WO2016106988A1 - 一种产紫杉二烯的重组真核菌株及利用该重组真核菌株制备紫杉二烯的方法 - Google Patents

一种产紫杉二烯的重组真核菌株及利用该重组真核菌株制备紫杉二烯的方法 Download PDF

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WO2016106988A1
WO2016106988A1 PCT/CN2015/075447 CN2015075447W WO2016106988A1 WO 2016106988 A1 WO2016106988 A1 WO 2016106988A1 CN 2015075447 W CN2015075447 W CN 2015075447W WO 2016106988 A1 WO2016106988 A1 WO 2016106988A1
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expressing
gene
expression cassette
gene expression
synthase
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元英进
翟芳
宋田青
李炳志
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天津大学
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/16—Yeasts; Culture media therefor
    • C12N1/18—Baker's yeast; Brewer's yeast
    • C12N1/185—Saccharomyces isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P5/00—Preparation of hydrocarbons or halogenated hydrocarbons
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/85—Saccharomyces
    • C12R2001/865—Saccharomyces cerevisiae

Definitions

  • the invention belongs to the technical field of bioengineering, and in particular relates to a recombinant eukaryotic strain producing taxadiene and a method for preparing taxadiene by using the recombinant eukaryotic strain.
  • Natural compounds are mainly obtained from the following ways: (1) extraction from plants; (2) synthesis by chemical synthesis; (3) synthesis by synthetic biology.
  • Synthetic biology is a new discipline for the production of large-scale and stable natural compounds. It is an important alternative for direct extraction from plants and chemical synthesis. This method is mainly related to the synthesis of natural compounds by genetic engineering. The gene is introduced into some commonly used model cell cells such as yeast, Escherichia coli or plants, in which a pathway for producing a natural compound is reconstituted to express a natural compound.
  • yeast yeast
  • Escherichia coli a pathway for producing a natural compound is reconstituted to express a natural compound.
  • Paclitaxel is an effective anticancer drug, and the traditional method of obtaining is: extraction from yew.
  • the raw resources of the raw yew are scarce, and the paclitaxel content of the yew is only about one in ten thousand, so the cost of extracting paclitaxel directly from the yew is too high to meet the demand.
  • researchers have also proposed that plant cell culture can be used to obtain paclitaxel.
  • plant cell culture can be used to obtain paclitaxel.
  • the efficiency of obtaining paclitaxel is not high and it is not suitable for large-scale production.
  • the step of synthesizing paclitaxel by chemical synthesis is complicated, and the yield per step is very low, resulting in high cost and low yield, which limits the application of the method.
  • the successful application of synthetic biology techniques in the biosynthesis of natural compounds opens up new avenues for the preparation of paclitaxel.
  • Taxadiene is one of the key precursors in paclitaxel biosynthesis pathway, and taxadiene is a class The diterpene compound is formed by enzymatic reaction of 4 molecules of isoprene-based pyrophosphate (IPP) by polymerization, cyclization or the like. Studies have found that taxane-derived taxane-derived taxadiene synthase can catalyze the synthesis of taxadiene from GGPP (geranylgeranyl pyrophosphate), which can be introduced into the cell of the chassis. Synthetic taxadiene.
  • IPP isoprene-based pyrophosphate
  • prokaryotic strains the most important one is the E. coli system.
  • Huang et al. of the Croteau group of Washington State University introduced the taxadiene synthase gene into E. coli by optimizing the endogenous DXP pathway of E. coli.
  • exogenous GGPP synthase gene the biosynthesis of taxadiene in E. coli cells was achieved, and the yield was 1.3 mg/L.
  • an object of the present invention is to provide a taxane-producing recombinant eukaryotic strain and a method for producing taxadiene using the recombinant eukaryotic strain.
  • the taxane-producing recombinant eukaryotic strain provided by the invention has high yield of taxadiene and provides a feasible method for the biosynthesis of taxadiene.
  • the present invention adopts the following technical solutions:
  • the present invention provides a taxane-producing recombinant eukaryotic strain, wherein a gene expression cassette expressing GGPP synthase A, a gene expression cassette expressing taxadiene synthase, and a 3-hydroxyl expression are introduced into the eukaryotic strain.
  • the gene expression cassette expressing GGPP synthase A comprises a promoter, a gene expressing GGPP synthase A, and the promoter of the gene expression cassette expressing GGPP synthase A is TDH1;
  • the gene expression cassette for expressing taxadiene synthase comprises a promoter and a gene expressing taxadiene synthase, and the promoter of the gene expression cassette expressing taxadiene synthase is ENO2;
  • the gene expression cassette for expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase comprises a promoter, a gene expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase, and the expression 3-hydroxy-
  • the promoter of the gene expression cassette of 3-methylglutaryl coenzyme A reductase is PDC1;
  • the fusion gene expression cassette of the gene expressing FPP synthase and the gene expressing GGPP synthase B includes a promoter, the fusion gene; and the promoter of the fusion gene expression cassette is GPM1.
  • GGPP is geranylgeranyl pyrophosphate.
  • the gene expressing GGPP synthase A means that the expression product of the gene has the activity of GGPP synthase A, which catalyzes the direct synthesis of GGPP by IPP, and the gene expressing GGPP synthase A can be wild type. It may also be mutated; it may be either an endogenous gene of a eukaryotic strain or a foreign gene of a eukaryotic strain, as long as the expression product retains the activity of the enzyme.
  • the gene expressing GGPP synthase A is derived from S. acidophilus, and is codon-optimized, and has the nucleotide represented by SEQ ID NO: 1. The sequence, labeled as the GGPPSsa gene.
  • the gene expressing taxadiene synthase means that the expression product of the gene has taxadiene synthase activity, which catalyzes GGPP synthesis of taxadiene, the gene expressing taxadiene synthase It may be either wild type or mutated as long as its expression product retains the activity of the enzyme.
  • the gene expressing taxadiene synthase is derived from the yew.
  • the taxadiene synthase-expressing gene is derived from the yew yew and is a truncated gene of taxadiene synthase of Taxus chinensis.
  • the taxadiene synthase-expressing gene is a truncated taxane synthase gene of Taxus chinensis, and is codon optimized having the SEQ ID
  • the nucleotide sequence shown by NO: 2 is labeled as the tTS gene.
  • the gene expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase means that the expression product of the gene has the activity of 3-hydroxy-3-methylglutaryl coenzyme A reductase, Catalysis
  • the intermediate product HMG-CoA (hydroxyl-methyl-glutaryl-CoA) in the mevalonate (MVA) pathway produces mevalonate.
  • the gene expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase can be either wild type or mutated; it can be either an endogenous gene of a eukaryotic strain or a foreign gene of a eukaryotic strain. As long as its expression product retains the activity of the enzyme.
  • the gene expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase is derived from yeast.
  • the gene expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase is derived from Saccharomyces cerevisiae YNL280C having the nucleotide sequence set forth in SEQ ID NO:3, Marked as the tHMGR gene.
  • the FPP is farnesyl pyrophosphate.
  • the gene expressing FPP synthase means that the expression product of the gene has FPP synthase activity, which catalyzes the formation of GPP by IPP and DMAPP, and then condenses with IPP to form a common precursor FPP of a semiquinone substance.
  • the gene expressing the FPP synthase may be wild type or mutated; it may be an endogenous gene of a eukaryotic strain or a foreign gene of a eukaryotic strain, as long as the expression product retains the activity of the enzyme. .
  • the gene expressing the FPP synthase in the recombinant eukaryotic strain provided by the present invention, is derived from yeast. In still other embodiments of the present invention, in the recombinant eukaryotic strain provided by the present invention, the gene expressing FPP synthase is derived from Saccharomyces cerevisiae YNL280C, which is labeled as ERG20 gene.
  • the gene expressing GGPP synthase B means that the expression product of the gene has GGPP synthase B activity, which catalyzes the synthesis of GGPP by FPP, and the gene expressing GGPP synthase B can be either wild type or It may be mutated; it may be either an endogenous gene of a eukaryotic strain or a foreign gene of a eukaryotic strain, as long as the expression product retains the activity of the enzyme.
  • the gene expressing GGPP synthase B is derived from yeast.
  • the gene expressing GGPP synthase B is derived from yeast. In still other embodiments of the present invention, in the recombinant eukaryotic strain provided by the present invention, the gene expressing GGPP synthase B is derived from Saccharomyces cerevisiae YNL280C, which is labeled as BTS1 gene.
  • the gene expressing FPP synthase and the gene expressing GGPP synthase B are introduced into the eukaryotic strain in the form of a fusion gene, and the expression is expressed.
  • the expression product of the fusion gene of FPP synthase and the gene expressing GGPP synthase B has both FPP synthase activity and GGPP synthase B activity, which can catalyze the formation of intermediate GPP, FPP by IPP and DMAPP, and finally synthesize. GGPP.
  • a gene expressing FPP synthase and a fusion gene expressing GGPP synthase B include a gene expressing FPP synthase, a gene expressing GGPP synthase, and a nucleotide sequence linker.
  • the nucleotide sequence linker functions to link a gene expressing FPP synthase and a gene expressing GGPP synthase.
  • the FPP synthase-expressing gene and the GGPP synthase B-expressing gene are derived from a gene that expresses GGPP synthase B, a linker, and a gene that expresses FPP synthase from 5'-3' Connected in turn.
  • the nucleotide sequence corresponding to the linker in the fusion gene is: 5'-GGTGGTGGTTCT-3', and the fusion gene is labeled as a BE gene having the sequence shown in SEQ ID NO: Nucleotide sequence.
  • the promoter TDH1 is derived from Saccharomyces cerevisiae strain S288c having the nucleotide sequence shown as SEQ ID NO: 8.
  • the promoter ENO2 is derived from Saccharomyces cerevisiae strain S288c having the nucleotide sequence shown as SEQ ID NO: 11.
  • the promoter PDC1 is derived from Saccharomyces cerevisiae strain S288c having the nucleotide sequence shown as SEQ ID NO: 10.
  • the promoter GPM1 is derived from Saccharomyces cerevisiae strain S288c having the nucleotide sequence shown as SEQ ID NO: 9.
  • a promoter is a sequence of deoxyribonucleic acid (DNA) that enables transcription of a gene.
  • the promoter can be recognized by RNA polymerase and transcription begins.
  • RNA ribonucleic acid
  • a promoter can interact with a transcription factor that determines the onset of transcription, controlling the initiation time and degree of expression of gene expression (transcription).
  • RNA ribonucleic acid
  • the introduced gene is generally introduced into the recipient cell in the form of a gene expression cassette.
  • the present invention unexpectedly discovered that when a gene expression cassette expressing GGPP synthase A and a gene expressing taxadiene synthase are expressed Expression of a cassette, a gene expression cassette expressing a 3-hydroxy-3-methylglutaryl coenzyme A reductase, a gene expressing a FPP synthase, and a gene expressing a gene of GGPP synthase B simultaneously introduced into a eukaryotic strain
  • the promoter of the gene expression cassette of GGPP synthase A is TDH1
  • the promoter of the gene expression cassette expressing taxadiene synthase is ENO2
  • the gene expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase is expressed.
  • the promoter of the cassette is PDC1
  • the promoter of the fusion gene expression cassette is GPM1
  • the yield of taxadiene in the shake flask fermentation stage of the recombinant eukaryotic strain corresponding to the promoter combination of each of the other gene expression cassettes is compared. Significantly improved, the difference was significant, P ⁇ 0.05.
  • the gene expression cassette expressing GGPP synthase A further comprises a terminator.
  • the terminator in the gene expression cassette expressing GGPP synthase A is selected from the group consisting of TEF1, ADH1, and TPI1.
  • the terminator of the gene expression cassette expressing GGPP synthase A is TEF1.
  • the terminator in the gene expression cassette expressing GGPP synthase A is not limited by the terminator provided by the present invention, and a person skilled in the art can select a terminator according to the actual situation.
  • the nucleotide sequence of the gene expression cassette expressing GGPP synthase A is: a promoter is sequentially connected from the 5′ end to the 3′ end, The gene and terminator of GGPP synthase A are expressed; the promoter is directly linked to the gene expressing GGPP synthase A, and the gene expressing GGPP synthase A is directly linked to the terminator.
  • the gene expression cassette expressing taxadiene synthase further comprises a terminator.
  • the terminator in the gene expression cassette expressing taxadiene synthase is selected from the group consisting of HXT7, PGI1, and FBA1.
  • the terminator in the gene expression cassette expressing taxadiene synthase is HXT7.
  • the terminator in the gene expression cassette expressing taxadiene synthase is not limited by the terminator provided by the present invention, and a person skilled in the art can select a terminator according to the actual situation.
  • the nucleotide sequence of the gene expression cassette expressing taxadiene synthase is: starting from the 5' end to the 3' end The gene encoding the taxadiene synthase and the terminator; the promoter is directly linked to the gene expressing taxadiene synthase, and the gene expressing taxadiene synthase is directly linked to the terminator.
  • a terminator is further included in the gene expression cassette expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase.
  • the terminator in the gene expression cassette expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase is selected from the group consisting of TEF2, GPD, PGK1.
  • the terminator in the gene expression cassette expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase is TEF2.
  • the terminator in the gene expression cassette expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase is not limited by the terminator provided by the present invention, and those skilled in the art can choose to terminate according to the actual situation. child.
  • the nucleotide sequence of the gene expression cassette expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase is: from 5' a promoter, a gene expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase, and a terminator are ligated to the 3' end; a promoter and expression of 3-hydroxy-3-methylglutaryl coenzyme A
  • the gene of the reductase is directly linked, and the gene expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase is directly linked to the terminator.
  • the fusion gene expression cassette expressing the FPP synthase gene and the gene expressing GGPP synthase B further comprises a terminator.
  • the terminator in the fusion gene expression cassette of the gene expressing FPP synthase and the gene expressing GGPP synthase B is selected from the group consisting of TPI1, TDH2, and CYC1. .
  • the terminator in the fusion gene expression cassette of the gene expressing FPP synthase and the gene expressing GGPP synthase B is TPI1.
  • the terminator in the fusion gene expression cassette of the gene expressing FPP synthase and the gene expressing GGPP synthase B is not limited by the terminator provided by the present invention, and those skilled in the art can select according to actual conditions. Terminator.
  • the nucleotide sequence of the fusion gene expression cassette expressing the FPP synthase gene and the gene expressing GGPP synthase B is: from the 5' end A promoter, a gene expressing FPP synthase, and a fusion gene and a terminator of a gene expressing GGPP synthase B are ligated to the 3' end; a fusion of a promoter and a gene expressing FPP synthase and a gene expressing GGPP synthase B
  • the genes are directly linked, and the fusion gene expressing the FPP synthase and the gene expressing the GGPP synthase B are directly linked to the terminator.
  • the eukaryotic strain used is a yeast. More preferably, in the recombinant eukaryotic strain provided by the present invention, the yeast used is Saccharomyces cerevisiae. In some embodiments of the invention, in the recombinant eukaryotic strain provided by the present invention, the Saccharomyces cerevisiae used is YSG50, CEN.PK2, BY4741 or YNL280C. In still other embodiments of the present invention, in the recombinant eukaryotic strain provided by the present invention, the Saccharomyces cerevisiae is specifically YNL280C, which is a S. cerevisiae single-knock strain derived from S288c.
  • the gene expression cassette expressing GGPP synthase A, the gene expression cassette expressing taxadiene synthase, and the expression of 3-hydroxy-3-methylglutaryl coenzyme A are reduced.
  • the gene expression cassette of the enzyme, the gene expressing the FPP synthase, and the fusion gene expression cassette expressing the gene of GGPP synthase B are each independently present in the recombinant eukaryotic strain, and the manner of existence is independently selected from the group consisting of: On the vector, or integrated into the genome of the eukaryotic strain.
  • the gene expression cassette expressing GGPP synthase A, the gene expression cassette expressing taxadiene synthase, and the expression of 3-hydroxy-3-methylglutaryl coenzyme A are reduced.
  • At least one of the fusion gene expression cassette of the enzyme gene expression cassette, the gene expressing the FPP synthase, and the gene expressing GGPP synthase B is integrated into the genome of the eukaryotic strain.
  • the integrated integration site is a multi-copy site of the genome of the eukaryotic strain used.
  • the multiple copy site is a Delta site.
  • a gene expression cassette expressing GGPP synthase A, a gene expression cassette expressing taxadiene synthase, and a 3-hydroxy-3-methyl group are expressed.
  • a gene expression cassette for aglutaryl-CoA reductase, a gene for expressing a FPP synthase, and a fusion gene expression cassette for a gene expressing GGPP synthase B are integrated into a multicopy site of the genome of the eukaryotic strain.
  • a gene expression cassette expressing GGPP synthase A, a gene expression cassette expressing taxadiene synthase, and a 3-hydroxy-3-methyl group are expressed.
  • a gene expression cassette for aglutaryl-CoA reductase, a gene for expressing a FPP synthase, and a fusion gene expression cassette for a gene expressing GGPP synthase B are integrated as a gene function module To the genome of a eukaryotic strain.
  • a gene expression cassette expressing GGPP synthase A, a gene expression cassette expressing taxadiene synthase, and a gene expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase The expression cassette, the gene expressing the FPP synthase, and the fusion gene expression cassette expressing the gene of GGPP synthase B are integrated into the multicopy site of the genome of the eukaryotic strain in the form of a gene function module.
  • the gene expression cassette expressing GGPP synthase A, the gene expression cassette expressing taxadiene synthase, and the expression 3-hydroxy-3-methylglutaryl coenzyme A in the recombinant eukaryotic strain provided by the present invention
  • the gene expression cassette of the reductase, the gene expressing the FPP synthase, and the fusion gene expression cassette expressing the gene of GGPP synthase B are integrated into the multicopy site of the genome of the eukaryotic strain in the form of a gene function module, and the GGPP synthase is expressed.
  • Gene expression cassette of A Gene expression cassette of A, gene expression cassette expressing taxadiene synthase, gene expression cassette expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase, gene expressing FPP synthase and expression of GGPP synthase
  • the order in which the fusion gene expression cassette of the gene of B is arranged in the gene function module is not fixed.
  • Each gene expression cassette can be joined by a short nucleotide sequence, such as a terminator, or directly.
  • the arrangement order of the above four gene expression cassettes in the gene function module is: a gene expression cassette expressing GGPP synthase A, and expressing yew Gene expression cassette for ene synthase, fusion gene expression cassette for gene expressing FPP synthase and gene for expressing GGPP synthase B, gene expression cassette for expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase, expression
  • the gene expression cassette of taxadiene synthase is arranged in tandem.
  • the recombinant eukaryotic strain provided by the present invention introduces a gene expression cassette expressing GGPP synthase A, a gene expressing FPP synthase, and a gene expressing GGPP synthase B into a eukaryotic strain.
  • the gene expression cassette expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase, and the gene expression cassette expressing taxadiene synthase the four gene expression cassettes are continuously connected to each other. The manner exists in the cells of eukaryotic strains and integrates into the genomic Delta locus of eukaryotic strains.
  • the gene expression module of the reductase gene expression cassette, the gene expressing the FPP synthase, and the fusion gene expression cassette expressing the gene of GGPP synthase B are integrated into the multicopy site of the genome of the eukaryotic strain in the form of a gene function module, the gene function module Also includes screening marker genes for easy recombination For screening of eukaryotic strains, the selection marker can be His, Lys, Leu or Ura.
  • the selection marker in the gene function module of the recombinant eukaryotic strain provided by the invention is the URA3 gene.
  • the selection marker gene in the gene function module of the recombinant eukaryotic strain provided by the present invention is not limited to the selection marker gene provided by the present invention, and a person skilled in the art can select a selection marker gene according to actual needs.
  • the gene function module integrated into the multicopy site of the eukaryotic strain comprises the following genes: a screening marker gene, a gene expression cassette expressing GGPP synthase A, Gene expression cassette for expressing taxadiene synthase, gene expression cassette for expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase, gene for expression of FPP synthase, and fusion gene for gene expressing GGPP synthase B
  • the expression cassette; its order in the genome of the recombinant eukaryotic strain (5'-3') is as follows:
  • DNA molecule 1 represents a screening marker gene
  • DNA molecule 2 represents a gene expression cassette expressing GGPP synthase A
  • DNA molecule 3 represents a gene expressing FPP synthase, and a fusion gene expression cassette expressing a gene of GGPP synthase B
  • a DNA molecule 4 represents a gene expression cassette expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase
  • DNA molecule 5 represents a gene expression cassette expressing taxadiene synthase.
  • DNA molecule 1 and DNA molecule 2 are directly connected; DNA molecule 2 and DNA molecule 3 are directly connected; DNA molecule 3 and DNA molecule 4 are directly connected; DNA molecule 4 and DNA molecule 5 are directly connected.
  • the present invention also provides a taxane-producing recombinant eukaryotic strain having the preservation number CGMCC No. 10074.
  • the recombinant eukaryotic strain is a recombinant Saccharomyces cerevisiae strain, which is obtained by genetic engineering technology using Saccharomyces cerevisiae as a starting strain.
  • the recombinant Saccharomyces cerevisiae strain, classified as Saccharomyces cerevisiae was deposited on November 28, 2014 at the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee, and the preservation number is CGMCC No. 10074.
  • CGMCC General Microbiology Center
  • the invention also provides a method for constructing a taxane-producing recombinant eukaryotic strain, comprising the following steps:
  • Step 1 Obtain a gene expression cassette expressing GGPP synthase A, a gene expression cassette expressing taxadiene synthase, a gene expression cassette expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase, and expressing FPP a fusion gene expression cassette of an enzyme gene and a gene expressing GGPP synthase B;
  • the gene expression cassette expressing GGPP synthase A comprises a promoter, a gene expressing GGPP synthase A; the promoter of a gene expression cassette expressing GGPP synthase A is TDH1;
  • the gene expression cassette for expressing taxadiene synthase comprises a promoter and a gene expressing taxadiene synthase; the promoter of the gene expression cassette expressing taxadiene synthase is ENO2;
  • a gene expression cassette for expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase includes a promoter, a gene expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase; and a 3-hydroxy-3- expression
  • the promoter of the gene expression cassette of methylglutaryl coenzyme A reductase is PDC1;
  • Step 2 Take a gene expression cassette expressing GGPP synthase A, a gene expression cassette expressing taxadiene synthase, a gene expression cassette expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase, and expressing FPP A fusion gene expression cassette of the gene of the enzyme and the gene expressing GGPP synthase B is introduced into the fungal strain, and is obtained.
  • the method further comprises homologous recombination in the eukaryotic strain, expressing the gene expression cassette of the GGPP synthase A, and expressing the purple Gene expression cassette for sinadiene synthase, gene expression cassette for expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase, gene for expression of FPP synthase, and fusion gene expression cassette for gene expressing GGPP synthase B
  • the integrated integration site in step 2 is a multi-copy site of a eukaryotic strain.
  • the multiple copy locus is a Delta locus.
  • the method for constructing a recombinant eukaryotic strain comprises:
  • Step 1 obtaining a fusion marker gene, a gene expression cassette expressing GGPP synthase A, a gene expressing FPP synthase, and a fusion gene expression cassette expressing a gene of GGPP synthase B, expression a gene expression cassette for 3-hydroxy-3-methylglutaryl coenzyme A reductase, and a gene expression cassette for expressing taxadiene synthase;
  • the gene expression cassette expressing GGPP synthase A comprises a promoter, a gene expressing GGPP synthase A; the promoter of a gene expression cassette expressing GGPP synthase A is TDH1;
  • the gene expression cassette for expressing taxadiene synthase comprises a promoter and a gene expressing taxadiene synthase; the promoter of the gene expression cassette expressing taxadiene synthase is ENO2;
  • a gene expression cassette for expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase includes a promoter, a gene expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase; and a 3-hydroxy-3- expression
  • the promoter of the gene expression cassette of methylglutaryl coenzyme A reductase is PDC1;
  • Step 2 The fusion marker gene, the gene expression cassette expressing the GGPP synthase A, the gene expressing the FPP synthase, and the gene expressing the GGPP synthase B, and the expression of the 3-hydroxy-3-methyl group are obtained.
  • the gene expression cassette of the glutaryl-CoA reductase and the gene expression cassette expressing the taxadiene synthase are introduced into the eukaryotic strain and integrated into the genome of the eukaryotic strain by homologous recombination.
  • the 5' end of the screening marker gene in the step 1 is ligated to the first homologous arm sequence of the eukaryotic strain chassis integration site.
  • the terminator is specifically CYC1.
  • the first homologous arm sequence of the eukaryotic strain substrate integration site is to provide a homology arm for the homologous recombination of the eukaryotic strain chassis cell genome and the recombinant DNA molecule.
  • the first homologous arm sequence is a sequence that is capable of homologous recombination with a multicopy site of a genome of a eukaryotic strain.
  • the first homologous arm sequence is a sequence capable of homologous recombination with the Delta site of the yeast genome, designated Delta1.
  • the 5' end of the gene expression cassette expressing GGPP synthase A in step 1 has a termination
  • the terminator serves to provide a homology arm for homologous recombination in a cell of a eukaryotic strain for screening a marker gene and a gene expression cassette expressing GGPP synthase A.
  • the terminator is linked to the 3' end of the selection marker gene.
  • the terminators are identical, such that the marker gene and the gene expression cassette expressing GGPP synthase A are homologously recombined in the cells of the eukaryotic strain chassis, and are sequentially connected before and after.
  • the terminator is specifically CYC1.
  • the 5' end of the fusion gene expression cassette of the gene expressing FPP synthase and the gene expressing GGPP synthase B in step 1 Linked to a terminator which functions as a fusion gene expression cassette for expression of a gene expression cassette of GGPP synthase A, a gene expressing FPP synthase, and a gene expressing GGPP synthase B in a cell of a eukaryotic strain chassis
  • Homologous recombination provides a homology arm that is identical to the 3' terminator in the gene expression cassette expressing GGPP synthase A, such that a gene expression cassette expressing GGPP synthase A, a gene expressing FPP synthase, and expression
  • the fusion gene expression cassette of the gene of GGPP synthase B is homologously recombined in the cells of the eukin
  • the 5' end of the gene expression cassette expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase in step 1 Attached to a terminator, the terminator functions as a fusion gene expression cassette for the gene expressing FPP synthase and a gene expressing GGPP synthase B, and a 3-hydroxy-3-methylglutaryl coenzyme A reductase.
  • the gene expression cassette provides a homology arm when homologous recombination occurs in a cell of a eukaryotic strain
  • the terminator is a 3' terminator in a fusion gene expression cassette of a gene expressing FPP synthase and a gene expressing GGPP synthase B.
  • a fusion gene expression cassette expressing a gene for FPP synthase and a gene expressing GGPP synthase B, and a gene expression cassette expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase are in a cell of a eukaryotic strain Homologous recombination occurs, which are connected in sequence.
  • the terminator is specifically TPI1.
  • the taxadiene synthase-expressing gene expression cassette of step 1 has a terminator attached to the 5' end and a eukaryotic strain cell integration site at the 3' end. Two homologous arm sequences. The role of the terminator is to generate homologous recombination in the cell of the eukaryotic strain for the gene expression cassette expressing the 3-hydroxy-3-methylglutaryl coenzyme A reductase and the gene expression cassette expressing the taxadiene synthase. A homology arm is provided.
  • the end The terminator was identical to the terminator at the 3' end of the gene expression cassette expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase.
  • the terminator is specifically TEF2.
  • the second homologous arm sequence of the eukaryotic strain chassis integration site is to provide a homology arm for the homologous recombination of the eukaryotic strain chassis cell genome and the recombinant DNA molecule.
  • the second homologous arm sequence is a sequence that is capable of homologous recombination with a multicopy site in the genome of a eukaryotic strainer cell.
  • the second homologous arm sequence is a sequence that homologously recombines with the Delta site of the yeast genome, designated Delta2.
  • a marker gene in accordance with the method for constructing a recombinant eukaryotic strain provided by the present invention, a marker gene, a gene expression cassette expressing GGPP synthase A, a gene expressing FPP synthase, and a gene expressing GGPP synthase B are selected.
  • the gene function module of the locus includes: a marker gene, a gene expression cassette expressing GGPP synthase A, a gene expressing FPP synthase, and a fusion gene expression cassette expressing a gene of GGPP synthase B, and expressing 3-hydroxy-3-methyl Gene expression cassette of glutaryl coenzyme A reductase, gene expression cassette expressing taxadiene synthase; its order in the genome of recombinant eukaryotic strains (5'-3') is as follows:
  • DNA molecule 1 represents a screening marker gene
  • DNA molecule 2 represents a gene expression cassette expressing GGPP synthase A
  • DNA molecule 3 represents a gene expressing FPP synthase, and a fusion gene expression cassette expressing a gene of GGPP synthase B
  • a DNA molecule 4 represents a gene expression cassette expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase
  • DNA molecule 5 represents a gene expression cassette expressing taxadiene synthase.
  • DNA molecule 1 and DNA molecule 2 are directly connected; DNA molecule 2 and DNA molecule 3 are directly connected; DNA molecule 3 and DNA molecule 4 are directly connected; DNA molecule 4 and DNA molecule 5 are directly connected.
  • the present invention also provides a method for preparing taxadiene by using the recombinant eukaryotic strain provided by the present invention, wherein the recombinant eukaryotic strain is introduced into the eukaryotic strain into a gene expression cassette expressing GGPP synthase A, and expressing the yew Gene expression cassette for diene synthase, gene expression cassette for expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase, gene for expressing FPP synthase, and gene for expressing GGPP synthase B Fusion gene expression cassette;
  • the gene expression cassette expressing GGPP synthase A comprises a promoter, a gene expressing GGPP synthase A, and the promoter of the gene expression cassette expressing GGPP synthase A is TDH1;
  • the gene expression cassette for expressing taxadiene synthase comprises a promoter and a gene expressing taxadiene synthase, and the promoter of the gene expression cassette expressing taxadiene synthase is ENO2;
  • the gene expression cassette for expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase comprises a promoter, a gene expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase, and the expression 3-hydroxy-
  • the promoter of the gene expression cassette of 3-methylglutaryl coenzyme A reductase is PDC1;
  • the fusion gene expression cassette of the gene expressing FPP synthase and the gene expressing GGPP synthase B comprises a promoter, the fusion gene; the promoter of the fusion gene expression cassette is GPM1;
  • the method for preparing taxadiene includes:
  • the recombinant eukaryotic strain is cultured, and the taxane is recovered from the culture.
  • the cultivation process comprises the following steps:
  • the recombinant yeast is taken, expanded, and then inoculated into a fermentation medium, and fermented and cultured in two phases to collect the taxadiene.
  • the carbon is supplemented by batch addition or addition during the two-phase culture process.
  • Source glucose The experimental results confirmed that the yield of taxadiene was further increased after the addition of glucose during the two-phase culture.
  • the carbon source is added in a fed-up manner, specifically, continuous addition to the fermentation liquid.
  • Glucose maintains the glucose concentration in the fermentation broth at 0g/L to 1g/L.
  • the fermentation medium comprises 20 g/L to 50 g/L of glucose, and 10 g/L of yeast. Powder, 20 g/L to 30 g/L peptone, 0 g/L to 8 g/L KH 2 PO 4 and 0 g/L to 6 g/L MgSO 4 .
  • the fermentation medium comprises 30 g/L to 50 g/L of glucose, and 10 g/L of yeast. Powder, 20 g/L to 30 g/L peptone, 0.5 g/L to 8 g/L KH 2 PO 4 and 0.5 g/L to 6 g/L MgSO 4 .
  • the temperature of the fermentation culture is 30 °C.
  • the temperature of the two-phase culture is 30 °C.
  • the two-phase culture time is from 70 h to 174 h. In still other embodiments of the invention, the two phase culture time is 138 h.
  • the present invention provides a taxane-producing recombinant eukaryotic strain and a method for preparing taxadiene using the recombinant eukaryotic strain.
  • the recombinant eukaryotic strain producing taxadiene is a gene expression cassette expressing GGPP synthase A, a gene expression cassette expressing taxadiene synthase, and a 3-hydroxy-3-methyl group in the eukaryotic strain.
  • the gene expression cassette expressing GGPP synthase A includes a promoter, and a GGPP synthase is expressed
  • the gene of A; the promoter of the gene expression cassette expressing GGPP synthase A is TDH1;
  • the gene expression cassette expressing taxadiene synthase includes a promoter, a gene expressing taxadiene synthase; and the expression of taxadiene
  • the promoter of the gene expression cassette of the enzyme is ENO2;
  • the gene expression cassette expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase includes a promoter and expression of 3-hydroxy-3-methylglutaryl coenzyme A.
  • the gene of the enzyme; the promoter of the gene expression cassette expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase is PDC1; the fusion gene expression cassette of the gene expressing FPP synthase and the gene expressing GGPP synthase B includes Promoter, the fusion gene; the promoter of the fusion gene expression cassette is GPM1.
  • the experimental results show that the taxane-producing recombinant eukaryotic strain provided by the present invention has a high yield of taxadiene, and the yield of taxadiene of all strains after shake flask fermentation is 161.82 mg/L, and the purple of the recombinant strain is purple.
  • the yield of cedardiene was significantly higher than that of other recombinant strains, and the difference was significant, P ⁇ 0.05.
  • the yield of taxadiene from recombinant Saccharomyces cerevisiae is further improved by optimization of fermentation conditions, providing a material basis for subsequent paclitaxel biosynthesis.
  • FIG. 1 shows the detection results of the GGPPSsa recombinant vector 1 in Example 1; wherein FIG. 1-A is a double-digested gel electrophoresis pattern of the GGPPSsa recombinant vector 1, the GGPPSsa recombinant vector 2, and the GGPPSsa recombinant vector 3; FIG. Nucleotide sequence information of the GGPPSsa gene expression cassette;
  • FIG. 2 shows the detection results of the BE recombinant vector 1 in Example 1, wherein FIG. 2 is a double-cleavage gel electrophoresis pattern of the BE recombinant vector 1, the BE recombinant vector 3, and the BE recombinant vector 4;
  • FIG. 3 shows the results of detection of tHMGR recombinant vector 1 in Example 1, wherein FIG. 3 is a double-cut-cut gel electrophoresis pattern of tHMGR recombinant vector 1 and tHMGR recombinant vector 2;
  • FIG. 4 shows the detection results of tTS recombinant vector 1 in Example 1, wherein FIG. 4 is a single-cut-cut gel electrophoresis pattern of tTS recombinant vector 1, tTS recombinant vector 2, and tTS recombinant vector 3;
  • Fig. 5 shows the results of genomic PCR verification of the recombinant strain in Example 1, wherein Fig. 5-A is the verification result of the recombinant strain 1; Fig. 5-B is the nucleus of the Delta site of the genome integrated into the strain of the recombinant strain 1 Glycosidic acid sequence information; Figure 5-C is the verification result of recombinant strain 2;
  • Figure 6 shows a standard curve of the taxadiene standard in Example 1.
  • Figure 7 shows the results of GC-TOF/MS detection of the taxadiene standard in Example 1, wherein Figure 7-A shows the MS detection result of the taxadiene standard; Figure 7-B shows the taxadiene standard. GC test results;
  • FIG. 8 shows the results of GC-TOF/MS detection of the sample obtained after the fermentation of the recombinant strain 1 in Example 1, wherein FIG. 8-A is the MS detection result of the sample; FIG. 8-B is the GC detection result of the sample;
  • FIG. 9 shows the results of GC-TOF/MS detection of the sample obtained after the fermentation of the recombinant strain 2 in Example 1, wherein FIG. 9-A is the MS detection result of the sample; FIG. 9-B is the GC detection result of the sample;
  • FIG. 10-A is the MS detection result of the sample
  • FIG. 10-B is the GC detection result of the sample
  • Figure 11 shows the results of GC-TOF/MS detection of the sample obtained by batch-feeding fermentation of the recombinant strain 1 in Example 3, wherein Figure 11-A shows the MS detection result of the sample; Figure 11-B shows the GC detection of the sample. result;
  • Figure 12 shows the results of GC-TOF/MS detection of the sample extracted by the recombinant strain 1 after fed-batch fermentation in Example 4, wherein Figure 12-A shows the MS detection result of the sample; Figure 12-B shows the GC detection of the sample. result.
  • Recombinant Saccharomyces Cerevisiae SyBE_Sc00011203 Classification and Name: Saccharomyces cerevisiae, deposited on November 28, 2014 at the General Microbiology Center (CGMCC) of China Microbial Culture Collection Management Committee, Address: Beichen West Road, Chaoyang District, Beijing No. 3, No. 3, Institute of Microbiology, Chinese Academy of Sciences, with the preservation number: CGMCC No. 10074.
  • CGMCC General Microbiology Center
  • the invention discloses a recombinant eukaryotic strain producing taxadiene and a method for preparing taxadiene by using the recombinant eukaryotic strain, and those skilled in the art can learn from the contents of the present invention and appropriately improve the process parameters. It is to be understood that all such alternatives and modifications are obvious to those skilled in the art and are considered to be included in the present invention.
  • the method and the application of the present invention have been described by the preferred embodiments, and it is obvious that the method and application described herein may be modified or appropriately modified and combined without departing from the scope of the present invention. The technique of the present invention is applied.
  • the recombinant eukaryotic strain producing taxadiene provided by the present invention and the reagents and raw materials used in the method for preparing taxadiene using the recombinant eukaryotic strain are commercially available.
  • Saccharomyces cerevisiae single knock strain YNL280C was purchased from Open Biosystems, Huntsville, AL; vector pRS425 was purchased from Addgene (American), and the ampicillin resistance gene on the vector was replaced by kanamycin resistance by yeast homologous recombination. Gene, get pRS425K.
  • E. coli TransT1 was purchased from Beijing Quanjin Biotechnology Co., Ltd.
  • GGPP synthase A codon optimized according to the GGPP synthase gene GGPPSsa derived from Sulfolobus acidophilus, synthesized by Jin Weizhi Company That is, it has the nucleotide sequence shown as SEQ ID NO: 1, and is labeled as GGPPSsa gene.
  • the gene for expressing taxadiene synthase codon optimized according to the taxadiene synthase gene derived from Taxus chinensis, and synthesized at Jinweizhi Company, labeled as tTS gene, which has The nucleotide sequence shown in SEQ ID NO: 2.
  • Gene expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase (endogenous gene): designing a pre-primer with a promoter homologous sequence, a post-primer with a terminator homologous sequence, for Saccharomyces cerevisiae
  • the single knocking strain YNL280C is used as a template and amplified by PCR to obtain a gene expressing 3-hydroxy-3-methylglutaryl coenzyme A reductase having the nucleotide sequence shown in SEQ ID NO: 3, labeled as tHMGR gene.
  • FPP synthase endogenous gene: a pre-primer with a promoter homologous sequence, a post-primer with a terminator homologous sequence, and a PCR amplification by S. cerevisiae single-stem strain YNL280C as a template A gene expressing FPP synthase was obtained, which was labeled as ERG20 gene.
  • Gene expressing GGPP synthase B (endogenous gene): designing a pre-primer with a promoter homologous sequence, a post-primer with a terminator homologous sequence, and PCR amplification by S. cerevisiae single-stem strain YNL280C as a template A gene expressing GGPP synthase B was obtained, which was designated as BTS1 gene.
  • a fusion gene of a gene expressing FPP synthase and a gene expressing GGPP synthase B OE-PCR (overlapping PCR) of ERG20 gene and BTS1 gene, and ERG20 gene and BTS1 gene were obtained by linker (5'-GGTGGTGGTTCT-3')
  • the marker gene URA3 gene was screened: it has the nucleotide sequence shown as SEQ ID NO: 5.
  • Promoter derived from YNL280C, the nucleotide information of the promoter and promoter corresponding to each gene is shown in Table 1.
  • Terminator from YNL280C.
  • the nucleotide sequence information of the terminator and terminator corresponding to each gene is shown in Table 1.
  • GGPPSsa gene The promoter of GGPPSsa gene was taken, and the terminator 1, promoter, GGPPSsa gene and terminator 2 were ligated by OE-PCR method, and the fragment containing Pst1 and BamH1 restriction sites was obtained, and ligated into vector pRS425K.
  • Three recombinant vectors were named: GGPPSsa recombinant vector 1, GGPPSsa recombinant vector 2, and GGPPSsa recombinant vector 3.
  • the recombinant vector constructed above was separately transformed into E. coli TransT1, colony PCR screening,
  • the plasmid was subjected to double enzyme digestion verification and sequencing verification to ensure that the target fragment was ligated correctly and the base sequence was not mutated.
  • GGPPSsa recombinant vector 1 GGPPSsa recombinant vector 2 and GGPPSsa recombinant vector 3 were respectively taken for double enzyme digestion verification, and the obtained detection results are shown in Fig. 1, wherein Fig. 1-A includes GGPPSsa recombinant vector 1, GGPPSsa recombinant vector 2 and GGPPSsa recombinant vector. 3 double-cut detection of the gel map. Among them, the gene expression cassette of GGPPSsa recombinant vector 1 is 2489 bp, and the vector length is 6749 bp, consistent with the corresponding DNA band size in the figure; sequencing verification is also correct.
  • each recombinant vector and each gene expression cassette expressing GGPP synthase A is shown in Table 2.
  • GGPPSsa Recombination carrier name Gene expression box name 5'-3' genes linked in turn GGPPSsa Recombination Carrier 1 GGPPSsa Gene Expression Box 1 Terminator CYC1, promoter TDH1, GGPPSsa gene, terminator TEF1 GGPPSsa Recombination Vector 2 GGPPSsa Gene Expression Box 2 Terminator CYC1, promoter TPI1, GGPPSsa gene, terminator TEF1 GGPPSsa Recombination Vector 3 GGPPSsa Gene Expression Box 3 Terminator CYC1, promoter HXT7, GGPPSsa gene, terminator TEF1
  • BE gene The promoter of BE gene was taken, and the terminator TEF1, promoter, BE gene and terminator TPI1 were ligated by OE-PCR method, and the fragment containing Pst1 and BamH1 restriction sites was obtained, and ligated into vector pRS425K.
  • Four recombinant vectors were named as: BE recombinant vector 1, BE recombinant vector 2, BE recombinant vector 3, and BE recombinant vector 4.
  • the recombinant vector constructed above was transformed into E. coli TransT1, colony PCR screening, plasmid extraction, double enzyme digestion verification and sequencing verification to ensure that the target fragment was correctly ligated and the base sequence was not mutated.
  • BE recombinant vector 1 Take BE recombinant vector 1 and perform double enzyme digestion verification. The results are shown in Figure 2.
  • the recombinant plasmid containing BE recombinant vector 1, BE recombinant vector 3 and BE recombinant vector 4 is digested and the gene expression cassette of BE recombinant vector 1 is used.
  • the length is 3751 bp, the length of the vector is 6749 bp, which is consistent with the corresponding DNA band size in the figure; the sequencing verification is also correct.
  • the correctness of the recombinant vector BE recombinant vector 2, BE recombinant vector 3, and BE recombinant vector 4 was verified by the same method. After detection, the BE gene expression cassette containing different promoters was successfully obtained in this example.
  • the correct recombinant strain was taken, and the plasmid was extracted and digested with Pst1 and BamH1.
  • the enzyme-cut product is recovered, that is, the BE gene expression cassette is obtained.
  • the information of each recombinant vector and each BE gene expression cassette is shown in Table 3.
  • Recombination carrier name Gene expression box name 5'-3' genes linked in turn BE recombinant vector 1 BE gene expression cassette 1 Terminator TEF1, promoter GPM1, BE gene and terminator TPI1 BE recombinant vector 2 BE gene expression cassette 2 Terminator TEF1, promoter HXK2, BE gene and terminator TPI1 BE recombinant vector 3 BE gene expression cassette 3 Terminator TEF1, promoter TEF1, BE gene and terminator TPI1 BE recombinant vector 4 BE gene expression cassette 4 Terminator TEF1, promoter TDH2, BE gene and terminator TPI1
  • tHMGR recombinant vector 1 The promoter of tHMGR gene was used, and the terminator TPI1, promoter, tHMGR gene and terminator TEF2 were ligated by OE-PCR method to obtain a fragment containing Pst1 and BamH1 restriction sites at both ends, and ligated into vector pRS425K.
  • Two recombinant vectors were named: tHMGR recombinant vector 1, tHMGR recombinant vector 2.
  • the recombinant vector constructed above was transformed into E. coli TransT1, colony PCR screening, plasmid extraction, double enzyme digestion verification and sequencing verification to ensure that the target fragment was correctly ligated and the base sequence was not mutated.
  • tHMGR recombinant vector 1 Take tHMGR recombinant vector 1 and perform double enzyme digestion detection. The results are shown in Figure 3.
  • the tHMGR recombinant vector 1 and the tHMGR recombinant vector 2 are double-digested.
  • the gene expression cassette of tHMGR recombinant vector 1 is 3086 bp.
  • the length is 6749 bp, which is consistent with the corresponding DNA band size in the figure; the sequencing verification is also correct.
  • the recombinant vector tHMGR recombinant vector 2 was taken and tested. After detection, the tHMGR gene expression cassette containing different promoters was successfully obtained in this example.
  • Recombination carrier name Gene expression box name 5'-3' genes linked in turn tHMGR recombinant vector 1 tHMGR gene expression cassette 1 Terminator TPI1, promoter PDC1, tHMGR gene and terminator TEF2 tHMGR recombinant vector 2 tHMGR gene expression cassette 2 Terminator TPI1, promoter PGK1, tHMGR gene and terminator TEF2
  • the promoter of the tTS gene was used, and the terminator TEF2, promoter, tTS gene, terminator HXT7 and the 239 bp homologous sequence Delta2 downstream of the Delta site were ligated by OE-PCR method to obtain a site containing Not1 restriction sites at both ends.
  • the fragment was ligated into the vector pRS425K to obtain three recombinant vectors, which were respectively named: tTS recombinant vector 1, tTS recombinant vector 2, and tTS recombinant vector 3.
  • the recombinant vector constructed above was transformed into E. coli TransT1, colony PCR screening, plasmid extraction, single enzyme digestion verification and sequencing verification to ensure that the target fragment was correctly linked and the base sequence was not mutated.
  • FIG. 4A shows the gelation map of tTS recombinant vector 1, tTS recombinant vector 2 and tTS recombinant vector 3 single enzyme digestion, tTS recombinant vector
  • the gene expression cassette of 1 is 4147 bp in length and the vector length is 6749 bp, which is consistent with the corresponding DNA band size in the figure; the sequencing verification is also correct.
  • the correctness of the tTS recombinant vector 2 and the tTS recombinant vector 3 was examined according to the same detection method. After detection, the tTS gene expression cassette containing different promoters was successfully obtained in this example.
  • each recombinant strain was taken, the plasmid was extracted, the Not1 enzyme was digested, and the digested product was recovered, and the gene expression cassette containing the tTS gene was obtained.
  • the information of each recombinant vector and each tTS gene expression cassette is shown in Table 5.
  • Recombination carrier name Gene expression box name 5'-3' genes linked in turn tTS recombinant vector 1 tTS gene expression cassette 1 Terminator TEF2, promoter ENO2, tTS gene, terminator HXT7, Delta2 tTS recombinant vector 2 tTS gene expression cassette 2 Terminator TEF2, promoter FBA1, tTS gene, terminator HXT7, Delta2 tTS recombinant vector 3 tTS gene expression cassette 3 Terminator TEF2, promoter TEF2, tTS gene, terminator HXT7, Delta2
  • the 248 bp homologous sequence Delta1, the screening marker gene URA3 gene and the final upstream of the Delta locus The CYC1 was spliced by OE-PCR method, and a fragment containing a Not1 restriction site at both ends was obtained, and ligated into the vector pRS425K to obtain a recombinant vector.
  • the recombinant vector constructed above was transformed into E. coli TransT1, colony PCR screening, plasmid extraction, single enzyme digestion verification and sequencing verification to ensure that the target fragment was correctly linked and the base sequence was not mutated. After detection, the expression cassette containing the screening marker gene URA3 gene was successfully obtained in this example.
  • the correct recombinant strain was obtained, the plasmid was extracted, the Not1 enzyme was digested, and the digested product was recovered, and the expression cassette containing the screening marker gene URA3 gene was obtained, and the gene information contained in the expression cassette was from 5' to 3' end.
  • the linkage is: Delta1, the screening marker gene URA3 gene and the terminator CYC1.
  • the recombinant yeast strain of the taxadiene biosynthesis pathway regulated by different promoter combinations is obtained by taking the above five gene expression cassettes and the chassis cell Saccharomyces cerevisiae YNL280C. Different promoter combinations were designed, and the 5 fragments were transformed into yeast by the lithium acetate method.
  • the above fragments were ligated by homologous sequences between the fragments and homologous to the Delta locus on the yeast genome.
  • the source sequence is recombined and integrated into the genome.
  • the transformed yeast was screened by SD-drop solid medium (synthetic yeast nitrogen source YNB 6.7 g/L, glucose 20 g/L, mixed amino acid powder 2 g/L, solid supplemented with 2% agar powder), and the obtained transformants were subjected to transformation. After the scribing was separated, the cells were transferred to a liquid medium for 24 hours, and the yeast genome was extracted as a template to carry out PCR verification to confirm the correct recombinant strain, and the plate was streaked or glycerol was preserved.
  • the recombinant Saccharomyces cerevisiae strain name and the recombinant gene information integrated in the recombinant Saccharomyces cerevisiae strain are shown in Table 6.
  • the recombinant strain 1 was taken, and the yeast genome was extracted as a template for PCR verification.
  • the four sets of verification primers used and the corresponding nucleotide information are shown in Table 7.
  • the size of the fragment amplified by the first set of primers should be 1020 bp
  • the size of the amplified fragment of the second set of primers is 2101 bp
  • the size of the amplified fragment of the third set of primers is 1591 bp
  • the size of the amplified set of the fourth set of primers is 2430 bp.
  • the PCR results of recombinant strain 1 are shown in Figure 5-A, where lane 1 represents the amplification results of the first set of primers; lane 2 represents the amplification results of the second set of primers; and lane 3 represents the amplification results of the third set of primers. Lane 4 represents the amplification results of the fourth set of primers.
  • Recombinant strain 2 was taken, and the yeast genome was extracted as a template for PCR verification.
  • Four sets of verification primers and corresponding nucleotide information were used in Table 8.
  • the size of the fragment amplified by the first set of primers should be 1025 bp
  • the size of the amplified fragment of the second set of primers is 2091 bp
  • the size of the amplified fragment of the third set of primers is 1591 bp
  • the size of the amplified set of the fourth set of primers is 2430 bp.
  • the PCR results of recombinant strain 2 are shown in Figure 5-C, where lane 1 represents the amplification results of the first set of primers; lane 2 represents the amplification results of the second set of primers; and lane 3 represents the amplification results of the third set of primers.
  • Lane 4 represents the amplification results of the fourth set of primers.
  • the amplification results of all the primer sets are in agreement with the theoretical size, indicating that the recombinant strain 2 in which the gene of interest is integrated is successfully obtained.
  • Seed medium synthetic yeast nitrogen source YNB 6.7g / L, glucose 20g / L, mixed amino acid powder 2g / L (specific formula reference [US] DC Amberg and other yeast genetic methods test guide), histidine 38mg / L, tryptophan 38 mg / L, leucine 190 mg / L, pH 5.8, sterilized at 115 ° C for 15 min.
  • Fermentation medium A glucose 20 g / L, yeast powder 10 g / L, peptone 20 g / L, pH 5.8, sterilized at 121 ° C for 20 min.
  • the upper emulsion phase was collected and centrifuged at 12,000 rpm for 20 min.
  • the upper organic phase was collected, and the water was removed using anhydrous sodium sulfate.
  • the filter was filtered using 0.22 ⁇ m. The final product taxane concentration was determined after membrane filtration.
  • the cell absorbance (OD 600 ) measured at 600 nm using a Model 722 spectrophotometer was used to characterize the cell concentration.
  • the taxadiene concentration was determined by GC-TOF/MS (Waters Corp., USA), and the silica gel capillary column was 30 m ⁇ 0.25 mm ⁇ 0.25 ⁇ m DB-5MS, J&W Scientific, Folsom, and the ionization method was electron bombardment ionization EI + , electron The beam energy is 70 eV and the ionization current is 40 ⁇ A.
  • the chromatographic conditions were as follows: the inlet temperature was set to 260 ° C, the column temperature starting temperature was maintained at 200 ° C for 3 min, and then the temperature was raised to 270 ° C at 4 ° C / min for 2 min.
  • Recombinant brewing strain recombinant strain 1 to recombinant strain 7 had the same growth trend in fermentation medium A, and there was no significant difference in growth status. At the end of fermentation, the final OD 600 of the bacteria reached about 25.
  • FIG. 8-A is the MS detection result of the sample, which is basically consistent with the peak of the standard product, indicating that the sample is taxadiene;
  • the peak corresponding to the peak time of 10.36 min is the peak of taxadiene, and the peak area is 3087; the dilution factor of the sample is 30 times; according to the peak area and standard
  • the curve and the dilution factor of the sample can be used to calculate the concentration of taxadiene in the sample, and then the yield of the taxoid of recombinant strain 1 can be calculated.
  • the sample obtained from recombinant strain 2 was tested, and the test result is shown in Fig. 9.
  • Fig. 9-A shows the MS detection result of the sample, which is basically consistent with the peak of the standard product, indicating that the sample is taxadiene; Fig.
  • the samples obtained from the recombinant strain 3 to the recombinant strain 8 were subjected to qualitative and quantitative detection, and the concentration of taxadiene in the sample was obtained, and the yield of taxadiene from the recombinant strain 3 to the recombinant strain 8 was calculated. .
  • the yield of taxadiene for each recombinant strain is shown in Table 9.
  • the selection of the promoter of each gene expression cassette affects the yield of the obtained recombinant eukaryotic strain of taxadiene.
  • the yield of taxadiene of the recombinant strain 1 was the highest, reaching 161.82 mg/L, and the difference was significant compared with other strains, P ⁇ 0.05.
  • the promoter of the GGPPSsa gene expression cassette is TDH1; the promoter of the BE gene expression cassette is GPM1; the promoter of the tHMGR gene expression cassette
  • the promoter of the PDC1;tTS gene expression cassette is ENO2
  • the yield of the obtained recombinant eukaryotic strain can be significantly increased.
  • the recombinant strain 1 (named SyBE_Sc00011203 according to the naming rules of the laboratory) was subjected to biological preservation, and the biological preservation information corresponding to the recombinant strain 1 was: Classification: Saccharomyces cerevisiae, deposited on November 28, 2014 at the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee.
  • CGMCC General Microbiology Center
  • the address of the depository is: No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing. Institute of Microbiology, Chinese Academy of Sciences, Zip Code: 100101, with the accession number: CGMCC No. 10074.
  • Example 2 Fermentation tank fermentation using a recombinant strain to produce taxadiene
  • Seed medium synthetic yeast nitrogen source YNB6.7g / L, glucose 20g / L, mixed amino acid powder 2g / L (specific formula reference [US] DC Amberg and other yeast genetic methods test guide), histidine 38mg / L, tryptophan 38 mg / L, leucine 190 mg / L, pH 5.8, sterilized at 115 ° C for 15 min.
  • Fermentation medium glucose 50g / L, yeast powder 10g / L, peptone 20g / L, KH 2 PO 4 0.5g / L, MgSO 4 0.5g / L; wherein, glucose 115 ° C sterilization 15min, yeast powder and peptone 121 Sterilize at °C for 20 min, the two are separately sterilized; KH 2 PO 4 and MgSO 4 are filter-sterilized; finally, the above ingredients are mixed.
  • the upper emulsion phase was collected and centrifuged at 12,000 rpm for 20 min.
  • the upper organic phase was collected, and the water was removed using anhydrous sodium sulfate.
  • the filter was filtered using 0.22 ⁇ m. The final product taxane concentration was determined after membrane filtration.
  • FIG. 10-A is the MS detection result of the sample, which is basically consistent with the peak of the standard product, indicating that the sample is taxadiene;
  • FIG. 10-B is the sample.
  • the GC test results were compared with the standard, and the peak corresponding to the peak time of 10.40 min was the peak of taxadiene, and the peak area was 6325; the dilution factor of the sample was 30 times; The area and the standard curve and the dilution factor of the sample can be used to obtain the concentration of taxadiene in the sample, and the yield of the taxoid of the recombinant strain 1 is calculated to be 242.64 mg/L.
  • Example 3 Fermentation tank fermentation using a recombinant strain to produce taxadiene
  • Seed medium synthetic yeast nitrogen source YNB6.7g / L, glucose 20g / L, mixed amino acid powder 2g / L (specific formula reference [US] DC Amberg and other yeast genetic methods test guide), histidine 38mg / L, tryptophan 38 mg / L, leucine 190 mg / L, pH 5.8, sterilized at 115 ° C for 15 min.
  • Fermentation medium glucose 30g / L, yeast powder 10g / L, peptone 25g / L, KH 2 PO 4 4g / L, MgSO 4 3g / L; wherein, glucose 115 ° C sterilization 15min, yeast powder and peptone 121 ° C
  • the bacteria were sterilized separately for 20 min; KH 2 PO 4 and MgSO 4 were filter-sterilized; finally, the above ingredients were mixed.
  • Feed solution glucose 800g / L, sterilized at 115 ° C for 15min.
  • the culture conditions were 350 rpm, 30 ° C, pH 5.8, aeration rate of 1 vvm, and culture for 138 h. Do a batch of glucose supplementation, add glucose solution to the original concentration of 30g / L. The cell growth curve was measured.
  • n-dodecane When the fermentation was carried out for 8 hours, 400 mL of n-dodecane was added to the fermentation broth for two-phase culture. When the fermentation was carried out, a 20% feed volume of n-dodecane was added at the same time to keep the organic ratio in the fermentation system unchanged at 20%. .
  • the upper emulsion phase was collected and centrifuged at 12,000 rpm for 20 min. The upper organic phase was collected, and the water was removed using anhydrous sodium sulfate. The final product was measured for the concentration of taxadiene using a 0.22 ⁇ m filter.
  • Fig. 11-A shows the MS detection result of the sample, which is basically consistent with the peak of the standard product, indicating that the sample is taxadiene;
  • Fig. 11-B shows the sample
  • the GC test results, compared with the standard, can be seen that the peak time is 10.37min.
  • the peak of the peak is the peak of taxadiene, the peak area is 5054; the dilution factor of the sample is 30 times; the concentration of taxadiene in the sample can be obtained according to the peak area and the standard curve and the dilution factor of the sample, and then calculated
  • the yield of taxadiene from recombinant strain 1 was 834 mg/L.
  • Example 4 Fermentation tank fermentation using a recombinant strain to produce taxadiene
  • Seed medium synthetic yeast nitrogen source YNB6.7g / L, glucose 20g / L, mixed amino acid powder 2g / L (specific formula reference [US] DC Amberg and other yeast genetic methods test guide), histidine 38mg / L, tryptophan 38 mg / L, leucine 190 mg / L, pH 5.8, sterilized at 115 ° C for 15 min.
  • Fermentation medium glucose 50g / L, yeast powder 10g / L, peptone 30g / L, KH 2 PO 4 8g / L, MgSO 4 6g / L; wherein, glucose 115 ° C sterilization 15min, yeast powder and peptone 121 ° C
  • the bacteria were sterilized separately for 20 min; KH 2 PO 4 and MgSO 4 were filter-sterilized; finally, the above ingredients were mixed.
  • Feed solution glucose 800g / L, sterilized at 115 ° C for 15min.
  • n-dodecane 400mL was added to the fermentation broth for two-phase culture.
  • the upper emulsion phase was collected and centrifuged at 12000 rpm for 20 min.
  • the upper organic phase was collected, and the water was removed using anhydrous sodium sulfate.
  • the filter was filtered using 0.22 ⁇ m.
  • the final product taxane concentration was determined after membrane filtration.
  • Fig. 12-A shows the MS detection result of the sample, which is basically consistent with the peak of the standard product, indicating that the sample is taxadiene
  • Fig. 12-B shows the sample GC test results, compared with the standard, the peak time is 10.37min
  • the corresponding peak is the peak of taxadiene, the peak area is 7465; the dilution factor of the sample is 30 times; the concentration of taxadiene in the sample can be obtained according to the peak area and the standard curve and the dilution factor of the sample, and further
  • the yield of taxadiene of recombinant strain 1 was calculated to be 1093 mg/L.

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Abstract

提供了一种产紫杉二烯的重组真核菌株及利用该重组真核菌株制备紫杉二烯的方法。该重组真核菌株为在该真核菌株中导入表达GGPP合酶A的基因表达盒、表达紫杉二烯合酶的基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、以及表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒；该表达GGPP合酶A的基因表达盒的启动子为TDH1；该表达紫杉二烯合酶的基因表达盒的启动子为ENO2；该表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒的启动子为PDC1；以及该融合基因表达盒的启动子为GPM1。

Description

一种产紫杉二烯的重组真核菌株及利用该重组真核菌株制备紫杉二烯的方法
本申请要求于2014年12月30日提交中国专利局、申请号为201410839706.7、发明名称为“一种产紫杉二烯的重组真核菌株及利用该重组真核菌株制备紫杉二烯的方法”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。
技术领域
本发明属于生物工程技术领域，特别涉及一种产紫杉二烯的重组真核菌株及利用该重组真核菌株制备紫杉二烯的方法。
背景技术
天然化合物主要从以下几个途径得到：(1)从植物中提取；(2)利用化学合成的方法合成；(3)利用合成生物学手段合成。合成生物学是生产大规模且性能稳定的天然化合物的新学科，为直接从植物中提取和化学全合成的一个重要替代方式，这种方式主要是通过基因工程手段将天然化合物合成途径的相关酶基因导入到酵母、大肠杆菌或者植物等一些常用的模式底盘细胞中，在其中重新构建可以生产天然化合物的途径，进而表达天然化合物。人类健康、能源、环境等领域的重大需求牵引着合成生物学的迅猛发展。
紫杉醇是一种效的抗癌药物，传统的获得方法为：从红豆杉中提取获得。但其原料红豆杉自然资源稀缺，且红豆杉中紫杉醇含量仅约为万分之一，所以直接从红豆杉中提取紫杉醇成本太高，也无法满足需求。也有研究学者提出通过植物细胞培养的方法来获得紫杉醇，但是因植物细胞生长周期较长，所以获得紫杉醇的效率也并不高，也不适合大规模生产。而化学合成法合成紫杉醇的步骤复杂，且每个步骤收率很低，导致成本很高、收率低，限制了该方法的应用。合成生物学技术在天然化合物的生物合成方面的成功应用为紫杉醇的制备开辟了新的途径。
紫杉二烯是紫杉醇生物合成途径中的关键前体之一，紫杉二烯是一类 二萜化合物，由4分子异戊二烯基焦磷酸(IPP)经聚合、环化等酶促反应生成。研究发现，短叶红豆杉来源的紫杉二烯合成酶可催化GGPP(geranylgeranyl pyrophosphate，牻牛儿基牻牛儿基焦磷酸)合成紫杉二烯，可通过将该基因导入底盘细胞中进行生物合成紫杉二烯。
目前，在微生物合成紫杉二烯的研究中，所利用的底盘细胞分为原核菌株和真核菌株。对于原核菌株，最主要的是大肠杆菌系统，根据研究报道，2001年，华盛顿州立大学Croteau课题组的Huang等人，将紫杉二烯合成酶基因导入大肠杆菌，通过优化大肠杆菌内源DXP途径，并引入外源GGPP合成酶基因，实现了紫杉二烯在大肠杆菌胞内的生物合成，产量为1.3mg/L；2010年，Ajikumar等利用多元模块代谢工程方法在大肠杆菌中合成紫杉二烯，经过发酵优化后，产量达到1020mg/L。但当此课题组进行后续紫杉二烯-5α-醇的合成时，却只检测到很少量的产物，这可能由于P450酶在缺乏电子传递系统的大肠杆菌的背景下很难有效发挥其催化功能。因此，原核菌株并不适合用于紫杉二烯的生物合成。因此真核系统成为该领域更具有研究前景的底盘细胞。
对于真核系统，目前的研究报道较少，紫杉二烯的产量也较小。Engels等在酵母中通过改造内源途径，对外源基因密码子优化等手段，得到紫杉二烯的产量仅为8.7mg/L。所以还需要新的高产紫杉二烯的重组真核菌株。
发明内容
有鉴于此，本发明的发明目的在于提供一种产紫杉二烯的重组真核菌株及利用该重组真核菌株制备紫杉二烯的方法。本发明提供的产紫杉二烯的重组真核菌株的紫杉二烯产量高，为紫杉二烯的生物合成提供了一种可行的方法。
为了实现本发明的发明目的，本发明采用如下的技术方案：
本发明提供了一种产紫杉二烯的重组真核菌株，在该真核菌株中导入表达GGPP合酶A的基因表达盒、表达紫杉二烯合酶的基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达FPP合酶的基因和 表达GGPP合酶B的基因的融合基因表达盒；
该表达GGPP合酶A的基因表达盒包括启动子、表达GGPP合酶A的基因，该表达GGPP合酶A的基因表达盒的启动子为TDH1；
该表达紫杉二烯合酶的基因表达盒包括启动子、表达紫杉二烯合酶的基因，该表达紫杉二烯合酶的基因表达盒的启动子为ENO2；
该表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒包括启动子、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因，该表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒的启动子为PDC1；
该表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒包括启动子、所述融合基因；所述融合基因表达盒的启动子为GPM1。
在本发明中，GGPP为牻牛儿基牻牛儿基焦磷酸。在本发明中，所述表达GGPP合酶A的基因是指该基因的表达产物具有GGPP合酶A的活性，其催化IPP直接合成GGPP，该表达GGPP合酶A的基因既可以是野生型的也可以是变异的；既可以是真核菌株内源基因，也可以是真核菌株外源基因，只要其表达产物保留该酶的活性即可。优选地，本发明提供的重组真核菌株中，表达GGPP合酶A的基因来源于嗜酸热硫化叶菌，且进行了密码子优化，其具有如SEQ ID NO:1所示的核苷酸序列，标记为GGPPSsa基因。
在本发明中，表达紫杉二烯合酶的基因是指该基因的表达产物具有紫杉二烯合酶的活性，其催化GGPP合成紫杉二烯，该表达紫杉二烯合酶的基因既可以是野生型的也可以是变异的，只要其表达产物保留该酶的活性即可。优选地，本发明提供的重组真核菌株中，表达紫杉二烯合酶的基因来源于短叶红豆杉。在本发明的一些实施例中，表达紫杉二烯合酶的基因来源于短叶红豆杉，且为截短的短叶红豆杉的紫杉二烯合酶的基因。在本发明的另外一些实施例中，该表达紫杉二烯合酶的基因为截短的短叶红豆杉的紫杉二烯合酶的基因，且进行了密码子优化，其具有如SEQ ID NO:2所示的核苷酸序列，标记为tTS基因。
在本发明中，表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因是指该基因的表达产物具有3-羟基-3-甲基戊二酰辅酶A还原酶的活性，其催化 甲羟戊酸(Mevalonate,MVA)途径中的中间产物HMG-CoA(hydroxyl-methyl-glutaryl-CoA)生成甲羟戊酸(mevalonate)。该表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因既可以是野生型的也可以是变异的；既可以是真核菌株内源基因，也可以是真核菌株外源基因，只要其表达产物保留该酶的活性即可。在本发明的一些实施例中，本发明提供的重组真核菌株中，表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因来源于酵母。在本发明的另外一些实施例中，表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因来源于酿酒酵母YNL280C，其具有如SEQ ID NO:3所示的核苷酸序列，标记为tHMGR基因。
在本发明中，所述FPP为法尼基焦磷酸。所述表达FPP合酶的基因是指该基因的表达产物具有FPP合酶的活性，其催化IPP和DMAPP通过缩合形成GPP，GPP再与IPP缩合形成半萜类物质的共同前体FPP。该表达FPP合酶的基因既可以是野生型的也可以是变异的；既可以是真核菌株内源基因，也可以是真核菌株外源基因，只要其表达产物保留该酶的活性即可。在本发明的一些实施例中，本发明提供的重组真核菌株中，表达FPP合酶的基因来源于酵母。在本发明的另外一些实施例中，本发明提供的重组真核菌株中，表达FPP合酶的基因来源于酿酒酵母YNL280C，标记为ERG20基因。
在本发明中，所述表达GGPP合酶B的基因是指该基因的表达产物具有GGPP合酶B的活性，其催化FPP合成GGPP，该表达GGPP合酶B的基因既可以是野生型的也可以是变异的；既可以是真核菌株内源基因，也可以是真核菌株外源基因，只要其表达产物保留该酶的活性即可。在本发明的一些实施例中，本发明提供的重组真核菌株中，表达GGPP合酶B的基因来源于酵母。在本发明的另外一些实施例中，本发明提供的重组真核菌株中，表达GGPP合酶B的基因来源于酵母。在本发明的另外一些实施例中，本发明提供的重组真核菌株中，表达GGPP合酶B的基因来源于酿酒酵母YNL280C，标记为BTS1基因。
在本发明中，本发明提供的重组真核菌株中，表达FPP合酶的基因、表达GGPP合酶B的基因以融合基因的形式被导入真核菌株中，该表达 FPP合酶的基因和表达GGPP合酶B的基因的融合基因的表达产物既具有FPP合酶的活性又具有GGPP合酶B的活性，可以催化IPP和DMAPP通过形成中间体GPP、FPP，最终合成GGPP。在本发明中，表达FPP合酶的基因和表达GGPP合酶B的融合基因，包括表达FPP合酶的基因、表达GGPP合酶的基因和核苷酸序列linker。该核苷酸序列linker起连接表达FPP合酶的基因和表达GGPP合酶的基因的作用。在本发明的一些实施例中，该表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因由表达GGPP合酶B的基因、linker和表达FPP合酶的基因从5’-3’依次连接而成。在本发明的另外一些实施例中，该融合基因中的linker对应的核苷酸序列为：5’-GGTGGTGGTTCT-3’，该融合基因标记为BE基因，其具有如SEQ ID NO:4所示的核苷酸序列。
在本发明中，启动子TDH1来源于酿酒酵母菌株S288c，其具有如SEQ ID NO:8所示的核苷酸序列。
在本发明中，启动子ENO2来源于酿酒酵母菌株S288c，其具有如SEQ ID NO:11所示的核苷酸序列。
在本发明中，启动子PDC1来源于酿酒酵母菌株S288c，其具有如SEQ ID NO:10所示的核苷酸序列。
在本发明中，启动子GPM1来源于酿酒酵母菌株S288c，其具有如SEQ ID NO:9所示的核苷酸序列。
在遗传学中，启动子是指一段能使基因进行转录的脱氧核糖核酸(DNA)序列。启动子可以被RNA聚合酶辨认，并开始转录。在核糖核酸(RNA)合成中，启动子可以和决定转录的开始的转录因子产生相互作用，控制基因表达(转录)的起始时间和表达的程度。在合成生物学的基因工程菌株的构建过程中，为了增加某一个代谢途径中某一些产物的累积或者新的产物的生成，需要将内源性或者外源性相关基因导入到受体细胞中，为了更有利于导入基因的表达，导入基因一般以基因表达盒的形式导入到受体细胞中。然而，在同时导入受体细胞中多个基因表达盒时，选择哪些启动子组合有利于最终产物的表达是无法预知的。本发明意外发现，当表达GGPP合酶A的基因表达盒、表达紫杉二烯合酶的基因表达 盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒同时导入真核菌株时，表达GGPP合酶A的基因表达盒的启动子为TDH1、表达紫杉二烯合酶的基因表达盒的启动子为ENO2、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒的启动子为PDC1、该融合基因表达盒的启动子为GPM1时，相比其他的各个基因表达盒的启动子组合所对应的重组真核菌株在摇瓶发酵阶段的紫杉二烯的产量大幅提高，差异显著，P＜0.05。
在本发明的另外一些实施例中，本发明提供的重组真核菌株中，表达GGPP合酶A的基因表达盒中还包含终止子。在本发明的另外一些实施例中，本发明提供的重组真核菌株中，表达GGPP合酶A的基因表达盒中的终止子选自TEF1、ADH1、TPI1。在本发明的一些实施例中，本发明提供的重组真核菌株中，表达GGPP合酶A的基因表达盒的终止子为TEF1。在本发明中，表达GGPP合酶A的基因表达盒中的终止子不受本发明提供的终止子的限制，本领域技术人员可以根据实际情况选择终止子。在本发明的另外一些实施例中，本发明提供的重组真核菌株中，表达GGPP合酶A的基因表达盒的核苷酸序列为：从5’端到3’端依次连接有启动子、表达GGPP合酶A的基因和终止子；启动子和表达GGPP合酶A的基因直接相连，表达GGPP合酶A的基因和终止子直接相连。
在本发明的另外一些实施例中，本发明提供的重组真核菌株中，表达紫杉二烯合酶的基因表达盒中还包含终止子。在本发明的另外一些实施例中，本发明提供的重组真核菌株中，表达紫杉二烯合酶的基因表达盒中的终止子选自HXT7、PGI1、FBA1。在本发明的另外一些实施例中，本发明提供的重组真核菌株中，表达紫杉二烯合酶的基因表达盒中的终止子为HXT7。在本发明中，表达紫杉二烯合酶的基因表达盒中的终止子不受本发明提供的终止子的限制，本领域技术人员可以根据实际情况选择终止子。在本发明的另外一些实施例中，本发明提供的重组真核菌株中，表达紫杉二烯合酶的基因表达盒的核苷酸序列为：从5’端到3’端依次连接有启动子、表达紫杉二烯合酶的基因和终止子；启动子和表达紫杉二烯合酶的基因直接相连，表达紫杉二烯合酶的基因和终止子直接相连。
在本发明的另外一些实施例中，本发明提供的重组真核菌株中，表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒中还包括终止子。在本发明的另外一些实施例中，本发明提供的重组真核菌株中，表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒中的终止子选自TEF2、GPD、PGK1。在本发明的另外一些实施例中，本发明提供的重组真核菌株中，表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒中的终止子为TEF2。在本发明中，表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒中的终止子不受本发明提供的终止子的限制，本领域技术人员可以根据实际情况选择终止子。在本发明的另外一些实施例中，本发明提供的重组真核菌株中，表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒的核苷酸序列为：从5’端到3’端依次连接有启动子、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因和终止子；启动子和表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因直接相连，表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因和终止子直接相连。
在本发明的另外一些实施例中，本发明提供的重组真核菌株中，表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒中还包含终止子。在本发明的另外一些实施例中，本发明提供的重组真核菌株中，表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒中的终止子选自TPI1、TDH2、CYC1。在本发明的另外一些实施例中，本发明提供的重组真核菌株中，表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒中的终止子为TPI1。在本发明中，表达FPP合酶的基因和所述表达GGPP合酶B的基因的融合基因表达盒中的终止子不受本发明提供的终止子的限制，本领域技术人员可以根据实际情况选择终止子。在本发明的另外一些实施例中，本发明提供的重组真核菌株中，表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒的核苷酸序列为：从5’端到3’端依次连接有启动子、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因和终止子；启动子和表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因直接相连，表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因和终止子直接相连。
优选地，本发明提供的重组真核菌株中，所用的真核菌株为酵母菌。更优选地，本发明提供的重组真核菌株中，所用的酵母菌为酿酒酵母。在本发明的一些实施例中，本发明提供的重组真核菌株中，所用的酿酒酵母为YSG50、CEN.PK2、BY4741或YNL280C。在本发明的另外一些实施例中，本发明提供的重组真核菌株中，酿酒酵母具体为YNL280C，其为来源于S288c的酿酒酵母单敲菌株。
优选地，本发明提供的重组真核菌株中，表达GGPP合酶A的基因表达盒、表达紫杉二烯合酶的基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒各自独立地存在于重组真核菌株之内，存在方式各自独立地选自：存在于游离型的载体上，或整合到所述真核菌株的基因组中。
优选地，本发明提供的重组真核菌株中，表达GGPP合酶A的基因表达盒、表达紫杉二烯合酶的基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒中至少一个整合在真核菌株的基因组中。
优选地，本发明提供的重组真核菌株中，整合的整合位点为所用真核菌株的基因组的多拷贝位点。
在本发明的一些实施例中，本发明提供的重组真核菌株中，多拷贝位点为Delta位点。
在本发明的一些实施例中，本发明提供的重组真核菌株中，表达GGPP合酶A的基因表达盒、表达紫杉二烯合酶的基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒都整合在真核菌株的基因组的多拷贝位点。
在本发明的一些实施例中，本发明提供的重组真核菌株中，表达GGPP合酶A的基因表达盒、表达紫杉二烯合酶的基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒以基因功能模块的形式整合 到真核菌株的基因组中。在本发明的另外一些实施例中，表达GGPP合酶A的基因表达盒、表达紫杉二烯合酶的基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒以基因功能模块的形式整合到真核菌株的基因组的多拷贝位点。
在本发明中，本发明提供的重组真核菌株中表达GGPP合酶A的基因表达盒、表达紫杉二烯合酶的基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒以基因功能模块的形式整合到真核菌株的基因组的多拷贝位点时，表达GGPP合酶A的基因表达盒、表达紫杉二烯合酶的基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒在基因功能模块中的排列顺序不固定。各个基因表达盒之间可以通过一段短的核苷酸序列连接，例如终止子，也可以直接相连。在本发明的另外一些实施例中，本发明提供的重组真核菌株中，以上四个基因表达盒在基因功能模块中的排列顺序为：表达GGPP合酶A的基因表达盒、表达紫杉二烯合酶的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达紫杉二烯合酶的基因表达盒的前后顺序排列。在本发明的另外一些实施例中，本发明提供的重组真核菌株中在真核菌株中导入表达GGPP合酶A的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达紫杉二烯合酶的基因表达盒后，这四个基因表达盒以连续的相互衔接的方式存在于真核菌株细胞之内，整合到真核菌株的基因组Delta位点上。
优选地，本发明提供的重组真核菌株中，当表达GGPP合酶A的基因表达盒、表达紫杉二烯合酶的基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒以基因功能模块的形式整合到真核菌株的基因组的多拷贝位点时，该基因功能模块中还包括筛选标记基因，以便于重组 真核菌株的筛选，筛选标记可以为His、Lys、Leu或Ura。在本发明的一些实施例中，本发明提供的重组真核菌株中的基因功能模块中的筛选标记为URA3基因。本发明提供的重组真核菌株中的基因功能模块中的筛选标记基因不局限于本发明提供的筛选标记基因，本领域技术人员可以根据实际需求选择筛选标记基因。
在本发明的一些实施例中，本发明提供的重组真核菌株中，整合到真核菌株多拷贝位点的基因功能模块包括以下基因：筛选标记基因、表达GGPP合酶A的基因表达盒、表达紫杉二烯合酶的基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒；其在重组真核菌株基因组中的排列顺序(5’-3’)如下所示：
DNA分子1—DNA分子2—DNA分子3—DNA分子4—DNA分子5
其中，DNA分子1代表筛选标记基因，DNA分子2代表表达GGPP合酶A的基因表达盒、DNA分子3代表表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒、DNA分子4代表表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、DNA分子5代表表达紫杉二烯合酶的基因表达盒。其中，DNA分子1、DNA分子2直接相连；DNA分子2、DNA分子3直接相连；DNA分子3、DNA分子4直接相连；DNA分子4、DNA分子5直接相连。
本发明还提供了一种产紫杉二烯的重组真核菌株，其保藏编号为CGMCC No.10074。该重组真核菌株为重组酿酒酵母菌株，以酿酒酵母作为出发菌株，通过基因工程技术构建获得。该重组酿酒酵母菌株，分类命名为酿酒酵母(Saccharomyces cerevisiae)，于2014年11月28日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏编号为：CGMCC No.10074。
本发明还提供了一种产紫杉二烯的重组真核菌株的构建方法，包括以下步骤：
步骤1：获得表达GGPP合酶A的基因表达盒、表达紫杉二烯合酶的基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒；
表达GGPP合酶A的基因表达盒包括启动子、表达GGPP合酶A的基因；表达GGPP合酶A的基因表达盒的启动子为TDH1；
表达紫杉二烯合酶的基因表达盒包括启动子、表达紫杉二烯合酶的基因；所述表达紫杉二烯合酶的基因表达盒的启动子为ENO2；
表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒包括启动子、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因；表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒的启动子为PDC1；
表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒包括启动子、所述融合基因；所述融合基因表达盒的启动子为GPM1；
步骤2：取表达GGPP合酶A的基因表达盒、表达紫杉二烯合酶的基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒，导入真菌菌株中，即得。
在本发明的一些实施例中，本发明提供的构建方法中，步骤2中导入真菌菌株中之后，还包括在真核菌株中发生同源重组，表达GGPP合酶A的基因表达盒、表达紫杉二烯合酶的基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒整合到真核菌株的基因组中的步骤。
在本发明的另外一些实施例中，本发明提供的构建方法中，步骤2中的整合的整合位点为真核菌株的多拷贝位点。在本发明的另外一些实施例中，给多拷贝位点为Delta位点。
在本发明的另外一些实施例中，本发明提供的重组真核菌株的构建方法，包括：
步骤1：获得筛选标记基因、表达GGPP合酶A的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒、表达 3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达紫杉二烯合酶的基因表达盒；
表达GGPP合酶A的基因表达盒包括启动子、表达GGPP合酶A的基因；表达GGPP合酶A的基因表达盒的启动子为TDH1；
表达紫杉二烯合酶的基因表达盒包括启动子、表达紫杉二烯合酶的基因；所述表达紫杉二烯合酶的基因表达盒的启动子为ENO2；
表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒包括启动子、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因；表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒的启动子为PDC1；
表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒包括启动子、所述融合基因；所述融合基因表达盒的启动子为GPM1；
步骤2：取步骤1所得筛选标记基因、表达GGPP合酶A的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达紫杉二烯合酶的基因表达盒，导入真核菌株中，经同源重组，整合到真核菌株基因组中，即得。
在本发明的另外一些实施例中，本发明提供的重组真核菌株的构建方法中，步骤1中的筛选标记基因的5’端连接有真核菌株底盘细胞整合位点第一同源臂序列，在其3’端连接有一个终止子。该终止子的作用是为筛选标记基因、表达GGPP合酶A的基因表达盒在真核菌株底盘细胞中发生同源重组时提供同源臂。在本发明的另外一些实施例中，该终止子具体为CYC1。该真核菌株底盘细胞整合位点第一同源臂序列的作用是为真核菌株底盘细胞基因组、重组DNA分子发生同源重组时提供同源臂。在本发明的另外一些实施例中，该第一同源臂序列为能与真核菌株基因组的多拷贝位点发生同源重组的序列。在本发明的另外一些实施例中，当真核菌株为酵母时，该第一同源臂序列为能与酵母基因组的Delta位点发生同源重组的序列，标记为Delta1。
在本发明的另外一些实施例中，本发明提供的重组真核菌株的构建方法中，步骤1中的表达GGPP合酶A的基因表达盒的5’端连接有一个终止 子，该终止子的作用是为筛选标记基因、表达GGPP合酶A的基因表达盒在真核菌株底盘细胞中发生同源重组时提供同源臂，该终止子与筛选标记基因3’端连接的终止子相同，使得筛选标记基因、表达GGPP合酶A的基因表达盒在真核菌株底盘细胞中发生同源重组，前后依次相连。在本发明的一些实施例中，该终止子具体为CYC1。
在本发明的另外一些实施例中，本发明提供的重组真核菌株的构建方法中，步骤1中的表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒的5’端连接有一个终止子，该终止子的作用是为表达GGPP合酶A的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒在真核菌株底盘细胞中发生同源重组时提供同源臂，该终止子与表达GGPP合酶A的基因表达盒中的3’端终止子相同，使得表达GGPP合酶A的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒在真核菌株底盘细胞中发生同源重组，前后依次相连。在本发明的一些实施例中，该终止子具体为TEF1。
在本发明的另外一些实施例中，本发明提供的重组真核菌株的构建方法中，步骤1中表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒的5’端连接有一个终止子，该终止子的作用是为表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒在真核菌株底盘细胞中发生同源重组时提供同源臂，该终止子与表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒中的3’端终止子相同，使得表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒在真核菌株底盘细胞中发生同源重组，前后依次连接。在本发明的另外一些实施例中，该终止子具体为TPI1。
在本发明的另外一些实施例中，步骤1中的表达紫杉二烯合酶的基因表达盒的5’端连接有一个终止子，3’端连接有一个真核菌株底盘细胞整合位点第二同源臂序列。该终止子的作用是为表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达紫杉二烯合酶的基因表达盒在真核菌株底盘细胞中发生同源重组时提供同源臂。在本发明的另外一些实施例中，该终 止子与表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒中的3’端的终止子相同。在本发明的另外一些实施例中，该终止子具体为TEF2。该真核菌株底盘细胞整合位点第二同源臂序列的作用是为真核菌株底盘细胞基因组、重组DNA分子发生同源重组时提供同源臂。在本发明的另外一些实施例中，该第二同源臂序列为能与真核菌株底盘细胞基因组中的多拷贝位点发生同源重组的序列。在本发明的另外一些实施例中，当真核菌株为酵母时，该第二同源臂序列为能与酵母基因组的Delta位点发生同源重组的序列，标记为Delta2。
在本发明的另外一些实施例中，按照本发明提供的重组真核菌株的构建方法，筛选标记基因、表达GGPP合酶A的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达紫杉二烯合酶的基因表达盒整合到真核菌株底盘细胞的基因组多拷贝位点的基因功能模块包括：筛选标记基因、表达GGPP合酶A的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达紫杉二烯合酶的基因表达盒；其在重组真核菌株基因组中的排列顺序(5’-3’)如下所示：
DNA分子1—DNA分子2—DNA分子3—DNA分子4—DNA分子5
其中，DNA分子1代表筛选标记基因，DNA分子2代表表达GGPP合酶A的基因表达盒、DNA分子3代表表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒、DNA分子4代表表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、DNA分子5代表表达紫杉二烯合酶的基因表达盒。其中，DNA分子1、DNA分子2直接相连；DNA分子2、DNA分子3直接相连；DNA分子3、DNA分子4直接相连；DNA分子4、DNA分子5直接相连。
本发明还提供了一种利用本发明提供的重组真核菌株制备紫杉二烯的方法，该重组真核菌株为在该真核菌株中导入表达GGPP合酶A的基因表达盒、表达紫杉二烯合酶的基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的 融合基因表达盒；
该表达GGPP合酶A的基因表达盒包括启动子、表达GGPP合酶A的基因，该表达GGPP合酶A的基因表达盒的启动子为TDH1；
该表达紫杉二烯合酶的基因表达盒包括启动子、表达紫杉二烯合酶的基因，该表达紫杉二烯合酶的基因表达盒的启动子为ENO2；
该表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒包括启动子、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因，该表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒的启动子为PDC1；
该表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒包括启动子、所述融合基因；所述融合基因表达盒的启动子为GPM1；
该制备紫杉二烯的方法包括：
培养重组真核菌株，从培养物中回收紫杉二烯，即得。
优选地，本发明提供的制备紫杉二烯的方法中，当真核菌株为酵母时，培养过程中，包括以下步骤：
取重组酵母菌，扩大培养后，接种于发酵培养基中，经发酵培养、两相培养，收集紫杉二烯，即得。
在本发明的另外一些实施例中，本发明提供的制备紫杉二烯的方法中，当真核菌株为酵母时，两相培养过程中，以批式补加或流加的方式补加碳源葡萄糖。实验结果证实，当在两相培养过程中补加葡萄糖之后，进一步提高了紫杉二烯的产量。
在本发明的另外一些实施例中，本发明提供的制备紫杉二烯的方法中，当真核菌株为酵母时，以流加的方式补加碳源，具体为向发酵液中连续流加葡萄糖，维持发酵液中的葡萄糖浓度在0g/L～1g/L。
在本发明的另外一些实施例中，本发明提供的制备紫杉二烯的方法中，当真核菌株为酵母时，发酵培养基包括20g/L～50g/L的葡萄糖、10g/L的酵母粉、20g/L～30g/L的蛋白胨、0g/L～8g/L的KH2PO4和0g/L～6g/L的MgSO4。
在本发明的另外一些实施例中，本发明提供的制备紫杉二烯的方法中，当真核菌株为酵母时，发酵培养基包括30g/L～50g/L的葡萄糖、10g/L 的酵母粉、20g/L～30g/L的蛋白胨、0.5g/L～8g/L的KH2PO4和0.5g/L～6g/L的MgSO4。
在本发明的另外一些实施例中，本发明提供的制备紫杉二烯的方法中，当真核菌株为酵母时，发酵培养的温度为30℃。
在本发明的另外一些实施例中，本发明提供的制备紫杉二烯的方法中，当真核菌株为酵母时，两相培养的温度为30℃。
在本发明的另外一些实施例中，本发明提供的制备紫杉二烯的方法中，当真核菌株为酵母时，两相培养的时间为70h～174h。在本发明的另外一些实施例中，两相培养的时间为138h。
本发明提供了一种产紫杉二烯的重组真核菌株及利用该重组真核菌株制备紫杉二烯的方法。该产紫杉二烯的重组真核菌株为在该真核菌株中导入表达GGPP合酶A的基因表达盒、表达紫杉二烯合酶的基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒；该表达GGPP合酶A的基因表达盒包括启动子、表达GGPP合酶A的基因；表达GGPP合酶A的基因表达盒的启动子为TDH1；表达紫杉二烯合酶的基因表达盒包括启动子、表达紫杉二烯合酶的基因；表达紫杉二烯合酶的基因表达盒的启动子为ENO2；表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒包括启动子、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因；表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒的启动子为PDC1；表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒包括启动子、该融合基因；该融合基因表达盒的启动子为GPM1。实验结果发现，本发明提供的产紫杉二烯的重组真核菌株的紫杉二烯的产量高，摇瓶发酵后所有菌株的紫杉二烯产量为161.82mg/L，该重组菌株的紫杉二烯产量显著高于其他重组菌株，差异显著，P＜0.05。在本发明的另外一些实施例中，通过对发酵条件的优化，进一步提高了重组酿酒酵母的紫杉二烯的产量，为后续紫杉醇生物合成提供了物质基础。
附图说明
图1示实施例1中GGPPSsa重组载体1的检测结果；其中图1-A为GGPPSsa重组载体1、GGPPSsa重组载体2、GGPPSsa重组载体3的双酶切验证凝胶电泳图；图1-B为GGPPSsa基因表达盒的核苷酸序列信息；
图2示实施例1中BE重组载体1的检测结果，其中图2为BE重组载体1、BE重组载体3、BE重组载体4的双酶切验证凝胶电泳图；
图3示实施例1中tHMGR重组载体1的检测结果，其中图3为tHMGR重组载体1、tHMGR重组载体2的双酶切验证凝胶电泳图；
图4示实施例1中tTS重组载体1的检测结果，其中图4为tTS重组载体1、tTS重组载体2、tTS重组载体3的单酶切验证凝胶电泳图；
图5示实施例1中重组菌株的基因组PCR验证结果，其中，图5-A为重组菌株1的验证结果；图5-B为重组菌株1中整合到该菌株的基因组的Delta位点的核苷酸序列信息；图5-C为重组菌株2的验证结果；
图6示实施例1中紫杉二烯标准品的标准曲线；
图7示实施例1中紫杉二烯标准品的GC-TOF/MS检测结果，其中图7-A为紫杉二烯标准品的MS检测结果；图7-B为紫杉二烯标准品的GC检测结果；
图8示实施例1中重组菌株1发酵后萃取所得样品的GC-TOF/MS检测结果，其中图8-A为样品的MS检测结果；图8-B为样品的GC检测结果；
图9示实施例1中重组菌株2发酵后萃取所得样品的GC-TOF/MS检测结果，其中图9-A为样品的MS检测结果；图9-B为样品的GC检测结果；
图10示实施例2中重组菌株1发酵后萃取所得样品的GC-TOF/MS检测结果，其中图10-A为样品的MS检测结果；图10-B为样品的GC检测结果；
图11示实施例3中重组菌株1批式补料发酵培养后萃取所得样品的GC-TOF/MS检测结果，其中图11-A为样品的MS检测结果；图11-B为样品的GC检测结果；
图12示实施例4中重组菌株1流加补料发酵培养后萃取所得样品的GC-TOF/MS检测结果，其中图12-A为样品的MS检测结果；图12-B为样品的GC检测结果。
生物保藏说明
重组酿酒酵母菌株SyBE_Sc00011203：分类命名：酿酒酵母(Saccharomyces cerevisiae)，于2014年11月28日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏单位地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，保藏编号为：CGMCC No.10074。
具体实施方式
本发明公开了一种产紫杉二烯的重组真核菌株及利用该重组真核菌株制备紫杉二烯的方法，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。
本发明提供的一种产紫杉二烯的重组真核菌株及利用该重组真核菌株制备紫杉二烯的方法中所用到的试剂和原料均可由市场购得。
为了使本技术领域的技术人员能够更好地理解本发明的技术方案，下面结合实施例，进一步阐述本发明：
实施例1 产紫杉二烯的重组酿酒酵母菌株的构建与紫杉二烯产量的研究
实验材料的来源：
酿酒酵母单敲菌株YNL280C购买于Open Biosystems，Huntsville，AL；载体pRS425购买于Addgene(American)，将该载体上的氨苄霉素抗性基因通过酵母同源重组的方法替换为卡那霉素抗性基因，得到pRS425K。
大肠杆菌TransT1购买于北京全式金生物技术有限公司。
表达GGPP合酶A的基因(外源基因)：根据嗜酸热硫化叶菌来源的GGPP合成酶基因GGPPSsa进行密码子优化，在金唯智公司进行合成得 到，其具有如SEQ ID NO:1所示的核苷酸序列，标记为GGPPSsa基因。表达紫杉二烯合酶的基因(外源基因)：根据短叶红豆杉来源的紫杉二烯合酶基因进行密码子优化，在金唯智公司进行合成得到，标记为tTS基因，其具有如SEQ ID NO:2所示的核苷酸序列。
表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因(内源基因)：设计带有启动子同源序列的前引物、带有终止子同源序列的后引物，以酿酒酵母单敲菌株YNL280C为模板，通过PCR扩增，得到表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因，其具有如SEQ ID NO:3所示的核苷酸序列，标记为tHMGR基因。
表达FPP合酶的基因(内源基因)：设计带有启动子同源序列的前引物、带有终止子同源序列的后引物，以酿酒酵母单敲菌株YNL280C为模板，通过PCR扩增，得到表达FPP合酶的基因，标记为ERG20基因。
表达GGPP合酶B的基因(内源基因)：设计带有启动子同源序列的前引物、带有终止子同源序列的后引物，以酿酒酵母单敲菌株YNL280C为模板，通过PCR扩增，得到表达GGPP合酶B的基因，标记为BTS1基因。
表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因：将ERG20基因和BTS1基因进行OE-PCR(重叠PCR)，得到ERG20基因和BTS1基因通过linker(5’-GGTGGTGGTTCT-3’)连接的融合基因，其具有如SEQ ID NO:4所示的核苷酸序列，标记为BE基因。
筛选标记基因URA3基因：其具有如SEQ ID NO:5所示的核苷酸序列。
Delta位点上游248bp同源序列Delta1：其具有如SEQ ID NO:6所示的核苷酸序列。
Delta位点下游239bp同源序列Delta2：其具有如SEQ ID NO:7所示的核苷酸序列。
启动子：来源于YNL280C，各个基因所对应的启动子和启动子的核苷酸信息见表1。
终止子：来源于YNL280C。各个基因所对应的终止子和终止子的核苷酸序列信息见表1。
表1 各个基因所对应的启动子、终止子及其核苷酸信息
Figure PCTCN2015075447-appb-000001
实验方法：
1、不同基因表达盒的构建：
(1)表达GGPP合酶A的基因表达盒的构建：
分别取GGPPSsa基因的启动子，将终止子1、启动子、GGPPSsa基因和终止子2通过OE-PCR方法拼接起来，得到两端包含Pst1和BamH1酶切位点的片段，连入载体pRS425K，得到3个重组载体，分别命名为：GGPPSsa重组载体1、GGPPSsa重组载体2、GGPPSsa重组载体3。
将上述构建的重组载体分别转化入大肠杆菌TransT1中，菌落PCR筛选，
提质粒，进行双酶切验证以及测序验证，以确保目的片段连接正确且碱基序列未发生突变。
分别取GGPPSsa重组载体1、GGPPSsa重组载体2和GGPPSsa重组载体3，进行双酶切验证，所得检测结果见图1，其中图1-A为包含GGPPSsa重组载体1、GGPPSsa重组载体2和GGPPSsa重组载体3双酶切验证的胶图。其中，GGPPSsa重组载体1的基因表达盒长度为2489bp，载体长度为 6749bp，与图中对应DNA条带大小相符；测序验证也正确。采用相同的方法验证GGPPSsa重组载体2、GGPPSsa重组载体3的正确性，经检测，本实施例成功获得了含不同启动子的表达GGPP合酶A的基因表达盒，GGPPSsa基因表达盒中的核苷酸序列信息如图1-B所示。
分别取验证正确的重组菌株，提取质粒，进行Pst1和BamH1双酶酶切，回收酶切产物，即得含表达GGPP合酶A的基因表达盒。各个重组载体、各个表达GGPP合酶A的基因表达盒的信息见表2。
表2 各个重组载体、各个表达GGPP合酶A的基因表达盒中所含有基因信息
重组载体名称 基因表达盒名称 5’-3’依次连接的各个基因
GGPPSsa重组载体1 GGPPSsa基因表达盒1 终止子CYC1、启动子TDH1、GGPPSsa基因、终止子TEF1
GGPPSsa重组载体2 GGPPSsa基因表达盒2 终止子CYC1、启动子TPI1、GGPPSsa基因、终止子TEF1
GGPPSsa重组载体3 GGPPSsa基因表达盒3 终止子CYC1、启动子HXT7、GGPPSsa基因、终止子TEF1
(2)BE基因表达盒的构建：
分别取BE基因的启动子，将终止子TEF1、启动子、BE基因和终止子TPI1通过OE-PCR方法拼接起来，得到两端包含Pst1和BamH1酶切位点的片段，连入载体pRS425K，得到4个重组载体，分别命名为：BE重组载体1、BE重组载体2、BE重组载体3、BE重组载体4。
将上述构建的重组载体分别转化入大肠杆菌TransT1中，菌落PCR筛选，提质粒，进行双酶切验证以及测序验证，以确保目的片段连接正确且碱基序列未发生突变。
取BE重组载体1，进行双酶切验证，检测结果见图2，其中包含BE重组载体1、BE重组载体3和BE重组载体4双酶切验证的胶图，BE重组载体1的基因表达盒长度为3751bp，载体长度为6749bp，与图中对应DNA条带大小相符；测序验证也正确。采用相同的方法验证重组载体BE重组载体2、BE重组载体3、BE重组载体4的正确性，经检测，本实施例成功获得了含不同启动子的BE基因表达盒。
分别取验证正确的重组菌株，提取质粒，进行Pst1和BamH1双酶酶切， 回收酶切产物，即得含BE基因表达盒。各个重组载体、各个BE基因表达盒的信息见表3。
表3 各个重组载体、各个BE基因表达盒中所含有基因信息
重组载体名称 基因表达盒名称 5’-3’依次连接的各个基因
BE重组载体1 BE基因表达盒1 终止子TEF1、启动子GPM1、BE基因和终止子TPI1
BE重组载体2 BE基因表达盒2 终止子TEF1、启动子HXK2、BE基因和终止子TPI1
BE重组载体3 BE基因表达盒3 终止子TEF1、启动子TEF1、BE基因和终止子TPI1
BE重组载体4 BE基因表达盒4 终止子TEF1、启动子TDH2、BE基因和终止子TPI1
(3)表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒的构建：
分别取tHMGR基因的启动子，将终止子TPI1、启动子、tHMGR基因和终止子TEF2通过OE-PCR方法拼接起来，得到两端包含Pst1和BamH1酶切位点的片段，连入载体pRS425K，得到2个重组载体，分别命名为：tHMGR重组载体1、tHMGR重组载体2。
将上述构建的重组载体分别转化入大肠杆菌TransT1中，菌落PCR筛选，提质粒，进行双酶切验证以及测序验证，以确保目的片段连接正确且碱基序列未发生突变。
取tHMGR重组载体1，进行双酶切检测，检测结果见图3，其中包含tHMGR重组载体1和tHMGR重组载体2双酶切验证的胶图，tHMGR重组载体1的基因表达盒长度为3086bp，载体长度为6749bp，与图中对应DNA条带大小相符；测序验证也正确。按照相同的检测方法，取重组载体tHMGR重组载体2，进行检测，经检测，本实施例成功获得了含不同启动子的tHMGR基因表达盒。
分别取验证正确的重组菌株，提取质粒，进行Pst1和BamH1双酶酶切，回收酶切产物，即得含tHMGR基因的基因表达盒。各个重组载体、各个tHMGR基因表达盒的信息见表4。
表4 各个重组载体、各个tHMGR基因表达盒中所含有基因信息
重组载体名称 基因表达盒名称 5’-3’依次连接的各个基因
tHMGR重组载体1 tHMGR基因表达盒1 终止子TPI1、启动子PDC1、tHMGR基因和终止子TEF2
tHMGR重组载体2 tHMGR基因表达盒2 终止子TPI1、启动子PGK1、tHMGR基因和终止子TEF2
(4)表达紫杉二烯合酶的基因表达盒的构建：
分别取tTS基因的启动子，将终止子TEF2、启动子、tTS基因、终止子HXT7和Delta位点下游239bp同源序列Delta2通过OE-PCR方法拼接起来，得到两端包含Not1酶切位点的片段，连入载体pRS425K，得到3个重组载体，分别命名为：tTS重组载体1、tTS重组载体2、tTS重组载体3。
将上述构建的重组载体分别转化入大肠杆菌TransT1中，菌落PCR筛选，提质粒，进行单酶切验证以及测序验证，以确保目的片段连接正确且碱基序列未发生突变。
取tTS重组载体1，进行单酶切检测，检测结果见图4，其中图4-A为包含tTS重组载体1、tTS重组载体2和tTS重组载体3单酶切验证的胶图，tTS重组载体1的基因表达盒长度为4147bp，载体长度为6749bp，与图中对应DNA条带大小相符；测序验证也正确。按照相同的检测方法检测tTS重组载体2、tTS重组载体3的正确性，经检测，本实施例成功获得了含不同启动子的tTS基因表达盒。
分别取验证正确的重组菌株，提取质粒，进行Not1酶切，回收酶切产物，即得含tTS基因的基因表达盒。各个重组载体、各个tTS基因表达盒的信息见表5。
表5 各个重组载体、各个tTS基因表达盒中所含有基因信息
重组载体名称 基因表达盒名称 5’-3’依次连接的各个基因
tTS重组载体1 tTS基因表达盒1 终止子TEF2、启动子ENO2、tTS基因、终止子HXT7、Delta2
tTS重组载体2 tTS基因表达盒2 终止子TEF2、启动子FBA1、tTS基因、终止子HXT7、Delta2
tTS重组载体3 tTS基因表达盒3 终止子TEF2、启动子TEF2、tTS基因、终止子HXT7、Delta2
(5)含筛选标记基因URA3基因表达盒的构建
将Delta位点上游248bp同源序列Delta1、筛选标记基因URA3基因和终 止子CYC1通过OE-PCR方法拼接起来，得到两端包含Not1酶切位点的片段，连入载体pRS425K，得重组载体。
将上述构建的重组载体分别转化入大肠杆菌TransT1中，菌落PCR筛选，提质粒，进行单酶切验证以及测序验证，以确保目的片段连接正确且碱基序列未发生突变。经检测，本实施例成功获得了含筛选标记基因URA3基因表达盒。
取验证正确的重组菌株，提取质粒，进行Not1酶切，回收酶切产物，即得含筛选标记基因URA3基因表达盒，该表达盒中所带有基因信息为从5’端到3’端依次连接有：Delta1、筛选标记基因URA3基因和终止子CYC1。2、不同启动子组合调控的紫杉二烯生物合成途径的重组酵母菌株的获得取上述5类基因表达盒、底盘细胞酿酒酵母YNL280C，设计不同的启动子组合方式，采用醋酸锂法将5片段进行酵母转化，利用酵母自身的同源重组原理将上述片段通过片段间的同源序列连接起来并通过与酵母基因组上Delta位点的同源序列发生重组而整合到基因组上。转化后酵母采用SD-drop固体培养基(合成酵母氮源YNB 6.7g/L，葡萄糖20g/L，混合氨基酸粉末2g/L，固体补加2％的琼脂粉)进行筛选，得到的转化子进行划线分纯后转移至液体培养基中培养24h，提取酵母基因组作为模板，进行PCR验证，确认正确的重组菌株，平板划线或甘油菌保存。
所得重组酿酒酵母菌株名称，及重组酿酒酵母菌株中整合的重组基因信息见表6。
表6 各个重组酿酒酵母菌株及重组酿酒酵母菌株中整合的重组基因信息
Figure PCTCN2015075447-appb-000002
Figure PCTCN2015075447-appb-000003
Figure PCTCN2015075447-appb-000004
注：其中“p”代表启动子，例如“TDH1p”代表启动子TDH1；
取重组菌株1，提取酵母基因组作为模板，进行PCR验证，其中所使用的四组验证引物以及对应的核苷酸信息见表7。
表7 重组菌株1PCR验证所用的引物组
Figure PCTCN2015075447-appb-000005
其中，使用第一组引物扩增得到的片段大小应为1020bp，第二组引物扩增片段大小为2101bp，第三组引物扩增片段大小为1591bp，第四组引物扩增片段大小为2430bp。重组菌株1的PCR验证结果见图5-A，其中，泳道1代表第一组引物的扩增结果；泳道2代表第二组引物的扩增结果；泳道3代表第三组引物的扩增结果；泳道4代表第四组引物的扩增结果。从图中可知，所有引物组扩增结果与理论大小相符，说明成功获得了整合了目的基因的重组菌株1，整合到重组菌株基因组Delta位点的核苷酸序列信息如图5-B所示。
取重组菌株2，提取酵母基因组作为模板，进行PCR验证，其中使用四组验证引物以及对应的核苷酸信息见表8。
表8 重组菌株2PCR验证所用的引物组
Figure PCTCN2015075447-appb-000006
Figure PCTCN2015075447-appb-000007
其中，使用第一组引物扩增的到的片段大小应为1025bp，第二组引物扩增片段大小为2091bp，第三组引物扩增片段大小为1591bp，第四组引物扩增片段大小为2430bp。重组菌株2的PCR验证结果见图5-C，其中，泳道1代表第一组引物的扩增结果；泳道2代表第二组引物的扩增结果；泳道3代表第三组引物的扩增结果；泳道4代表第四组引物的扩增结果。从图中可知，所有引物组扩增结果与理论大小相符，说明成功获得了整合了目的基因的重组菌株2。
采用相同的检测方法验证重组菌株3至重组菌株8的正确性，得成功获得了各个重组菌株。将确认正确的重组菌株，平板划线或甘油菌保存。3、在摇瓶上研究各个重组菌株的发酵性能
培养方法：
种子培养基：合成酵母氮源YNB 6.7g/L，葡萄糖20g/L，混合氨基酸粉末2g/L(具体配方参考[美]D.C.安伯格等酵母遗传学方法试验指南)，组氨酸38mg/L，色氨酸38mg/L，亮氨酸190mg/L，pH5.8，115℃灭菌15min。发酵培养基A：葡萄糖20g/L，酵母粉10g/L，蛋白胨20g/L，pH5.8，121℃灭菌20min。
将上述重组菌株1至重组菌株8分别接种于5mL种子培养基中，在30℃、220rpm培养24h，以初始菌体浓度OD600＝0.2转接于新鲜的5mL种子培养基中，于30℃、220rpm条件下培养12h，以初始菌体浓度OD600＝0.1分别接种于50mL发酵培养基A中，于30℃、220rpm条件下培养70h，测定菌体生长曲线。在发酵10h时向发酵液中加入2.5mL正十二烷进行两相培养，发酵结束后收集上层乳浊液相12000rpm离心20min，收集上层有机相，使 用无水硫酸钠除去水分，使用0.22μm滤膜过滤后测定终产物紫杉二烯浓度。
分析方法：
以722型分光光度计在600nm处测定的菌体吸光值(OD600)表征菌体浓度。
紫杉二烯浓度采用GC-TOF/MS(Waters Corp.，USA)测定，硅胶毛细管柱为30m×0.25mm×0.25μm DB-5MS，J&W Scientific，Folsom，电离方式为电子轰击电离EI+，电子束能量70eV，离子化电流40μA。色谱条件为：进样口温度设置为260℃，柱温起始温度200℃保持3min，然后以4℃/min的速度升温至270℃，保持2min。
分离纯化紫杉二烯标准品，配置浓度梯度，运用GC-TOF/MS绘制标准曲线，所得标准曲线见图6，得标准曲线满足：y＝55.867x-2711.9(R2＝0.999)，确定线性范围为75mg/L-250mg/L。标准品的GC-TOF/MS检测图片见图7，其中，图7-A为紫杉二烯标准品的MS检测结果；图7-B为紫杉二烯标准品的GC检测结果。重组菌株发酵后萃取得到的样品进行测定，按拟合曲线公式计算产量。
实验结果：
重组酿酒菌株重组菌株1至重组菌株7在发酵培养基A中生长趋势一致，生长状况无明显差别，发酵结束时菌体最终OD600达到25左右。
重组酿酒酵母重组菌株1至重组菌株8在发酵培养基A中进行发酵培养之后，取重组菌株发酵后萃取所得样品进行GC-TOF/MS检测，以定性和定量所得样品。取重组菌株1所得样品进行检测，检测结果见图8，其中图8-A为该样品的MS检测结果，与标准品出峰情况基本一致，说明该样品为紫杉二烯；图8-B为该样品的GC检测结果，与标准品对比，可知出峰时间为10.36min所对应的峰为紫杉二烯的峰，其峰面积为3087；样品稀释倍数为30倍；根据峰面积和标准曲线以及样品的稀释倍数即可计算得该样品中紫杉二烯的浓度，进而计算出重组菌株1的紫杉二烯的产量。取重组菌株2所得样品进行检测，检测结果见图9，其中图9-A为该样品的MS检测结果，与标准品出峰情况基本一致，说明该样品为紫杉二烯；图9-B为 该样品的GC检测结果，与标准品对比，可知出峰时间为10.38min所对应的峰为紫杉二烯的峰，其峰面积为5503；样品稀释倍数为10倍；根据峰面积和标准曲线以及样品的稀释倍数即可获得该样品中紫杉二烯的浓度，进而计算出重组菌株2的紫杉二烯的产量。按照相同的方法，取重组菌株3至重组菌株8所得样品进行定性和定量检测，即可获得该样品中紫杉二烯的浓度，计算出重组菌株3至重组菌株8的紫杉二烯的产量。各个重组菌株的紫杉二烯的产量见表9。
表9 各个重组菌株的紫杉二烯产量
Figure PCTCN2015075447-appb-000008
根据表9中实验结果可知，在真核菌株中导入GGPPSsa基因、BE基因、tHMGR基因、tTS基因时，各个基因表达盒的启动子的选择会影响所得重组真核菌株的紫杉二烯的产量；在本发明获得的重组菌株1至重组菌株8中，重组菌株1的的紫杉二烯的产量最高，达到了161.82mg/L，与其他菌株相比，差异显著，P＜0.05。由此可见，在真核菌株中导入GGPPSsa基因、BE基因、tHMGR基因、tTS基因时，GGPPSsa基因表达盒的启动子为TDH1；BE基因表达盒的启动子为GPM1；tHMGR基因表达盒的启动子为PDC1；tTS基因表达盒的启动子为ENO2时，能够显著提高所得重组真核菌株的紫杉二烯产量。
将重组菌株1(根据实验室的命名规则，将该菌株命名为SyBE_Sc00011203)进行生物保藏，重组菌株1所对应的生物保藏信息为： 分类命名：酿酒酵母(Saccharomyces cerevisiae)，于2014年11月28日保藏在中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏单位地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所，邮编：100101，保藏编号为：CGMCC No.10074。
实施例2 利用重组菌株进行发酵罐发酵生产紫杉二烯
实验材料：实施例制备获得的重组菌株1。
实验方法：
种子培养基：合成酵母氮源YNB6.7g/L，葡萄糖20g/L，混合氨基酸粉末2g/L(具体配方参考[美]D.C.安伯格等酵母遗传学方法试验指南)，组氨酸38mg/L，色氨酸38mg/L，亮氨酸190mg/L，pH5.8，115℃灭菌15min。发酵培养基：葡萄糖50g/L，酵母粉10g/L，蛋白胨20g/L，KH2PO40.5g/L，MgSO40.5g/L；其中，葡萄糖115℃灭菌15min，酵母粉与蛋白胨121℃灭菌20min，两者分开灭菌；KH2PO4和MgSO4过滤除菌；最后将上述成分混合。
培养方法：将重组菌株1接种于5mL种子培养基中，在30℃、220rpm培养24h，以初始菌体浓度OD600＝0.2转接于新鲜的100mL种子培养基中，于30℃、220rpm条件下培养12h，以初始菌体浓度OD600＝0.5接种于50mL发酵培养基中。于30℃、220rpm条件下培养70h，测定菌体生长曲线。在发酵10h时向发酵液中加入2.5mL正十二烷进行两相培养，发酵结束后收集上层乳浊液相12000rpm离心20min，收集上层有机相，使用无水硫酸钠除去水分，使用0.22μm滤膜过滤后测定终产物紫杉二烯浓度。
3、分析方法：
与实施例1中的分析方法相同。
4、实验结果：
取所得样品进行检测，检测结果见图10，其中图10-A为该样品的MS检测结果，与标准品出峰情况基本一致，说明该样品为紫杉二烯；图10-B为该样品的GC检测结果，与标准品对比，可知出峰时间为10.40min所对应的峰为紫杉二烯的峰，其峰面积为6325；样品稀释倍数为30倍；根据峰 面积和标准曲线以及样品的稀释倍数即可获得该样品中紫杉二烯的浓度，进而计算出重组菌株1的紫杉二烯的产量为242.64mg/L。
实施例3 利用重组菌株进行发酵罐发酵生产紫杉二烯
实验材料：实施例制备获得的重组菌株1。
实验方法：
种子培养基：合成酵母氮源YNB6.7g/L，葡萄糖20g/L，混合氨基酸粉末2g/L(具体配方参考[美]D.C.安伯格等酵母遗传学方法试验指南)，组氨酸38mg/L，色氨酸38mg/L，亮氨酸190mg/L，pH5.8，115℃灭菌15min。发酵培养基：葡萄糖30g/L，酵母粉10g/L，蛋白胨25g/L，KH2PO44g/L，MgSO43g/L；其中，葡萄糖115℃灭菌15min，酵母粉与蛋白胨121℃灭菌20min，两者分开灭菌；KH2PO4和MgSO4过滤除菌；最后将上述成分混合。补料溶液：葡萄糖800g/L，115℃灭菌15min。
培养方法：将重组菌株1接种于5mL种子培养基中，在30℃、220rpm培养24h，以初始菌体浓度OD600＝0.2转接于新鲜的100mL种子培养基中，于30℃、220rpm条件下培养12h，以初始菌体浓度OD600＝0.5接种于2L发酵培养基(5L发酵罐)中。培养条件为350rpm，30℃，pH5.8，通气量1vvm，培养138h。做批式补葡萄糖培养，补加葡萄糖溶液至原浓度30g/L。测定菌体生长曲线。在发酵8h时向发酵液中加入400mL正十二烷进行两相培养，发酵过程中补料时同时加入20％补料体积的正十二烷使发酵体系中有机相比例保持在20％不变。发酵过程中取样收集上层乳浊液相12000rpm离心20min，收集上层有机相，使用无水硫酸钠除去水分，使用0.22μm滤膜过滤后测定终产物紫杉二烯浓度。
3、分析方法：
与实施例1中的分析方法相同。
4、实验结果：
取所得样品进行检测，检测结果见图11，其中图11-A为该样品的MS检测结果，与标准品出峰情况基本一致，说明该样品为紫杉二烯；图11-B为该样品的GC检测结果，与标准品对比，可知出峰时间为10.37min所对 应的峰为紫杉二烯的峰，其峰面积为5054；样品稀释倍数为30倍；根据峰面积和标准曲线以及样品的稀释倍数即可获得该样品中紫杉二烯的浓度，进而计算出重组菌株1的紫杉二烯的产量为834mg/L。
实施例4 利用重组菌株进行发酵罐发酵生产紫杉二烯
实验材料：实施例制备获得的重组菌株1。
实验方法：
种子培养基：合成酵母氮源YNB6.7g/L，葡萄糖20g/L，混合氨基酸粉末2g/L(具体配方参考[美]D.C.安伯格等酵母遗传学方法试验指南)，组氨酸38mg/L，色氨酸38mg/L，亮氨酸190mg/L，pH5.8，115℃灭菌15min。发酵培养基：葡萄糖50g/L，酵母粉10g/L，蛋白胨30g/L，KH2PO48g/L，MgSO46g/L；其中，葡萄糖115℃灭菌15min，酵母粉与蛋白胨121℃灭菌20min，两者分开灭菌；KH2PO4和MgSO4过滤除菌；最后将上述成分混合。补料溶液：葡萄糖800g/L，115℃灭菌15min。
培养方法：将重组菌株1接种于5mL种子培养基中，在30℃、220rpm培养24h，以初始菌体浓度OD600＝0.2转接于新鲜的100mL种子培养基中，于30℃、220rpm条件下培养12h，以初始菌体浓度OD600＝0.5接种于2L发酵培养基(5L发酵罐)中，培养条件为350rpm，30℃，pH5.8，通气量1vvm，培养174h。做流加葡萄糖培养，控制培养基中葡萄糖浓度在0-1g/L。测定菌体生长曲线。在发酵8h时向发酵液中加入400mL正十二烷进行两相培养，发酵过程中取样收集上层乳浊液相12000rpm离心20min，收集上层有机相，使用无水硫酸钠除去水分，使用0.22μm滤膜过滤后测定终产物紫杉二烯浓度。
3、分析方法：
与实施例1中的分析方法相同。
4、实验结果：
取所得样品进行检测，检测结果见图12，其中图12-A为该样品的MS检测结果，与标准品出峰情况基本一致，说明该样品为紫杉二烯；图12-B为该样品的GC检测结果，与标准品对比，可知出峰时间为10.37min 所对应的峰为紫杉二烯的峰，其峰面积为7465；样品稀释倍数为30倍；根据峰面积和标准曲线以及样品的稀释倍数即可获得该样品中紫杉二烯的浓度，进而计算出重组菌株1的紫杉二烯的产量为1093mg/L。
以上对本发明所提供的一种产紫杉二烯的重组真核菌株及利用该重组真核菌株制备紫杉二烯的方法进行了详细介绍。本文应用了具体个例对本发明的原理及实施方式进行了阐述，以上实施例的说明只是用于帮助理解本发明的方法及其核心思想。应当指出，对于本技术领域技术人员来说，在不脱离本发明原理的前提下，还可以对本发明进行若干改进和修饰，这些改进和修饰也落入本发明权利要求的保护范围内。
Figure PCTCN2015075447-appb-000009
Figure PCTCN2015075447-appb-000010
Figure PCTCN2015075447-appb-000011
Figure PCTCN2015075447-appb-000012
Figure PCTCN2015075447-appb-000013
Figure PCTCN2015075447-appb-000014
Figure PCTCN2015075447-appb-000015
Figure PCTCN2015075447-appb-000016
Figure PCTCN2015075447-appb-000017
Figure PCTCN2015075447-appb-000018
Figure PCTCN2015075447-appb-000019
Figure PCTCN2015075447-appb-000020
Figure PCTCN2015075447-appb-000021
Figure PCTCN2015075447-appb-000022
Figure PCTCN2015075447-appb-000023

Claims (12)

  1. 一种产紫杉二烯的重组真核菌株，其特征在于，在所述真核菌株中导入表达GGPP合酶A的基因表达盒、表达紫杉二烯合酶的基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒；
    所述表达GGPP合酶A的基因表达盒包括启动子、所述表达GGPP合酶A的基因；所述表达GGPP合酶A的基因表达盒的启动子为TDH1；
    所述表达紫杉二烯合酶的基因表达盒包括启动子、所述表达紫杉二烯合酶的基因；所述表达紫杉二烯合酶的基因表达盒的启动子为ENO2；
    所述表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒包括启动子、所述表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因；所述表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒的启动子为PDC1；
    所述表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒包括启动子、所述融合基因；所述融合基因表达盒的启动子为GPM1。
  2. 根据权利要求1所述的重组真核菌株，其特征在于，所述真核菌株为酵母菌。
  3. 根据权利要求2所述的重组真核菌株，其特征在于，所述酵母菌为酿酒酵母。
  4. 根据权利要求3所述的重组真核菌株，其特征在于，所述酿酒酵母为YNL280C。
  5. 根据权利要求1至4中任意一项所述的重组真核菌株，其特征在于，所述表达GGPP合酶A的基因表达盒、所述表达紫杉二烯合酶的基因表达盒、所述表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、所述表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒各自独立地存在于所述重组真核菌株之内，存在方式各自独立地选自：存在于游离型的载体上，或整合到所述真核菌株的基因组中。
  6. 根据权利要求1至5中任意一项所述的重组真核菌株，其特征在于，所述表达GGPP合酶A的基因表达盒、所述表达紫杉二烯合酶的基 因表达盒、所述表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、所述表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒中至少一个整合在所述真核菌株的基因组中。
  7. 根据权利要求6所述的重组真核菌株，其特征在于，所述整合的整合位点为所述真核菌株的基因组的多拷贝位点。
  8. 根据权利要求7所述的重组真核菌株，其特征在于，所述多拷贝位点为Delta位点。
  9. 一种产紫杉二烯的重组真核菌株，其保藏编号为CGMCC No.10074。
  10. 一种产紫杉二烯的重组真核菌株的构建方法，其特征在于，包括以下步骤：
    步骤1：获得表达GGPP合酶A的基因表达盒、表达紫杉二烯合酶的基因表达盒、表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒；
    所述表达GGPP合酶A的基因表达盒包括启动子、所述表达GGPP合酶A的基因；所述表达GGPP合酶A的基因表达盒的启动子为TDH1；
    所述表达紫杉二烯合酶的基因表达盒包括启动子、所述表达紫杉二烯合酶的基因；所述表达紫杉二烯合酶的基因表达盒的启动子为ENO2；
    所述表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒包括启动子、所述表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因；所述表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒的启动子为PDC1；
    所述表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒包括启动子、所述融合基因；所述融合基因表达盒的启动子为GPM1；
    步骤2：取所述表达GGPP合酶A的基因表达盒、所述表达紫杉二烯合酶的基因表达盒、所述表达3-羟基-3-甲基戊二酰辅酶A还原酶的基因表达盒、所述表达FPP合酶的基因和表达GGPP合酶B的基因的融合基因表达盒，导入所述真菌菌株中，即得。
  11. 利用如权利要求1至9中任一项所述的重组真核菌株制备紫杉二烯的方法，其特征在于，包括：
    培养所述重组真核菌株，从培养物中回收紫杉二烯，即得。
  12. 根据权利要求11所述的制备紫杉二烯的方法，其特征在于，当所述真核菌株为酵母菌时，包括：
    取所述重组酵母菌，扩大培养后，接种于发酵培养基中，经发酵培养、两相培养，收集紫杉二烯，即得。
PCT/CN2015/075447 2014-12-30 2015-03-31 一种产紫杉二烯的重组真核菌株及利用该重组真核菌株制备紫杉二烯的方法 WO2016106988A1 (zh)

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