore, the wild yeast is Saccharomyces cerevisiae CEN.PK2-1C or Saccharomyces cerevisiae BY4741.

[0020] The present invention also provides a method for preparing the above-mentioned recombinant bacteria, comprising the following steps:

[0021] (1) Integrating the farnesyl diphosphate synthase ERG20 gene and the (-)-α-bisabolol synthase mEeBBS gene expression cassette into wild yeast to obtain strain AB01; the (-)-α-bisabolol synthase mEeBBS gene sequence is shown in SEQ ID NO.4;

[0022] (2) integrating the tHMG1 gene and IDI1 gene expression cassettes into the genome of strain AB01 to obtain strain AB02;

[0023] (3) Replace the promoter of the squalene synthase ERG9 gene with a weak promoter P HXT1Afterwards, it was integrated into the genome of strain AB02 to obtain strain AB10;

[0024] (4) integrating the farnesyl diphosphate synthase ERG20 gene and the (-)-α-bisabolol synthase mEeBBS gene expression cassette into at least five separate genomic sites of strain AB10 to obtain the yeast engineered strain AB24;

[0025] (5) integrating the ERG10 gene and tHMG1 gene expression cassette, the ERG12 gene and ERG13 gene expression cassette, the ERG19 gene and ERG8 gene expression cassette, and the tHMG1 gene and IDI1 gene expression cassette into the genome of strain AB24 to obtain strain AB30;

[0026] (6) integrating the expression cassettes of acetyl-CoA synthetase genes ACS1 and ACS2 into the genome of strain AB30 to obtain strain AB32;

[0027] (7) Replace the promoter of the phosphofructokinase PFK1 gene with the promoter P TEF1 Then, it was integrated into the genome of strain AB32 to obtain strain AB55, thus obtaining the recombinant bacterium.

[0028] The present invention also provides the use of the above-mentioned recombinant strain in the preparation of (-)-α-bisabolol and its preparation.

[0029] The present invention also provides a method for producing (-)-α-bisabolol, comprising the step of fermenting using the above-mentioned recombinant bacteria; preferably, comprising the following steps:

[0030] (1) taking the recombinant bacterium according to any one of claims 1 to 14, inoculating it into a seed culture medium, and culturing it for 20 to 24 hours;

[0031] (2) taking the seed liquid, inoculating it into the fermentation medium, adding 0% to 20% by volume of n-dodecane, and fermenting for 48 to 96 hours, during which time the feed medium is supplemented;

[0032] The seed culture medium is an aqueous solution containing the following additives: 10-25 g / L of peptone, 5-15 g / L of yeast powder and 20-35 g / L of glucose;

[0033] The fermentation medium is an aqueous solution containing the following additives: 10-35 g / L of glucose or glycerol, 2-3 g / L of potassium dihydrogen phosphate, 2.5-3.0 g / L of dipotassium hydrogen phosphate, 10-28 g / L of yeast powder, and 10-20 g / L of yeast peptone.

[0034] The feed medium is an aqueous solution containing the following additives: 500-800 g / L glucose, 9 g / L KH2PO4, 2.5 g / L MgSO4, 3.5 g / L K2SO4, 0.28 g / L Na2SO4, 10 ml / L trace element solution and 12 ml / L vitamin solution.

[0035] Furthermore, the volume ratio of the seed solution, fermentation medium and n-dodecane is 2:25:5; the culture is shaking culture, the temperature is 30° C., and the rotation speed is 200 rpm.

[0036] Furthermore, the seed culture medium is an aqueous solution containing the following additives: 20 g / L peptone, 10 g / L yeast powder, and 20 g / L glucose;

[0037] The fermentation medium is an aqueous solution containing the following additives: 20 g / L glucose or glycerol, 2.2 g / L potassium dihydrogen phosphate, 2.9 g / L dipotassium hydrogen phosphate, 10 g / L yeast powder, and 20 g / L peptone.

[0038] The feed medium is an aqueous solution containing the following additives: 500-800 g / L glucose, 9 g / L KH2PO4, 2.5 g / L MgSO4, 3.5 g / L K2SO4, 0.28 g / L Na2SO4, 10 ml / L trace element solution and 12 ml / L vitamin solution.

[0039] Beneficial effects of the present invention: The present invention provides a genetically engineered bacterium producing (-)-α-bisabolol, and obtains a recombinant yeast with high yield of (-)-α-bisabolol by modifying the (-)-α-bisabolol synthase EeBBS gene, weakening squalene synthesis, enhancing the expression of the farnesyl diphosphate synthase ERG20 gene and the mEeBBS gene, strengthening the MVA pathway, enhancing the acetic acid utilization ability and regulating the glycolysis pathway, so that the yield in the shake flask stage of the production process exceeds 2 g / L, and the yield in the fed-batch fermentation stage exceeds 30 g / L, which is suitable for practical promotion and application.

[0040] The (-)-α-bisabolol synthase EeBBS gene of the present invention is derived from Eremanthus erythropappus, the farnesyl diphosphate synthase ERG20 gene is derived from Saccharomyces cerevisiae, 7 genes of the MVA pathway: ERG10, ERG13, tHMG1, ERG12, ERG8, ERG19 and IDI1, are all derived from Saccharomyces cerevisiae, the acetyl-CoA synthetase synthesis genes are ACS1 and ACS2 genes, are all derived from Saccharomyces cerevisiae, and the phosphofructokinase gene PFK is derived from Saccharomyces cerevisiae.

[0041] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0042] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 : pBBS17 plasmid map.

[0044] Figure 2 :pBBS17-ERG20-mEeBBS plasmid map.

[0045] Figure 3 : ERG20 and mEeBBS expression cassettes were integrated into the yeast genome map.

[0046] Figure 4 : GC-MS analysis of (-)-α-bisabolol synthesized by standard products and yeast engineered strains (A, standard product image, B, yeast engineered strain image).

[0047] Figure 5 : The accumulation of (-)-α-bisabolol in the recombinant yeast after 72 h of culture in shake flasks. DETAILED DESCRIPTION

[0048] The raw materials and equipment used in the present invention are all known products, which are obtained by purchasing commercially available products.

[0049] Sequence information involved in the present invention:

[0050] (a) Amino acid sequence of (-)-α-bisabolol synthase EeBBS (SEQ ID NO.1):

[0051] *

[0052] (ii) the promoter P HXT1 Nucleotide sequence (SEQ ID NO.2):

[0053]

[0054] (3) The promoter P TEF1 has the nucleotide sequence (SEQ ID NO.3):

[0055] GACCGCGAATCCTTACATCACACCCAATCCCCCACAAGTGATCCCCCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGAGACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCCATTGATATTTAAGTTAATAAACGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGTTTTAATTACAAA

[0056] (4) The nucleotide sequence of the mutant mEeBBS (SEQ ID NO.4)

[0057] MSTAFPVAALVSASASFSSSEPLPSPPPTTTATKQRNTTNHHSSIWGDQFLTYHESEDILKEKLLVDELKEQVRKELISASVDDHLELIDAIQRLGVAYYYENEIEEALQHIYVTYGDHWIHTDNLQSISLWFRLLRQQGFNVSSGIFKNH MNDKGNFKESISNDVQGMLELYEAAYMRVEGEEILDYALEFTKTNLGILANDPSCDSSLRSQIQQALKQPLRKRLPRLEAVRYIPIYKQQACHNDVLLKLAKLDFNVLQSMHKKELSHICKWWKDLNMQEKLPYVRDRLVEGYFWILGIYF EPHHARTRMFLIKTCMWLVVIDDTFDNYGAYEELEQFTEAVERWSISCLDELPEYMKLVYQELVNVHQEMEESLEKEGKAFQIHYVKEMAKECTRSLLLEAKWLKDGYMPTLEEYLSNSLITCAYAV MVARSYVGREDVMIREETFKWVATHPPLVKASCLILRLMDDIATHKEEQERGHVASSIECYIRENVGATEEEACELFSKQIEDAWKVINRECLRPMDVPFPLVMPAINLARICDALYVKGNDGFNHAG V EVINYIKSLVVHPLVV*

[0058] (The underlined part is the mutation site)

[0059] (V) Primer information involved

[0060] Table 1 Nucleotide sequences of primers

[0061]

[0062]

[0063] Example 1: Screening for highly active (-)-α-bisabolol synthase EeBBS mutants

[0064] (1) Chemical synthesis (Shanghai Qingke Biotechnology Co., Ltd.) of the (-)-α-bisabolol synthase gene EeBBS from Eremanthus erythropappus; using EeBBS-F / EeBBS-R as primers, the (-)-α-bisabolol synthase gene EeBBS was PCR amplified using an error-prone PCR kit, the PCR product was detected by 1.0% agarose gel electrophoresis, and the gene fragment was purified using a clean-up kit;

[0065] The target plasmid pBBS17 ( Figure 1 ) for double enzyme digestion, the digestion product was detected by 1.0% agarose gel electrophoresis and the linearized vector fragment was recovered and purified by gel cutting; then the purified gene fragment was connected to the linearized plasmid pBBS17 using the one-step cloning kit of Novozymes (37°C, 30min); the connection product was transformed into E.coli DH5α to obtain a transformation product; the transformation product was spread on LB solid culture medium (containing ampicillin at a final concentration of 100mg / L), and after a single clone was obtained, the plasmid was extracted and sequenced after culture at 37°C and 220rpm for 8-12h. If the verification was correct, the (-)-α-bisabolol synthase EeBBS mutant library plasmid pBBS17-EeBBS was obtained;

[0066] The EeBBS mutant library plasmid constructed above was transformed into Saccharomyces cerevisiae BY4741 to obtain a transformation product; the transformation product was spread on SD-URA solid culture medium and inverted in a constant temperature incubator at 30°C for about 48 hours to obtain a transformant, which is the recombinant yeast engineering strain Saccharomyces cerevisiae BY4741pBBS17-EeBBS.

[0067] (2) The above-mentioned recombinant yeast engineering strain Saccharomyces cerevisiae BY4741pBBS17-EeBBS was taken, inoculated into a seed culture medium, and cultured for 24 hours. The seed liquid was taken and inoculated into a fermentation medium at 1% (v / v), and then covered with 20% (v / v) n-dodecane. The culture was shaken at 30°C and 200 rpm for 72 hours. The yield of each engineering strain was detected and compared, and it was determined that the highly active (-)-α-bisabolol synthase mEeBBS mutant contained L530P / S547V / E558V mutations. The nucleotide sequence of the mutant mEeBBS gene is shown in SEQ ID NO.4.

[0068] The formula of the seed culture medium is: peptone 20g / L, yeast powder 10g / L, glucose 20g / L;

[0069] The formula of the fermentation medium is 20 g / L of galactose and glycerol, 2.2 g / L of potassium dihydrogen phosphate, 2.9 g / L of dipotassium hydrogen phosphate, 10 g / L of yeast powder, and 20 g / L of peptone.

[0070] Example 2 Construction of the recombinant genetically engineered bacteria producing (-)-α-bisabolol of the present invention

[0071] (1) Expression of the farnesyl diphosphate synthase ERG20 gene and the (-)-α-bisabolol synthase mEeBBS gene

[0072] Using the genome of Saccharomyces cerevisiae CEN.PK2-1C or BY4741 as a template, the PCR product of the farnesyl diphosphate synthase ERG20 gene was obtained by PCR using primers ERG20-F / ERG20-R. The PCR product was detected by 1.0% agarose gel electrophoresis and the gene fragment was purified using a clean-up kit;

[0073] The target plasmid pBBS17-mEeBBS was double-digested with NotI and BcuI, and the digestion product was detected by 1.0% agarose gel electrophoresis and the linearized vector fragment was recovered and purified by gel cutting; then the purified gene fragment was connected with the linearized plasmid pBBS17-mEeBBS using a one-step cloning kit of Novozymes (37°C, 30min); the connection product was transformed into E. coli DH5α to obtain a transformation product; the transformation product was spread on LB solid medium (containing ampicillin at a final concentration of 100 mg / L), and after a single clone was obtained, the plasmid was extracted and sequenced after culture at 37°C and 220rpm for 8 to 12 hours. If the verification was correct, the expression plasmid pBBS17-ERG20-mEeBBS of the farnesyl diphosphate synthase ERG20 gene and the (-)-α-bisabolol synthase mEeBBS gene was obtained. Figure 2 );

[0074] Using the genome of Saccharomyces cerevisiae CEN.PK2-1C or BY4741 as a template, PCR products of homology arms GAL1-7-UP and GAL1-7-Dn were obtained by PCR using primers GAL1-7-UF / GAL1-7-UR and GAL1-7-DF / GAL1-7-DR. The PCR products were detected by 1.0% agarose gel electrophoresis and the gene fragments were purified using a clean-up kit;

[0075] Using plasmid pBBS17-ERG20-mEeBBS as a template, PCR products of ERG20 and mEeBBS expression cassettes were obtained by PCR using primers GAL1-7-F / GAL1-7-R. The PCR products were detected by 1.0% agarose gel electrophoresis and the gene fragments were purified using a clean-up kit;

[0076] The purified products of homology arms GAL1-7-UP and GAL1-7-Dn and the purified products of ERG20 and mEeBBS expression cassettes were integrated into Saccharomyces cerevisiae CEN.PK2-1C using CRISPR / Cas9 gene editing technology to obtain the yeast engineering strain AB01 ( Figure 3 ).

[0077] Further, taking the construction method of plasmid pBBS17-ERG20-mEeBBS as an example, using the genome of Saccharomycescerevisiae CEN.PK2-1C or BY4741 as a template, primers tHMG1-F / tHMG1-R and IDI1-F / IDI1-R were used to obtain the expression plasmid pBBS17-tHMG1-IDI1 of the hydroxymethylglutaryl-CoA reductase tHMG1 gene and the isopentenyl diphosphate isomerase enzyme IDI1 gene;

[0078] Furthermore, taking the construction method of yeast engineering strain AB01 as an example, the expression cassettes of hydroxymethylglutaryl-CoA reductase tHMG1 gene and isopentenyl diphosphate isomerase IDI1 gene were integrated into the genome of the yeast engineering strain to obtain yeast engineering strain AB02.

[0079] (2) Weakening the ability to synthesize squalene

[0080] Furthermore, the genome of Saccharomyces cerevisiae CEN.PK2-1C or BY4741 was used as a template and PCR was performed using primers P HXT1 -F / P HXT1 -R get promoter P HXT1 The PCR product was detected by 1.0% agarose gel electrophoresis and the gene fragment was purified by clean-up kit;

[0081] Using the genome of Saccharomyces cerevisiae CEN.PK2-1C or BY4741 as a template, PCR products of homology arms ProERG9-UP and ProERG9-Dn were obtained by PCR using primers ProERG9-UF / ProERG9-UR and ProERG9-DF / ProERG9-DR. The PCR products were detected by 1.0% agarose gel electrophoresis and the gene fragments were purified using a clean-up kit;

[0082] Furthermore, taking the construction method of yeast engineering strain AB01 as an example, the promoter of squalene synthase ERG9 gene was replaced with a weak promoter P HXT1 And integrated into the genome of yeast engineering strain to obtain yeast engineering strain AB10.

[0083] (3) Enhanced expression of the farnesyl diphosphate synthase ERG20 gene and the (-)-α-bisabolol synthase mEeBBS gene

[0084] Furthermore, taking the construction method of yeast engineering strain AB01 as an example, plasmid pBBS17-ERG20-mEeBBS was used as a template, PCR products of ERG20 and mEeBBS expression cassettes were obtained by PCR, and CRISPR / Cas9 gene editing technology was used to integrate the expression cassettes of farnesyl diphosphate synthase ERG20 gene and (-)-α-bisabolol synthase mEeBBS gene into 5 genomic sites of the yeast engineering strain, respectively, to obtain yeast engineering strain AB24.

[0085] (4) Enhanced the expression of 7 genes in the MVA pathway.

[0086] Furthermore, taking the construction method of plasmid pBBS17-ERG20-mEeBBS as an example, the genome of Saccharomyces cerevisiae CEN.PK2-1C or BY4741 was used as a template and plasmid pBBS17 was used as a vector to construct expression plasmids of seven genes ERG10, ERG13, tHMG1, ERG12, ERG8, ERG19 and IDI1 of the MVA pathway, namely pBBS17-ERG10-tHMG1, pBBS17-ERG12-ERG13, pBBS17-ERG19-ERG8 and pBBS17-tHMG1-IDI1, respectively.

[0087] Furthermore, taking the construction method of yeast engineering strain AB01 as an example, the expression plasmids pBBS17-ERG10-tHMG1, pBBS17-ERG12-ERG13, pBBS17-ERG19-ERG8 and pBBS17-tHMG1-IDI1 of the above-mentioned 7 genes of the MVA pathway were used as templates, and PCR products of the corresponding expression cassettes of the 7 genes of the MVA pathway were obtained by PCR. The corresponding expression cassettes of the 7 genes of the MVA pathway were integrated into the genome of the yeast engineering strain using CRISPR / Cas9 gene editing technology to obtain the yeast engineering strain AB30.

[0088] (5) Enhance the ability to utilize acetic acid.

[0089] Further, taking the construction method of plasmid pBBS17-ERG20-mEeBBS as an example, the genome of Saccharomycescerevisiae CEN.PK2-1C or BY4741 was used as a template, the PCR products of acetyl-CoA synthetase genes ACS1 and ACS2 were obtained by PCR, and the plasmid pBBS17 was used as a vector to obtain the expression plasmid pBBS17-ACS1-ACS2 of acetyl-CoA synthetase genes ACS1 and ACS2;

[0090] Furthermore, taking the construction method of yeast engineering strain AB01 as an example, the expression plasmid pBBS17-ACS1-ACS2 of the above-mentioned acetyl-CoA synthetase genes ACS1 and ACS2 was used as a template, and the PCR products of the acetyl-CoA synthetase gene ACS1 and ACS2 expression cassettes were obtained by PCR. The acetyl-CoA synthetase gene ACS1 and ACS2 expression cassettes were integrated into the genome of the yeast engineering strain using CRISPR / Cas9 gene editing technology to obtain the yeast engineering strain AB32.

[0091] (6) Phosphofructokinase (PFK) that regulates the glycolysis pathway.

[0092] Furthermore, the genome of Saccharomyces cerevisiae CEN.PK2-1C or BY4741 was used as a template and PCR was performed using primers P TEF1 -F / P TEF1 -R get promoter P TEF1 The PCR product was detected by 1.0% agarose gel electrophoresis and the gene fragment was purified by clean-up kit;

[0093] Furthermore, taking the construction method of yeast engineering strain AB01 as an example, the genome of Saccharomyces cerevisiae CEN.PK2-1C or BY4741 was used as a template, and the PCR products of homology arms PFK1-UP and PFK1-Dn were obtained by PCR, and the promoter of the phosphofructokinase PFK1 gene of the glycolytic pathway was replaced with the promoter P TEF1 And integrated into the genome of yeast engineering strain to obtain yeast engineering strain AB55.

[0094] The yeast engineering strain AB55 constructed in the present invention has been deposited in the China Center for Type Culture Collection (CCTCC), with the deposit number: CCTCC M 20221785.

[0095] Example 3 Preparation of (-)-α-Bisabolol

[0096] 1) Shake flask fermentation

[0097] The recombinant yeast engineering strains AB10 / AB24 / AB30 / AB32 / AB55 constructed in Example 2 were inoculated into a seed culture medium, and cultured on a shaker at 30°C and 200 rpm for 24 h. The seed solution was inoculated into a fermentation medium at 1% (v / v), and then covered with 20% (v / v) n-dodecane, and cultured on a shaker at 30°C and 200 rpm for 72 h.

[0098] The formula of the seed culture medium is: peptone 20g / L, yeast powder 10g / L, glucose 20g / L;

[0099] The formula of the fermentation medium is glucose and glycerol 20 g / L, potassium dihydrogen phosphate 2.2 g / L, dipotassium hydrogen phosphate 2.9 g / L, yeast powder 10 g / L, and peptone 20 g / L.

[0100] 2) Fed-batch fermentation

[0101] Take the recombinant yeast engineering strain AB55 constructed in Example 2, inoculate it in a seed culture medium, and culture it on a shaker at 30°C and 200rpm for 24h. Take the seed solution and inoculate it into a 5L fermenter containing 2L fermentation medium at 10% (v / v), and then cover it with 20% (v / v) n-dodecane. The fed batch fermentation of the present invention is carried out at 30°C, and the pH in the fermentation system is maintained at 5.0 by automatically adding 5M ammonium hydroxide. The air flow rate range is 1vvm to 2vvm (air volume / working volume / minute). When the glucose concentration in the culture medium is reduced to 1g / L, start adding the feed culture medium solution and adjust the residual glucose concentration to below 1g / L. Ferment for more than 96h to obtain a (-)-α-bisabolol yield of more than 30g / L.

[0102] The feed culture medium of the present invention comprises: 500-800 g / L glucose, 9 g / L KH2PO4, 2.5 g / L MgSO4, 3.5 g / L K2SO4, 0.28 g / L Na2SO4, 10 ml / L trace element solution and 12 ml / L vitamin solution.

[0103] The beneficial effects of the present invention are demonstrated by experimental examples below.

[0104] Experimental Example 1: Application of recombinant Escherichia coli in the synthesis of (-)-α-bisabolol

[0105] (1) Culture medium preparation

[0106] YPD medium composition: peptone 20g / L, yeast powder 10g / L, glucose 20g / L, solvent is deionized water, pH value is natural. YPD plate is YPD liquid medium with agar added to the final concentration of 2g / L.

[0107] Seed culture medium: peptone 20g / L, yeast powder 10g / L, glucose 20g / L;

[0108] Fermentation medium: glucose and glycerol 20g / L, potassium dihydrogen phosphate 2.2g / L, dipotassium hydrogen phosphate 2.9g / L, yeast powder 10g / L, peptone 20g / L.

[0109] Feed medium: 500-800 g / L glucose, 9 g / L KH2PO4, 2.5 g / L MgSO4, 3.5 g / L K2SO4, 0.28 g / L Na2SO4, 10 ml / L trace element solution and 12 ml / L vitamin solution.

[0110] (2) Production of (-)-α-Bisabolol at shake flask level

[0111] The recombinant yeast engineering strains AB10 / AB24 / AB30 / AB32 / AB55 constructed in Example 2 were selected for fermentation test in shake flasks. The specific steps of the shake flask fermentation experiment are as follows:

[0112] A single colony streaked on a YPD solid medium for 48 hours was selected and inoculated into a seed medium, and then cultured at 30°C and 200 rpm on a shaker for 20 to 24 hours. The cultured seeds were inoculated into a fermentation medium at 1% (v / v), and then 20% (v / v) n-dodecane was added, and cultured at 30°C and 200 rpm on a shaker for 72 hours. After the fermentation was completed, the supernatant of the fermentation liquid was taken to obtain (-)-α-bisabolol, which can make the yield of (-)-α-bisabolol in the fermentation liquid of the recombinant yeast engineering strain AB10 / AB24 / AB30 / AB32 / AB55 reach more than 2 g / L. Figure 5 .

[0113] (3) Production of (-)-α-bisabolol in 5L fermentation tank

[0114] Take the recombinant yeast engineering strain AB55 constructed in Example 2, inoculate it in a seed culture medium, and culture it on a shaker at 30°C and 200rpm for 24h. Take the seed solution and inoculate it into a 5L fermenter containing 2L fermentation medium at 10% (v / v), and then cover it with 20% (v / v) n-dodecane. The fed batch fermentation of the present invention is carried out at 30°C, and the pH in the fermentation system is maintained at 5.0 by automatically adding 5M ammonium hydroxide. The air flow rate range is 1vvm to 2vvm (air volume / working volume / minute). When the glucose concentration in the culture medium is reduced to 1g / L, start adding the feed culture medium solution and adjust the residual glucose concentration to below 1g / L. Take the supernatant after about 96h of fermentation to obtain a (-)-α-bisabolol yield of more than 30g / L.

[0115] (4) Determination of (-)-α-bisabolol content:

[0116] Standard sample preparation: Prepare 9g / L (-)-α-bisabolol standard sample, dilute to concentrations of 10, 30, 50, 70, and 90mg / L respectively, and pass 1mL through the membrane for testing;

[0117] Sample preparation: Take 1 mL of fermentation liquid and centrifuge at 12000 rpm for 5 min to separate the two phases; the separated organic phase is passed through a membrane for testing;

[0118] GC-MS detection method: the column temperature control program is 50℃ for 3min; the temperature is increased to 280℃ at 20℃ / min and maintained for 5min; the injection port temperature is 200℃; the injection mode split ratio is 10:1; the split flow rate is 10mL / min; the chromatographic column is Agilent HP-5MS UI (30m*250um*0.25um); the column flow rate is: 1mL / min;

[0119] See the detection results and GC-MS spectrum of (-)-α-bisabolol in the fermentation broth obtained by fermentation of (-)-α-bisabolol standard and recombinant yeast engineering strain AB01. Figure 4 The recombinant yeast engineering strains AB10 / AB24 / AB30 / AB32 / AB55 were inoculated into the fermentation medium with glucose as the carbon source and fermented for 72 hours. The yield of (-)-α-bisabolol in the fermentation broth of the recombinant yeast engineering strains AB10 / AB24 / AB30 / AB32 / AB55 reached more than 2 g / L. Figure 5 .

[0120] In summary, the present invention obtains a recombinant yeast with high yield of (-)-α-bisabolol by random mutation screening to obtain a high-activity (-)-α-bisabolol synthase mEeBBS gene, weakens squalene synthesis, enhances the expression of the farnesyl diphosphate synthase ERG20 gene and the mEeBBS gene, strengthens the MVA pathway, enhances the acetic acid utilization ability and regulates the glycolysis pathway. The production process of the recombinant strain of the present invention has a yield of more than 2g / L in the shake flask stage, and a yield of more than 30g / L in the 5L fermenter fed batch fermentation stage, which is suitable for practical promotion and application.

Claims

1. A recombinant bacterium for producing (-)-α-bisabolol, characterized in that: It is deposited in China Center for Type Culture Collection (CCTCC) with the accession number CCTCC M 20221785.

2. The recombinant bacterium according to claim 1, characterized in that Compared with wild yeast, the recombinant bacteria have increased (-)-α-bisabolol synthase EeBBS activity, weakened squalene synthesis, enhanced expression of farnesyl diphosphate synthase ERG20 gene and (-)-α-bisabolol synthase mEeBBS gene, strengthened MVA pathway, enhanced acetic acid utilization ability, and enhanced glycolysis pathway.

3. The recombinant bacterium according to claim 2, characterized in that The activity of the expressed (-)-α-bisabolol synthase EeBBS is improved because the expressed (-)-α-bisabolol synthase EeBBS is mutated into (-)-α-bisabolol synthase mEeBBS, and the mutations are: L530P, S547V and E558V mutations.

4. The recombinant bacterium according to claim 3, characterized in that The recombinant bacteria contains a high-activity mutant mEeBBS gene of the (-)-α-bisabolol synthase EeBBS gene, and the sequence is shown in SEQ ID NO.

4.

5. The recombinant bacterium according to claim 2, characterized in that The weakening of squalene synthesis is the down-regulation of the transcription level of the squalene synthesis gene ERG9 in wild yeast.

6. The recombinant bacterium according to claim 5, characterized in that The promoter of the squalene synthesis gene ERG9 in the recombinant bacteria is P HXT1 .

7. The recombinant bacterium according to claim 6, characterized in that The P HXT1 The nucleotide sequence is shown in SEQ ID NO.

2.

8. The recombinant bacterium according to claim 2, characterized in that The recombinant bacteria contain no less than 5 copies of the farnesyl diphosphate synthase ERG20 gene and the (-)-α-bisabolol synthase mEeBBS gene.

9. The recombinant bacterium according to claim 2, characterized in that The MVA pathway enhancement is the overexpression of genes in the MVA pathway, and the genes in the MVA pathway are ERG10, ERG13, tHMG1, ERG12, ERG8, ERG19 and IDI1.

10. The recombinant bacterium according to claim 9, characterized in that The gene overexpression of the MVA pathway is to transfer the genes of the MVA pathway into wild yeast.

11. The recombinant bacterium according to claim 2, characterized in that The enhancement of acetic acid utilization ability is achieved by overexpression of acetyl-CoA synthetase synthesis genes; the acetyl-CoA synthetase synthesis genes are ACS1 and ACS2.

12. The recombinant bacterium according to claim 11, characterized in that The acetyl-CoA synthetase synthesis gene overexpression is to transfer the acetyl-CoA synthetase synthesis gene into wild yeast.

13. The recombinant bacterium according to claim 2, characterized in that The enhancement of the glycolytic pathway is to enhance the expression of the phosphofructokinase PFK gene.

14. The recombinant bacterium according to claim 13, characterized in that The promoter of the phosphofructokinase PFK gene in the recombinant bacteria is P TEF1 .

15. The recombinant bacterium according to claim 14, characterized in that The P TEF1 The nucleotide sequence is shown in SEQ ID NO.

3.

16. The recombinant bacterium according to any one of claims 2 to 15, characterized in that The wild yeast is Saccharomyces cerevisiae CEN.PK2-1C or Saccharomyces cerevisiae BY4741.

17. The method for preparing a recombinant bacterium according to any one of claims 1 to 16, characterized in that: The steps include: (1) Integrating the farnesyl diphosphate synthase ERG20 gene and the (-)-α-bisabolol synthase mEeBBS gene expression cassette into wild yeast to obtain strain AB01; the (-)-α-bisabolol synthase mEeBBS gene sequence is shown in SEQ ID NO.4; (2) integrating the tHMG1 gene and IDI1 gene expression cassettes into the genome of strain AB01 to obtain strain AB02; (3) Replace the promoter of the squalene synthase ERG9 gene with a weak promoter P HXT1 Afterwards, it was integrated into the genome of strain AB02 to obtain strain AB10; (4) integrating the farnesyl diphosphate synthase ERG20 gene and the (-)-α-bisabolol synthase mEeBBS gene expression cassette into at least five separate genomic sites of strain AB10 to obtain the yeast engineered strain AB24; (5) integrating the ERG10 gene and tHMG1 gene expression cassette, the ERG12 gene and ERG13 gene expression cassette, the ERG19 gene and ERG8 gene expression cassette, and the tHMG1 gene and IDI1 gene expression cassette into the genome of strain AB24 to obtain strain AB30; (6) integrating the expression cassettes of acetyl-CoA synthetase genes ACS1 and ACS2 into the genome of strain AB30 to obtain strain AB32; (7) Replace the promoter of the phosphofructokinase PFK1 gene with the promoter P TEF1 Then, it was integrated into the genome of strain AB32 to obtain strain AB55, thus obtaining the recombinant bacterium.

18. Use of the recombinant strain according to any one of claims 1 to 16 in the preparation of (-)-α-bisabolol and its preparation.

19. A method for producing (-)-α-bisabolol, characterized in that: The method comprises the step of fermenting using the recombinant bacteria according to any one of claims 1 to 16.

20. The method of claim 19, wherein: The method comprises the following steps: (1) taking the recombinant bacterium according to any one of claims 1 to 14, inoculating it into a seed culture medium, and culturing it for 20 to 24 hours; (2) taking the seed liquid, inoculating it into the fermentation medium, adding 0% to 20% by volume of n-dodecane, and fermenting for 48 to 96 hours, during which time the feed medium is supplemented; The seed culture medium is an aqueous solution containing the following additives: 10-25 g / L of peptone, 5-15 g / L of yeast powder and 20-35 g / L of glucose; The fermentation medium is an aqueous solution containing the following additives: 10-35 g / L of glucose or glycerol, 2-3 g / L of potassium dihydrogen phosphate, 2.5-3.0 g / L of dipotassium hydrogen phosphate, 10-28 g / L of yeast powder, and 10-20 g / L of yeast peptone; The feed medium is an aqueous solution containing the following additives: 500-800 g / L glucose, 9 g / L KH2PO4, 2.5 g / L MgSO4, 3.5 g / L K2SO4, 0.28 g / L Na2SO4, 10 ml / L trace element solution and 12 ml / L vitamin solution.

21. The method according to claim 20, characterized in that: The volume ratio of the seed solution, fermentation medium and n-dodecane is 2:25:5; the culture is shaking culture, the temperature is 30° C., and the rotation speed is 200 rpm.

22. The method according to claim 20, characterized in that: The seed culture medium is an aqueous solution containing the following additives: 20 g / L peptone, 10 g / L yeast powder, and 20 g / L glucose; The fermentation medium is an aqueous solution containing the following additives: 20 g / L glucose or glycerol, 2.2 g / L potassium dihydrogen phosphate, 2.9 g / L dipotassium hydrogen phosphate, 10 g / L yeast powder, and 20 g / L peptone; The feed medium is an aqueous solution containing the following additives: 500-800 g / L glucose, 9 g / L KH2PO4, 2.5 g / L MgSO4, 3.5 g / L K2SO4, 0.28 g / L Na2SO4, 10 ml / L trace element solution and 12 ml / L vitamin solution.

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