Disclosure of Invention
In view of the above, the technical problem to be solved by the present invention is to provide a mutant of beta-farnesene synthase, and an altered strain and application thereof.
The invention mutates the farnesene synthetase (AaFS) from sweet wormwood obtained by gene synthesis, obtains the genetic engineering strain containing the mutant by gene integration, and can effectively improve the content of beta-farnesene on the basis of ensuring 96h fermentation time of the obtained genetic engineering strain.
The amino acid sequence of the farnesene synthase from sweet wormwood is shown as SEQ ID NO. 1, and the mutant comprises at least one of the following mutation sites:
T434I、D248N、K197L、K197T、K197Y、K197A、K197M、K197H、K197F、K197G、K197R、K197E、K197Q、K197S、K197W、K197I、K197P、K197V、K197C、K197D、K197N、T526A、T526E、T526F、T526L、T526H、T526G、T526P、T526S、T526Y、T526K、T526V、T526M、T526N、T526D、T526C、T526I、T526W、T526Q、T526R、S11F、P28Q、H33N、K58E、K70R、I100V、H116Y、V150I、F180Y、D185G、R227K、D248N、S260N、R407K、I423V、T434I、A442G、S455T、S475T、E544D Or K561I.
In some embodiments, the mutation site in the mutant is at least one of K197A, T526I, H116Y, R227K, D N or T434I.
As a practical case, the mutant comprises mutation sites K197A and T526N, K197A and T526I, K197M and T526N, K197M and T526I, D248N and R227K, D248N and H116Y, D248N and T434I, R227K and H116Y, R227K and T434I, or H116Y and T434I.
In some embodiments, the mutation site comprises at least one of K197A, T526I, T I or R227K.
According to the invention, through screening mutants, the GL0116: aaFST434I mutant strain and the GL0116: aaFSR227K-T434I mutant strain are obtained, the beta-farnesene content is 2.32 g/L and 2.23 g/L respectively, and the beta-farnesene content is improved by 58.3% and 52.2% respectively compared with the non-mutant strain. Since AaFS is a specific synthetase for β -farnesene, only β -farnesene was detected by the gas phase method, and no α -farnesene was produced. Preferably, the mutation sites in the mutant include R227K and T434I.
Furthermore, the invention also provides nucleic acids encoding the mutants.
The invention optimizes the code of the coding nucleic acid according to the characteristics of saccharomycetes. Among many optimization schemes, the AaFSR227K-T434I mutant coded by the nucleic acid sequence shown in SEQ ID No.2 has the most excellent expression effect and beta-farnesene yield.
Preferably, the nucleic acid has a nucleotide sequence as shown in SEQ ID NO. 2;
Or a sequence having 1 or more nucleotides substituted, deleted, added and/or substituted on the basis of the nucleic acid sequence shown in SEQ ID NO. 2;
or a sequence having 80% or more identity to the nucleic acid sequence shown in SEQ ID NO. 2.
The identity is 80% or more, including 80% or more, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, 99.5% or more, 99.6% or more, 99.7% or more, 99.8% or more, or 99.9% or more.
Further, the invention also provides an expression cassette comprising a promoter and a nucleic acid as described above.
In some embodiments, the promoter is a eukaryotic promoter or a prokaryotic promoter, as the invention is not limited in this regard. For example, the promoter is TEF1 promoter、GAP promoter、GAL1 promoter、GAL10 promoter、ADH1 promoter、PGK1 promoter、TEF1 promoter、CYC1 promoter、HIS3 promoter、URA3 promoter、LEU2 promoter、MET25 promoter. in the present invention, and replicons, terminators, and/or enhancers may also be included in the expression cassette. For example, the replicon is 2. Mu. Ori, pBR322 ori, or F1 ori, and the enhancer is selected from UASGAL, UASG, HSE (a thermal element), CUP1-UAS, UASINO, UASCTR, UASSTE, UASPH05, UASTYR1, or UASRNR. For example, the terminator is selected from ADH1 terminator、CYC1 terminator、CYC1 terminator、PGK1 terminator、TDH3 terminator、CDC20T terminator、HRR25T terminator、FOB1T terminator、HDA1T terminator、DEP1T terminator、PDA1T terminator、SLD2T terminator.
The invention also provides a plasmid vector comprising a nucleic acid as described above or an expression cassette as described above.
In the invention, the plasmid vector is a cloning vector or an expression vector. In the present invention, the plasmid vector is used for storage, amplification of the nucleic acid or the expression cassette or for expression of the mutant, which is not limited in the present invention. In some embodiments, the vector is a plasmid vector, including but not limited to PY26、YEp13、YEp24、YEp351、YEp352、YEp353、YEp354、YEp355、YEp356、YEp356r、YEp357、YEp357r、YEp358、YEp363、YEp364、YEp365、YEp366、YEp366r、YEp367、YEp367r、YEp368.
Further, the present invention provides a transformant transformed or transfected with a plasmid vector as described above, or having integrated in its genome a nucleic acid as described above or an expression cassette as described above.
In the present invention, the host is used for amplification or storage of the nucleic acid fragment, expression cassette or plasmid vector as described above, and may be used for expression of the mutant as described above, or for preparation of β -farnesene, which is not limited thereto. For example, the transformant is a eukaryotic host including, but not limited to, yeast, insect cells, kidney epithelial cells, or a prokaryotic host including, but not limited to, E.coli.
As a practical case, the host used as the transformant for amplification or storage is E.coli, and the host used as the transformant for the production of beta-farnesene is yeast. For example, the hosts are Hansenula polymorpha (Hansenula polymorpha), pichia pastoris, debaryomyces hansenii (Hansenula polymorpha), kluyveromyces lactis (Kluyveromyces lactis), yarrowia lipolytica (yarrowia lipolytica), saccharomyces cerevisiae var, diasticus (saccharifying yeast), schizosaccharomyces pombe (Schizosaccharomyces pombe), rhodotorula glutinis (rhodosporidium). In some embodiments, the host of the transformant is Saccharomyces cerevisiae.
The invention screens and optimizes the host, and the result shows that different chassis bacteria have obvious influence on the yield of beta-farnesene. Preferably, the host of the transformant of the present invention has a genotype of CEN.PK113-7D:: ACS1-6:: ERG 10::: ERG13:: tHMG1:: HMG2:: ERG 12::: ERG8:: ERG19: IDI1:: ERG20, or its genotype is CEN.PK113-7D::ACS1-6::ERG10::ERG13::tHMG1::HMG2::ERG12::ERG8::ERG19::IDI1::ERG20::ERG8-20::Idi1::tHMG::adh::Acs::Acl::Zwf::Gnd::Pdr5::Pdr10::Osh3::VhbΔDpp1ΔLpp1ΔGpd1::Fs:: ΔBts1.
Further, the invention also provides a preparation method of the beta-farnesene, which comprises the step of culturing the transformant to obtain a product containing the beta-farnesene.
The method provided by the invention can shorten the fermentation time, the composition of the culture medium is simple, the shake flask fermentation time is shortened from the original 120h to 96h by adopting a double-phase fermentation method, and the yield of beta-farnesene can reach 31.67 g/L.
The culture medium comprises water and (NH4)2SO4 3 g/L、KH2PO4 3.5 g/L、MgSO4·7H2O 6.2 g/L、 glucose 80 g/L, galactose 9.9 g/L, trace elements 10 mL/L, vitamin 120 mu L/mL, cuSO4·5H2 O0.6 mL/L and 10% (V/V) n-dodecane.
The invention carries out mutation on farnesene synthetase (AaFS) from sweet wormwood to obtain GL0116: aaFST434I mutant strain and GL0116: aaFS R227K-T434I mutant strain, and after further screening and optimizing chassis bacteria, the yield of beta-farnesene reaches 31.67 g/L.
Detailed Description
The invention provides a beta-farnesene synthase mutant, an altered strain and application, and a person skilled in the art can properly improve the technological parameters by referring to the content of the text. It is expressly noted that all such similar substitutions and modifications will be apparent to those skilled in the art, and are deemed to be included in the present invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those skilled in the relevant art that the invention can be practiced and practiced with modification and alteration and combination of the methods and applications herein without departing from the spirit and scope of the invention.
Unless defined otherwise herein, scientific and technical terms used in connection with the present invention shall have the meaning as understood by one of ordinary skill in the art.
Furthermore, unless otherwise indicated herein, terms in the singular herein shall include the plural and terms in the plural shall include the singular. More specifically, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise.
The terms "comprising," "including," and "having" are used interchangeably and are intended to mean that the elements of the subject matter described may be present in addition to the elements listed. It should also be understood that the use of "including," comprising, "and" having "descriptions herein also provides an" consisting of.
The term "and/or" as used herein includes the meaning of "and", "or" and "all or any other combination of the elements linked by the term of interest".
The term "at least one" means one or more, and "a plurality" means two or more. "at least one of" or the like means any combination of these items, including any combination of single item(s) or plural items(s).
The invention mutates farnesene synthetase (AaFS) from sweet wormwood, and screens mutants with good phenotype by using two mutation strategies, wherein one strategy is to predict proper mutation sites by combining enzyme and substrate molecules and the other strategy is to select corresponding sites of directional mutation by carrying out homologous comparison on amino acid sequences corresponding to the farnesene synthetase and according to conservative sequence comparison. Then, designing the corresponding primer of the mutation site obtained by two strategies, and constructing a plasmid vector containing a strong promoter and farnesene synthetase by using a PCR in-vitro amplification technology and a molecular cloning technology. And integrating different plasmid vectors obtained after sequencing into GL0116 strain, and quantitatively detecting the yield of beta-farnesene through shake flask fermentation. In addition, the invention also provides a biphase fermentation method of the saccharomyces cerevisiae engineering strain.
The test materials adopted by the invention are all common commercial products and can be purchased in the market.
4.1 Culture medium and related solution
LB medium, namely Tryptone 10 g/L, yeast Extract 5 g/L and NaCl 10 g/L, and 2% (M/V) agar powder is added separately into the LB solid medium, and the mixture is sterilized at 121 ℃ and 20min after uniform mixing.
YPD minimal medium comprising Tryptone 20 g/L, yeast Extract 10 g/L, glucose 20 g/L, and YPD solid medium comprising 2% (M/V) agar powder, mixing, and sterilizing at 115deg.C and 20min.
Shake flask fermentation medium ：(NH4)2SO4 3 g/L、KH2PO4 3.5 g/L、MgSO4·7H2O 6.2 g/L、 glucose 80 g/L, galactose 9.9 g/L, trace elements 10 mL/L, cuSO4·5H2 O0.6 mL/L, mixing, adjusting pH to 6.3-6.5, packaging into shake flask 500 mL (without baffle, with liquid loading amount of 100 mL), and adding 10% (V/V) n-dodecane respectively. 20 min were sterilized at 115 ℃. In addition, when inoculating the fermentation medium, it was necessary to add 120. Mu.L/mL of vitamin stock solution (previously prepared) separately to the shake flasks, and to control the initial OD=6 of each flask to an inoculum size of 10% (v/v).
Vitamin mother liquor comprises 0.05 g/L of biotin, B5 1g/L, B3 1g/L, inositol 25 g/L, B1 1g/L, B6 1g/L and p-aminobenzoic acid 0.2 g/L. Biotin is dissolved in ddH2 O of 750 mL, then 10mL of 5M NaOH is added, the rest of the components are added, after complete dissolution, the pH is adjusted to 6.3-6.5 by 1M HCl, finally the volume is fixed to 1L, the mixture is preserved at 4 ℃, and filtration sterilization is needed in the subsequent use.
Firstly preparing EDTA solution from microelement mother liquor ：0.5M EDTA 80 ml/L,ZnSO4·7H2O 5.75 g/L,MnCl2·4H2O 0.32 g/L,CoCl2·6H2O 0.47 g/L,Na2MoO4·2H2O 0.48 g/L,CaCl2·2H2O 2.9 g/L,FeSO4·7H2O 2.8 g/L., adding sodium hydroxide into the EDTA solution to adjust the pH value so as to improve the solubility, then sequentially adding the rest medicines, adjusting the pH value to about 4.0 by using the sodium hydroxide after the residual medicines are completely dissolved, finally fixing the volume to 1L, and preserving the mixture in a dark place at the temperature of 4 ℃.
4.2 Using antibiotic species and concentrations
The types and concentrations of antibiotics used are shown in Table 1:
TABLE 1 types and concentrations of antibiotics used in this experiment
4.3 Related sequences
SEQ ID NO.1 amino acid sequence of Artemisia annua-derived farnesene synthetase:
MSTLPISSVSSSSSTSPLVVDDKDSTKPDVIRHTMNFNASIWGDQFLTYDEPEDLVMKKQLVEELKEEVKKELITIKGSNEPMQHVKLIELIDAVQRLGIAYHFEEEIEEALQHIHVTYGEQWVDKENLQSISLWFRLLRQQGFNVSSGVFKDFMDEKGKFKESLCNDAQGILALYEAAFMRVEDETILDNALEFTKVHLDIIAKDPSCDTSLRTQIHQALKQPLRRRLARIEALHYMPIYQQETSHDEVLLKLAKLDFSVLQSMHKKELSHICKWWKDLDLQNKLPYVRDRVVEGYFWILSIYYEPQHARTRMFLMKTCMWLVVLDDTFDNYGTYEELEIFTQAVERWSISCLDMLPEYMKLIYQELLNLHVEMEESLEKEGKTYQIHYVKEMAKELVRNYLVEARWLKEGYMPTLEEYMSISMVTGTYGLMTARSYVGRADIVTEDTFKWVSSYPPIVKASCVIIRLMDDIVSHKEEQERGHVASSIECYSKESGASEEEACEYISRKVEDAWKVINRESLRPTAVPFPLLMPAINLARMCEVLYSVNDGFTHAEGDMKSYMKSFFVHPMVV*
SEQ ID NO. 2, aaFSR227K-T434I mutant amino acid sequence
MSTLPISSVSSSSSTSPLVVDDKDSTKPDVIRHTMNFNASIWGDQFLTYDEPEDLVMKKQLVEELKEEVKKELITIKGSNEPMQHVKLIELIDAVQRLGIAYHFEEEIEEALQHIHVTYGEQWVDKENLQSISLWFRLLRQQGFNVSSGVFKDFMDEKGKFKESLCNDAQGILALYEAAFMRVEDETILDNALEFTKVHLDIIAKDPSCDTSLRTQIHQALKQPLRKRLARIEALHYMPIYQQETSHDEVLLKLAKLDFSVLQSMHKKELSHICKWWKDLDLQNKLPYVRDRVVEGYFWILSIYYEPQHARTRMFLMKTCMWLVVLDDTFDNYGTYEELEIFTQAVERWSISCLDMLPEYMKLIYQELLNLHVEMEESLEKEGKTYQIHYVKEMAKELVRNYLVEARWLKEGYMPTLEEYMSISMVTGTYGLMIARSYVGRADIVTEDTFKWVSSYPPIVKASCVIIRLMDDIVSHKEEQERGHVASSIECYSKESGASEEEACEYISRKVEDAWKVINRESLRPTAVPFPLLMPAINLARMCEVLYSVNDGFTHAEGDMKSYMKSFFVHPMVV*
SEQ ID NO. 3, aaFSR227K-T434I mutant nucleic acid sequence
atgtcaaccttgcctatttcttctgtctcatcctcttcatctacctctccattggtcgtagacgataaggactctactaaaccagacgtcatcaggcacaccatgaatttcaacgcttctatatggggagaccagtttttaacttacgacgaacctgaggatttggtcatgaaaaaacagttggtcgaagaattgaaggaggaggtcaagaaggagttgattacaatcaagggatcaaacgaacctatgcagcacgttaagttgatcgaattaatagatgctgtccaaagattgggtatagcctaccacttcgaggaggaaatcgaggaggctttacaacatatacacgtcacatacggtgaacagtgggtcgataaagagaatttgcagtctatctcattgtggttcaggttgttaaggcaacaaggttttaatgtttcatctggagttttcaaggactttatggacgagaaaggtaaattcaaggagtctttgtgcaacgatgctcagggtattttagcattgtatgaggccgcatttatgagggttgaagacgagactatcttagataacgcattggagttcaccaaggtccacttagacattattgctaaagacccatcatgtgacacctctttgagaactcaaatacaccaggcattaaagcaacctttgaggaagaggttggctagaatcgaagcattacactatatgccaatatatcagcaggaaacctcacacgacgaagttttgttaaagttagcaaaattggacttctctgtcttgcagtcaatgcataagaaggagttgtctcatatctgcaagtggtggaaggatttagatttacaaaataagttgccatacgtcagagatagggttgtagagggatacttctggatcttgtctatatactatgagcctcagcacgccagaaccagaatgttcttaatgaagacctgcatgtggttagtagtattagacgacaccttcgacaattatggaacatacgaggaattggagatctttactcaagccgttgagagatggtctatttcttgcttggacatgttgccagagtatatgaagttgatctaccaggagttactgaacttgcacgtcgaaatggaggaatctttggagaaagagggaaagacataccagattcactatgtcaaggaaatggccaaagagttggtaaggaactatttggttgaggccagatggttgaaagagggttatatgcctaccttggaggagtacatgtcaatctcaatggttactggtacctatggtttgatgatagccagatcatacgtcggaagagctgatatcgtaacagaggataccttcaagtgggtttcttcataccctcctatcgtcaaggcctcttgcgtcataatcaggttgatggatgacattgtttctcataaggaggaacaggagaggggtcacgtagcctcatcaatagagtgctattcaaaagagtctggtgcatcagaggaagaggcatgtgaatacatctctagaaaagtagaggatgcctggaaggtcattaacagggagtcattgagacctactgctgtaccttttcctttgttgatgcctgctatcaacttggcaaggatgtgcgaagttttgtattcagtaaacgatggtttcactcacgccgaaggtgatatgaaatcatatatgaaatcttttttcgtacatcctatggtagtataa
SEQ ID NO. 4, aaFST434I mutant amino acid sequence
MSTLPISSVSSSSSTSPLVVDDKDSTKPDVIRHTMNFNASIWGDQFLTYDEPEDLVMKKQLVEELKEEVKKELITIKGSNEPMQHVKLIELIDAVQRLGIAYHFEEEIEEALQHIHVTYGEQWVDKENLQSISLWFRLLRQQGFNVSSGVFKDFMDEKGKFKESLCNDAQGILALYEAAFMRVEDETILDNALEFTKVHLDIIAKDPSCDTSLRTQIHQALKQPLRRRLARIEALHYMPIYQQETSHDEVLLKLAKLDFSVLQSMHKKELSHICKWWKDLDLQNKLPYVRDRVVEGYFWILSIYYEPQHARTRMFLMKTCMWLVVLDDTFDNYGTYEELEIFTQAVERWSISCLDMLPEYMKLIYQELLNLHVEMEESLEKEGKTYQIHYVKEMAKELVRNYLVEARWLKEGYMPTLEEYMSISMVTGTYGLMIARSYVGRADIVTEDTFKWVSSYPPIVKASCVIIRLMDDIVSHKEEQERGHVASSIECYSKESGASEEEACEYISRKVEDAWKVINRESLRPTAVPFPLLMPAINLARMCEVLYSVNDGFTHAEGDMKSYMKSFFVHPMVV*
SEQ ID NO. 5, aaFST434I mutant nucleic acid sequence
atgtcaaccttgcctatttcttctgtctcatcctcttcatctacctctccattggtcgtagacgataaggactctactaaaccagacgtcatcaggcacaccatgaatttcaacgcttctatatggggagaccagtttttaacttacgacgaacctgaggatttggtcatgaaaaaacagttggtcgaagaattgaaggaggaggtcaagaaggagttgattacaatcaagggatcaaacgaacctatgcagcacgttaagttgatcgaattaatagatgctgtccaaagattgggtatagcctaccacttcgaggaggaaatcgaggaggctttacaacatatacacgtcacatacggtgaacagtgggtcgataaagagaatttgcagtctatctcattgtggttcaggttgttaaggcaacaaggttttaatgtttcatctggagttttcaaggactttatggacgagaaaggtaaattcaaggagtctttgtgcaacgatgctcagggtattttagcattgtatgaggccgcatttatgagggttgaagacgagactatcttagataacgcattggagttcaccaaggtccacttagacattattgctaaagacccatcatgtgacacctctttgagaactcaaatacaccaggcattaaagcaacctttgaggagaaggttggctagaatcgaagcattacactatatgccaatatatcagcaggaaacctcacacgacgaagttttgttaaagttagcaaaattggacttctctgtcttgcagtcaatgcataagaaggagttgtctcatatctgcaagtggtggaaggatttagatttacaaaataagttgccatacgtcagagatagggttgtagagggatacttctggatcttgtctatatactatgagcctcagcacgccagaaccagaatgttcttaatgaagacctgcatgtggttagtagtattagacgacaccttcgacaattatggaacatacgaggaattggagatctttactcaagccgttgagagatggtctatttcttgcttggacatgttgccagagtatatgaagttgatctaccaggagttactgaacttgcacgtcgaaatggaggaatctttggagaaagagggaaagacataccagattcactatgtcaaggaaatggccaaagagttggtaaggaactatttggttgaggccagatggttgaaagagggttatatgcctaccttggaggagtacatgtcaatctcaatggttactggtacctatggtttgatgatagccagatcatacgtcggaagagctgatatcgtaacagaggataccttcaagtgggtttcttcataccctcctatcgtcaaggcctcttgcgtcataatcaggttgatggatgacattgtttctcataaggaggaacaggagaggggtcacgtagcctcatcaatagagtgctattcaaaagagtctggtgcatcagaggaagaggcatgtgaatacatctctagaaaagtagaggatgcctggaaggtcattaacagggagtcattgagacctactgctgtaccttttcctttgttgatgcctgctatcaacttggcaaggatgtgcgaagttttgtattcagtaaacgatggtttcactcacgccgaaggtgatatgaaatcatatatgaaatcttttttcgtacatcctatggtagtataa
PY26 plasmid map corresponding gene sequence
gacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagtatgatccaatatcaaaggaaatgatagcattgaaggatgagactaatccaattgaggagtggcagcatatagaacagctaaagggtagtgctgaaggaagcatacgataccccgcatggaatgggataatatcacaggaggtactagactacctttcatcctacataaatagacgcatataagtacgcatttaagcataaacacgcactatgccgttcttctcatgtatatatatatacaggcaacacgcagatataggtgcgacgtgaacagtgagctgtatgtgcgcagctcgcgttgcattttcggaagcgctcgttttcggaaacgctttgaagttcctattccgaagttcctattctctagaaagtataggaacttcagagcgcttttgaaaaccaaaagcgctctgaagacgcactttcaaaaaaccaaaaacgcaccggactgtaacgagctactaaaatattgcgaataccgcttccacaaacattgctcaaaagtatctctttgctatatatctctgtgctatatccctatataacctacccatccacctttcgctccttgaacttgcatctaaactcgacctctacattttttatgtttatctctagtattactctttagacaaaaaaattgtagtaagaactattcatagagtgaatcgaaaacaatacgaaaatgtaaacatttcctatacgtagtatatagagacaaaatagaagaaaccgttcataattttctgaccaatgaagaatcatcaacgctatcactttctgttcacaaagtatgcgcaatccacatcggtatagaatataatcggggatgcctttatcttgaaaaaatgcacccgcagcttcgctagtaatcagtaaacgcgggaagtggagtcaggctttttttatggaagagaaaatagacaccaaagtagccttcttctaaccttaacggacctacagtgcaaaaagttatcaagagactgcattatagagcgcacaaaggagaaaaaaagtaatctaagatgctttgttagaaaaatagcgctctcgggatgcatttttgtagaacaaaaaagaagtatagattctttgttggtaaaatagcgctctcgcgttgcatttctgttctgtaaaaatgcagctcagattctttgtttgaaaaattagcgctctcgcgttgcatttttgttttacaaaaatgaagcacagattcttcgttggtaaaatagcgctttcgcgttgcatttctgttctgtaaaaatgcagctcagattctttgtttgaaaaattagcgctctcgcgttgcatttttgttctacaaaatgaagcacagatgcttcgttcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttacctcactcattaggcaccccaggctttacactttatgcttccggctcctatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctcgctattacgccagctgaattggaaagaggtttagacattggctcttcattgagcttagaacccttttgggcagaaagatatccgtcgaacaacgttctattaggaatggcggataaattatttaaattatatcacactaattttcctcctgtggtagccctaagaacttttggtttgaatctgacgaataagatcggtccagttaagaatatgatcattgacacattaggaggaaatgagaaatgagaggtatgtaaatagaaatagactagctccacttttaagaattatttatgcaattaaatacatgggtgaccaaaagagcgggcggatacacgcgtcaccacaagcagaataaaaggtaaacctgaaattgttttaacataaaatgaaaaatgcttgtttgcaaccctatatagaatcataaaacattcgtgactataaaatgaataaactaaactattctaagaaaatgaaataaatgacaaaaaaacgtgttttttggactagaaggcttaatcaaaagctttatttagaagtgtcaacaacgtatctaccaacgatttgacccttttccatcttttcgtaaatttctggcaaggtagacaagccgacaaccttgattggagacttgaccaaacctctggcgaagaattgttaattaaagatctccgcggtccggagttaactgatcagcggccgcgctagttctagaaaattatattgaattttcaaaaattcttactttttttttggatggacgcaaagaagtttaataatcatattacatggcaataccaccatatacatatccatatctaatcttacttatatgttgtggaaatgtaaagagccccattatcttagcctaaaaaaaccttctctttggaactttcagtaatacgcttaactgctcattgctatattgaagtacggattagaagccgccgagcgggcgacagccctccgacggaagactctcctccgtgcgtcctggtcttcaccggtcgcgttcctgaaacgcagatgtgcctcgcgccgcactgctccgaacaataaagattctacaatactagcttttatggttatgaagaggaaaaattggcagtaacctggccccacaaaccttcaaatgaacgaatcaaattaacaaccataggatgataatgcgattagttttttagccttatttctggggtaattaatcagcgaagcgatgatttttgatctattaacagatatataaatgcaaaagctgcataaccactttaactaatactttcaacattttcggtttgtattacttcttattcaaatgtcataaaagtatcaacaaaaaattgttaatatacctctatactttaacgtcaaggagaaaaaactatacggattctagaactagtggatcccccgggctgcaggaattcgatatcaagcttatcgataccgtcgacctcgaggcgatttaatctctaattattagttaaagttttataagcatttttatgtaacgaaaaataaattggttcatattattactgcactgtcacttaccatggaaagaccagacaagaagttgccgacagtctgttgaattggcctggttaggcttaagtctgggtccgcttctttacaaatttggagaatttctcttaaacgatatgtatattcttttcgttggaaaagatgtcttccaaaaaaaaaaccgatgaattagtggaaccaaggaaaaaaaaagaggtatccttgattaaggaacactgtttaaacagtgtggtttccaaaaccctgaaactgcattagtgtaatagaagactagacacctcgatacaaataatggttactcaattcaaaactgccagcgaattcgactctgcaattgctcaagacaagctagttgtcgtagatttctacgccacttggtgcggtccatgtaaaatgattgctccaatgattgaaaggccggtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaatttcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatagggtaataactgatataattaaattgaagctctaatttgtgagtttagtatacatgcatttacttataatacagtttttcatgagatgcctgcaagcaattcgttctgtatcaggcgcaggagcgtcccgtccgggtcgaccaaagcggccatcgtgcctccccactcctgcagttcgggggcatggatgcgcggatagccgctgctggtttcctggatgccgacggatttgcactgccggtagaactccgcgaggtcgtccagcctcaggcagcagctgaaccaactcgcgaggggatcgagcccggggtgggcgaagaactccagcatgagatccccgcgctggaggatcatccagccggcgtcccggaaaacgattccgaagcccaacctttcatagaaggcggcggtggaatcgaaatctcgtgatggcaggttgggcgtcgcttggtcggtcatttcgaattcgagctcgcccttagattagattgctatgctttctttctaatgagcaagaagtaaaaaaagttgtaatagaacaagaaaaatgaaactgaaacttgagaaattgaagaccgtttattaacttaaatatcaatgggaggtcatcgaaagagaaaaaaatcaaaaaaaaaaattttcaagaaaaagaaacgtgataaaaatttttattgcctttttcgacgaagaaaaagaaacgaggcggtctcttttttcttttccaaacctttagtacgggtaattaacgacaccctagaggaagaaagaggggaaatttagtatgctgtgcttgggtgttttgaagtggtacggcgatgcgcggagtccgagaaaatctggaagagtaaaaaaggagtagaaacattttgaagctatggtgtgtggcatatgcgtatatataccaatctaagtctgtgctccttccttcgttcttccttctgttcggagattaccgaatcaaaaaaatttcaaagaaaccgaaatcaaaaaaaagaataaaaaaaaaatgatgaattgaattgaaaagctgtggtatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcga（SEQ ID No.6）
PY26-AaFS plasmid map corresponding gene sequence
gacgaaagggcctcgtgatacgcctatttttataggttaatgtcatgataataatggtttcttagtatgatccaatatcaaaggaaatgatagcattgaaggatgagactaatccaattgaggagtggcagcatatagaacagctaaagggtagtgctgaaggaagcatacgataccccgcatggaatgggataatatcacaggaggtactagactacctttcatcctacataaatagacgcatataagtacgcatttaagcataaacacgcactatgccgttcttctcatgtatatatatatacaggcaacacgcagatataggtgcgacgtgaacagtgagctgtatgtgcgcagctcgcgttgcattttcggaagcgctcgttttcggaaacgctttgaagttcctattccgaagttcctattctctagaaagtataggaacttcagagcgcttttgaaaaccaaaagcgctctgaagacgcactttcaaaaaaccaaaaacgcaccggactgtaacgagctactaaaatattgcgaataccgcttccacaaacattgctcaaaagtatctctttgctatatatctctgtgctatatccctatataacctacccatccacctttcgctccttgaacttgcatctaaactcgacctctacattttttatgtttatctctagtattactctttagacaaaaaaattgtagtaagaactattcatagagtgaatcgaaaacaatacgaaaatgtaaacatttcctatacgtagtatatagagacaaaatagaagaaaccgttcataattttctgaccaatgaagaatcatcaacgctatcactttctgttcacaaagtatgcgcaatccacatcggtatagaatataatcggggatgcctttatcttgaaaaaatgcacccgcagcttcgctagtaatcagtaaacgcgggaagtggagtcaggctttttttatggaagagaaaatagacaccaaagtagccttcttctaaccttaacggacctacagtgcaaaaagttatcaagagactgcattatagagcgcacaaaggagaaaaaaagtaatctaagatgctttgttagaaaaatagcgctctcgggatgcatttttgtagaacaaaaaagaagtatagattctttgttggtaaaatagcgctctcgcgttgcatttctgttctgtaaaaatgcagctcagattctttgtttgaaaaattagcgctctcgcgttgcatttttgttttacaaaaatgaagcacagattcttcgttggtaaaatagcgctttcgcgttgcatttctgttctgtaaaaatgcagctcagattctttgtttgaaaaattagcgctctcgcgttgcatttttgttctacaaaatgaagcacagatgcttcgttcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttacctcactcattaggcaccccaggctttacactttatgcttccggctcctatgttgtgtggaattgtgagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctcgctattacgccagctgaattggaaagaggtttagacattggctcttcattgagcttagaacccttttgggcagaaagatatccgtcgaacaacgttctattaggaatggcggataaattatttaaattatatcacactaattttcctcctgtggtagccctaagaacttttggtttgaatctgacgaataagatcggtccagttaagaatatgatcattgacacattaggaggaaatgagaaatgagaggtatgtaaatagaaatagactagctccacttttaagaattatttatgcaattaaatacatgggtgaccaaaagagcgggcggatacacgcgtcaccacaagcagaataaaaggtaaacctgaaattgttttaacataaaatgaaaaatgcttgtttgcaaccctatatagaatcataaaacattcgtgactataaaatgaataaactaaactattctaagaaaatgaaataaatgacaaaaaaacgtgttttttggactagaaggcttaatcaaaagctttatttagaagtgtcaacaacgtatctaccaacgatttgacccttttccatcttttcgtaaatttctggcaaggtagacaagccgacaaccttgattggagacttgaccaaacctctggcgaagaattgttaattaaagatctccgcggtccggagttaactgatcagcggccgcgctagttctagaaaattatattgaattttcaaaaattcttactttttttttggatggacgcaaagaagtttaataatcatattacatggcaataccaccatatacatatccatatctaatcttacttatatgttgtggaaatgtaaagagccccattatcttagcctaaaaaaaccttctctttggaactttcagtaatacgcttaactgctcattgctatattgaagtacggattagaagccgccgagcgggcgacagccctccgacggaagactctcctccgtgcgtcctggtcttcaccggtcgcgttcctgaaacgcagatgtgcctcgcgccgcactgctccgaacaataaagattctacaatactagcttttatggttatgaagaggaaaaattggcagtaacctggccccacaaaccttcaaatgaacgaatcaaattaacaaccataggatgataatgcgattagttttttagccttatttctggggtaattaatcagcgaagcgatgatttttgatctattaacagatatataaatgcaaaagctgcataaccactttaactaatactttcaacattttcggtttgtattacttcttattcaaatgtcataaaagtatcaacaaaaaattgttaatatacctctatactttaacgtcaaggagaaaaaactatacggattctagaactagtggatcatgtcaaccttgcctatttcttctgtctcatcctcttcatctacctctccattggtcgtagacgataaggactctactaaaccagacgtcatcaggcacaccatgaatttcaacgcttctatatggggagaccagtttttaacttacgacgaacctgaggatttggtcatgaaaaaacagttggtcgaagaattgaaggaggaggtcaagaaggagttgattacaatcaagggatcaaacgaacctatgcagcacgttaagttgatcgaattaatagatgctgtccaaagattgggtatagcctaccacttcgaggaggaaatcgaggaggctttacaacatatacacgtcacatacggtgaacagtgggtcgataaagagaatttgcagtctatctcattgtggttcaggttgttaaggcaacaaggttttaatgtttcatctggagttttcaaggactttatggacgagaaaggtaaattcaaggagtctttgtgcaacgatgctcagggtattttagcattgtatgaggccgcatttatgagggttgaagacgagactatcttagataacgcattggagttcaccaaggtccacttagacattattgctaaagacccatcatgtgacacctctttgagaactcaaatacaccaggcattaaagcaacctttgaggagaaggttggctagaatcgaagcattacactatatgccaatatatcagcaggaaacctcacacgacgaagttttgttaaagttagcaaaattggacttctctgtcttgcagtcaatgcataagaaggagttgtctcatatctgcaagtggtggaaggatttagatttacaaaataagttgccatacgtcagagatagggttgtagagggatacttctggatcttgtctatatactatgagcctcagcacgccagaaccagaatgttcttaatgaagacctgcatgtggttagtagtattagacgacaccttcgacaattatggaacatacgaggaattggagatctttactcaagccgttgagagatggtctatttcttgcttggacatgttgccagagtatatgaagttgatctaccaggagttactgaacttgcacgtcgaaatggaggaatctttggagaaagagggaaagacataccagattcactatgtcaaggaaatggccaaagagttggtaaggaactatttggttgaggccagatggttgaaagagggttatatgcctaccttggaggagtacatgtcaatctcaatggttactggtacctatggtttgatgaccgccagatcatacgtcggaagagctgatatcgtaacagaggataccttcaagtgggtttcttcataccctcctatcgtcaaggcctcttgcgtcataatcaggttgatggatgacattgtttctcataaggaggaacaggagaggggtcacgtagcctcatcaatagagtgctattcaaaagagtctggtgcatcagaggaagaggcatgtgaatacatctctagaaaagtagaggatgcctggaaggtcattaacagggagtcattgagacctactgctgtaccttttcctttgttgatgcctgctatcaacttggcaaggatgtgcgaagttttgtattcagtaaacgatggtttcactcacgccgaaggtgatatgaaatcatatatgaaatcttttttcgtacatcctatggtagtataaccccgggctgcaggaattcgatatcaagcttatcgataccgtcgacctcgaggcgatttaatctctaattattagttaaagttttataagcatttttatgtaacgaaaaataaattggttcatattattactgcactgtcacttaccatggaaagaccagacaagaagttgccgacagtctgttgaattggcctggttaggcttaagtctgggtccgcttctttacaaatttggagaatttctcttaaacgatatgtatattcttttcgttggaaaagatgtcttccaaaaaaaaaaccgatgaattagtggaaccaaggaaaaaaaaagaggtatccttgattaaggaacactgtttaaacagtgtggtttccaaaaccctgaaactgcattagtgtaatagaagactagacacctcgatacaaataatggttactcaattcaaaactgccagcgaattcgactctgcaattgctcaagacaagctagttgtcgtagatttctacgccacttggtgcggtccatgtaaaatgattgctccaatgattgaaaggccggtacccaattcgccctatagtgagtcgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatggcgcgacgcgccctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaatttcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatagggtaataactgatataattaaattgaagctctaatttgtgagtttagtatacatgcatttacttataatacagtttttcatgagatgcctgcaagcaattcgttctgtatcaggcgcaggagcgtcccgtccgggtcgaccaaagcggccatcgtgcctccccactcctgcagttcgggggcatggatgcgcggatagccgctgctggtttcctggatgccgacggatttgcactgccggtagaactccgcgaggtcgtccagcctcaggcagcagctgaaccaactcgcgaggggatcgagcccggggtgggcgaagaactccagcatgagatccccgcgctggaggatcatccagccggcgtcccggaaaacgattccgaagcccaacctttcatagaaggcggcggtggaatcgaaatctcgtgatggcaggttgggcgtcgcttggtcggtcatttcgaattcgagctcgcccttagattagattgctatgctttctttctaatgagcaagaagtaaaaaaagttgtaatagaacaagaaaaatgaaactgaaacttgagaaattgaagaccgtttattaacttaaatatcaatgggaggtcatcgaaagagaaaaaaatcaaaaaaaaaaattttcaagaaaaagaaacgtgataaaaatttttattgcctttttcgacgaagaaaaagaaacgaggcggtctcttttttcttttccaaacctttagtacgggtaattaacgacaccctagaggaagaaagaggggaaatttagtatgctgtgcttgggtgttttgaagtggtacggcgatgcgcggagtccgagaaaatctggaagagtaaaaaaggagtagaaacattttgaagctatggtgtgtggcatatgcgtatatataccaatctaagtctgtgctccttccttcgttcttccttctgttcggagattaccgaatcaaaaaaatttcaaagaaaccgaaatcaaaaaaaagaataaaaaaaaaatgatgaattgaattgaaaagctgtggtatggtgcactctcagtacaatctgctctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcga（SEQ ID No.7）
4.4 The related strains
Saccharomyces cerevisiae strain (GL 0116) constructed based on Saccharomyces cerevisiae CEN.PK113-7D, genotype CEN.PK113-7D:: ACS1-6:: ERG10:: ERG 13::: tHMG 1::: HMG2:: ERG12:: ERG8:: ERG19:: IDI 1:::: ERG20, designated GL0116.
A strain of Saccharomyces cerevisiae (2739), constructed based on Saccharomyces cerevisiae CEN.PK113-7D, has genotype CEN.PK113-7D::ACS1-6::ERG10::ERG13::tHMG1::HMG2::ERG12::ERG8::ERG19::IDI1::ERG20::ERG8-20::Idi1::tHMG::adh::Acs::Acl::Zwf::Gnd::Pdr5::Pdr10::Osh3::VhbΔDpp1ΔLpp1ΔGpd1::Fs:: ΔBts1, designated 2739.
The application is further illustrated by the following examples. It should be understood that, in various embodiments of the present application, the sequence number of each process described above does not mean that the execution sequence of some or all of the steps may be executed in parallel or executed sequentially, and the execution sequence of each process should be determined by its functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.
EXAMPLE 1 construction of the corresponding vector containing the farnesene synthase (AaFS) from Artemisia annua
Primers were designed to construct plasmid vectors, as shown in Table 2:
TABLE 2 construction of primers required for expression vectors
(1) Linear cyclisation PY26
The PY26 plasmid (original plasmid sequence is shown as the foregoing) is used as a PCR template, PY26-KH-F/R is used as an upstream primer and a downstream primer, a PCR system is configured, as shown in Table 3,
TABLE 3 PCR amplification System
The working procedure was set with a PCR instrument, the reaction procedure was 95℃pre-denaturation for 5 min, 95℃denaturation for 30 s, 60℃annealing for 30 s, 72℃extension (30 s/Kb), repeated denaturation annealing extension for 30-33 cycles, and finally 72℃extension for 5 min and incubation at 16 ℃. And (3) after the PCR is finished, agarose gel electrophoresis is carried out, a gel recovery kit or a liquid PCR recovery kit is used for recovering the fragments after the target fragments are obtained, and then the corresponding concentration of the fragments is detected.
(2) Obtaining the target gene fragment
A PCR system was prepared using a plasmid (original AaFS gene sequence shown above) for synthesizing AaFS genes as a template and AaFS-F/R as the upstream and downstream primers, as shown in Table 3. The subsequent PCR reaction system and recovery method are the same as those in (1).
(3) The fragment is connected with the vector to construct the corresponding expression vector
The fragment recovered above was ligated with the linearized vector fragment using a ligase, and the ligation system is shown in Table 4.
TABLE 4 connection System
Mixing the above systems uniformly, placing at 50deg.C for 15-30 min, and immediately maintaining at 4deg.C. And transferring the reacted mixture into escherichia coli competent DH5 alpha for chemotransformation.
Placing in ice bath for 30min, heat-shocking at 42 deg.C for 45-90 s min, rapidly placing in ice for 2-3 min, adding 800-1000 μl LB culture medium, placing in 37 deg.C, recovering 50-60 min in 200 rpm constant temperature shaker, coating on corresponding resistance plate, and culturing in 37 deg.C constant temperature incubator with inversion.
(4) Obtaining correct clones
And (3) performing colony PCR verification on the culture dish after the night culture, and preparing a PCR system by taking the colony as a corresponding template and PY26-YZ-F/R as an upstream primer and a downstream primer, wherein the reaction system is the same as that in (1) shown in the table 3. And (3) obtaining a transformant with the same size as the target fragment through agarose gel electrophoresis, and carrying out delivery measurement. Single colony can be selected after sequencing and base comparison is successful, and plasmid extraction is carried out.
(5) And (5) plasmid extraction.
EXAMPLE 2 homology modeling and molecular docking of southernwood-derived farnesene synthase
(1) Homology modeling of farnesene synthase
The homologous modeling is carried out through an SWISS-MODEL online website, and the amino acid sequence corresponding to the farnesene synthetase (AaFS) synthesized by the gene is used for comparing the modeling of the existing MODEL, so that the obtained comparison MODEL is E7BTW7.1.A, and the comparison MODEL is shown in figure 1. The homology with the target protein is higher, the homology modeling model is evaluated by a PROCHECK tool, a protein simulation structure with higher matching degree is selected as a reference standard for subsequent analysis, and finally, the protein simulation structure is displayed in a Ramachannan conformational diagram (Ramachannan conformational diagram) form, and is shown in fig. 2.
It can be seen from fig. 2 that the ratio of the optimal regions of the modeled protein structure reaches 92.1% and exceeds the 90% rationality threshold. Therefore, the model has reasonable result, and can carry out the subsequent simulation analysis related to molecular docking and the like.
EXAMPLE 3 site-directed saturation mutagenesis was constructed and fermentation validation was performed
(1) Molecular docking simulation
The experiment adopts Autodock to simulate semi-flexible molecular docking of AaFS protein and ligand FPP small molecule, before molecular docking, dehydration and hydrotreatment are carried out on the tested protein and small molecule, then molecular docking is carried out on farnesene synthetase and FPP compound, and docking results are treated by Pymol and displayed in a visual window.
The predicted box sizes with different sizes are utilized, and after molecular docking simulation, two sites with low binding free energy, namely site 197 and site 526, are respectively obtained, and the hydrogen bond sizes between the specific site amino acid and the peripheral amino acid are shown in fig. 3 and 4.
(2) Plasmid vector for constructing various mutants
Site-directed saturation mutagenesis was subsequently performed on both sites and PCR amplification was performed by designing the corresponding mutation primers (see Table 5 for specific mutation primers) to linearize the plasmid vector already constructed in example 1.
TABLE 5 primers required for site-directed saturation mutagenesis
| Primer name | Primer sequence (5 '. Fwdarw.3') |
| K197G-F | cgcattggagttcaccggcgtccacttagac |
| K197A-F | cgcattggagttcaccgcagtccacttagac |
| K197V-F | cgcattggagttcaccgtcgtccacttagac |
| K197L-F | cgcattggagttcaccctcgtccacttagac |
| K197I-F | cgcattggagttcaccatcgtccacttagac |
| K197P-F | cgcattggagttcaccccagtccacttagac |
| K197F-F | cgcattggagttcaccttcgtccacttagac |
| K197Y-F | cgcattggagttcacctacgtccacttagac |
| K197W-F | cgcattggagttcacctgggtccacttagac |
| K197S-F | cgcattggagttcaccagcgtccacttagac |
| K197T-F | cgcattggagttcaccacggtccacttagac |
| K197C-F | cgcattggagttcacctgcgtccacttagac |
| K197M-F | cgcattggagttcaccatggtccacttagac |
| K197N-F | cgcattggagttcaccaatgtccacttagac |
| K197Q-F | cgcattggagttcacccaggtccacttagac |
| K197D-F | cgcattggagttcaccgacgtccacttagac |
| K197E-F | cgcattggagttcaccgaagtccacttagac |
| K197R-F | cgcattggagttcacccgtgtccacttagac |
| K197H-F | cgcattggagttcacccatgtccacttagac |
| AaFS-197TB-R | ggtgaactccaatgcgttatctaagatagtctcg |
| T526G-F | gagaatctttgagaccaggcgctgttccatttccattg |
| T526A-F | gagaatctttgagaccagcagctgttccatttccattg |
| T526V-F | gagaatctttgagaccagtcgctgttccatttccattg |
| T526L-F | gagaatctttgagaccactcgctgttccatttccattg |
| T526I-F | gagaatctttgagaccaatcgctgttccatttccattg |
| T526P-F | gagaatctttgagaccaccagctgttccatttccattg |
| T526F-F | gagaatctttgagaccattcgctgttccatttccattg |
| T526Y-F | gagaatctttgagaccatacgctgttccatttccattg |
| T526W-F | gagaatctttgagaccatgggctgttccatttccattg |
| T526S-F | gagaatctttgagaccaagcgctgttccatttccattg |
| T526K-F | gagaatctttgagaccaaaggctgttccatttccattg |
| T526C-F | gagaatctttgagaccatgcgctgttccatttccattg |
| T526M-F | gagaatctttgagaccaatggctgttccatttccattg |
| T526N-F | gagaatctttgagaccaaatgctgttccatttccattg |
| T526Q-F | gagaatctttgagaccacaggctgttccatttccattg |
| T526D-F | gagaatctttgagaccagacgctgttccatttccattg |
| T526E-F | gagaatctttgagaccagaagctgttccatttccattg |
| T526R-F | gagaatctttgagaccacgtgctgttccatttccattg |
| T526H-F | gagaatctttgagaccacatgctgttccatttccattg |
| AaFS526TB-R | tggtctcaaagattctctattaataactttccaagc |
| 526TB-YZ-F | gatggtctatctcttgcttggatatgttg |
| 526TB-YZ-R | ggatatgtatatggtggtattgccatg |
| 197TB-YZ-F | ctccattggtcgtagacgataaggactc |
| 197TB-YZ-R | ctggttctggcgtgctgagg |
The primers in the table are used for PCR amplification, the corresponding reaction system is shown in the table 3, the reaction program is shown in the example 1, the target fragment is obtained after agarose gel electrophoresis, digestion and recovery are carried out, then the target fragment is transferred into escherichia coli transformation competent DH5 alpha, the transformation is carried out by the transformation method in the example 1, and finally successful transformants are obtained, and plasmid vectors corresponding to various mutants are constructed.
(3) Integration of the mutant plasmid vector into Saccharomyces cerevisiae
The plasmid vectors of the mutants obtained above were integrated in Saccharomyces cerevisiae strain (GL 0116) and the AaFS mutants were integrated using yeast electrotransformation, the specific yeast electrotransformation procedure was as follows:
① Taking 1mL test tube overnight to culture first-stage seeds, and transferring 50 mL shake flasks for second-stage seed culture the next day;
② Measuring OD600 to 0.8-1.0 about 4-5 hours, and carrying out ice bath on the secondary seeds for 10-15 min hours;
③ Centrifuging 6000 g at 4 ℃ for 6 min, discarding the supernatant, and collecting thalli;
④ Washing the cells twice with 35-50 mL sterile water, centrifuging at 6000g at 4deg.C for 4-6 min, discarding supernatant, and collecting the cells;
⑤ Repeating ④ steps, washing with sterile water twice, re-suspending with 35-50 mL 10% glycerol solution, centrifuging at 6000: 6000 g at 4deg.C for 4-6: 6 min, discarding supernatant, and collecting thallus;
⑥ Adding a certain amount of 10% glycerol, slowly and uniformly mixing with a gun head, and sub-packaging into 50 mu L of each tube for standby;
⑦ Adding 500 mu g of RNA plasmid or 500 mu g of gRNA and fragments thereof to about 3-7 mu L, uniformly mixing with a gun head, adding into an electric rotating cup which is dried and precooled in advance, and placing on ice;
⑧ Setting voltage clicking (breakdown time is 4.9-5.5 ms, and empty contrast is 6.0 ms) of 2.5 kv, placing an electric rotating cup in an electric rotating instrument for electric rotating, immediately adding 700-900 mu L YPD culture based on the electric rotating cup after the electric rotating is finished, transferring to a2 mL centrifuge tube, taking a proper amount of bacterial liquid coating plate after recovery culture at 30 ℃ for 1 h, and then placing a culture dish in a 30 ℃ constant temperature incubator for culture.
After colonies were grown on the dishes, colony PCR was performed. The single colony of yeast is selected and placed in 20-30 mu L of cell lysate, and after being evenly mixed, the single colony of yeast is placed in a PCR instrument, and is subjected to 98 ℃ pyrolysis for 1h to be used as a template, and then the Fly Mix reagent is used for PCR, and the corresponding PCR system is shown in Table 6.
TABLE 6 Saccharomyces cerevisiae colony PCR reaction System
The working procedure was set with a PCR instrument, the reaction procedure was 95℃pre-denaturation for 2 min, 95℃denaturation for 20 s, 58℃annealing for 20 s, 72℃extension (10 s/Kb), repeated denaturation annealing extension for 30-35 cycles, and finally 72℃extension for 5min and incubation at 16 ℃. And (3) after the PCR is finished, agarose gel electrophoresis is carried out, and the corresponding transformants with consistent target length are obtained through comparison, namely, the integration is successful. Then, the correct transformant is selected for culture, and the subsequent fermentation experiment is carried out.
(4) Performing shake flask fermentation experiment
The obtained site-directed mutant fermentation strain is subjected to shake flask site-directed fermentation, the strain to be fermented is subjected to single colony selection, transferred into a test tube and cultured at 30 ℃ overnight. Then, the bacterial liquid in the test tube was transferred to YPD shake flask (250 mL, liquid loading amount 50 mL) and cultured at 30℃for 1-2 days to obtain seed liquid. The seed solution is then transferred to a shake flask fermentation medium (the specific fermentation medium formulation is shown in FIG. 4.1) and cultured at 30℃for 4-5 days for detection. According to the fermentation procedure, the 197 th and 526 th sites of farnesene synthase (AaFS) are subjected to saturation mutation and fermented by shaking.
(5) Quantitative detection of beta-farnesene yield
The experiment carries out gas phase detection by setting an internal standard method, and the corresponding detection steps are as follows:
① Pretreatment of fermentation samples, namely shaking up fermentation liquor, taking 30mL into a centrifuge tube, and centrifuging 8 min under the condition of 6000-8000 rpm/min. After centrifugation, 100. Mu.L of the upper oil phase was carefully taken together with 900. Mu.L of the liquid A tetradecane internal standard, at which time the sample was diluted ten times, filtered through a 0.22. Mu.M nylon membrane and loaded into a liner tube for gas phase detection.
② The gas phase detection comprises the steps of confirming that the chromatographic column is HP-5, connecting the gas chromatographic column to an instrument according to the requirement of short inlet and outlet, and then operating according to a series of steps of starting up, opening a hydrogen-air integrated machine (ensuring normal water level), opening a gas cylinder (reverse opening and closing), and ensuring normal both of needle washing liquid and waste liquid.
③ And (3) sample detection, namely after the corresponding method is called out, starting the instrument, loading samples after the base line is stable, and finally entering a shutdown program, and ending the experiment.
④ And (3) data processing, namely after the sample is detected, determining the peak area (the peak at the time belongs to beta-farnesene) about 13 min in a processing method of an area normalization method, and then determining the beta-farnesene yield of the corresponding sample by drawing a standard curve.
Quantitative detection of beta-farnesene yield was performed on the above-described fermentation strain according to the above-described method steps, and specific yield results are shown in the following FIGS. 5 to 6:
As can be seen from FIGS. 5 and 6, after the saturation mutation of the 197 th site, GL0116: aaFSK197A is increased by 40.6% compared with the yield of the control group, and after the saturation mutation of the 526 th site, GL0116: aaFST526N is increased by 27.8% compared with the yield of the control group.
EXAMPLE 4 conservative sequence alignment of farnesene synthase (AaFS)
(1) Alignment of sequences
Protein similarity search is carried out from NCBI online websites by using a BLAST function, aaFS target protein sequences related in the patent are uploaded, several farnesene synthetases with high homology are selected according to the homology, and drawing is carried out in weblog websites, so that a sequence comparison diagram of the farnesene synthetase (AaFS) is obtained, and the sequence comparison diagram is shown in fig. 7.
(2) Selection of subsequent directed mutation sites
In combination with the amino acid sequence of the FS original sequence, the following mutation sites 11, 28, 58, 70, 100, 116, 150, 180, 185, 227, 248, 260, 407, 423, 434, 442, 455, 475, 544 and 561 can be selected for mutation experiments, and the mutation sites are S11F、P28Q、H33N、K58E、K70R、I100V、H116Y、V150I、F180Y、D185G、R227K、D248N、S260N、R407K、I423V、T434I、A442G、S455T、S475T、E544D、K561I.
(3) Plasmid vector for constructing various mutants
Site-directed saturation mutagenesis was subsequently performed on both sites and PCR amplification was performed by designing the corresponding mutation primers (see Table 7 for specific mutation primers) to linearize the plasmid vector already constructed in example 1.
TABLE 7 primers for construction of directed mutant plasmid vectors
| Primer name | Sequence (5 '. Fwdarw.3') |
| S11F-F | ctatttcttctgtctcattctcttcatc |
| S11F-R | gagaggtagatgaagagaatgagacagaag |
| P28Q-F | cgataaggactctactaaacaggacgtc |
| P28Q-R | ctgatgacgtcctgtttagtagagtcc |
| H33N-F | caggacgtcatcaggaacaccatg |
| H33N-R | gcgttgaaattcatggtgttcctg |
| K58E-F | ctgaggatttggtcatggagaaacagttg |
| K58E-R | cgaccaactgtttctccatgacc |
| K70R-F | gaaggaggaggtccggaagg |
| K70R-R | gtaatcaactccttccggacctcc |
| I100V-F | ctgtccaaagattgggtgtagcctac |
| I100V-R | gaagtggtaggctacacccaatc |
| H116Y-F | gaggctttacaacatatatacgtcacatacgg |
| H116Y-R | cactgttcaccgtatgtgacgtatatatg |
| V150I-F | caaggttttaatgtttcatctggaatattcaaggac |
| V150I-R | ctcgtccataaagtccttgaatattccagatg |
| F180Y-F | gcattgtatgaggccgcatacatg |
| F180Y-R | cttcaaccctcatgtatgcggc |
| D185G-F | atgagggttgaaggagagactatcttag |
| D185G-R | caatgcgttatctaagatagtctctccttc |
| R227K-F | cattaaagcaacctttgaggaagaggttg |
| R227K-R | cgattctagccaacctcttcctcaaag |
| D248N-F | gcaggaaacctcacacaacgaag |
| D248N-R | ctaactttaacaaaacttcgttgtgtgagg |
| S260N-F | gcaaaattggacttcaacgtcttgc |
| S260N-R | cttatgcattgactgcaagacgttgaag |
| R407K-F | ctatttggttgaggccaagtggttg |
| R407K-R | catataaccctctttcaaccacttggcc |
| I427V-F | ccttggaggagtacatgtcagtctcaatg |
| I427V-R | ggtaccagtaaccattgagactgacatg |
| T434I-F | ggtacctatggtttgatgatagccagatc |
| T434I-R | cttccgacgtatgatctggctatcatc |
| A442G-F | cagatcatacgtcggaagaggagatatc |
| A442G-R | ggtatcctctgttacgatatctcctcttcc |
| S455T-F | gataccttcaagtgggtttctacataccctc |
| S455T-R | ccttgacgataggagggtatgtagaaacc |
| S475T-F | ggttgatggatgacattgttactcataagg |
| S475T-R | ctcctgttcctccttatgagtaacaatgtc |
| E544D-F | caacttggcaaggatgtgcgacgttttg |
| E544D-R | gtttactgaatacaaaacgtcgcacatcc |
| K561I-F | ctcacgccgaaggtgatatgatatcata |
| K561I-R | ggatgtacgaaaaaagatttcatatatgatatcatatcacc |
| DDFS-YZ-F | ccaaacctctggcgaagaattgt |
| DDFS-YZ-R | ccgcggagatcttatactaccatagg |
The primers in the table are used for PCR amplification, the corresponding reaction system is shown in the table 3, the reaction program is shown in the example 1, the target fragment is obtained after agarose gel electrophoresis, digestion and recovery are carried out, then the target fragment is transferred into escherichia coli transformation competent DH5 alpha, the transformation is carried out by the transformation method in the example 1, and finally successful transformants are obtained, and plasmid vectors corresponding to various mutants are constructed.
(4) Integration of the mutant plasmid vector into Saccharomyces cerevisiae
Plasmid vectors of the above-obtained mutants were integrated in Saccharomyces cerevisiae strain (GL 0116), and the AaFS mutants were integrated using yeast electrotransformation, and the specific yeast electrotransformation process and subsequent electrotransformation verification process were as described in example 3.
(5) Performing shake flask fermentation experiment
The obtained 21 strains with directional mutation are subjected to shake flask fermentation and quantitative detection, and the fermentation method and the detection method are shown in example 3.
The results of the quantitative detection of the beta-farnesene yield after shaking flask fermentation by performing directed mutation in farnesene synthase (AaFS) are shown in FIG. 8.
As can be seen from FIG. 8, after the directed mutation of the farnesene synthase (AaFs), the beta-farnesene content of a part of the strain is greatly improved. Wherein, GL0116 is a AaFSH116Y mutant strain, GL0116 is a AaFSR227K mutant is a 37.3% higher than a control group, GL0116 is a AaFSD248N mutant is a 45.6% higher than a control group, and GL0116 is a AaFST434I mutant (mutant base of farnesene synthase is shown in SEQ ID No. 3) is a 58.3% higher than the control group.
EXAMPLE 5 additive mutagenesis of a mutant effective Strain
(1) Construction of the superimposed mutant plasmid vector
The variants which perform well in example 3 and example 4 were subjected to the addition mutation according to the previous procedure, i.e. K197A-T526N、K197A-T526I、K197M-T526N、K197M-T526I、D248N-R227K、D248N-H116Y、D248N-T434I、R227K-H116Y、R227K-T434I、H116Y-T434I., by PCR amplification by designing the corresponding mutation primers (specific mutation primers are shown in tables 5 and 7), linearizing the plasmid vectors carried by the strains which have been fermented to completion and have good mutation effects in example 3 and example 4.
(4) Integration of the mutant plasmid vector into Saccharomyces cerevisiae
Plasmid vectors of the above-obtained various superposition mutants were integrated in Saccharomyces cerevisiae strain (GL 0116), and the AaFS mutants were integrated using yeast electrotransformation, and the specific yeast electrotransformation process and subsequent electrotransformation verification process were as shown in example 3.
(5) Performing shake flask fermentation experiment
The 10 strains with superimposed mutation are subjected to shake flask fermentation and quantitative detection, the fermentation method and the detection method are shown in example 3, and the content of the beta-farnesene obtained after fermentation and gas phase detection is shown in figure 9.
As can be seen from FIG. 9, after the mutation sites are combined in pairs to carry out the superposition mutation on the farnesene synthase (Fs), some strains after the superposition mutation have the beta-farnesene content which is improved but not high, and in addition, the fermentation effect of partial superposition mutant strains is not increased and reduced. After comparing the yields of the ten mutant strains, GL0116 is found that AaFSR227K-T434I mutant strain (the mutation base sequence of farnesene synthetase is shown as SEQ ID No. 2) has the highest content, reaching 2.23 g/L, and the yield is improved by 52.2% compared with that of a control group.
EXAMPLE 6 experiments on mutant effective strains in genetically engineered strains
(1) Integration of the mutant plasmid vector into 2739 Saccharomyces cerevisiae
The plasmid vectors of the mutants and the superposition mutants obtained in the above manner were integrated in 2739 gene engineering strains, and the AaFS mutants were integrated by using yeast electrotransformation, and the specific yeast electrotransformation process and the subsequent electrotransformation verification process were the same as those shown in example 3.
(2) Performing shake flask fermentation experiment
The 10 strains with superimposed mutation are subjected to shake flask fermentation and quantitative detection, the fermentation method and the detection method are shown in example 3, and the content of the beta-farnesene obtained after fermentation and gas phase detection is shown in figure 10.
As can be seen from FIG. 10, after various mutants of farnesene synthase (AaFS) with good expression are integrated in 2739 strain, the beta-farnesene yield of a part of the mutants corresponding to the strain is improved. After comparison, the content of AaFST434I mutant strain (the mutation base sequence of farnesene synthetase is shown as SEQ ID No. 3) is highest, which reaches 31.67 g/L.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.