CN117965507A - Recombinant microorganism for obtaining alpha-farnesene and beta-farnesene and construction method thereof - Google Patents
Recombinant microorganism for obtaining alpha-farnesene and beta-farnesene and construction method thereof Download PDFInfo
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- CN117965507A CN117965507A CN202311866971.XA CN202311866971A CN117965507A CN 117965507 A CN117965507 A CN 117965507A CN 202311866971 A CN202311866971 A CN 202311866971A CN 117965507 A CN117965507 A CN 117965507A
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- farnesene
- recombinant microorganism
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Abstract
The invention discloses a recombinant microorganism for obtaining alpha-farnesene and beta-farnesene and a construction method thereof, belonging to the field of synthetic biology. The invention obtains the alpha-farnesene synthase and beta-farnesene synthase with excellent performance, including the alpha-farnesene synthase or the beta-farnesene synthase with the sequences shown as SEQ ID NO. 1-6. The recombinant microorganism of the invention contains 5 farnesene synthase genes on the basis of CEN.PK2-1D, contains additional MVA pathway genes ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1, IDI1, and an additional ERG20 and a tHMG1 gene.
Description
Technical Field
The invention belongs to the field of synthetic biology, and particularly relates to a recombinant microorganism for obtaining alpha-farnesene and beta-farnesene and a construction method thereof.
Background
Farnesene is a sesquiterpene compound and has wide application in agriculture and industrial production. At present, the farnesene can be used as the existing petroleum-based diene monomer substitute for the production of tackifying resins, so that the softening point, the stability and the thermal stability of the tackifying resins are improved. Meanwhile, farnesene is a main component of a plurality of plant volatile substances, and is commonly used as a pest attractant in combination with insect attractants for agricultural production according to a signal linkage mechanism between plants and pests and natural advantages of the plant volatile substances in pest trapping and killing. Polymerization process Nintene is also used as a gum base component in place of man-made polymers derived from petroleum sources, and is also used in foods such as chewing gum bases because it is a food acceptable polymer. Farnesene is currently obtained by plant extraction, chemical synthesis and biosynthetic methods. The microbial fermentation is not limited by factors such as plant growth, numerous impurities and the like, has lower cost and less environmental pollution compared with a chemical synthesis method, and is an economic and green synthesis mode.
At present, the microbial synthesis of farnesene mainly adopts escherichia coli, saccharomyces cerevisiae and yarrowia lipolytica, and certain progress is obtained. The enzymes used at present for producing alpha-farnesene are basically all of apple sources, and beta-farnesene synthase is of sweet wormwood sources.
Disclosure of Invention
It is an object of the present invention to provide a farnesene synthase having excellent properties and the use of the farnesene synthase in the construction of a recombinant microorganism producing farnesene.
A farnesene synthase with excellent performance is alpha-farnesene synthase or beta-farnesene synthase; wherein the alpha-farnesene synthase is tea tree-derived alpha-farnesene synthase with the amino acid sequence shown in SEQ ID NO.1 or is based on tea tree-derived alpha-farnesene synthase with the amino acid sequence shown in SEQ ID NO.1, and contains one or two of the following mutations: W281C, C455N; or the alpha-farnesene synthase is a pear-derived alpha-farnesene synthase with an amino acid sequence shown as SEQ ID NO.3, or a pear-derived alpha-farnesene synthase mutant containing one or more of the following mutations based on the pear-derived alpha-farnesene synthase with the amino acid sequence shown as SEQ ID NO. 3: G252E, D10G, A T; the beta-farnesene synthase is a chamomile-derived beta-farnesene synthase with the amino acid sequence shown in SEQ ID NO.2, or a chamomile-derived beta-farnesene synthase mutant containing one or more of the following mutations based on the chamomile-derived beta-farnesene synthase with the amino acid sequence shown in SEQ ID NO. 2: f11S, M35: 35T, T319:319S, I434:434: 434T, I460:460: 460V, K59: 59R, S204:204Y.
The farnesene synthase synthesized farnesene has more excellent performance, can be used for producing farnesene or constructing recombinant microorganisms for producing farnesene, and improves the yield of farnesene.
The second object of the invention is to provide a recombinant microorganism producing farnesene, and a construction method and application of the recombinant microorganism in producing farnesene.
A recombinant microorganism producing farnesene is a recombinant microorganism producing alpha-farnesene or a recombinant microorganism producing beta-farnesene. Wherein, the copy number of the gene in the recombinant microorganism producing the alpha-farnesene is ERG10: ERG13: tHMG1: ERG12: ERG8: MVD1: IDI1: ERG20: aFS = 2:2: x:2:2:2:2:2: x, X is an integer greater than or equal to 1; the copy number of the gene in the recombinant microorganism producing beta-farnesene is ERG10: ERG13: tHMG1: ERG12: ERG8: MVD1: IDI1: ERG20: bFS = 2:2: x:2:2:2:2:2: x, X is an integer of 1 or more. Wherein ERG10 is a gene encoding acetoacetyl-CoA thiolase, ERG13 is a gene encoding HMG-CoA synthase, tHMG1 is a gene encoding HMG-CoA reductase, ERG12 is a gene encoding mevalonate kinase, ERG8 is a gene encoding mevalonate-5-phosphate kinase, MVD1 is a gene encoding mevalonate pyrophosphate decarboxylase, IDI1 is a gene encoding isoprene pyrophosphate isomerase, ERG20 is a gene encoding farnesene pyrophosphate synthase, aFS is a gene encoding alpha-farnesene synthase, and bFS is a gene encoding beta-farnesene synthase. Preferably, the aFS-coded alpha-farnesene synthase is tea tree-derived alpha-farnesene synthase with an amino acid sequence shown as SEQ ID NO.1 or pear-derived alpha-farnesene synthase with an amino acid sequence shown as SEQ ID NO.3 or an alpha-farnesene synthase mutant; the bFS encoded beta-farnesene synthase is a chamomile-derived beta-farnesene synthase with an amino acid sequence shown in SEQ ID No.2 or a beta-farnesene synthase mutant, more preferably, the aFS encoded alpha-farnesene synthase has an amino acid sequence shown in SEQ ID No.4 (on the basis of pear-derived alpha-farnesene synthase, contains a G252E mutation); bFS (on the basis of a beta-farnesene synthase from chamomile, containing the F11S, M35T, T319S, I434T, I V mutation) or bFS (on the basis of a beta-farnesene synthase from chamomile, containing the F11S, M T, T319S, I434T, I460V, K Y mutation) as shown in SEQ ID NO. 6.
More preferably, the Access/GENE ids of ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1, IDI1, ERG20 in NCBI are shown in the following table.
| Gene | Accession/GENE id |
| ERG10 | 856079 |
| ERG13 | 854913 |
| tHMGR | 854900, Truncating 4-1659bp |
| ERG12 | NM_001182715.1 |
| ERG8 | CP046093.1，689693..691048 |
| MVD1 | NM_001183220.1 |
| IDI1 | NM_001183931.1 |
| ERG20 | 853272 |
Preferably, the recombinant microorganism producing farnesene takes saccharomyces cerevisiae as a host; more preferably, the recombinant microorganism producing farnesene takes Saccharomyces cerevisiae CEN.PK2-1D strain as host.
Preferably, the recombinant microorganism producing alpha-farnesene contains 5 coding genes of alpha-farnesene synthase shown in SEQ ID NO.3 or SEQ ID NO.4 on the basis of Saccharomyces cerevisiae CEN.PK2-1D, and contains additional MVA pathway genes (ERG 10, ERG13, THMG1, ERG12, ERG8, MVD1, IDI 1) and an additional ERG20 gene and an additional tHMG1 gene. More preferably, the recombinant microorganism producing alpha-farnesene is obtained by transferring genes shown in the following table into Saccharomyces cerevisiae CEN.PK2-1D through plasmids, and integrating the related genes into specific positions of Saccharomyces cerevisiae CEN.PK2-1D chromosomes.
Preferably, the recombinant microorganism producing beta-farnesene contains 5 coding genes of beta-farnesene synthase shown in SEQ ID NO.5 or SEQ ID NO.6 on the basis of Saccharomyces cerevisiae CEN.PK2-1D, and contains additional MVA pathway genes (ERG 10, ERG13, THMG1, ERG12, ERG8, MVD1, IDI 1) and an additional ERG20 gene and an additional tHMG1 gene. More preferably, the recombinant microorganism producing beta-farnesene is obtained by transferring genes shown in the following table into Saccharomyces cerevisiae CEN.PK2-1D through plasmids, and integrating related genes into specific positions of Saccharomyces cerevisiae CEN.PK2-1D chromosome.
In the above table, the sequences of the relevant promoters, genes or terminators are as follows:
>pTEF1：AGCTCATAGCTTCAAAATGTTTCTACTCCTTTTTTACTCTTCCAGATTTTCTCGGACTCCGCGCATCGCCGTACCACTTCAAAACACCCAAGCACAGCATACTAAATTTCCCCTCTTTCTTCCTCTAGGGTGTCGTTAATTACCCGTACTAAAGGTTTGGAAAAGAAAAAAGAGACCGCCTCGTTTCTTTTTCTTCGTCGAAAAAGGCAATAAAAATTTTTATCACGTTTCTTTTTCTTGAAAATTTTTTTTTTGATTTTTTTCTCTTTCGATGACCTCCCATTGATATTTAAGTTAATAAACGGTCTTCAATTTCTCAAGTTTCAGTTTCATTTTTCTTGTTCTATTACAACTTTTTTTACTTCTTGCTCATTAGAAAGAAAGCATAGCAATCTAATCTAAGTTTTCTAGAACTAGTGGATCCCCCGGGaaaa;
>cas9：ATGGACAAGAAGTACTCCATTGGGCTCGATATCGGCACAAACAGCGTCGGtTGGGCCGTCATTACGGACGAGTACAAGGTGCCGAGCAAAAAATTCAAAGTTCTGGGCAATACCGATCGCCACAGCATAAAGAAGAACCTCATTGGCGCCCTCCTGTTCGACTCCGGGGAGACGGCCGAAGCCACGCGGCTCAAAAGAACAGCACGGCGCAGATATACCCGCAGAAAGAATCGGATCTGCTACCTGCAGGAGATCTTTAGTAATGAGATGGCTAAGGTGGATGACTCTTTCTTCCATAGGCTGGAGGAGTCCTTTTTGGTGGAGGAGGATAAAAAGCACGAGCGCCACCCAATCTTTGGCAATATCGTGGACGAGGTGGCGTACCATGAAAAGTACCCAACCATATATCATCTGAGGAAGAAGCTTGTAGACAGTACTGATAAGGCTGACTTGCGGTTGATCTATCTCGCGCTGGCGCATATGATCAAATTTCGGGGACACTTCCTCATCGAGGGGGACCTGAACCCAGACAACAGCGATGTCGACAAACTCTTTATCCAACTGGTTCAGACTTACAATCAGCTTTTCGAAGAGAACCCGATCAACGCATCCGGAGTTGACGCCAAAGCAATCCTGAGCGCTAGGCTGTCCAAATCCCGGCGGCTCGAAAACCTCATCGCACAGCTCCCTGGGGAGAAGAAGAACGGCCTGTTTGGTAATCTTATCGCCCTGTCACTCGGGCTGACCCCCAACTTTAAATCTAACTTCGACCTGGCCGAAGATGCCAAGCTTCAACTGAGCAAAGACACCTACGATGATGATCTCGACAATCTGCTGGCCCAGATCGGCGACCAGTACGCAGACCTTTTTTTGGCGGCAAAGAACCTGTCAGACGCCATTCTGCTGAGTGATATTCTGCGAGTGAACACGGAGATCACCAAAGCTCCGCTGAGCGCTAGTATGATCAAGCGCTATGATGAGCACCACCAAGACTTGACTTTGCTGAAGGCCCTTGTCAGACAGCAACTGCCTGAGAAGTACAAGGAAATTTTCTTCGATCAGTCTAAAAATGGCTACGCCGGATACATTGACGGCGGAGCAAGCCAGGAGGAATTTTACAAATTTATTAAGCCCATCTTGGAAAAAATGGACGGCACCGAGGAGCTGCTGGTAAAGCTTAACAGAGAAGATCTGTTGCGCAAACAGCGCACTTTCGACAATGGAAGCATCCCCCACCAGATTCACCTGGGCGAACTGCACGCTATCCTCAGGCGGCAAGAGGATTTCTACCCCTTTTTGAAAGATAACAGGGAAAAGATTGAGAAAATCCTCACATTTCGGATACCCTACTATGTAGGCCCCCTCGCCCGGGGAAATTCCAGATTCGCGTGGATGACTCGCAAATCAGAAGAGACCATCACTCCCTGGAACTTCGAGGAAGTCGTGGATAAGGGGGCCTCTGCCCAGTCCTTCATCGAAAGGATGACTAACTTTGATAAAAATCTGCCTAACGAAAAGGTGCTTCCTAAACACTCTCTGCTGTACGAGTACTTCACAGTTTATAACGAGCTCACCAAGGTCAAATACGTCACAGAAGGGATGAGAAAGCCAGCATTCCTGTCTGGAGAGCAGAAGAAAGCTATCGTGGACCTCCTCTTCAAGACGAACCGGAAAGTTACCGTGAAACAGCTCAAAGAAGACTATTTCAAAAAGATTGAATGTTTCGACTCTGTTGAAATCAGCGGAGTGGAGGATCGCTTCAACGCATCCCTGGGAACGTATCACGATCTCCTGAAAATCATTAAAGACAAGGACTTCCTGGACAATGAGGAGAACGAGGACATTCTTGAGGACATTGTCCTCACCCTTACGTTGTTTGAAGATAGGGAGATGATTGAAGAACGCTTGAAAACTTACGCTCATCTCTTCGACGACAAAGTCATGAAACAGCTCAAGAGGCGCCGATATACAGGATGGGGGCGGCTGTCAAGAAAACTGATCAATGGGATCCGAGACAAGCAGAGTGGAAAGACAATCCTGGATTTTCTTAAGTCCGATGGATTTGCCAACCGGAACTTCATGCAGTTGATCCATGATGACTCTCTCACCTTTAAGGAGGACATCCAGAAAGCACAAGTTTCTGGCCAGGGGGACAGTCTTCACGAGCACATCGCTAATCTTGCAGGTAGCCCAGCTATCAAAAAGGGAATACTGCAGACCGTTAAGGTCGTGGATGAACTCGTCAAAGTAATGGGAAGGCATAAGCCCGAGAATATCGTTATCGAGATGGCCCGAGAGAACCAAACTACCCAGAAGGGACAGAAGAACAGTAGGGAAAGGATGAAGAGGATTGAAGAGGGTATAAAAGAACTGGGGTCCCAAATCCTTAAGGAACACCCAGTTGAAAACACCCAGCTTCAGAATGAGAAGCTCTACCTGTACTACCTGCAGAACGGCAGGGACATGTACGTGGATCAGGAACTGGACATCAATCGGCTCTCCGACTACGACGTGGATCATATCGTGCCCCAGTCTTTTCTCAAAGATGATTCTATTGATAATAAAGTGTTGACAAGATCCGATAAAAATAGAGGGAAGAGTGATAACGTCCCCTCAGAAGAAGTTGTCAAGAAAATGAAAAATTATTGGCGGCAGCTGCTGAACGCCAAACTGATCACACAACGGAAGTTCGATAATCTGACTAAGGCTGAACGAGGTGGCCTGTCTGAGTTGGATAAAGCCGGCTTCATCAAAAGGCAGCTTGTTGAGACACGCCAGATCACCAAGCACGTGGCCCAAATTCTCGATTCACGCATGAACACCAAGTACGATGAAAATGACAAACTGATTCGAGAGGTGAAAGTTATTACTCTGAAGTCTAAGCTGGTCTCAGATTTCAGAAAGGACTTTCAGTTTTATAAGGTGAGAGAGATCAACAATTACCACCATGCGCATGATGCCTACCTGAATGCAGTGGTAGGCACTGCACTTATCAAAAAATATCCCAAGCTTGAATCTGAATTTGTTTACGGAGACTATAAAGTGTACGATGTTAGGAAAATGATCGCAAAGTCTGAGCAGGAAATAGGCAAGGCCACCGCTAAGTACTTCTTTTACAGCAATATTATGAATTTTTTCAAGACCGAGATTACACTGGCCAATGGAGAGATTCGGAAGCGACCACTTATCGAAACAAACGGAGAAACAGGAGAAATCGTGTGGGACAAGGGTAGGGATTTCGCGACAGTCCGGAAGGTCCTGTCCATGCCGCAGGTGAACATCGTTAAAAAGACCGAAGTACAGACCGGAGGCTTCTCCAAGGAAAGTATCCTCCCGAAAAGGAACAGCGACAAGCTGATCGCACGCAAAAAAGATTGGGACCCCAAGAAATACGGCGGATTCGATTCTCCTACAGTCGCTTACAGTGTACTGGTTGTGGCCAAAGTGGAGAAAGGGAAGTCTAAAAAACTCAAAAGCGTCAAGGAACTGCTGGGCATCACAATCATGGAGCGATCAAGCTTCGAAAAAAACCCCATCGACTTTCTCGAGGCGAAAGGATATAAAGAGGTCAAAAAAGACCTCATCATTAAGCTTCCCAAGTACTCTCTCTTTGAGCTTGAAAACGGCCGGAAACGAATGCTCGCTAGTGCGGGCGAGCTGCAGAAAGGTAACGAGCTGGCACTGCCCTCTAAATACGTTAATTTCTTGTATCTGGCCAGCCACTATGAAAAGCTCAAAGGGTCTCCCGAAGATAATGAGCAGAAGCAGCTGTTCGTGGAACAACACAAACACTACCTTGATGAGATCATCGAGCAAATAAGCGAATTCTCCAAAAGAGTGATCCTCGCCGACGCTAACCTCGATAAGGTGCTTTCTGCTTACAATAAGCACAGGGATAAGCCCATCAGGGAGCAGGCAGAAAACATTATCCACTTGTTTACTCTGACCAACTTGGGCGCGCCTGCAGCCTTCAAGTACTTCGACACCACCATAGACAGAAAGCGGTACACCTCTACAAAGGAGGTCCTGGACGCCACACTGATTCATCAGTCAATTACGGGGCTCTATGAAACAAGAATCGACCTCTCTCAGCTCGGTGGAGACAGCAGGGCTGACCCCAAGAAGAAGAGGAAGGTGTGA;
>tCYC1：TCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAGACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACGCTCGAAGGCTTTAATTTGC;
>tGAL10pGAL7：Tttgccagcttactatccttcttgaaaatatgcactctatatcttttagttcttaattgcaacacatagatttgctgtataacgaattttatgctattttttaaatttggagttcagtgataaaagtgtcacagcgaatttcctcacatgtagggaccgaattgtttacaagttctctgtaccaccatggagacatcaaagattgaaaatctatggaaagatatggacggtagcaacaagaatatagcacgagccgcgaagttcatttcgttacttttgatatcgctcacaactattgcgaagcgcttcagtgaaaaaatcataaggaaaagttgtaaatattattggtagtattcgtttggtaaagtagagggggtaatttttcccctttattttgttcatacattcttaaattgctttgcctctccttttggaaagctatacttcggagcactgttgagcgaaggctcattagatatattttctgtcattttccttaacccaaaaataagggaaagggtccaaaaagcgctcggacaactgttgaccgtgatccgaaggactggctatacagtgttcacaaaatagccaagctgaaaataatgtgtagctatgttcagttagtttggctagcaaagatataaaagcaggtcggaaatatttatgggcattattatgcagagcatcaacatgataaaaaaaaacagttgaatattccctcaaaa;
>tADH1：GCGAATTTCTTATGATTTATGATTTTTATTATTAAATAAGTTATAAAAAAAATAAGTGTATACAAATTTTAAAGTGACTCTTAGGTTTTAAAACGAAAATTCTTATTCTTGAGTAACTCTTTCCTGTAGGTCAGGTTGCTTTCTCAGGTATAGCATGAGGTCGCTCTTATTGACCACACCTCTACCGG;
>pGAL1pGAL10：Tatagttttttctccttgacgttaaagtatagaggtatattaacaattttttgttgatacttttatgacatttgaataagaagtaatacaaaccgaaaatgttgaaagtattagttaaagtggttatgcagcttttgcatttatatatctgttaatagatcaaaaatcatcgcttcgctgattaattaccccagaaataaggctaaaaaactaatcgcattattatcctatggttgttaatttgattcgttgatttgaaggtttgtggggccaggttactgccaatttttcctcttcataaccataaaagctagtattgtagaatctttattgttcggagcagtgcggcgcgaggcacatctgcgtttcaggaacgcgaccggtgaagaccaggacgcacggaggagagtcttccgtcggagggctgtcgcccgctcggcggcttctaatccgtacttcaatatagcaatgagcagttaagcgtattactgaaagttccaaagagaaggtttttttaggctaagataatggggctctttacatttccacaacatataagtaagattagatatggatatgtatatggtggtattgccatgtaatatgattattaaacttctttgcgtccatccaaaaaaaaagtaagaatttttgaaaattcaatataa;
>tHMG1：ACTTAGTCATACGTCATTGGTATTCTCTTGAAAAAGAAGCACAACAGCACCATGTGTTACGTAAAATATTTACTTTATAGTTTGTACGTCATAATTTCTTCCATATTACAAGTTCGTGCATATATAGAAAGAATTCTGTTGTTGTAATTGTCATAACTATTGAGCTTTACCTGAAAATTCAACGAAAAAAACTCAAAAACCACATGCTTCTCTTGAGTCATGCGGTTCCTTTCCCTTATGAGTGAAAATCTTCCTTTTTTAGCTATGTGCGCCATCCGATAAATGTAGGAGCAATGAAGC;
>tERG20：AACTAACGCTAATCGATAAAACATTAGATTTCAAACTAGATAAGGACCATGTATAAGAACTATATACTTCCAATATAATATAGTATAAGCTTTAAGATAGTATCTCTCGATCTACCGTTCCACGTGACTAGTCCAAGGATTTTTTTTAA;
>tPGK1：ATTGAATTGAATTGAAATCGATAGATCAATTTTTTTCTTTTCTCTTTCCCCATCCTTTACGCTAAAATAATAGTTTATTTTATTTTTTGAATATTTTTTATTTATATACGTATATATAGACTATTATTTATCTTTTAATGATTATTAAGATTTTTATTAAAAAAAATTACGCTCCTCTTTTAATGCCTTTATGCAGTTTTTTTTCCCATTCGATATTTCTATGTTCGGGTTCAGCGTATTTTAAGTTTAATAACTCGAAAATTCTGCGTTCGTT;
>pURA3：TTCAATTCATCATTTTTTTTTTATTCTTTTTTTTGATTTCGGTTTCTTTGAAATTTTTTTGATTCGGTAATCTCCGAACAGAAGGAAGAACGAAGGAAGGAGCACAGACTTAGATTGGTATATATACGCATATGTAGTGTTGAAGAAACATGAAATTGCCCAGTATTCTTAACCCAACTGCACAGAACAAAAACCTGCAGGAAACGAAGATAAATC;
>tURA3：aaaactgtattataagtaaatgcatgtatactaaactcacaaattagagcttcaatttaattatatcagttattaccc。
Furthermore, the recombinant microorganism for producing the farnesene can knock out GAL80 gene, after knocking out Gal80, the strain can realize the synthesis of the farnesene under the condition of not adding galactose for induction, so that the experimental flow can be reduced, and the fermentation cost can be reduced.
The construction method of the recombinant microorganism for producing farnesene comprises cloning genes ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1, IDI1, ERG20, pyc-aFS/Mac-bFS onto a plurality of plasmids, transferring the plasmids into a host, and screening to obtain the recombinant microorganism for expressing each gene. Preferably, the method for constructing the farnesene-producing recombinant microorganism comprises the following steps: the plasmids shown in the table above were constructed, and the plasmids were transferred into Saccharomyces cerevisiae CEN.PK2-1D, so that the related genes were integrated into the chromosome of Saccharomyces cerevisiae CEN.PK2-1D, to obtain a farnesene-producing recombinant microorganism.
The recombinant microorganisms producing farnesene can be used for producing farnesene.
It is a further object of the present invention to provide a method for constructing a library of mutants, comprising the steps of: and (3) performing PCR amplification on the key genes by using low-fidelity DNA polymerase taq polymerase and adjusting the concentration of magnesium ions in the system to obtain DNA fragments containing mutation, and cloning the DNA fragments into an expression vector. Transferring the expression vector obtained by the construction into an expression host, such as saccharomyces cerevisiae, and the like, and coating and screening a plate to obtain a mutant strain library.
The fourth object of the present invention is to provide a method for efficiently screening forward mutants of farnesene synthesis related enzyme, comprising the steps of: inoculating the strain from the screening plate to a high-flux seed culture plate (including but not limited to a 96-well plate and a 384-well plate), transferring the strain to a fermentation culture plate (including but not limited to a 96-well plate and a 384-well plate) when the strain grows to a logarithmic growth phase, extracting the high-flux product when the yield of the strain reaches the maximum, transferring the extracted product to a new plate (including but not limited to the 96-well plate and the 384-well plate), adding a broad-spectrum color developing agent (including but not limited to iodine, vanillin concentrated sulfuric acid color developing agent, cerium molybdate, anisaldehyde and potassium permanganate), reacting (at normal temperature or high temperature or low temperature, standing or shaking) for a sufficient time, and detecting the maximum absorption value by an enzyme-labeling instrument. Sequencing to determine the positive mutation site. The invention provides an alpha-farnesene synthase or beta-farnesene synthase mutant with one or more point mutations and improved performance obtained by random mutation screening. Wherein the extractant is isopropyl palmitate, white oil (liquid paraffin), methyl oleate or rapeseed oil. The invention also provides application of the extractant in extraction of farnesene based on the fact that isopropyl palmitate, white oil (liquid paraffin) methyl oleate or rapeseed oil are used as the extractant, and the yield of farnesene is not affected.
The invention aims to provide a method for constructing a farnesene high-yield strain, which comprises the following steps: the farnesene synthase containing at least one positive mutation is introduced into a chassis strain which can be used for farnesene production, and the yield of farnesene is further improved by adjusting the proportion of the MVA pathway key genes tHMG1 and the farnesene synthase, so that the farnesene high-yield strain is obtained. Thus, the recombinant microorganism can be used for culturing to obtain high-yield farnesene.
The sixth object of the present invention is to provide a method for constructing and obtaining a high-yield strain of another product by a one-step method based on a certain high-yield strain, comprising the following steps: the strain with high yield of the product A is taken as a chassis, the synthesized gene of the product is targeted by the gRNA through a crispr gene editing method, the donor DNA contains the synthesized gene of the product B, and the one-time replacement of the key gene is realized through a crispr gene editing technology, so that the strain with high yield of the product B is obtained.
Several different sources of farnesene synthase were validated and compared in the present invention, and as a result, performance was found to be superior to the farnesene synthase currently in common use. And the engineering transformation is carried out to obtain the beta-farnesene synthase with improved performance, and finally, the farnesene synthetic yeast strain with high yield is obtained.
Drawings
FIG. 1 is a schematic construction diagram of plasmid pZY 600.
FIG. 2 is a schematic diagram of the construction of plasmid pZY 413.
FIG. 3 is a schematic diagram of the construction of plasmid pZY 412L.
FIG. 4 is a schematic diagram of the construction of plasmid pZY 414.
FIG. 5 is a schematic diagram of the construction of plasmid pZY 900.
FIG. 6 is a schematic diagram of the construction of the AFS and BFS series of plasmids.
FIG. 7 is a graph showing the results of farnesene production by fermentation of different strains.
FIG. 8 is a schematic diagram of the construction of plasmids pAFS1-P2, pAFS1-P3, pAFS1-P4, pAFS1-P5, pAFS1-P6, pAFS 1-P7.
FIG. 9 is a graph showing the results of the production of beta-farnesene high producing strain.
Detailed Description
The following examples serve to further illustrate the invention but are not to be construed as limiting the invention. The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated.
EXAMPLE 1 construction of the required vector for Chassis Yeast Strain
(1) Plasmid pZY600
The function of plasmid pZY600 is to integrate Cas9 protein for subsequent strain engineering using Crispr-Cas9 gene editing technology. Relevant features of plasmid pZY 600: delta ChrXII-2: hygro_ptef1_cas 9_ tCYC (fig. 1), wherein ptef1_cas9_ tCYC refers to Cas9 expressing the expression controlled by the TEF1 promoter, the terminator is cyc1, and the screening marker is hygromycin; delta chrXII-2 refers to the position of the chromosome into which the expression cassette is inserted.
The specific construction process of the plasmid pZY600 comprises the following steps: the plasmid pZY403 is used as a template, and the primers G6001-F and G6001-R are used for PCR amplification to obtain a fragment G6001 (ChrXII-2 site homology left and right arms for integrating Cas9 genes, a NotI enzyme cutting site is introduced, and a plasmid skeleton containing ampicillin resistance and ura labels) is used for preparing the plasmid; using pZY403 as template, using primers G6002-F and G6002-R to obtain fragment G6002 (hygromycin resistance gene and its promoter TEF terminator TEF) by PCR amplification; fragment G6003 (Gene Cas9 and its promoter TEF1 terminator CYC 1) was obtained by PCR amplification using p43802 (addgene) as template and primers G6003-F and G6003-R. The fragment is recombined in saccharomyces cerevisiae in vivo to construct pZY600 by DNA assemble (yeast assembly), and then amplified in escherichia coli, and after enzyme digestion verification and correct sequencing, pZY600 is obtained. The NotI enzyme is tangentially processed to obtain a fragment with a target gene, and the sequence of the fragment is shown as SEQ ID NO. 7.
Wherein, the construction of plasmid pZY403 is: the yeast 30000B genome is used as a template, and the primers Z4031-F and Z4031-R are used for PCR amplification to obtain a fragment Z4031 (ChrXII-2 homologous left arm for integrating ERG13 gene); PCR amplification using the yeast 30000B genome as template and primers Z4032-F and Z4032-R to obtain fragment Z4032 (promoter); PCR amplification using the yeast 30000B genome as template and primers Z4033-F and Z4033-R to obtain fragment Z4033 (ERG 13 gene); PCR amplification using the yeast 30000B genome as template and primers Z4034-F and Z4034-R to obtain fragment Z4034 (terminator); the plasmid pZY402 (see CN 111019850A for details) is used as a template, and the primers Z4035-F and Z4035-R are used for PCR amplification to obtain a fragment Z4035 (screening mark); the yeast 30000B genome is used as a template, and the primers Z4036-F and Z4036-R are used for PCR amplification to obtain a fragment Z4036 (ChrXII-2 site homologous right arm for integrating ERG13 gene); PCR amplification using pZY402 (see CN 111019850A for details) as template and primers Z4037-F and Z4037-R to obtain fragment Z4037 (introduced NotI cleavage site, plasmid backbone containing ampicillin resistance and ura tag); . The fragment is recombined in saccharomyces cerevisiae in vivo to construct pZY403 by DNAassemble (yeast assembly), and then amplified in escherichia coli, and pZY403 is obtained after enzyme digestion verification and correct sequencing.
The sequences of the primers used for constructing the above plasmids are shown in the following table:
(2) Plasmid pZY413 and gRNA plasmid pZY607
The plasmid pZY413 has the function of over-expressing related genes in the MVA pathway, and provides a high-efficiency FPP precursor synthesis chassis strain for subsequent gene screening. Relevant features of plasmid pZY 413: delta ChrXI-3: ERG8_pGAl1pGAl10_tHMG1_pGAl7_ERG12 (FIG. 2), the promoters GAL1, GAL7, GAL10 were used to control the expression of the genes ERG8, ERG12, THMG1, respectively, and the inserted chromosomal locus was ChrXI-3.
The specific construction process of the plasmid pZY413 comprises the following steps: fragment 4131 (for integration of the homologous right arm of ERG8, tHMG1, ERG12 genes, introduction of NotI cleavage site and part of plasmid backbone containing ampicillin resistance and ura tag) was obtained by PCR amplification using plasmid pZY402 (see CN 111019850A for details) as template with primers 4131-F and 4121-R; the pZY402 is used as a template, and the primers 4122-F and 4132-R are used for PCR amplification to obtain a segment 4132 (the rest part of plasmid skeleton containing ampicillin resistance and ura label is used for integrating part of the ChrXI-3 homologous right arm of ERG8, tHMG1 and ERG12 genes, and a NotI enzyme cutting site is introduced); PCR amplification using pZY402 as template and primers 4133-F and 4133-R to obtain segment 4133 (for integrating the remainder of ChrXI-3 site homology right arm of ERG8, tHMG1, ERG12 genes, part of target genes ERG8, tHMG1, ERG12 and promoter terminator thereof); the fragment 4134 (remainder of the genes ERG8, tHMG1, ERG12 and promoter terminator thereof) was obtained by PCR amplification using pZY402 as a template with primers 4134-F and 4134-R. The fragment is recombined in saccharomyces cerevisiae in vivo to construct pZY413 by a DNAassemble method, and then amplified in escherichia coli, and pZY413 is obtained after enzyme digestion verification and correct sequencing. The NotI enzyme is tangentially processed to obtain a fragment with a target gene, and the sequence of the fragment is shown as SEQ ID NO. 8.
PZY607 is a gRNA plasmid targeting ChrXI-3 locus, a tool plasmid for target recognition cleavage of Saccharomyces cerevisiae genomic DNA in Crispr-Cas9 gene editing technology, which replaces the guide-RNA sequence with ATATGTCTCTAATTTTGGAA on the basis of plasmid p43803 (http:// www.addgene.org/43803 /). Using plasmid p43803 as template, using primers 6071-F and G6031-R to obtain fragment 6071 (20 bp target sequence for targeted recognition, gRNA scafold, terminator SUP4 and part of plasmid backbone containing ampicillin resistance and ura tag) by PCR amplification; fragment 6072 (the remainder of the plasmid backbone containing the ampicillin resistance and ura tag, the 20bp target sequence for targeted recognition and promoter SNR 52) was obtained by PCR amplification using p43803 as template with primers G6032-F and 6072-R. The above fragment was recombined in Saccharomyces cerevisiae by DNA assemble to construct pZY607.
The sequences of the primers used for constructing the above plasmids are shown in the following table:
(3) Plasmid pZY412L and gRNA plasmid pZY606
The plasmid pZY412L has the function of over-expressing related genes in the MVA pathway, and provides a high-efficiency FPP precursor synthesis chassis strain for subsequent gene screening. Relevant features of plasmid pZY 412L: delta ChrXII-4: IDI1_pGAl1pGAl10_ERG10_pGAl7_MVD1 (FIG. 3), the expression genes IDI1, MVD1, ERG10 were controlled by the promoters GAL1, GAL7, GAL10, respectively, and the inserted chromosomal locus was ChrXII-4.
Specific construction process of plasmid pZY 412L: the fragment pZY412SPD (a nonsensical DNA sequence for replacing the g418 resistance gene in pZY410 (see CN 111019850A for details), containing the cohesive ends of SalI and SpeI cleavage sites) was obtained by PCR template-free annealing using primers 412-F and 412-R, in a reaction system of 10. Mu.L, 4.5. Mu.L for each primer and 1. Mu.L for 10 XT 4 DNA LIGASE buffer, followed by PCR: 95 ℃ for 5min; dropping PCR at 95 ℃,1min, -1 ℃/cycle,70cycle;25 ℃ for 5min;12℃and infinity (the fragment can be taken out for temporary storage at-40℃after template-free denaturation annealing PCR). The large fragment pZY412VPD (vector fragment containing the genes MVD1, ERG10, IDI1 of interest and the ampicillin resistance and ura selection markers) of 12835bp was recovered by digestion of pZY401 (see CN 111019850A for details) with SalI and SpeI followed by gel recovery. And (3) carrying out enzyme ligation (about 20 hours) on the fragment pZY412SPD and the fragment pZY412VPD by using T4 DNA ligase at the temperature of 16 ℃ according to the mol ratio of 3:1, transferring the enzyme ligation product into competent cells of escherichia coli DH10B for amplification, and obtaining the plasmid of pZY412L after enzyme digestion verification and correct sequencing. The NotI enzyme is tangentially processed to obtain a fragment with a target gene, and the sequence of the fragment is shown as SEQ ID NO. 9.
PZY606 is a gRNA plasmid targeting the Chr XII-4 locus, a tool plasmid for target recognition and cutting of Saccharomyces cerevisiae genomic DNA in Crispr-Cas9 gene editing technology, and a guide-RNA sequence is replaced by GCTTCAAGAATTGAGTAAAC on the basis of a plasmid p 43803. Fragment 6061 (20 bp target sequence for targeted recognition, gRNAscafold, terminator SUP4 and a part of plasmid backbone containing ampicillin resistance and ura tag) was obtained by PCR amplification using plasmid p43803 as template with primers 6061-F and G6031-R; fragment 6062 (the remainder of the plasmid backbone containing the ampicillin resistance and ura tag, the 20bp target sequence for targeted recognition and promoter SNR 52) was obtained by PCR amplification using p43803 as template with primers G6032-F and 6062-R. The above fragment was recombined in Saccharomyces cerevisiae by DNAassemble to construct pZY606.
The sequences of the primers used for constructing the above plasmids are shown in the following table:
(4) Plasmid pZY414 and gRNA plasmid pZY608
The plasmid pZY414 has the function of over-expressing related genes in the MVA pathway, and provides a high-efficiency FPP precursor synthesis chassis strain for subsequent gene screening. Related features of plasmid pZY 414: delta ChrX-3: pGAl1_ERG13_pGAl10_tHMG1, the expressed genes ERG13, THMG1 were controlled by the promoters GAL1, GAL10, respectively, and the inserted chromosomal site was ChrX-3.
The specific construction process of the plasmid pZY414 comprises the following steps: the Saccharomyces cerevisiae CEN.PK2-1D (30000B) genome is used as a template, and a segment 4141 (ChrX-3 site homology right arm for integrating ERG13 and tHMG1 genes and introducing NotI enzyme cutting site) is obtained by PCR amplification with primers 4141-F and 4141-R; PCR amplification using plasmid pRS426 as template and primers 4142-F and 4142-R to give fragment 4142 (plasmid backbone containing ampicillin resistance and ura tag); using 30000B genome as template, using primers 4143-F and 4143-R to obtain segment 4143 (ChrX-3 site homologous right arm for integrating ERG13 and tHMG1 gene, introducing NotI enzyme cutting site); the fragment 4144 (the genes of interest ERG13, tHMG1 and their promoter terminator) was obtained by PCR amplification using pZY410 (see CN 111019850A for details) as a template and primers 4144-F and 4144-R. The fragment is recombined in saccharomyces cerevisiae in vivo to construct pZY414 by a DNAassemble method, and then amplified in escherichia coli, and plasmid of the pZY414 is obtained after enzyme digestion verification and correct sequencing. The NotI enzyme is tangentially processed to obtain a fragment with a target gene, and the sequence of the fragment is shown as SEQ ID NO. 10.
PZY608 is a gRNA plasmid targeting ChrX-3 locus, a tool plasmid for target recognition cleavage of Saccharomyces cerevisiae genomic DNA in Crispr-Cas9 gene editing technology, which replaces the guide-RNA sequence with CTAATGTGTCCGCGTTTCTA on the basis of plasmid p 43803. Using plasmid p43803 as template, using primers 6081-F and G6031-R to obtain fragment 6081 (20 bp target sequence for targeted recognition, gRNAscafold, terminator SUP4 and part of plasmid backbone containing ampicillin resistance and ura tag) by PCR amplification; fragment 6082 (the remainder of the plasmid backbone containing the ampicillin resistance and ura tag, the 20bp target sequence for targeted recognition and promoter SNR 52) was obtained by PCR amplification using p43803 as template with primers G6032-F and 6082-R. The fragment is recombined in saccharomyces cerevisiae in vivo to construct pZY608 by a DNAassemble method, and then amplified in escherichia coli, and pZY608 is obtained after enzyme digestion verification and correct sequencing.
The sequences of the primers used for constructing the above plasmids are shown in the following table:
EXAMPLE 2 Chassis Yeast Strain construction
The linearized fragment of plasmid pZY600 is transferred into Saccharomyces cerevisiae strain CEN.PK2-1D, integrated into chromosome according to homologous recombination, the integration site is ChrXII-2, and the screening marker is hygromycin (hygror), so as to successfully construct background strain JCR1.
The linearized fragment of plasmid pZY413 was transformed into Saccharomyces cerevisiae JCR1 together with plasmid pZY607, and the selection marker was uracil (URA 3), and the plasmid was removed by 5-FOA reverse screen to obtain strain JCR23.
The linearized fragment of plasmid pZY414 was transformed into Saccharomyces cerevisiae JCR23 together with plasmid pZY608, and the selection marker was uracil (URA 3), and the plasmid was removed by 5-FOA reverse screen to obtain strain JCR25.
The linearized fragment of plasmid pZY412L was transformed into Saccharomyces cerevisiae JCR25 together with plasmid pZY606, and the selection marker was uracil (URA 3), and the plasmid was removed by 5-FOA reverse screening to obtain strain JCR27.
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Example 3 construction of expression vectors containing farnesene synthases from different sources
(1) Plasmid pZY900
Relevant features of plasmid pZY 900: Δleu2: LEU2 (URA 3) _TCYC1_LacZ_pGAL10pGAl1_ERG20_ tERG20 (FIG. 5), the expressed genes ERG20 and LacZ were controlled by the promoters GAL1 and GAL10, respectively, the selection marker was Leu2, and the inserted chromosomal site was Leu2.
The specific construction process of the plasmid pZY900 comprises the following steps: the yeast S288c genome is used as a template, and fragments 9001 (left homologous arm of Leu 2), 9002 (terminator tTDH 2), 9006 (gene ERG20 and terminator tERG 20) and 9007 (right arm of Leu 2) are obtained by amplification of 900-1F/1R, 900-2F/2R, 900-6F/6R and 900-7F/7R respectively; the genome of yeast 30000B was used as a template, and the fragments 9003 (terminator tCYC 1) and 9005 (promoters pGAL1 and Pgal) were obtained by amplification with primers 900-3F/3R and 900-5F/5R, respectively; the plasmid backbone (introducing MssI cleavage sites, selectable markers) was obtained by amplification with primers 900-8F/8R. The fragment is recombined in saccharomyces cerevisiae in vivo to construct pZY900 by DNAassemble (yeast assembly), and then amplified in escherichia coli, and after enzyme digestion verification and sequencing are correct, the pZY900 is obtained, and the sequence is shown as SEQ ID NO. 11.
The sequences of the primers used for constructing the above plasmids are shown in the following table:
(2) Plasmid AFS series, BFS series
△LEU2：LEU2(URA3)_TCYC1_TS_pGAL10pGAL1_ERG20_tERG20
The specific construction process of the plasmids AFS and BFS comprises the following steps: the amino acid sequences of the alpha-farnesene synthase and the beta-farnesene synthase from different sources can be queried in GeneBank (Accession numbers see table below), the nucleotide coding each enzyme is optimized according to the codon preference of Saccharomyces cerevisiae, and the optimized sequences are synthesized by gold Style company. Wherein, the nucleotide sequence for coding the pear source alpha-farnesene synthase is shown in SEQ ID NO.12, the nucleotide sequence for coding the tea tree source alpha-farnesene synthase is shown in SEQ ID NO.13, and the nucleotide sequence for coding the chamomile source beta-farnesene synthase is shown in SEQ ID NO.14. The gene was amplified by PCR to have BsaI cleavage sites, and then assembled with the universal plasmid pZY900 by Goden gate assembly to obtain an expression vector. The sources of farnesene synthase in plasmids pAFS1-6 are pear, cherry, populus trichocarpa, castor, tea tree and apple in sequence; the farnesene synthase in the plasmids pBFS1-8 is derived from flos Matricariae Chamomillae, sunflower, flos Chrysanthemi Indici, herba et flos Pyrethri Cinerariifolii, herba Cynara, lettuce, herba Artemisiae Annuae, and Fusarium graminearum.
The sequences of the primers used for constructing the above plasmids are shown in the following table:
EXAMPLE 4 construction of farnesene producing Strain
The plasmids constructed in example 3 were transferred into strain JCR27, and the screening plates were Sc-ura (synthetic yeast nitrogen source YNB 6.7g/L, glucose 20g/L, uracil-deficient mixed amino acid powder 1.3g/L,2% agar powder).
These strains were cultivated by shake flask fermentation, in particular as follows: from the transformation plate, the monoclonal was picked into PA flasks containing 5mL of seed medium formulated as: YNB (6.7 g/L), yeast powder (10 g/L), glucose (20 g/L), and uracil-deficient mixed amino acid powder (1.3 g/L). Seed shaking culture at 30deg.C overnight, transferring, and covering 20% of organic phase (n-dodecane or isopropyl myristate, isopropyl palmitate, white oil (liquid paraffin), methyl oleate or rapeseed oil) with original OD 600 =0.1, shaking and fermenting at 30deg.C, wherein the fermentation medium comprises: peptone (20 g/L), yeast powder (10 g/L), glucose (10 g/L), galactose (10 g/L). And (3) after fermentation, collecting samples, and detecting the product composition by using an organic phase GCMS. Alpha-farnesene synthase from which products can be detected includes apple (Md-aFS), cherry (Pyr-aFS), pear (Pyc-aFS), tea tree (Cas-aFS), populus tomentosa (Pot-aFS) sources, beta-farnesene synthase from sweet wormwood (Aa-bFS), chamomile (Mac-bFS), wild chrysanthemum (Chi-aFS), fusarium graminearum (FgJ 03939) sources. The yield results are shown in FIG. 7.
To our knowledge, this is the first time that pear, tea tree derived α -farnesene synthase was expressed in microorganisms and the product was successfully detected. The product was also successfully detected by expressing wild chrysanthemum-derived beta-farnesene synthase in the microorganism for the first time. The activity of the pear-derived alpha-farnesene synthase and the chamomile-derived beta-farnesene synthase is higher, wherein the tea-tree-derived alpha-farnesene synthase which is not subjected to heterologous expression in the previous research also has better performance.
EXAMPLE 5 construction of a library of farnesene synthase mutations
The method is that random mutation is adopted, i.e. primers are designed, a low-fidelity DNA polymerase is used for amplifying a farnesene synthetic gene, the concentration of magnesium ions (2 mM-12 mM) is regulated, a key gene is amplified by PCR, a DNA fragment containing mutation is obtained, the DNA fragment and an expression vector pZY900 (after BsaI cutting) are transferred into a strain JCR27 through a lithium acetate transformation method, a strain containing mutant is obtained through yeast assembly, and Sc-ura screening plates are coated.
For random mutation of pear-derived alpha-farnesene synthase, primers are pAFS-mutation-F and pAFS1-mutation-R; for random mutation of beta-farnesene synthase from chamomile, the primers were pBFS-mutation-F and pBFS1-mutation-R; the primers for random mutation of tea tree-derived alpha-farnesene synthase are pAFS-mutation-F and pAFS5-mutation-R.
The sequences of the primers are shown in the following table:
example 6 high throughput screening and characterization of strains containing farnesene synthase mutants
Positive clones were selected on the plates in example 5 to a high throughput seed culture plate containing Sc-ura deficient medium, strains containing wild-type farnesene synthase were selected as controls, cultured at 999rpm at 30℃and transferred to a fermentation culture plate containing fermentation medium when the strains grew to logarithmic growth phase, a covering agent was added to reduce volatilization of the products, high throughput product extraction was performed by adding an extractant (n-dodecane, isopropyl myristate, liquid paraffin, white oil, isopropyl palmitate) until the yield of the strains reached the highest, the products obtained by the extraction were transferred to new plates, a broad-spectrum developer (vanillin-concentrated sulfuric acid developer) was added, and the maximum absorbance was detected by an enzyme-labeling instrument (65 ℃ for 20 min). Selecting a strain with absorbance higher than that of a wild strain, extracting a yeast plasmid, transferring the escherichia coli, amplifying the extracted plasmid, and determining a positive mutation site by first-generation sequencing. The results show that for a beta-farnesene synthase from chamomile sources, amino acid mutations with increased enzymatic activity include the following fold increases in yield of F11S, M35T, T319S, I T, I460V, K R, S Y below.
The obtained beneficial mutations were combined variously, the obtained plasmids were transformed into strain JCR27, and further evaluated by 96-well plate fermentation, and finally the combination with the maximum fold increase was obtained, wherein pBFS45 (F11S, M35T, T319S, I434T, I460V) and pBFS46 (F11S, M35T, T319S, I434T, I460V, K59R, S Y) had the maximum increase in yield, the optimal mutant yield of beta-farnesene synthase was increased by nearly 2 times compared with the wild type, and the yields reached 430mg/L and 470mg/L, respectively. The yields of the two mutants are similar, and the two mutants are selected for subsequent construction.
The procedure for constructing plasmid pBFS45 is as follows. Mac-bFS-I460V was obtained by amplification from pBFS plasmid (i.e., using pBFS plasmid as a template, the same applies hereinafter) using primer pBFS-1F/pBFS-2R, mac-bFS-I434T was obtained by amplification from pBFS18 plasmid using primer pBFS-3F/R, mac-bFS-T319S was obtained by amplification from pBFS plasmid using primer pBFS-4F/R, mac-bFS-M35T was obtained by amplification from pBFS plasmid using primer pBFS-5F/R, and Mac-bFS-F11S was obtained by amplification from pBFS9 plasmid using primer pBFS 45-6F/R. These fragments were then ligated together by overlap extension PCR and cloned in pZY900 by Golden Gate assembly to give plasmid pBFS45.
The primer sequences used to construct plasmid pBFS45 are shown in the following table:
The procedure for constructing plasmid pBFS, 46, is as follows. Mac-bFS-A1341G was obtained by amplification from pBFS plasmid (i.e., using pBFS plasmid as a template, the same applies hereinafter) using primer pBFS-1F/pBFS-1R, mac-bFS-S204Y was obtained by amplification from pBFS plasmid using primer pBFS-2F/R, mac-bFS-K59R was obtained by amplification from pBFS plasmid using primer pBFS-3F/R, and Mac-bFS-K59R was obtained by amplification from pBFS plasmid using primer pBFS-4F/pBFS 45-6R. These fragments were then ligated together by overlap extension PCR and cloned in pZY900 by Golden Gate assembly to give plasmid pBFS.
The primer sequences used to construct plasmid pBFS and 46 are shown in the following table:
the same mutation method performs mutation screening on the alpha-farnesene synthase from pear sources and tea trees, and as a result, the improved mutant results obtained from the pear sources are as follows.
The mutant yield of tea tree source is 1.52 times of wild type, and shake flask yield reaches 257.85mg/L.
EXAMPLE 7 construction of plasmids required for farnesene high producing Strain
(1) Realizes the high yield of alpha-farnesene, constructs plasmids pAFS1-P2, pAFS1-P3, pAFS1-P4, pAFS1-P5, pAFS1-P6 and pAFS1-P7, and the construction schematic diagram of the plasmids is shown in FIG. 8.
The pear-derived alpha-farnesene synthase gene was optimized according to the Saccharomyces cerevisiae codon (SEQ ID NO. 12) and synthesized by gold SpA. The obtained plasmid was used as a template for Pyc-afs gene amplification.
Ura3 left arm, tCYC1, pGAL10-pGAL1, tPGK1, ura3 right arm was obtained from CEN.PK2-1D (CEN.PK2-1D yeast genomic DNA, hereinafter the same) by amplification with primers pAFS1-P2-1F/R, pAFS1-P2-3F/R, pAFS1-P2-5F/R, pAFS1-P2-7F/R, pAFS 1-P2-8F/R. His3 marker was obtained by amplification from pRS423 plasmid with primer pAFS1-P2-2F/R, tHMG1 was obtained by amplification from S288C (S288C yeast genomic DNA, the same applies hereinafter) with primer pAFS-P2-4F/R, pyc-aFS was obtained by amplification from primer pAFS1-P2-6F/R, plasmid backbone was obtained by amplification from pRS426 plasmid with primer pAFS-P2-9F/R, and then these fragments were assembled by the method of DNA assemble to obtain pAFS-P2.
A Ura3 left arm, tCYC1, pGAL10-pGAL1, tPGK1, ura3 right arm was amplified from CEN.PK2-1D using primers pAFS1-P2-1F/R, pAFS1-P2-3F/pAFS1-P3-3R, pAFS1-P3-5F/pAFS1-P2-5R, pAFS1-P2-7F/R, pAFS1-P2-8F/R, his3 marker was amplified from pRS423 using primers pAFS1-P2-2F/R, pyc-aFS was amplified from pRS426 using primers pAFS1-P2-9F/R, and then these fragments were assembled to yield pAFS-P3.
Ura3 left arm, tCYC1, pGAL10-pGAL1, tPGK1, ura3 right arm was obtained from CEN.PK2-1D amplification with primer pAFS-P2-1F/R, pAFS1-P2-3F/pAFS1-P4-3R, pAFS1-P4-5F/pAFS1-P2-5R, pAFS1-P2-7F/R, pAFS 1-P2-8F/R. His3 marker was amplified from pRS423 with primer pAFS-P2-2F/R, pyc-aFS was amplified with primer pAFS1-P4-4F/R, pAFS1-P2-6F/R, plasmid backbone was amplified from pRS426 with primer pAFS1-P2-9F/R, and these fragments were assembled to pAFS-P4.
YPRCDELTA15 left arm, tCYC1, pGAL10-pGAL1, tPGK1, YPRCDELTA15 right arm was obtained from CEN.PK2-1D amplification with primers pAFS-P5-1F/R, pAFS1-P5-3F/R, pAFS1-P5-5F/R, pAFS1-P5-7F/R, pAFS 1-P5-8F/R. Trp1 marker was amplified from pRS424 using primers pAFS1-P5-2F/R, pyc-aFS was amplified using primers pAFS1-P5-4F/R, pAFS1-P5-6F/R, plasmid backbone was amplified from pRS426 using primers pAFS1-P5-9F/R, and these fragments were assembled to pAFS-P5.
YPRCDELTA15 left arm, pGAL10-pGAL1, tPGK1, YPRCDELTA right arm was amplified from CEN.PK2-1D using primers pAFS-P5-1F/R, pAFS1-P6-5F/pAFS1-P5-5R, pAFS1-P5-7F/R, pAFS 1-P5-8F/R. Trp1 marker was amplified from pRS424 using primer pAFS-P5-2F/pAFS 1-P6-2R, tGPM1 from S288C using primer pAFS1-P6-3F/R, pyc-aFS from pRS426 using primer pAFS1-P5-6F/R, plasmid backbone was obtained using primer pAFS-P5-9F/R, and these fragments were assembled to obtain pAFS-P6.
YPRCDELTA15 left arm, tCYC1, pGAL10-pGAL1, tPGK1, YPRCDELTA right arm were obtained from CEN.PK2-1D amplification with primers pAFS-1-P5-1F/R, pAFS1-P5-3F/pAFS1-P7-3R, pAFS1-P7-5F/pAFS1-P5-5R, pAFS1-P5-7F/R, pAFS 1-P5-8F/R. Trp1 marker was amplified from pRS424 using primers pAFS-P5-2F/R, tHMG1 was amplified from S288C using primers pAFS1-P7-4F/R, pyc-aFS was amplified from pRS426 using primers pAFS1-P5-6F/R, plasmid backbone was amplified from pRS426 using primers pAFS1-P5-9F/R, and then these fragments were assembled to pAFS-P7.
The primer sequences used to construct the above plasmids are shown in the following table:
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pAFS38-P2, pAFS-P3, pAFS-P4, pAFS-P5, pAFS38-P6, pAFS-P7 are constructed in the same manner as pAFS1-P2, pAFS1-P3, pAFS1-P4, pAFS1-P5, pAFS1-P6, pAFS1-P7 except that the template P pAFS1 in the case of construction pAFS1-P2, pAFS1-P3, pAFS1-P4, pAFS1-P5, pAFS1-P6, pAFS1-P7 is replaced with pAFS38.
(2) Realizes the high yield of beta-farnesene, constructs plasmids pBFS, pBFS45-P2, pBFS45-P3, pBFS45-P4, pBFS45-P5, pBFS45-P6 and pBFS45-P7. Plasmids pBFS, pBFS46-P2, pBFS46-P3, pBFS46-P4, pBFS46-P5, pBFS46-P6, pBFS-P7 were constructed.
Construction of plasmid pBFS45 is described in example 6.
Ura3 left arm, tCYC1, pGAL10-pGAL1, tPGK1, ura3 right arm was obtained from CEN.PK2-1D amplification with primer pAFS-P2-1F/R, pAFS1-P2-3F/R, pAFS1-P2-5F/pBFS45-P2-5R, pBFS-P2-7F/pAFS 1-P2-7R, pAFS 1-P2-8F/R. His3 marker was amplified from pRS423 with primer pAFS-P2-2F/R, tHMG1 was amplified from S288C with primer pAFS-P2-4F/R, mac-bFS was amplified from pBFS with primer pBFS-P2-6F/pBFS-P2-6R (F11S, M35T, T319S, I434T, I460V), plasmid backbone was amplified from pRS426 with primer pAFS-P2-9F/R, and these fragments were assembled to give pBFS-45-P2.
Ura3 left arm, tCYC1, pGAL10-pGAL1, tPGK1, ura3 right arm were amplified from CEN.PK2-1D using primer pAFS1-P2-1F/R、pAFS1-P2-3F/pAFS1-P3-3R、pAFS1-P3-5F/pBFS45-P3-5R、pBFS45-P3-7F/pAFS1-P2-7R、pAFS1-P2-8F/R, his3 marker was amplified from pRS423 using primer pAFS-P2-2F/R, mac-bFS (F11S, M35T, T319S, I434T, I460V) was amplified from pRS426 using primer pBFS-P3-6F/R, plasmid backbone was obtained from pRS426 using primer pAFS-P2-9F/R, and these fragments were assembled to obtain pBFS-P3.
Ura3 left arm, tCYC1, pGAL10-pGAL1, tPGK1, ura3 right arm were obtained from CEN.PK2-1D amplification with primer pAFS1-P2-1F/R、pAFS1-P2-3F/pBFS45-P4-3R、pBFS45-P4-5F/pBFS45-P4-5R、pBFS45-P4-7F/pAFS1-P2-7R、pAFS1-P2-8F/R. His3 marker was amplified from pRS423 with primer pAFS1-P2-2F/R, mac-bFS (F11S, M35T, T319S, I434T, I460V) was amplified from pBFS with primer pBFS-P4-4F/R, pBFS-P4-6F/R, plasmid backbone was amplified from pRS426 with primer pAFS-P2-9F/R, and these fragments were assembled to pBFS-P4.
YPRCDELTA15 left arm, tCYC1, pGAL10-pGAL1, tPGK1, YPRCDELTA right arm were obtained from CEN.PK2-1D amplification with primer pAFS1-P5-1F/R、pAFS1-P5-3F/pAFS1-P7-3R、pAFS1-P7-5F/pBFS45-P5-5R、pBFS45-P5-7F/pAFS1-P5-7R、pAFS1-P5-8F/R. Trp1 marker was amplified from pRS424 using primers pAFS-P5-2F/R, tHMG1 was amplified from S288C using primers pAFS1-P7-4F/pAFS1-P7-4R, mac-bFS was amplified from pBFS45 using primers pBFS-P5-6F/R (F11S, M35T, T319S, I434T, I460V), plasmid backbone was amplified from pRS426 using primers pAFS-P5-9F/R, and these fragments were assembled to pBFS45-P5.
YPRCDELTA was amplified from CEN.PK2-1D using primers pAFS-P5-1F/R, pAFS1-P6-5F/pBFS45-P6-5R, pBFS-P6-7F/pAFS 1-P5-7R, pAFS1-P5-8F/R to yield YPRCDELTA left arm, pGAL10-pGAL1, tPGK1, YPRCDELTA15 right arm. Trp1 marker was amplified from pRS424 using primers pAFS-P5-2F/pAFS 1-P6-2R, tGPM1 from S288C using primers pAFS1-P6-3F/R, mac-bFS (F11S, M35T, T319S, I434T, I460V) from pBFS45 using primers pBFS45-P6-6F/R, plasmid backbone was amplified from pRS426 using primers pAFS1-P5-9F/R, and then these fragments assembled to pBFS-P6.
YPRCDELTA15 left arm, tCYC1, pGAL10-pGAL1, tPGK1, YPRCDELTA right arm were obtained from CEN.PK2-1D amplification with primers pAFS-1-P5-1F/R, pAFS1-P5-3F/pBFS45-P7-3R, pBFS45-P7-5F/R, pBFS45-P7-7F/pAFS1-P5-7R, pAFS 1-P5-8F/R. Trp1 marker was amplified from pRS424 using primers pAFS1-P5-2F/R, mac-bFS (F11S, M35T, T319S, I434T, I460V) was amplified from pBFS using primers pBFS45-P7-4F/R, pBFS-P7-6F/R, plasmid backbone was amplified from pRS426 using primers pAFS1-P5-9F/R, and these fragments were assembled to pBFS-45-P7.
Construction of plasmid pBFS to 46 is described in example 6.
PBFS46-P2, pBFS-P3, pBFS-P4, pBFS-P5, pBFS-46-P6, pBFS-P7 were constructed in the same manner as pBFS-45-P2, pBFS-45-P3, pBFS-45-P4, pBFS-45-P5, pBFS-45-P6, pBFS-45-P7 except that the template pBFS45 in the case of constructing pBFS-P2, pBFS-45-P3, pBFS-P4, pBFS-45-P5, pBFS-P6, pBFS-45-P7 was replaced with pBFS46.
Wherein the nucleotide sequence of Mac-bFS containing F11S, M T, T319S, I434T, I460V mutation optimized according to Saccharomyces cerevisiae codon is shown in SEQ ID NO. 16; the nucleotide sequence of Mac-bFS containing the F11S, M T, T319S, I434T, I460V, K59R, S204Y mutation, optimized according to the Saccharomyces cerevisiae codon, is shown in SEQ ID NO. 17.
The primer sequences used to construct the above plasmids are shown in the following table:
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EXAMPLE 8 construction of beta-farnesene high producing Strain
The plasmid pBFS45 was linearized with Mssi, the fragment carrying the gene of interest was recovered, transformed into Saccharomyces cerevisiae JCR27, integrated on the chromosome according to homologous recombination, with the integration site LEU2 and the selection marker leucine, and strain JVA122 was constructed. Linearized plasmids pBFS-P2, pBFS-P3 and pBFS-P4, and fragments with target genes are recovered and respectively transformed into saccharomyces cerevisiae JVA122, and are integrated on a chromosome according to homologous recombination, the integration site is URA3, and the screening markers are histidine, so that strains JVA124, JVA125 and JVA129 are constructed. Linearization pBFS-P5, recovery of the fragment with the target gene, transformation to Saccharomyces cerevisiae JVA124, JVA125, JVA129, integration into chromosome according to homologous recombination, integration site YPRCDELTA, screening marker tryptophan, construction of strain JVA127, JVA130, JVA134. Linearizing pBFS to P6, recovering fragments with target genes, respectively converting the fragments into Saccharomyces cerevisiae JVA124, JVA125 and JVA129, integrating the fragments into chromosomes according to homologous recombination, wherein the integration site is YPRCDELTA, screening the fragments with the markers of tryptophan, and constructing strains JVA128, JVA131 and JVA135. Linearizing pBFS to P7, recovering fragments with target genes, respectively converting the fragments into Saccharomyces cerevisiae JVA124, JVA125 and JVA129, integrating the fragments into chromosomes according to homologous recombination, wherein the integration site is YPRCDELTA, screening the fragments with the markers of tryptophan, and constructing strains JVA127, JVA138 and JVA139. After shaking flask fermentation, the strains can show good performance, the beta-farnesene yield can reach higher level, and the yield range is 200mg/L-800mg/L (figure 9). The strain JVA139 with the highest yield was selected for GAL80 gene knockout (construction of knockout cassette pZY 521: the use of galactose in the fermentation process was eliminated by using the genome of yeast 30000B as a template, using primer 5201-F/5211-R amplification to obtain the left homology arm of GAL80, pZY900 as a template, using primer 5212-F/5212-R amplification to obtain screening marker URA3, and using primer 5213-F/5203-R amplification to obtain the right homology arm of GAL 80).
The primer sequences are shown in the following table:
| Primer(s) | Sequence (5 '-3') |
| 5201-F | caatggtctaggtagtggcattcg |
| 5211-R | CGACTCACTATAGGGCGAATTGGGTACgacgggagtggaaagaacgg |
| 5212-F | tcccgttctttccactcccgtcGTACCCAATTCGCCCTATAGTGAG |
| 5212-R | gccaagcacagggcaagatgcttTCACAGCTTGTCTGTAAGCGGA |
| 5213-F | GCATCCGCTTACAGACAAGCTGTGAaagcatcttgccctgtgctt |
| 5203-R | gagaccaccaagaatacagaagctattat |
As can be seen from FIG. 9, there was a clear difference in yield between strains containing different numbers of genes (Thmg A and farnesene synthase), 5 beta-farnesene synthases were contained on the basis of the wild type strain CEN.PK2-1D strain, additional MVA pathway genes (ERG 10, ERG13, THMG1, ERG12, ERG8, MVD1, IDI 1) and one additional ERG20, one additional Thmg1 strain with the highest beta-farnesene yield, among strains not knocked out gal 80. Therefore, the adjustment of the ratio of Thmg to farnesene synthase has great significance for high yield.
Similarly, linearized plasmids pBFS, pBFS-P4, pBFS-P7 were sequentially transferred into strain JCR27 to obtain strain JVA139-pBFS, and GAL80 gene knockout construction was performed to obtain strain JVA140-pBFS46.
EXAMPLE 9 construction of alpha-farnesene high producing Strain
The construction of the alpha-farnesene high-producing strain can be performed by conventional homologous recombination in a manner similar to the construction of the beta-farnesene high-producing strain in example 8, and the corresponding strain JSA126-JSA138 can be obtained by the construction. The strain with high yield of beta-farnesene can be used as a chassis, crispr-cas9 gene editing technology is adopted, and alpha-farnesene synthase is used for replacing beta-farnesene synthase at one time, so that the strain with high yield of alpha-farnesene synthase is obtained. Construction of plasmid pAFS-gRNA: the plasmid pKlURA is used as a template, PD pAFS-gRNA is obtained by PCR amplification by using primers pAFS-gRNA-F and pAFS-gRNA-R, pCAS9 is used as a carrier framework, plasmid pAFS-gRNA is constructed by using a Golden Gate method, and pAFS-gRNA is obtained after enzyme digestion verification and correct sequencing.
The primer sequences are shown in the following table:
| Primer(s) | Sequence (5 '-3') |
| pAFS-gRNA-F | aaaggtctcaGATCGCTGGCATCAACAATGGGAAGTTTTAGAGCTAGAAATAGCA |
| pAFS-gRNA-R | AAAGGTCTCAAAACTCTAGACTTTTTCGATGATGTAGTTTCT |
Linearizing plasmids pAFS, pAFS1-P4 and pAFS1-P5, recovering fragments with target genes, transferring the fragments into a strain JVA139 together with pAFS-gRNA corresponding to beta-farnesene high-yield strain construction plasmids (pAFS 1 vs pBFS45, pAFS1-P4 vs pBFS45-P4 and pAFS-P5 vs pBFS-P7), marking 5-FOA plates after single cloning is carried out, and constructing to obtain a strain JSA 132Crispr.
The strain JSA132Crispr is subjected to shake flask fermentation, and the yield of alpha-farnesene can reach a higher level, and the yield is 473+/-35 mg/L. The GAL80 gene was knocked out (construction of a knock-out cassette pZY521, construction of a strain JSA145 with primers 5201-F/5211-R amplification to obtain a left homology arm of GAL80 using a genome of yeast 30000B as a template, amplification of pZY900 as a template with primers 5212-F/5212-R to obtain a screening marker URA3, amplification of primers 5213-F/5203-R to obtain a right homology arm of GAL 80;) by eliminating the use of galactose during fermentation. Strain JSA145 contains 5 α -farnesene synthases (Pyc-aFS) on the basis of the wild-type strain CEN.PK2-1D, contains additional MVA pathway genes (ERG 10, ERG13, THMG1, ERG12, ERG8, MVD1, IDI 1) and an additional ERG20, an additional Thmg1, and knocks out GAL80 gene.
Similarly, linearized plasmids pAFS, pAFS-P4 and pAFS-P5, a fragment carrying the desired gene was recovered, and transformed into strain JVA139 together with pAFS-gRNA corresponding to the beta-farnesene high-producing strain construction plasmids (pAFS 38 vs pBFS45, pAFS38-P4 vs pBFS45-P4, pAFS-P5 vs pBFS 45-P7), and after a single clone was obtained, 5-FOA plates were streaked, and strain JSA132-pAFS38-Crispr was constructed, and shake flask yield was 580mg/L. The strain JSA 145-pAFS-Crispr is obtained by knocking out GAL80 gene, and the shake flask yield reaches 598mg/L. The strain JSA 145-pAFS-Crispr contains 5 alpha-farnesene synthase (Pyc-aFS (G252E)) genes (SEQ ID NO. 18) on the basis of the wild-type strain CEN.PK2-1D, contains additional MVA pathway genes (ERG 10, ERG13, THMG1, ERG12, ERG8, MVD1, IDI 1) and an additional ERG20, an additional Thmg1, and knocks out GAL80 gene.
EXAMPLE 10 fermenter fermentation of farnesene high producing Strain
The fermentation medium described in reference (van Hoek,P.;de Hulster,E.;van Di jken,J.P.;Pronk,J.T.Fermentative capacity in high-cell-density fed-batch cultures of baker's yeast.Biotechnol.Bioeng.2000,68,517-523.) was fed-batch fermented with respect to the strains constructed, the α -farnesene-synthesizing strain, the β -farnesene strain (strains JSA145, JSA 145-pAFS-Crispr, JVA 140-pBFS), and a covering agent, which may be n-dodecane, isopropyl myristate, isopropyl palmitate, liquid paraffin, white oil, was added during the fermentation to effect in situ extraction. The dissolved oxygen is controlled to be more than 20 percent in the fermentation process, the pH value is 5, the glucose concentration is 1-2g/L, and the ethanol concentration is 5-10g/L. Finally, on a fermentation tank, the yield of the alpha-farnesene of the strain JSA145 reaches 28g/L, and the yield of the alpha-farnesene of the strain JSA 145-pAFS-Crispr reaches 36g/L. The yield of the beta-farnesene of the strain JVA140 reaches 50g/L, and the yield of the beta-farnesene of the strain JVA140-pBFS46 reaches 55g/L.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (9)
1. A farnesene synthase characterized by: the farnesene synthase is an alpha-farnesene synthase mutant,
The alpha-farnesene synthase is based on the pear-derived alpha-farnesene synthase with the amino acid sequence shown in SEQ ID NO.3, and contains one or two of the following mutations: G252E, D10G, A T.
2. Use of a farnesene synthase according to claim 1 for the production of farnesene or for the construction of a farnesene-producing recombinant microorganism.
3. A recombinant microorganism producing farnesene, characterized by: the recombinant microorganism is a recombinant microorganism for producing alpha-farnesene;
The copy number of the gene in the recombinant microorganism producing the alpha-farnesene is ERG10: ERG13: tHMG1: ERG12: ERG8: MVD1: IDI1: ERG20: aFS = 2:2: x:2:2:2:2:2: x, X is an integer of 1 or more,
Wherein ERG10 is a gene encoding acetoacetyl-CoA thiolase, ERG13 is a gene encoding HMG-CoA synthase, tHMG1 is a gene encoding HMG-CoA reductase, ERG12 is a gene encoding mevalonate kinase, ERG8 is a gene encoding mevalonate-5-phosphate kinase, MVD1 is a gene encoding mevalonate pyrophosphate decarboxylase, IDI1 is a gene encoding isoprene pyrophosphate isomerase, ERG20 is a gene encoding farnesene pyrophosphate synthase, aFS is a gene encoding alpha-farnesene synthase which is the alpha-farnesene synthase mutant of claim 1.
4. A farnesene-producing recombinant microorganism according to claim 3, wherein: the amino acid sequence of aFS coded alpha-farnesene synthase is shown as SEQ ID NO. 4.
5. A farnesene-producing recombinant microorganism according to claim 3, wherein: the recombinant microorganism takes Saccharomyces cerevisiae as a host.
6. A farnesene-producing recombinant microorganism according to claim 3, wherein:
the recombinant microorganism for producing alpha-farnesene contains 5 coding genes of alpha-farnesene synthase shown in SEQ ID NO.4 on the basis of Saccharomyces cerevisiae CEN.PK2-1D, and contains additional MVA pathway genes ERG10, ERG13, THMG1, ERG12, ERG8, MVD1 and IDI1, and an additional ERG20 gene and an additional tHMG1 gene.
7. The farnesene-producing recombinant microorganism according to any one of claims 3-6, wherein: the recombinant microorganism knocks out GAL80 gene.
8. The method for constructing a farnesene-producing recombinant microorganism according to any one of claims 3 to 7, wherein: the construction method comprises cloning genes ERG10, ERG13, tHMG1, ERG12, ERG8, MVD1, IDI1, ERG20, pyc-aFS onto a plurality of plasmids, transferring the linearized fragments containing the target genes into a host, and screening to obtain recombinant microorganisms expressing the genes.
9. Use of a farnesene-producing recombinant microorganism according to any one of claims 3-7 for the production of farnesene.
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