CN116716196A - Recombinant bacterium for producing (-) -alpha-bisabolol and preparation method and application thereof - Google Patents

Recombinant bacterium for producing (-) -alpha-bisabolol and preparation method and application thereof Download PDF

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CN116716196A
CN116716196A CN202310076063.4A CN202310076063A CN116716196A CN 116716196 A CN116716196 A CN 116716196A CN 202310076063 A CN202310076063 A CN 202310076063A CN 116716196 A CN116716196 A CN 116716196A
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gene
bisabolol
recombinant bacterium
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alpha
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朱占涛
翟诗岚
保明狄
李文
马小燕
赵华
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Shanghai Ruikang Biotechnology R & D Co ltd
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Abstract

The invention provides a recombinant bacterium for producing (-) -alpha-bisabolol, which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC M20221785. The invention also provides a method for obtaining the recombinant bacterium by utilizing the (-) -alpha-bisabolol synthetase high-activity mutant mEeBBS L530P/S547V/E558V, weakening squalene synthesis, enhancing the expression of farnesyl diphosphate synthase ERG20 gene and EeBBS gene, enhancing MVA pathway, enhancing acetic acid utilization capacity and regulating glycolysis pathway, and the method has higher (-) -alpha-bisabolol yield, has the shake flask stage yield exceeding 2g/L and the fed-batch fermentation stage yield exceeding 30g/L, and is suitable for practical popularization and application.

Description

Recombinant bacterium for producing (-) -alpha-bisabolol and preparation method and application thereof
Technical Field
The invention belongs to the technical field of gene recombination fermentation, and in particular relates to a recombinant bacterium for producing (-) -alpha-bisabolol, a preparation method and application thereof.
Background
(-) -alpha-Bisabolol, also known as saposhnikovia root alcohol, is a natural monocyclic unsaturated sesquiterpene alcohol with the chemical formula C 15 H 26 O, CAS registry number 23089-26-1, has a relative molecular mass of 222.37 and a density of 0.9211g/mL. Is used as an active ingredient in cosmetics due to its stability, good skin compatibility, skin healing properties, etc. (-) -alpha-bisabolol also has the effects of preserving, anti-inflammatory, healing ulcers, dissolving gallstones and the like, and is attractive in the pharmaceutical industry.
Currently, methods for producing α -bisabolol include plant extraction, chemical synthesis, and microbial synthesis. Commercial α -bisabolol is produced mainly from essential oils extracted from cambridge brazil (Eremanthus erytropappus) by steam distillation. The alpha-bisabolol with high purity can also be obtained by separating and extracting other plants rich in alpha-bisabolol such as chamomile, chamomile and the like. However, the extraction and purification of α -bisabolol from plant sources is limited by environmental seasonal fluctuations, slow production cycle, high purification costs, and low extraction yield. Therefore, it is difficult to realize large-scale industrial production by the plant extraction method. The (-) -alpha-bisabolol has the defects of high direct chemical synthesis difficulty, low biological activity, low purity and the like due to the complex chiral chemical structure of the (-) -alpha-bisabolol, and is not suitable for large-scale production. With the development of synthetic biology, the heterologous expression of α -bisabolol synthase in microorganisms, the use of microbial fermentation to obtain α -bisabolol, is a competitive way. Compared with plant extraction and chemical synthesis, the microbial synthesis has the advantages of low cost, easily available raw materials, no medicament residue and the like, and is the most potential method.
The current literature reports that the yield of alpha-bisabolol by batch fermentation of yarrowia lipolytica Y.lipolytica by group Ji Xiaojun of Nanjing university in 2020 reaches 364.23mg/L. Saccharomyces cerevisiae cell factory created by Jiangsu King Biotechnology Co., ltd in 2020 can produce 10.26g/L alpha-bisabolol at fermentation tank level of 100h with yield of 0.1026g/L/h. The E.coli engineering strain created by Korean institute of life science and technology in 2021 can produce 23.4g/L (-) -alpha-bisabolol at 96h fermenter level with a yield of 0.2438g/L/h. Although the heterologous production of (-) -alpha-bisabolol by microorganisms has achieved a certain result, further improvement of the productivity of recombinant strains is required to meet the industrial production applications.
Disclosure of Invention
The invention aims at providing a novel recombinant strain for producing (-) -alpha-bisabolol.
The invention provides a recombinant bacterium for producing (-) -alpha-bisabolol, which is preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC M20221785.
Further, compared with wild yeast, the recombinant bacterium has the advantages of improved expressed (-) -alpha-bisabolol synthase EeBBS activity, weakened squalene synthesis, enhanced expression of farnesyl diphosphate synthase ERG20 gene and (-) -alpha-bisabolol synthase mEeBBS gene, enhanced MVA pathway, enhanced acetic acid utilization capacity and enhanced glycolytic pathway.
Still further, the above-described increase in activity of expressed (-) - α -bisabolol synthase EeBBS is a mutation of expressed (-) - α -bisabolol synthase EeBBS to (-) - α -bisabolol synthase mEeBBS, the mutation being: three mutations of L530P, S547V and E558V.
Furthermore, the recombinant bacterium contains a high-activity mutant mEeBBS gene of (-) -alpha-bisabolol synthetase EeBBS gene, and the sequence of the mutant mEeBBS gene is shown as SEQ ID NO. 4.
Further, the weakening of squalene synthesis is to lower the transcription level of squalene synthesis gene ERG9 in wild yeast.
Furthermore, the promoter for promoting squalene synthesis gene ERG9 in the recombinant bacterium is P HXT1 The method comprises the steps of carrying out a first treatment on the surface of the Preferably, the P HXT1 The nucleotide sequence of (2) is shown as SEQ ID NO. 2.
Further, the recombinant bacterium contains a farnesyl diphosphate synthase ERG20 gene and a (-) -alpha-bisabolol synthase mEeBBS gene with copy numbers not lower than 5.
Further, the MVA pathway enhancement described above is overexpression of genes of the MVA pathway, which are ERG10, ERG13, hmg1, ERG12, ERG8, ERG19 and IDI1.
Further, the above-mentioned gene overexpression of the MVA pathway is a gene transferred into the MVA pathway in wild yeast.
Further, the enhancement of the acetic acid utilization ability is overexpression of an acetyl-CoA synthetase synthesis gene; the acetyl coenzyme A synthetase synthesis genes are ACS1 and ACS2.
Further, the above-mentioned overexpression of acetyl-CoA synthetase synthase gene is a transfer of acetyl-CoA synthetase synthase gene in wild yeast.
Further, the glycolytic pathway enhancement is to enhance the expression of phosphofructokinase PFK gene.
Furthermore, the promoter for promoting phosphofructokinase PFK gene in the recombinant bacterium is P TEF1 The method comprises the steps of carrying out a first treatment on the surface of the Preferably, the P TEF1 The nucleotide sequence of (2) is shown as SEQ ID NO. 3.
Further, the wild yeast is yeast Saccharomyces cerevisiae CEN.PK2-1C or Saccharomyces cerevisiae BY4741.
The invention also provides a preparation method of the recombinant bacterium, which comprises the following steps:
(1) Integrating the farnesyl diphosphate synthase ERG20 gene and (-) -alpha-bisabolol synthase mEeBBS gene expression cassette into wild saccharomycetes to obtain a strain AB01; the (-) -alpha-bisabolol synthase mEeBBS gene sequence is shown in SEQ ID NO. 4;
(2) Integrating the tHMG1 gene and the IDI1 gene expression cassette into the genome of the strain AB01 to obtain a strain AB02;
(3) Replacement of the squalene synthetase ERG9 Gene promoter with the weak promoter P HXT1 Then, the strain AB10 is obtained by integrating the genome of the strain AB02;
(4) Integrating the farnesyl diphosphate synthase ERG20 gene and (-) -alpha-bisabolol synthase mEeBBS gene expression cassette into at least 5 separate genomic loci of the strain AB10 to obtain a yeast engineering strain AB24;
(5) Integrating the ERG10 gene and the tHMG1 gene expression cassette, the ERG12 gene and the ERG13 gene expression cassette, the ERG19 gene and the ERG8 gene expression cassette, and the tHMG1 gene and the IDI1 gene expression cassette into the genome of the strain AB24 to obtain a strain AB30;
(6) Integrating the expression cassettes of acetyl-CoA synthetase genes ACS1 and ACS2 into the genome of the strain AB30 to obtain a strain AB32;
(7) Replacement of the promoter of the phosphofructokinase PFK1 Gene with the promoter P TEF1 And integrating the recombinant strain into the genome of the strain AB32 to obtain a strain AB55, thus obtaining the recombinant strain.
The invention also provides the application of the recombinant strain in preparing (-) -alpha-bisabolol and preparations thereof.
The invention also provides a method for producing (-) -alpha-bisabolol, which comprises the step of fermenting by using the recombinant bacterium; preferably, the method comprises the following steps:
(1) Inoculating the recombinant bacterium of any one of claims 1 to 14 into a seed culture medium, and culturing for 20 to 24 hours;
(2) Inoculating the seed liquid into a fermentation culture medium, adding n-dodecane with the volume ratio of 0-20%, and fermenting and culturing for 48-96 hours, and supplementing a feed supplement culture medium during the fermentation culture;
the seed culture medium is an aqueous solution containing the following additives: 10-25 g/L peptone, 5-15 g/L yeast powder and 20-35 g/L glucose;
the fermentation medium is an aqueous solution containing the following additives: 10 to 35g/L of glucose or glycerol, 2 to 3g/L of monopotassium phosphate, 2.5 to 3.0g/L of dipotassium phosphate, 10 to 28g/L of yeast powder and 10 to 20g/L of yeast peptone.
The feed medium is an aqueous solution containing the following additives: 500-800 g/L glucose and 9g/L KH 2 PO 4 、2.5g/L MgSO 4 、3.5g/L K 2 SO 4 、0.28g/L Na 2 SO 4 10ml/L trace element solution and 12ml/L vitamin solution.
Further, the volume ratio of the seed solution to the fermentation medium to the n-dodecane is 2:25:5; the culture is shaking culture, the temperature is 30 ℃, and the rotating speed is 200rpm.
Further, the seed culture medium is an aqueous solution containing the following additives: 20g/L of peptone, 10g/L of yeast powder and 20g/L of glucose;
the fermentation medium is an aqueous solution containing the following additives: glucose or glycerin 20g/L, potassium dihydrogen phosphate 2.2g/L, dipotassium hydrogen phosphate 2.9g/L, yeast powder 10g/L, peptone 20g/L.
The feed medium is an aqueous solution containing the following additives: 500-800 g/L glucose and 9g/L KH 2 PO 4 、2.5g/L MgSO 4 、3.5g/L K 2 SO 4 、0.28g/L Na 2 SO 4 10ml/L trace element solution and 12ml/L vitamin solution.
The invention has the beneficial effects that: the invention provides a genetically engineered bacterium for producing (-) -alpha-bisabolol, which is a recombinant saccharomycete for obtaining high-yield (-) -alpha-bisabolol by modifying (-) -alpha-bisabolol synthase EeBBS genes, weakening squalene synthesis, enhancing expression of farnesyl diphosphate synthase ERG20 genes and mEeBBS genes, enhancing MVA pathway, enhancing acetic acid utilization capacity, regulating glycolysis pathway and the like, so that the yield of the recombinant saccharomycete exceeds 2g/L in a shaking bottle stage and exceeds 30g/L in a fed-batch fermentation stage in the production process, and is suitable for practical popularization and application.
The (-) -alpha-bisabolol synthase EeBBS gene is derived from Eremanthus erythropappus, the farnesyl diphosphate synthase ERG20 gene is derived from Saccharomyces cerevisiae, and 7 genes of the MVA pathway are derived from: 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, both derived from Saccharomyces cerevisiae, and the phosphofructokinase gene PFK derived from Saccharomyces cerevisiae.
It should be apparent that, in light of the foregoing, various modifications, substitutions and alterations can be made herein without departing from the spirit and scope of the invention as defined by the appended claims.
The above-described aspects of the present invention will be described in further detail below with reference to specific embodiments in the form of examples. It should not be understood that the scope of the above subject matter of the present invention is limited to the following examples only. All techniques implemented based on the above description of the invention are within the scope of the invention.
Drawings
Fig. 1: pBBS17 plasmid map.
Fig. 2: pBBS17-ERG20-mEeBBS plasmid map.
Fig. 3: the ERG20 and mEeBBS expression cassettes are integrated into the yeast genome map.
Fig. 4: GC-MS analysis of the standard and yeast engineering strains to synthesize (-) - α -bisabolol (A, standard panels, B, yeast engineering strain panels).
Fig. 5: the recombinant microzyme is cultured on a shake flask for 72 hours, and then (-) -alpha-bisabolol accumulation amount is obtained.
Detailed Description
The raw materials and equipment used in the invention are all known products and are obtained by purchasing commercial products.
Sequence information related to the present invention:
the amino acid sequence of (-) -alpha-bisabolol synthase EeBBS (SEQ ID NO. 1):
MSTAFPVAALVSASASFSSSEPLPSPPPTTTATKQRNTTNHHSSIWGDQFLTYHESEDILKEKLLVDELKEQVRKELISASVDDHLELIDAIQRLGVAYYYENEIEEALQHIYVTYGDHWIHTDNLQSISLWFRLLRQQGFNVSSGIFKNHMNDKGNFKESISNDVQGMLELYEAAYMRVEGEEILDYALEFTKTNLGILANDPSCDSSLRSQIQQALKQPLRKRLPRLEAVRYIPIYKQQACHNDVLLKLAKLDFNVLQSMHKKELSHICKWWKDLNMQEKLPYVRDRLVEGYFWILGIYFEPHHARTRMFLIKTCMWLVVIDDTFDNYGAYEELEQFTEAVERWSISCLDELPEYMKLVYQELVNVHQEMEESLEKEGKAFQIHYVKEMAKECTRSLLLEAKWLKDGYMPTLEEYLSNSLITCAYAVMVARSYVGREDVMIREETFKWVATHPPLVKASCLILRLMDDIATHKEEQERGHVASSIECYIRENVGATEEEACELFSKQIEDAWKVINRECLRPMDVPFLLVMPAINLARICDALYSKGNDGFNHAGEEVINYIKSLVVHPLVV*
(II) the promoter P HXT1 Nucleotide sequence (SEQ ID NO. 2):
ATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGTGCAGGTCTCATCTGGAATATAATTCCCCCCTCCTGAAGCAAATTTTTCCTTTGAGCCGGAATTTTTGATATTCCGAGTTCTTTTTTTCCATTCGCGGAGGTTAT TCCATTCCTAAACGAGTGGCCACAATGAAACTTCAATTCATATCGACCGACTATTTTTCTCCGAACCAAAAAAATAGCAGGGCGAGATTGGAGCTGCGGAAAAAAGAGGAAAAAATTTTTTCGTAGTTTTCTTGTGCAAATTAGGGTGTAAGGTTTCTAGGGCTTATTGGTTCAAGCAGAAGAGACAACAATTGTAGGTCCTAAATTCAAGGCGGATGTAAGGAGTATTGGTTTCGAAAGTTTTTCCGAAGCGGCATGGCAGGGACTACTTGCGCATGCGCTCGGATTATCTTCATTTTTGCTTGCAAAAACGTAGAATCATGGTAAATTACATGAAGAATTCTCTTTTTTTTTTTTTTTTTTTTTTTTTTACCTCTAAAGAGTGTTGACCAACTGAAAAAACCCTTCTTCAAGAGAGTTAAACTAAGACTAACCATCATAACTTCCAAGGAATTAATCGATATCTTGCACTCCTGATTTTTCTTCAAAGAGACAGCGCAAAGGATTATGACACTGTTGCATTGAGTCAAAAGTTTTTCCGAAGTGACCCAGTGCTCTTTTTTTTTTTCCGTGAAGGACTGACAAATATGCGCACAAGATCCAATACGTAATGGAAATTCGGAAAAACTAGGAAGAAATGCTGCAGGGCATTGCCGTGCCGATCTTTTGTCTTTCAGATATATGAGAAAAAGAATATTCATCAAGTGCTGATAGAAGAATACCACTCATATGACGTGGGCAGAAGACAGCAAACGTAAACATGAGCTGCTGCGACATTTGATGGCTTTTATCCGACAAGCCAGGAAACTCCACCATTATCTAATGTAGCAAAATATTTCTTAACACCCGAAGTTGCGTGTCCCCCTCACGTTTTTAATCATTTGAATTAGTATATTGAAATTATATATAAAGGCAACAATGTCCCCATAATCAATTCCATCTGGGGTCTCATGTTCTTTCCCCACCTTAAAATCTATAAAGATATCATAATCGTCAACTAGTTGATATACGTAAAATC
(III) the promoter P TEF1 Nucleotide sequence of (SEQ ID NO. 3):
GACCGCGAATCCTTACATCACACCCAATCCCCCACAAGTGATCCCCCACACACCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGAGACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCCATTGATATTTAAGTTAATAAACGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGTTTTAATTACAAA
(IV) nucleotide sequence of mutant mEeBBS (SEQ ID NO. 4)
MSTAFPVAALVSASASFSSSEPLPSPPPTTTATKQRNTTNHHSSIWGDQFLTYHESEDILKEKLLVDELKEQVRKELISASVDDHLELIDAIQRLGVAYYYENEIEEALQHIYVTYGDHWIHTDNLQSISLWFRLLRQQGFNVSSGIFKNHMNDKGNFKESISNDVQGMLELYEAAYMRVEGEEILDYALEFTKTNLGILANDPSCDSSLRSQIQQALKQPLRKRLPRLEAVRYIPIYKQQACHNDVLLKLAKLDFNVLQSMHKKELSHICKWWKDLNMQEKLPYVRDRLVEGYFWILGIYF EPHHARTRMFLIKTCMWLVVIDDTFDNYGAYEELEQFTEAVERWSISCLDELPEYMKLVYQELVNVHQEMEESLEKEGKAFQIHYVKEMAKECTRSLLLEAKWLKDGYMPTLEEYLSNSLITCAYAVMVARSYVGREDVMIREETFKWVATHPPLVKASCLILRLMDDIATHKEEQERGHVASSIECYIRENVGATEEEACELFSKQIEDAWKVINRECLRPMDVPFPLVMPAINLARICDALYVKGNDGFNHAGVEVINYIKSLVVHPLVV*
(wherein the underlined part is the site of mutation)
(fifth) primer information related
TABLE 1 nucleotide sequences of primers
Example 1 screening of highly active (-) - α -bisabolol synthase EeBBS mutants
(1) Synthesizing (-) -alpha-bisabolol synthetase gene EeBBS from Eremanthus erythropappus by combination (Shanghai qinghao biosciences Co., ltd.); using EeBBS-F/EeBBS-R as a primer, carrying out PCR amplification on (-) -alpha-bisabolol synthase gene EeBBS by using an error-prone PCR kit, detecting a PCR product by using 1.0% agarose gel electrophoresis, and purifying a gene fragment by using a clean-up kit;
double digestion of the target plasmid pBBS17 (FIG. 1) with BamH1 and HindIII, detection of the digested products by 1.0% agarose gel electrophoresis and recovery of the linearized vector fragment by gel digestion; then, the purified gene fragment is connected with a linearization plasmid pBBS17 by adopting a one-step cloning kit of Norfluzab (37 ℃ C., 30 min); converting the connection product into E.coli DH5 alpha to obtain a conversion product; coating the transformation product on LB solid culture medium (containing 100mg/L ampicillin) to obtain monoclonal, shake-flask culturing at 37 ℃ and 220rpm for 8-12 h, extracting plasmid, and sequencing and verifying to obtain (-) -alpha-bisabolol synthase EeBBS mutant library plasmid pBBS17-EeBBS;
transforming the constructed EeBBS mutant library plasmid to Saccharomyces cerevisiae BY4741 to obtain a transformation product; the transformed product is coated on SD-URA solid culture medium, and is inversely cultured for about 48 hours in a constant temperature incubator at 30 ℃ to obtain a transformant, and the transformant is recombinant yeast engineering strain Saccharomyces cerevisiae BY4741pBBS 17-EeBBS.
(2) Taking the recombinant yeast engineering strain Saccharomyces cerevisiae BY4741pBBS17-EeBBS, inoculating to a seed culture medium, culturing for 24 hours, taking seed liquid, inoculating to a fermentation culture medium according to 1% (V/V), covering 20% (V/V) n-dodecane, shake culturing for 72 hours at 30 ℃ by a shaking table at 200rpm, detecting and comparing the yield of each engineering strain, and determining that the high-activity (-) -alpha-bisabolol synthase mEeBBS mutant contains L530P/S547V/E558V mutation, wherein the nucleotide sequence of the mutant mEeBBS gene is shown as SEQ ID No. 4.
Wherein, the formula of the seed culture medium is as follows: 20g/L of peptone, 10g/L of yeast powder and 20g/L of glucose;
the fermentation medium comprises galactose and glycerol 20g/L, potassium dihydrogen phosphate 2.2g/L, dipotassium hydrogen phosphate 2.9g/L, yeast powder 10g/L, and peptone 20g/L.
EXAMPLE 2 construction of recombinant genetically engineered bacterium producing (-) -alpha-bisabolol of the invention
(1) Expression of farnesyl diphosphate synthase ERG20 Gene and (-) -alpha-bisabolol synthase mEeBBS Gene
Using Saccharomyces cerevisiae CEN.PK2-1C or BY4741 genome as template, obtaining a farnesyl diphosphate synthase ERG20 gene PCR product BY PCR using a primer ERG20-F/ERG20-R, detecting the PCR product BY 1.0% agarose gel electrophoresis and purifying the gene fragment BY using a clean-up kit;
double digestion is carried out on the target plasmid pBBS17-mEeBBS by using two enzymes of NotI and BcuI, and the digested products are detected by 1.0% agarose gel electrophoresis and the linearized vector fragment is recovered and purified by gel digestion; then adopting a one-step cloning kit of the Norflu to connect the purified gene fragment with a linearization plasmid pBBS17-mEeBBS (37 ℃ C., 30 min); converting the connection product into E.coli DH5 alpha to obtain a conversion product; coating the transformation product on LB solid culture medium (containing 100mg/L ampicillin) to obtain monoclonal, shake-flask culturing at 37deg.C and 220rpm for 8-12 h, extracting plasmid, sequencing, and verifying to obtain expression plasmid pBBS17-ERG20-mEeBBS of farnesyl diphosphate synthase ERG20 gene and (-) -alpha-bisabolol synthase mEeBBS gene (figure 2);
using Saccharomyces cerevisiae cen.pk2-1C or BY4741 genome as a template, obtaining PCR products of homologous arms GAL1-7-UP and GAL1-7-Dn BY PCR using primers GAL1-7-UF/GAL1-7-UR, GAL1-7-DF/GAL1-7-DR, detecting the PCR products BY 1.0% agarose gel electrophoresis and purifying the gene fragment BY using clean-UP kit;
using plasmid pBBS17-ERG20-mEeBBS as a template, obtaining PCR products of ERG20 and mEeBBS expression cassettes by PCR using primers GAL1-7-F/GAL1-7-R, detecting the PCR products by 1.0% agarose gel electrophoresis, and purifying gene fragments by using a clean-up kit;
the purified products of the homology arms GAL1-7-UP and GAL1-7-Dn and the purified products of the ERG20 and mEeBBS expression cassettes were integrated into Saccharomyces cerevisiae CEN.PK2-1C using CRISPR/Cas9 gene editing technology to obtain yeast engineering strain AB01 (FIG. 3).
Further, taking the construction method of the plasmid pBBS17-ERG20-mEeBBS as an example, using Saccharomyces cerevisiae CEN.PK2-1C or BY4741 genome as a template, using primers tHMG1-F/tHMG1-R and IDI1-F/IDI1-R to obtain an expression plasmid pBBS17-tHMG1-IDI1 of the hydroxymethylglutaryl-CoA reductase tHMG1 gene and the isopentenyl-diphosphate isomerase IDI1 gene;
further, taking the construction method of the yeast engineering strain AB01 as an example, integrating the expression cassettes of the hydroxymethylglutaryl-CoA reductase tHMG1 gene and the isopentenyl diphosphate isomerase IDI1 gene into the genome of the yeast engineering strain to obtain the yeast engineering strain AB02.
(2) Weakening squalene synthesis capacity
Further, primer P was used BY PCR using Saccharomyces cerevisiae CEN.PK2-1C or BY4741 genome as a template HXT1 -F/P HXT1 R gives the promoter P HXT1 Detecting the PCR product by 1.0% agarose gel electrophoresis and purifying the gene fragment by using a clean-up kit;
using Saccharomyces cerevisiae CEN.PK2-1C or BY4741 genome as template, obtaining PCR products of homology arms ProERG9-UP and ProERG9-Dn BY PCR using primers ProERG9-UF/ProERG9-UR and ProERG9-DF/ProERG9-DR, detecting the PCR products BY 1.0% agarose gel electrophoresis and purifying gene fragments BY using clean-UP kit;
further, taking the construction method of the yeast engineering strain AB01 as an example, the squalene synthetase ERG9 gene promoter is replaced by a weak promoter P HXT1 And integrating the strain into the genome of the yeast engineering strain to obtain the yeast engineering strain AB10.
(3) Enhancing expression of farnesyl diphosphate synthase ERG20 gene and (-) -alpha-bisabolol synthase mEeBBS gene
Further, taking a construction method of a yeast engineering strain AB01 as an example, taking a plasmid pBBS17-ERG20-mEeBBS as a template, obtaining PCR products of ERG20 and mEeBBS expression cassettes by PCR, and integrating expression cassettes of farnesyl diphosphate synthase ERG20 genes and (-) -alpha-bisabolol synthase mEeBBS genes into 5 genome loci of the yeast engineering strain respectively by using a CRISPR/Cas9 gene editing technology to obtain the yeast engineering strain AB24.
(4) Enhancing the expression of 7 genes of the MVA pathway.
Further, taking the construction method of the plasmid pBBS17-ERG20-mEeBBS as an example, taking Saccharomyces cerevisiae CEN.PK2-1C or BY4741 genome as a template, respectively constructing expression plasmids of 7 genes ERG10, ERG13, tHMG1, ERG12, ERG8, ERG19 and IDI1 of the MVA pathway BY taking the plasmid pBBS17 as a vector, and respectively carrying out the construction of pBBS17-ERG10-tHMG1, pBBS17-ERG12-ERG13, pBBS17-ERG19-ERG8 and pBBS17-tHMG1-IDI1.
Taking a construction method of the yeast engineering strain AB01 as an example, respectively taking expression plasmids pBBS17-ERG10-tHMG1, pBBS17-ERG12-ERG13, pBBS17-ERG19-ERG8 and pBBS17-tHMG1-IDI1 of 7 genes of the MVA pathway as templates, obtaining PCR products of corresponding expression cassettes of the 7 genes of the MVA pathway by PCR, and integrating the corresponding expression cassettes of the 7 genes of the MVA pathway into a genome of the yeast engineering strain by using a CRISPR/Cas9 gene editing technology to obtain the yeast engineering strain AB30.
(5) Enhancing acetic acid utilization.
Further, taking a construction method of a plasmid pBBS17-ERG20-mEeBBS as an example, taking Saccharomyces cerevisiae CEN.PK2-1C or BY4741 genome as a template, obtaining PCR products of acetyl-CoA synthetase genes ACS1 and ACS2 through PCR, and taking the plasmid pBBS17 as a carrier to obtain expression plasmids pBBS17-ACS1-ACS2 of the acetyl-CoA synthetase genes ACS1 and ACS2;
taking a construction method of a yeast engineering strain AB01 as an example, taking the expression plasmids pBBS17-ACS1-ACS2 of the acetyl-CoA synthetase genes ACS1 and ACS2 as templates, obtaining PCR products of the expression cassettes of the acetyl-CoA synthetase genes ACS1 and ACS2 by PCR, and integrating the expression cassettes of the acetyl-CoA synthetase genes ACS1 and ACS2 into a genome of the yeast engineering strain by using a CRISPR/Cas9 gene editing technology to obtain the yeast engineering strain AB32.
(6) Phosphofructokinase PFK that regulates the glycolytic pathway.
Further, primer P was used BY PCR using Saccharomyces cerevisiae CEN.PK2-1C or BY4741 genome as a template TEF1 -F/P TEF1 R gives the promoter P TEF1 Detecting the PCR product by 1.0% agarose gel electrophoresis and purifying the gene fragment by using a clean-up kit;
further, taking a construction method of a yeast engineering strain AB01 as an example, taking a Saccharomyces cerevisiae CEN.PK2-1C or BY4741 genome as a template, obtaining PCR products of homology arms PFK1-UP and PFK1-Dn through PCR, and replacing a promoter of a phosphofructokinase PFK1 gene of a glycolytic pathway with a promoter P TEF1 And integrating the strain into the genome of the yeast engineering strain to obtain the yeast engineering strain AB55.
The yeast engineering strain AB55 constructed by the invention is preserved in China Center for Type Culture Collection (CCTCC), and the preservation number is as follows: cctccc M20221785.
EXAMPLE 3 preparation of (-) -alpha-bisabolol
1) Shaking flask fermentation culture
Taking recombinant yeast engineering strains AB10/AB24/AB30/AB32/AB55 constructed in example 2, inoculating to a seed culture medium, shaking and culturing at 30 ℃ for 24 hours by a shaking table at 200rpm, taking a seed solution, inoculating to a fermentation culture medium according to 1% (v/v), covering with 20% (v/v) n-dodecane, and shaking and culturing at 30 ℃ for 72 hours by a shaking table at 200rpm to obtain the recombinant yeast engineering strain;
wherein, the formula of the seed culture medium is as follows: 20g/L of peptone, 10g/L of yeast powder and 20g/L of glucose;
the fermentation medium comprises glucose and glycerol 20g/L, potassium dihydrogen phosphate 2.2g/L, dipotassium hydrogen phosphate 2.9g/L, yeast powder 10g/L and peptone 20g/L.
2) Fed-batch fermentation
The recombinant yeast engineering strain AB55 constructed in example 2 was inoculated into a seed culture medium, shake-cultured at 30℃and 200rpm for 24 hours, the seed solution was inoculated into a 5L fermenter containing 2L of fermentation medium at 10% (v/v), and then covered with 20% (v/v) n-dodecane. The fed-batch fermentation according to the invention is carried out at 30℃and the pH in the fermentation system is maintained at 5.0 by automatic addition of 5M ammonium hydroxide. The air flow ranges from 1vvm to 2vvm (air volume/working volume/minute). When the glucose concentration in the culture medium is reduced to 1g/L, the feeding culture medium solution is fed in, the residual glucose concentration is regulated to be less than 1g/L, and the fermentation is carried out for more than 96 hours, so that the (-) -alpha-bisabolol yield exceeds 30g/L.
The feed medium of the invention: 500-800 g/L glucose and 9g/L KH 2 PO 4 、2.5g/L MgSO 4 、3.5g/L K 2 SO 4 、0.28g/L Na 2 SO 4 10ml/L trace element solution and 12ml/L vitamin solution.
The following experiments prove the beneficial effects of the invention.
Experimental example 1, application of recombinant Escherichia coli in synthesis of (-) -alpha-bisabolol
(1) Preparation of culture medium
YPD medium composition: 20g/L peptone, 10g/L yeast powder and 20g/L glucose, and the solvent is deionized water, and the pH value is natural. YPD plates were prepared by adding 2g/L agar to YPD liquid medium.
Seed culture medium: 20g/L of peptone, 10g/L of yeast powder and 20g/L of glucose;
fermentation medium: glucose and glycerin 20g/L, potassium dihydrogen phosphate 2.2g/L, dipotassium hydrogen phosphate 2.9g/L, yeast powder 10g/L, peptone 20g/L.
Feed medium: 500-800 g/L glucose and 9g/L KH 2 PO 4 、2.5g/L MgSO 4 、3.5g/L K 2 SO 4 、0.28g/L Na 2 SO 4 10ml/L trace element solution and 12ml/L vitamin solution.
(2) Shake flask horizontal (-) -alpha-bisabolol production
The recombinant yeast engineering strain AB10/AB24/AB30/AB32/AB55 constructed in example 2 is selected and subjected to fermentation experiment tests in shake flasks, and the specific shake flask fermentation experiment steps are as follows:
single colony inoculated seed culture medium is selected and cultured in YPD solid culture medium for 48 hr, and shaking culture is performed at 30 deg.c and 200rpm for 20-24 hr. Inoculating the cultured seeds into a fermentation culture medium according to 1% (v/v), adding 20% (v/v) n-dodecane, shaking and culturing at 30 ℃ and 200rpm for 72 hours, and collecting the supernatant of the fermentation broth after the fermentation is finished to obtain (-) -alpha-bisabolol, so that the yield of the (-) -alpha-bisabolol in the fermentation broth of the recombinant yeast engineering strain AB10/AB24/AB30/AB32/AB55 can reach more than 2g/L, and the yield is shown in figure 5.
(3) Production of (-) -alpha-bisabolol by 5L fermenter level
The recombinant yeast engineering strain AB55 constructed in example 2 was inoculated into a seed culture medium, shake-cultured at 30℃and 200rpm for 24 hours, the seed solution was inoculated into a 5L fermenter containing 2L of fermentation medium at 10% (v/v), and then covered with 20% (v/v) n-dodecane. The fed-batch fermentation according to the invention is carried out at 30℃and the pH in the fermentation system is maintained at 5.0 by automatic addition of 5M ammonium hydroxide. The air flow ranges from 1vvm to 2vvm (air volume/working volume/minute). When the glucose concentration in the culture medium is reduced to 1g/L, the feeding culture medium solution is fed in, the residual glucose concentration is regulated to be less than 1g/L, fermentation is carried out for about 96 hours, and the supernatant is taken, so that the yield of (-) -alpha-bisabolol exceeds 30g/L.
(4) Determination of (-) - α -bisabolol content:
preparing a standard sample: preparing 9g/L (-) -alpha-bisabolol standard samples, respectively diluting the standard samples into standard samples with the concentration of 10, 30, 50, 70 and 90mg/L, and passing 1mL through a membrane to be measured;
sample preparation: taking 1mL of fermentation liquor, centrifuging for 5min at the rotating speed of 12000rpm, and separating two phases; separating the obtained organic phase, and passing the organic phase through a membrane to be detected;
GC-MS detection method: the column temperature control program is that the temperature is kept at 50 ℃ for 3min; heating to 280 ℃ at 20 ℃/min and keeping for 5min; the temperature of the sample inlet is 200 ℃; the split ratio of the sample injection mode is 10:1; the split flow is 10mL/min; the chromatographic column is Agilent HP-5MS UI (30 m.times.250 um.times.0.25 um); the column flow rate is: 1mL/min;
the detection result and GC-MS spectrum of (-) -alpha-bisabolol in the fermentation broth obtained by fermenting the (-) -alpha-bisabolol standard substance and the recombinant yeast engineering strain AB01 are shown in figure 4. The recombinant yeast engineering strains AB10/AB24/AB30/AB32/AB55 are respectively inoculated into a fermentation medium taking glucose as a carbon source for fermentation for 72 hours, so that the yield of (-) -alpha-bisabolol in the fermentation liquid of the recombinant yeast engineering strains AB10/AB24/AB30/AB32/AB55 can reach more than 2g/L, and the yield is shown in figure 5.
In conclusion, the recombinant saccharomycetes for obtaining high-yield (-) -alpha-bisabolol through random mutation screening, weakening squalene synthesis, enhancing the expression of the ERG20 gene and the mEeBBS gene of farnesyl diphosphate synthase, enhancing MVA pathway, enhancing acetic acid utilization capacity, regulating glycolysis pathway and the like. The production of the recombinant strain in the shaking bottle stage exceeds 2g/L, and the production of the recombinant strain in the fed-batch fermentation stage exceeds 30g/L in a 5L fermentation tank, so that the recombinant strain is suitable for practical popularization and application.

Claims (19)

1. The recombinant bacterium for producing (-) -alpha-bisabolol is characterized by being preserved in China Center for Type Culture Collection (CCTCC) with the preservation number of CCTCC M20221785.
2. The recombinant bacterium according to claim 1, wherein the recombinant bacterium has an increased activity of expressed (-) - α -bisabolol synthase EeBBS, a weakened squalene synthesis, an increased expression of farnesyl diphosphate synthase ERG20 gene and (-) - α -bisabolol synthase mEeBBS gene, an increased MVA pathway, an increased acetic acid utilization capacity, and an increased glycolytic pathway, as compared to a wild-type yeast.
3. The recombinant bacterium of claim 2, wherein the increased activity of expressed (-) - α -bisabolol synthase EeBBS is a mutation of expressed (-) - α -bisabolol synthase EeBBS to (-) - α -bisabolol synthase mEeBBS, the mutation being: three mutations of L530P, S547V and E558V.
4. The recombinant bacterium according to claim 3, wherein the recombinant bacterium comprises a highly active mutant mEeBBS gene of (-) -alpha-bisabolol synthase EeBBS gene, and the sequence is shown as SEQ ID NO. 4.
5. The recombinant bacterium of claim 2, wherein the attenuation of squalene synthesis is a down-regulation of the transcription level of squalene synthesis gene ERG9 in wild-type yeast.
6. The recombinant bacterium according to claim 5, wherein the promoter for promoting squalene synthesis gene ERG9 in the recombinant bacterium is P HXT1 The method comprises the steps of carrying out a first treatment on the surface of the Preferably, the P HXT1 The nucleotide sequence of (2) is shown as SEQ ID NO. 2.
7. The recombinant bacterium according to claim 2, wherein the recombinant bacterium comprises a farnesyl diphosphate synthase ERG20 gene and a (-) - α -bisabolol synthase mEeBBS gene having a copy number of not less than 5.
8. The recombinant bacterium of claim 2, wherein the MVA pathway enhancement is overexpression of genes of the MVA pathway, the genes of the MVA pathway being ERG10, ERG13, hmg1, ERG12, ERG8, ERG19 and IDI1.
9. The recombinant bacterium according to claim 8, wherein the gene overexpression of the MVA pathway is a gene transferred into the MVA pathway in wild yeast.
10. The recombinant bacterium according to claim 2, wherein the enhancement of acetic acid utilization ability is overexpression of an acetyl-coa synthetase synthesis gene; the acetyl coenzyme A synthetase synthesis genes are ACS1 and ACS2.
11. The recombinant bacterium according to claim 10, wherein the overexpression of the acetyl-coa synthase synthesis gene is a transfer of the acetyl-coa synthase synthesis gene in a wild yeast.
12. The recombinant bacterium of claim 2, wherein the glycolytic pathway enhancement is an enhancement of expression of phosphofructokinase PFK gene.
13. The recombinant bacterium of claim 12, wherein said recombinant bacterium comprisesThe promoter for promoting phosphofructokinase PFK gene in bacteria is P TEF1 The method comprises the steps of carrying out a first treatment on the surface of the Preferably, the P TEF1 The nucleotide sequence of (2) is shown as SEQ ID NO. 3.
14. The recombinant bacterium according to any one of claims 2 to 13, wherein the wild-type yeast is yeast Saccharomyces cerevisiae cen.pk2-1C or Saccharomyces cerevisiae BY4741.
15. The method for producing a recombinant bacterium according to any one of claims 1 to 14, comprising the steps of:
(1) Integrating the farnesyl diphosphate synthase ERG20 gene and (-) -alpha-bisabolol synthase mEeBBS gene expression cassette into wild saccharomycetes to obtain a strain AB01; the (-) -alpha-bisabolol synthase mEeBBS gene sequence is shown in SEQ ID NO. 4;
(2) Integrating the tHMG1 gene and the IDI1 gene expression cassette into the genome of the strain AB01 to obtain a strain AB02;
(3) Replacement of the squalene synthetase ERG9 Gene promoter with the weak promoter P HXT1 Then, the strain AB10 is obtained by integrating the genome of the strain AB02;
(4) Integrating the farnesyl diphosphate synthase ERG20 gene and (-) -alpha-bisabolol synthase mEeBBS gene expression cassette into at least 5 separate genomic loci of the strain AB10 to obtain a yeast engineering strain AB24;
(5) Integrating the ERG10 gene and the tHMG1 gene expression cassette, the ERG12 gene and the ERG13 gene expression cassette, the ERG19 gene and the ERG8 gene expression cassette, and the tHMG1 gene and the IDI1 gene expression cassette into the genome of the strain AB24 to obtain a strain AB30;
(6) Integrating the expression cassettes of acetyl-CoA synthetase genes ACS1 and ACS2 into the genome of the strain AB30 to obtain a strain AB32;
(7) Replacement of the promoter of the phosphofructokinase PFK1 Gene with the promoter P TEF1 And integrating the recombinant strain into the genome of the strain AB32 to obtain a strain AB55, thus obtaining the recombinant strain.
16. Use of the recombinant strain according to any one of claims 1 to 14 for the preparation of (-) - α -bisabolol and its formulations.
17. A method for producing (-) - α -bisabolol, comprising the step of fermenting using the recombinant bacterium of any one of claims 1 to 14; preferably, the method comprises the following steps:
(1) Inoculating the recombinant bacterium of any one of claims 1 to 14 into a seed culture medium, and culturing for 20 to 24 hours;
(2) Inoculating the seed liquid into a fermentation culture medium, adding n-dodecane with the volume ratio of 0-20%, and fermenting and culturing for 48-96 hours, and supplementing a feed supplement culture medium during the fermentation culture;
the seed culture medium is an aqueous solution containing the following additives: 10-25 g/L peptone, 5-15 g/L yeast powder and 20-35 g/L glucose;
the fermentation medium is an aqueous solution containing the following additives: 10 to 35g/L of glucose or glycerol, 2 to 3g/L of monopotassium phosphate, 2.5 to 3.0g/L of dipotassium phosphate, 10 to 28g/L of yeast powder and 10 to 20g/L of yeast peptone.
The feed medium is an aqueous solution containing the following additives: 500-800 g/L glucose and 9g/L KH 2 PO 4 、2.5g/L MgSO 4 、3.5g/L K 2 SO 4 、0.28g/L Na 2 SO 4 10ml/L trace element solution and 12ml/L vitamin solution.
18. The method according to claim 17: the method is characterized in that: the volume ratio of the seed liquid to the fermentation medium to the n-dodecane is 2:25:5; the culture is shaking culture, the temperature is 30 ℃, and the rotating speed is 200rpm.
19. The method according to claim 17: the method is characterized in that: the seed culture medium is an aqueous solution containing the following additives: 20g/L of peptone, 10g/L of yeast powder and 20g/L of glucose;
the fermentation medium is an aqueous solution containing the following additives: glucose or glycerin 20g/L, potassium dihydrogen phosphate 2.2g/L, dipotassium hydrogen phosphate 2.9g/L, yeast powder 10g/L, peptone 20g/L.
The feed medium is an aqueous solution containing the following additives: 500-800 g/L glucose and 9g/L KH 2 PO 4 、2.5g/L MgSO 4 、3.5g/L K 2 SO 4 、0.28g/L Na 2 SO 4 10ml/L trace element solution and 12ml/L vitamin solution.
CN202310076063.4A 2023-02-07 2023-02-07 Recombinant bacterium for producing (-) -alpha-bisabolol and preparation method and application thereof Pending CN116716196A (en)

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