WO2018082588A1 - 一种重组菌及其用途 - Google Patents

一种重组菌及其用途 Download PDF

Info

Publication number
WO2018082588A1
WO2018082588A1 PCT/CN2017/109029 CN2017109029W WO2018082588A1 WO 2018082588 A1 WO2018082588 A1 WO 2018082588A1 CN 2017109029 W CN2017109029 W CN 2017109029W WO 2018082588 A1 WO2018082588 A1 WO 2018082588A1
Authority
WO
WIPO (PCT)
Prior art keywords
nucleic acid
encoding
yeast
gemasene
synthase
Prior art date
Application number
PCT/CN2017/109029
Other languages
English (en)
French (fr)
Inventor
张学礼
黄璐琦
戴住波
王冬
张丽丽
郭娟
刘怡
Original Assignee
中国科学院天津工业生物技术研究所
中国中医科学院中药研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院天津工业生物技术研究所, 中国中医科学院中药研究所 filed Critical 中国科学院天津工业生物技术研究所
Priority to JP2019544968A priority Critical patent/JP7129990B2/ja
Priority to EP17867973.4A priority patent/EP3536792A4/en
Priority to US16/347,552 priority patent/US11421199B2/en
Publication of WO2018082588A1 publication Critical patent/WO2018082588A1/zh
Priority to US17/812,157 priority patent/US20220396759A1/en
Priority to US18/456,333 priority patent/US20240010969A1/en

Links

Images

Classifications

    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • 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
    • 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
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/1085—Transferases (2.) transferring alkyl or aryl groups other than methyl groups (2.5)
    • 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
    • C12P5/002—Preparation of hydrocarbons or halogenated hydrocarbons cyclic
    • 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
    • C12P5/007—Preparation of hydrocarbons or halogenated hydrocarbons containing one or more isoprene units, i.e. terpenes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y205/00—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5)
    • C12Y205/01—Transferases transferring alkyl or aryl groups, other than methyl groups (2.5) transferring alkyl or aryl groups, other than methyl groups (2.5.1)
    • C12Y205/0101—(2E,6E)-Farnesyl diphosphate synthase (2.5.1.10), i.e. geranyltranstransferase
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y402/00—Carbon-oxygen lyases (4.2)
    • C12Y402/03—Carbon-oxygen lyases (4.2) acting on phosphates (4.2.3)
    • C12Y402/03023—Germacrene-A synthase (4.2.3.23)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/32—Amino acids
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/34—Sugars
    • 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
    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/30—Organic components
    • C12N2500/38—Vitamins
    • 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 relates to the field of biochemical industry, in particular to a recombinant bacteria and a synthesis of ⁇ -elemene based on a recombinant microorganism method.
  • Beta-elemene (beta-elemene) is a volatile sesquiterpene compound with tulip flavor. It is a raw material for new cancer drugs in the country. At present, it is mainly isolated and extracted from plants such as turmeric and medlar, but this method has many disadvantages, including low content and large difference, difficult product purification, long plant growth cycle, and serious damage to biological resources, especially wild resources. .
  • farnesyl pyrophosphate FPP
  • GMAS gemasene synthase
  • Gemene A is thermally unstable and prone to intramolecular thermal rearrangement to ⁇ -elemen Alkenes have been partially used in the production of beta-elemene precursor gemasene A by recombinant bacteria, but the yields are low, which does not meet the requirements of industrial applications.
  • the recombinant bacteria provided by the present invention contain or express gemasene synthase or gemasene A in vivo.
  • the gemasene synthase fusion protein includes the gemasene synthase and farnesyl pyrophosphate synthase.
  • the above recombinant bacteria are classified into one or more according to the source of the fusion protein gene host, and the nucleic acid encoding the fusion protein includes the nucleic acid encoding the gemasene synthase and the farnesyl pyrophosphate synthase Encoding nucleic acid;
  • the fusion protein has one or more encoding nucleic acids
  • At least two of the gemasene A synthetase encoding nucleic acid source hosts are different, and at least two of the farnesyl pyrophosphate synthase encoding nucleic acid source hosts are different.
  • the gene-derived host described in the present invention is different from the original source host of the gene, and the germacrene A synthase gene of the present invention can be derived from a plant or microorganism known to contain gemasene A synthetase.
  • the clone is obtained, for example, may be selected from the group consisting of sunflower (Helianthus annuus L.), Tanacetum parthenium, lettuce (Lactuca sativa Linn.), Artemisia carvifolia, cyanobacteria, etc., the farnesyl pyrophosphate
  • the farnesyl diphosphate synthase gene can be cloned from a plant or microorganism known to contain gemasene synthase, and for example, can be selected from Salvia miltiorrhiza, yeast (Yeast), Acanthopanax senticosus (Rupr) .Maxim.) Harms), Eucommia ulmoides Oliv., etc.
  • the nucleic acid encoding the gemasene synthase comprises the nucleic acid represented by SEQ ID NO. 3 or the nucleic acid represented by positions 13-1686 of SEQ ID NO.
  • the nucleic acid encoding the farnesyl pyrophosphate synthase includes the nucleic acid shown in SEQ ID NO. 2 or the nucleic acid shown in positions 1-156 of SEQ ID NO.
  • the fusion protein further comprises a linker peptide for linking the gemasene synthase and the farnesyl pyrophosphate synthase;
  • the linker peptide is selected from the group consisting of GGGS, YGQ (3A001), PGGH (4A001), YRSQI (5A002), VIPFSS (6A005), FLYLKF (6B004), WRFSPKLQ (8A005) or HHVQESQCISTV (12A003).
  • the in vivo contains or expresses a gemalene A synthetase or a gemasene synthase fusion protein, which is an encoding nucleic acid of the gemasene synthase or an encoding core of the fusion protein.
  • a gemalene A synthetase or a gemasene synthase fusion protein which is an encoding nucleic acid of the gemasene synthase or an encoding core of the fusion protein.
  • the nucleic acid expression cassette containing the gemasene synthase comprises a promoter, an encoding nucleic acid of the gemasene synthase, and a terminator;
  • the nucleic acid expression cassette containing the fusion protein comprises a promoter, an encoding nucleic acid of the fusion protein, and a terminator;
  • the promoter is selected from TEF1 or MF1 or PGK1; the terminator is CYC1 or ADH1;
  • the promoter is TEF1, and the terminator is CYC1;
  • the promoter is MF1, and the terminator is CYC1;
  • the promoter is PGK1 and the terminator is ADH1.
  • the promoter TEF1 comprises the sequence shown in SEQ ID NO. 4; the promoter MF1 comprises the sequence shown in SEQ ID NO. 1; and the terminator CYC1t comprises the sequence shown in SEQ ID NO.
  • the recombinant strain further expresses one or more marker genes; and/or the marker gene is selected from his3 or trpl.
  • the nucleic acid expression cassette containing the gemasene synthase is introduced into the yeast by a vector encoding the nucleic acid expression cassette encoding the gemeene A synthetase;
  • the nucleic acid expression cassette containing the fusion protein is introduced into the yeast by expressing the vector encoding the nucleic acid expression cassette containing the fusion protein.
  • the nucleic acid expression cassette encoding the gemasene synthase is introduced into the yeast by a plasmid;
  • the nucleic acid expression cassette encoding the fusion protein is introduced into the yeast by a plasmid form and/or integrated into a chromosome.
  • the fusion protein is selected from at least one of the following: SynSmFPS-GGGS-STpGMAS, SynSmFPS-YGQ-STpGMAS, SynSmFPS-PGGH-STpGMAS, SynSmFPS-YRSQI-STpGMAS, SynSmFPS-VIPFIS-STpGMAS, SynSmFPS-FLYLKF-STpGMAS, SynSmFPS-WRFSPKLQ-STpGMAS, SynSmFPS-HHVQESQCISTV-STpGMAS, SynSmFPS-WRFSPKLQ-STpGMAS, ERG20-GGGS-LsLTC2;
  • the fusion protein is preferably SynSmFPS-8A005-STpGMAS;
  • fusion proteins are three fusion proteins: SynSmFPS-WRFSPKLQ(8A005)-STpGMAS, ERG20-GGGS-LsLTC2, SynSmFPS-GGGS-STpGMAS;
  • the expression cassette expressing the fusion protein-encoding nucleic acid is selected from at least one of the following:
  • the expression cassette expressing the fusion protein-encoding nucleic acid is preferably: P MF1 -SynSmFPS-8A005-STpGMAS-T CYC1;
  • Fusion protein expression cassette encoding nucleic acid is particularly preferably three kinds: P MF1 -SynSmFPS-8A005-STpGMAS -T CYC1, P PGK1 -ERG20-GGGS-LsLTC2-T ADH1 and P TEF1 -SynSmFPS-GGGS-STpGMAS- T CYC1.
  • the vector expressing the gemeene A synthetase encoding nucleic acid expression cassette is selected from the following:
  • the vector expressing the gemeene A synthetase encoding nucleic acid expression cassette is selected from the following:
  • the above-described fusion protein gene integrated in the chromosome an expression cassette is selected from P TEF1 -SynSmFPS-GGGS-STpGMAS- T CYC1 and P PGK1 -ERG20-GGGS-LsLTC2- T ADH1.
  • the yeast is a strain obtained by increasing the content and/or activity of an alcohol dehydrogenase, an aldehyde dehydrogenase, and an acetyl-CoA synthetase in the starting yeast.
  • the bacteria obtained by increasing the content and/or activity of the alcohol dehydrogenase, acetaldehyde dehydrogenase and acetyl-CoA synthetase in the starting yeast are nucleic acids and acetaldehyde which increase the alcohol dehydrogenase in the starting yeast. a copy number of the nucleic acid encoding the dehydrogenase and the nucleic acid encoding the acetyl-CoA synthetase;
  • the copy number of the nucleic acid encoding the nucleic acid of the alcohol dehydrogenase, the nucleic acid encoding the acetaldehyde dehydrogenase, and the encoding nucleic acid of the acetyl-CoA synthetase in the yeast is the expression of the nucleic acid encoding the alcohol dehydrogenase
  • the cassette, the nucleic acid expression cassette encoding the acetaldehyde dehydrogenase, the nucleic acid expression cassette encoding the acetyl-CoA synthetase, and the other said marker gene (his3) were introduced into the starting yeast by homologous recombination.
  • the starting yeast is Saccharomyces cerevisiae; and/or the Saccharomyces cerevisiae is NK2-SQ.
  • One of the marker genes is TRP1; the other of the marker genes is HIS3.
  • the above-described alcohol dehydrogenase gene ADH2 comprises the sequence shown in SEQ ID NO. 6, and the acetaldehyde dehydrogenase gene ALD6 comprises the sequence shown in SEQ ID NO. 7, and the acetyl-CoA synthetase gene ACS1 comprises SEQ The sequence shown in ID NO.
  • the recombinant bacteria are as follows:
  • the recombinant ELE-001 is a strain obtained by introducing pRS313-LEU2-P TEF1 -STpGMAS-T CYC1 into yeast FPP-001;
  • the recombinant ELE-002 is a strain obtained by introducing pRS425-LEU2-P TEF1 -STpGMAS-T CYC1 into yeast FPP-001;
  • the recombinant ELE-011 is a strain obtained by introducing pRS425-LEU2-P TEF1 -SynSmFPS-GGGS-STpGMAS-T CYC1 into yeast FPP-001;
  • the recombinant ELE-012 is a strain obtained by introducing pRS425-LEU2-P TEF1 -SynSmFPS-3A001-STpGMAS-T CYC1 into yeast FPP-001;
  • the recombinant ELE-013 is a strain obtained by introducing pRS425-LEU2-P TEF1 -SynSmFPS-4A001-STpGMAS-T CYC1 into yeast FPP-001;
  • the recombinant ELE-014 is a strain obtained by introducing pRS425-LEU2-P TEF1 -SynSmFPS-5A002-STpGMAS-T CYC1 into yeast FPP-001;
  • the recombinant ELE-015 is a strain obtained by introducing pRS425-LEU2-P TEF1 -SynSmFPS-6A005-STpGMAS-T CYC1 into yeast FPP-001;
  • the recombinant ELE-016 is a strain obtained by introducing pRS425-LEU2-P TEF1 -SynSmFPS-6B004-STpGMAS-T CYC1 into yeast FPP-001;
  • the recombinant ELE-017 is a strain obtained by introducing pRS425-LEU2-P TEF1 -SynSmFPS-8A005-STpGMAS-T CYC1 into yeast FPP-001;
  • the recombinant ELE-018 is a strain obtained by introducing pRS425-LEU2-P TEF1 -SynSmFPS-12A003-STpGMAS-T CYC1 into yeast FPP-001;
  • the recombinant ELE-019 is a strain obtained by introducing pRS425-LEU2-P MF1 -SynSmFPS-8A005-STpGMAS-T CYC1 into yeast FPP-001;
  • Recombinant strain ELE-020 as the pRS425-LEU2-P MF1 -SynSmFPS- 8A005-STpGMAS-T CYC1, after introduction yeast FPP-001, and then introduced by homologous recombination P PGK1 -ERG20-GGGS-LsLTC2- T ADH1, P TEF1 -SynSmFPS-GGGS-STpGMAS-T CYC1 , rDNA-TRP1-up and rDNA-TRP1-down.
  • Yeast FPP-001 as described above will NDT80-HIS3-up, P PGK1 -ADH2-T ADH1, P TDH3 -ACS1-T TPI1, P TEF1 -ALD6-T CYC1 NDT80-HIS3-down and introduced into S. cerevisiae strain obtained.
  • the recombinant ELE-020 is Saccharomyces cerevisiae CGMCC No. 14829, which is also the scope of protection of the present invention.
  • the ELE-020 recombinant strain was deposited on October 20, 2017 at the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee.
  • CGMCC General Microbiology Center
  • the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.
  • a third object of the present invention is to provide a method for producing gemasene A.
  • the method provided by the present invention comprises the steps of: fermenting the above recombinant bacteria to obtain gemasene A;
  • a fourth object of the present invention is to provide a process for producing ?-elemene.
  • the method provided by the invention comprises the following steps:
  • the fermentation is performed by first culturing the recombinant bacteria in a seed culture medium to obtain a seed liquid; and then injecting the seed liquid into a fermentation medium for fermentation culture, and the fermentation culture product is recorded as fermentation. system.
  • a feed medium is added to the fermentation system; preferably, when the dissolved oxygen value in the fermentation system is greater than 60%, a supplement is added to the fermentation system.
  • the glucose concentration in the feed medium to the fermentation system was 5 g/L.
  • the seed medium and the fermentation medium formulation per L volume comprises: 25 g glucose, 15 g ammonium sulfate, 6.15 g magnesium sulfate heptahydrate, 0.72 g zinc sulfate heptahydrate, 8 g potassium dihydrogen phosphate, 2 mL chlorine.
  • the calcium chloride mother liquor is 19.2 g / L aqueous solution of calcium chloride dihydrate
  • the formulation of the trace metal salt mother liquor per L volume comprises: 19.1 g disodium edetate; 10.2 g zinc sulfate heptahydrate; 0.5 g manganese chloride tetrahydrate; 0.86 g cobalt chloride hexahydrate; 0.78 g five Copper sulphate; 0.56 g of sodium molybdate dihydrate; 5.12 g of iron sulphite heptahydrate; the rest is water.
  • each L volume of vitamin mother liquor comprises: 0.05 g biotin; 0.2 g sodium p-aminobenzoate; 1 g niacin; 1 g calcium pantothenate; 1 g pyridoxine hydrochloride; 1 g thiamine hydrochloride; 25 g inositol;
  • the formulation of the feed medium per L volume contained 800 g glucose, 5.125 g magnesium sulfate heptahydrate, 3.5 g potassium sulfate, 0.28 g sodium sulfate, 9 g potassium dihydrogen phosphate and 1 g tryptophan; the balance was water.
  • solid or liquid selection medium is SD-Ura-His-Leu medium
  • the culture condition in the above step b) is 30 ° C, 250 rpm; the inoculation step is a flame ring inoculation;
  • the method for culturing the seed liquid in the above fermentation method is specifically: after the recombinant bacteria is activated, picking up the monoclonal on the plate to the test tube containing the SD-Ura-His-Leu medium, incubating at 30 ° C, shaking at 250 rpm overnight; taking 500 ⁇ L The bacterial solution was incubated in a 250 mL flask containing 50 mL SD-Ura-His-Leu medium at 30 ° C, shaking at 250 rpm for 24 h; 2 mL of the bacterial solution was pipetted into 3 1 L flasks containing 100 mL of seed medium, 30 Incubate for 48 h at 250 C with shaking at °C.
  • the organic solvent is n-dodecane; and the heating condition is: heating at 100 to 380 ° C for 1 hour.
  • Figure 1 shows the gemasene A biosynthetic pathway.
  • Figure 2 is a GC-MS test chart.
  • Figure 1 shows the gemasene A biosynthetic pathway.
  • the DNA polymerase was equipped with an amplification system (TAKARA).
  • the amplification system was: 5 ⁇ PS Buffer 10 ⁇ L, dNTPMix 4 ⁇ L, 1 ⁇ L of primers, and 1 ⁇ L of genomic DNA template.
  • Polymerase (2.5 U/ ⁇ L) 0.5 ⁇ L, supplemented with distilled water to a total volume of 50 ⁇ L.
  • the amplification conditions were: pre-denaturation at 98 ° C for 3 minutes (1 cycle); denaturation at 98 ° C for 10 seconds, annealing at 55 ° C for 15 seconds, extension at 72 ° C for 2.5 minutes (30 cycles); extension at 72 ° C for 10 minutes (1 cycle) .
  • Table 1 is the primer sequence
  • Nanjing Kingsray Biotechnology Co., Ltd. is based on SynSmFPS (SEQ ID NO. 2, derived from Danshen) and STpGMAS (SEQ ID NO.3, derived from the white chrysanthemum) gene sequence, design full-length primers, use OVERLAP method to form template DNA, and then use PCR amplification method to obtain SynSmFPS (SEQ ID NO. 2) and STpGMAS ( The double-stranded DNA of SEQ ID NO.
  • RNA was extracted by CTAB method (cetyltrimethylammonium bromide method): 1 ml of 2*CTAB extract (2% CTAB, 100 mM Tris-HCl) was added to a 1.5 ml centrifuge tube.
  • CTAB method cetyltrimethylammonium bromide method
  • First-strand reverse transcription-PCR Take the RNase-free PCR tube and equip the first strand reverse transcription kit (Bao Bioengineering (Dalian) Co., Ltd.) with system: Radom 6Mers 2ul, dNTP 1ul, total RNA 1ul (200ng ), H2O 6ul, Total 10ul, instantaneous centrifugation, PCR at 65 ° C for 5 min, quenching on ice; then adding the following system reaction (first chain reverse transcription kit comes with): 5 *primer Buffer 4ul, RNAs Inhibiter 0.5ul, R-Transcription1 ul, H 2 O 4.5ul, instantaneous centrifugation, reaction by PCR: 30 ° C for 10 min, 42 ° C for 60 min, 70 ° C for 15 min, 4 ° C heat preservation.
  • the primers in Table 2 were used to amplify about 1068 bp ERG20-GGGS (SEQ ID NO. 11 13-1686 is ERG20) and 1688 bp GGGS-LsLTC2 (SEQ ID). No. 1-156 in NO. 12 is LsLTC2).
  • Phusion High-Fidelity PCR Master Mix with HF Buffer purchased from NEB (Beijing) Co., Ltd.
  • the amplification system is: 5 ⁇ Phusion HF Buffer 10 ⁇ L, dNTP (10 mM each dNTP) 1 ⁇ L 20 ng of DNA template, 1.5 ⁇ L of primer (10 ⁇ M), 0.5 ⁇ L of Phusion High-Fidelity DNA Polymerase (2.5 U/ ⁇ L), and distilled water to a total volume of 50 ⁇ L.
  • the amplification conditions were: pre-denaturation at 98 ° C for 3 minutes (1 Cycling; denaturation at 98 °C for 10 seconds, annealing at 58 °C for 10 seconds, extension at 72 °C for 1 minute (30 cycles); extension at 72 °C for 10 minutes (1 cycle).
  • ADH2 amplified in the above "I. Preparation of target gene” and plasmid pM2-tHMG1 (described in China) were separately obtained from SexA1 (purchased from NEB (Beijing) Co., Ltd.) and Asc1 (purchased from NEB (Beijing) Co., Ltd.).
  • Asc1 purchased from NEB (Beijing) Co., Ltd.
  • double digestion the 1052 bp ADH2 digestion product and the 4738 bp plasmid pM2-tHMG1 were digested, and the ADH2 digestion product was ligated with the plasmid pM2-tHMG1 to obtain the recombinant plasmid pM2-ADH2.
  • the ACS1 amplified in the above "I. Preparation of the target gene” and the plasmid pM4-AtCPR1 (described in Chinese patent ZL201310399947.X) were double-digested with SexA1 and Asc1, respectively, and the 2201 bp ACS1 digestion product and 5061 bp were obtained. Plasmid pM4-AtCPR1 after digestion The backbone was ligated to the plasmid pM4-AtCPR1 and the recombinant plasmid pM4-ACS1 was obtained.
  • the ALD6 amplified by the above-mentioned "I. Preparation of target gene” and the plasmid pM3-ERG9 (described in Chinese patent application ZL201310399947.X) were double-digested with SexA1 and Asc1, respectively, and 1511 bp ALD6 digested product and 4598 bp plasmid were obtained.
  • the pM3-ERG9 was digested into the backbone; the ALD6 digested product was digested with the plasmid pM3-ERG9 and ligated to obtain the recombinant plasmid pM3-ALD6.
  • SexA1 digests the TEF1 amplified in the above "I. Preparation of target gene", and obtains a 440 bp TEF1 digestion product;
  • Asc1 digests the CYC1 amplified in the above "I. Preparation of target gene", and obtains a 322 bp CYC1 digestion product;
  • SexA1 and Asc1 digested pUC57-STpGMAS, and recovered 1694 bp of STpGMAS;
  • the amplification conditions were: pre-denaturation at 98 ° C for 3 minutes (1 cycle); denaturation at 98 ° C for 10 seconds, annealing at 58 ° C for 10 seconds, extension at 72 ° C for 1.5 minutes (30 cycles); extension at 72 ° C for 10 minutes (1 cycle) .
  • a 2456 bp PCR amplification product was obtained.
  • the amplified product was purified, and then digested with SacII, and the target fragment SacII-TEF1-STpGMAS-CYC1-SacII was recovered by tapping, and used.
  • Plasmid pRS313 was digested with SacII (Sikorski, R.S.and Hieter, P. 1989, Genetics 122(1): 19-27) and pRS425 (Sikorski, RS and Hieter, P.
  • the primer of Table 3 was used to amplify the 6692 bp plasmid backbone pRS313-TEF1-STpGMAS-CYC1;
  • primers in Table 3 were used to amplify LEU2 (1808 bp).
  • the amplification system was: 5 ⁇ Phusion HF Buffer 10 ⁇ L, dNTP (10 mM each dNTP) 1 ⁇ L, DNA template 20 ng, primer (10 ⁇ M) each 1.5 ⁇ L, Phusion High-Fidelity DNA Polymerase (2.5 U/ ⁇ L) 0.5 ⁇ L, supplement Distilled water to a total volume of 50 ⁇ L.
  • the amplification conditions were: pre-denaturation at 98 ° C for 3 minutes (1 cycle); denaturation at 98 ° C for 10 seconds, annealing at 58 ° C for 10 seconds, extension at 72 ° C for 4 minutes (30 cycles); extension at 72 ° C for 10 minutes (1 cycle) .
  • the target fragment was purified by tapping. 2 ⁇ L of 10 ⁇ T4 DNA Ligase Reaction Buffer (NEB) and 1 ⁇ L of T4 Polynucleotide kinase (NEB) were added to the product LEU2 fragment, supplemented with distilled water to 20 ⁇ L, phosphorylated at 37 ° C for 1 h, and recovered with pRS313-P TEF1 -STpGMAS- T CYC1 was ligated with T4 DNA ligase (NEB), transformed, and sequenced to obtain plasmid: pRS313-LEU2-P TEF1 -STpGMAS-T CYC1 .
  • NEB 10 ⁇ T4 DNA Ligase Reaction Buffer
  • NEB T4 Polynucleotide kinase
  • the amplification system was: 5 ⁇ Phusion HF Buffer 10 ⁇ L, dNTP (10 mM each dNTP) 1 ⁇ L, DNA template 20 ng, primer (10 ⁇ M) each 1.5 ⁇ L, Phusion High-Fidelity DNA Polymerase (2.5 U/ ⁇ L) 0.5 ⁇ L, supplement Distilled water to a total volume of 50 ⁇ L.
  • the amplification conditions were: pre-denaturation at 98 ° C for 3 minutes (1 cycle); denaturation at 98 ° C for 10 seconds, annealing at 58 ° C for 10 seconds, extension at 72 ° C for 1 minute (30 cycles); extension at 72 ° C for 10 minutes (1 cycle) .
  • SynSmFPS-GGGS and GGGS-STpGMAS were used together as a template, and the 2767 bp SynSmFPS-GGGS-STpGMAS fragment was amplified by using the primers in Table 4 (SexA1-SynSmFPS and STpGMAS-Asc1).
  • the amplification system was: 5 ⁇ Phusion HF Buffer 10 ⁇ L, dNTP (10 mM each dNTP) 1 ⁇ L, DNA template SynSmFPS-GGGS and GGGS-STpGMAS each 20 ng, primer (10 ⁇ M) each 1.5 ⁇ L, Phusion High-Fidelity DNA Polymerase (2.5 U / ⁇ L) 0.5 ⁇ L, supplemented with distilled water to a total volume of 50 ⁇ L.
  • the amplification conditions were: pre-denaturation at 98 ° C for 3 minutes (1 cycle); denaturation at 98 ° C for 10 seconds, annealing at 58 ° C for 10 seconds, extension at 72 ° C for 2 minutes (30 cycles); extension at 72 ° C for 10 minutes (1 cycle) .
  • the amplified product was purified, and then digested with SexA1 and Asc1, and the target fragment SexA1-SynSmFPS-GGGS-STpGMAS-Asc1 (2760 bp) was recovered by tapping, and used.
  • the plasmid pRS425-LEU2-P TEF1 -STpGMAS-T CYC1 constructed in the above "4" was digested with SexA1 and Asc1, and a large 7602 bp fragment was recovered by tapping to obtain the vector pRS425-LEU2-P TEF1 -...-T CYC1.
  • the plasmid pRS425-LEU2-P TEF1 -SynSmFPS-GGGS-STpGMAS-T CYC1 constructed by the above-mentioned "1, preparation of target gene” and the above-mentioned "5" item, respectively, using BamH1 (purchased from TaKaRa) and SexA1, respectively.
  • Double digestion; 814bp target promoter gene MF1 and 9988bp vector fragment pRS425-LEU2-...-SynSmFPS-GGGS-STpGMAS-T CYC1 were purified by tapping and added to the ligation system (50ng each): 2 ⁇ L 10 ⁇ T4 DNA Ligase Reaction Buffer (NEB), 1 ⁇ L of T4 DNA Ligase (NEB, 400,000 cohesive end units/ml), supplemented with distilled water to 20 ⁇ L, reacted at room temperature for 2 hours to obtain a ligation product, transformed into Trans10 competent cells, and verified by plasmid sequencing.
  • the resulting plasmid with the correct sequence was named pRS425-LEU2-P MF1 -SynSmFPS-GGGS-STpGMAS-T CYC1 .
  • ERG20-GGGS and GGGS-LsLTC2 were used together as a template, and the primers of Table 5 (SexA1-ERG20 and LsLTC2-Asc1) were amplified to obtain an about 2744 bp ERG20-GGGS-LsLTC2 fragment.
  • the amplification system was: 5 ⁇ Phusion HF Buffer 10 ⁇ L, dNTP (10 mM each dNTP) 1 ⁇ L, DNA template ERG20-GGGS and GGGS-LsLTC2 each 20 ng, primer (10 ⁇ M) each 1.5 ⁇ L, Phusion High-Fidelity DNA Polymerase (2.5 U / ⁇ L) 0.5 ⁇ L, supplemented with distilled water to a total volume of 50 ⁇ L.
  • the amplification conditions were: pre-denaturation at 98 ° C for 3 minutes (1 cycle); denaturation at 98 ° C for 10 seconds, annealing at 58 ° C for 10 seconds, extension at 72 ° C for 2 minutes (30 cycles); extension at 72 ° C for 10 minutes (1 cycle) .
  • the amplified product was purified, and then digested with SexA1 and Asc1, and the target fragment SexA1-ERG20-GGGS-LsLTC2-Asc1 (about 2744 bp) was recovered by tapping, and then ligated with the vector pZ2-tHMG1 and ligated to obtain a recombination. Plasmid pM2-ERG20-GGGS-LsLTC2.
  • the amplification system was: 5 ⁇ Phusion HF Buffer 10 ⁇ L, dNTP (10 mM each dNTP) 1 ⁇ L, DNA template 20 ng, primer (10 ⁇ M) 1.5 ⁇ L each, Phusion High-Fidelity DNA Polymerase (2.5 U/ ⁇ L) 0.5 ⁇ L, supplemented with distilled water To a total volume of 50 ⁇ L.
  • the amplification conditions were: pre-denaturation at 98 ° C for 3 minutes (1 cycle); denaturation at 98 ° C for 10 seconds, annealing at 58 ° C for 10 seconds, extension at 72 ° C for 1 minute (30 cycles); extension at 72 ° C for 10 minutes (1 cycle) .
  • the amplified product NDT80 was cloned into pEASY-Blunt Simple cloning vector (pEASY cloning vector, TransGen Biotech Co., Ltd.) and transformed into Trans10 competent cells, and plasmid sequencing was performed to obtain plasmid pEASY-NDT80.
  • NK2-SQ genomic DNA and pRS314 (Sikorski, RS and Hieter, P. 1989, Genetics 122(1): 19-27) template
  • primers in Table 8 were used to amplify about rDNA (SEQ ID NO. 9) and TRP1 (SEQ ID NO. 10).
  • the amplification system was: 5 ⁇ Phusion HF Buffer 10 ⁇ L, dNTP (10 mM each dNTP) 1 ⁇ L, DNA template 20 ng, primer (10 ⁇ M) 1.5 ⁇ L each, Phusion High-Fidelity DNA Polymerase (2.5 U/ ⁇ L) 0.5 ⁇ L, supplemented with distilled water To a total volume of 50 ⁇ L.
  • the amplification conditions were: pre-denaturation at 98 ° C for 3 minutes (1 cycle); denaturation at 98 ° C for 10 seconds, annealing at 58 ° C for 10 seconds, extension at 72 ° C for 1 minute (30 cycles); extension at 72 ° C for 10 minutes (1 cycle) .
  • the amplified product rDNA was cloned into pEASY-Blunt Simple cloning vector and transformed into Trans10 competent cells, and the plasmid was verified by sequencing to obtain plasmid pEASY-rDNA.
  • PmeI was digested with pEASY-rDNA, and the 5122 bp target fragment (30 ng) was purified by tapping, 4 ⁇ L of NEB buffer, 1 ⁇ L of CIP dephosphorylation enzyme (NEB), supplemented with distilled water to 40 ⁇ L, treated at 37 ° C for 1 h, and added to a final concentration of 10 ⁇ mol of EDTA, 65 The reaction was terminated at °C for 30 min, and the 5122 bp target fragment pEASY-rDNA was recovered by tapping, and used.
  • NEB CIP dephosphorylation enzyme
  • TRP1 Purification of TRP1 (30 ng) by tapping, adding 4 ⁇ L of 10 ⁇ T4 DNA Ligase Reaction Buffer (NEB), 1 ⁇ L of T4 Polynucleotide kinase (NEB), supplemented with distilled water to 40 ⁇ L, phosphorylated at 37 ° C for 1 h, recovered after tapping, and used with pEASY-rDNA T4 DNA ligase (NEB) was ligated, transformed into Trans10 competent cells, and verified by sequencing to obtain plasmid pEASY-rDNA-TRP1.
  • NEB 10 ⁇ T4 DNA Ligase Reaction Buffer
  • NEB T4 Polynucleotide kinase
  • phosphorylated at 37 ° C for 1 h recovered after tapping, and used with pEASY-rDNA T4 DNA ligase (NEB) was ligated, transformed into Trans10 competent cells, and verified by sequencing to obtain plasmid pEASY-rDNA-
  • the starting bacteria were separately cultured in the corresponding medium (Table 13) at 30 ° C, 250 rpm overnight, 1 mL (OD about 0.6-1.0) was dispensed into 1.5 mL EP tube, centrifuged at 10000 g for 1 min at 4 ° C, and the supernatant was discarded. Wash with sterile water (4 ° C), centrifuge under the same conditions, and discard the supernatant.
  • treatment solution (10 mM LiAc (lithium acetate); 10 mM DTT (dithiothreitol); 0.6 M sorbitol (sorbitol); 10 mM Tris-HCl (trishydroxymethylaminomethane buffer solution, pH 7.5) ), DTT was added when the treatment solution was used, and it was allowed to stand at 25 ° C for 20 min.
  • LiAc lithium acetate
  • DTT dithiothreitol
  • sorbitol sorbitol
  • Tris-HCl trishydroxymethylaminomethane buffer solution, pH 7.5
  • NDT80-HIS3-up and NDT80-HIS3-down are the upstream homologous arm and the downstream homologous arm of HIS3, respectively; the fragments were amplified as follows:
  • the amplification system was: 5 ⁇ Phusion HF Buffer 10 ⁇ L, dNTP (10 mM each dNTP) 1 ⁇ L, DNA template 20 ng, primer (10 ⁇ M) 1.5 ⁇ L each, Phusion High-Fidelity DNA Polymerase (2.5 U/ ⁇ L) 0.5 ⁇ L, supplemented with distilled water To a total volume of 50 ⁇ L. Expand The conditions were as follows: pre-denaturation at 98 ° C for 3 minutes (1 cycle); denaturation at 98 ° C for 10 seconds, annealing at 58 ° C for 10 seconds, extension at 72 ° C for 2 minutes (30 cycles); extension at 72 ° C for 10 minutes (1 cycle) ), the product is recovered by tapping.
  • the screening medium composition was: 0.8% yeast selection medium SD-Ura-Trp-His (Beijing Fenino (Functional Genome) Technology Co., Ltd.), 2% glucose, 0.01% Trp.
  • the correct positive clone was identified by PCR and designated as strain FPP-001.
  • the starting strain Saccharomyces cerevisiae FPP-001 was cultured overnight in SD-Ura-His liquid medium to prepare a competent state. Then, the plasmids pRS313-LEU2-P TEF1 -STpGMAS-T CYC1 and pRS425-LEU2-P TEF1 -STpGMAS-T CYC1 were added separately, mixed and transferred to an electric rotor, 2.7kv shock was 5.7ms, and 1mL 1M sorbitol was added, 30 After resuscitation at °C for 1 h, it was applied to SD-Ura-His-Leu medium at 30 ° C for more than 36 h.
  • the screening medium composition was: 0.8% yeast selection medium SD-Ura-Trp-Leu-His (Beijing Fenino (Functional Genome) Technology Co., Ltd.), 2% glucose, 0.01% Trp.
  • the correct positive clones were identified by PCR and named as strain ELE-001 (transferred into plasmid pRS313-LEU2-P TEF1 -STpGMAS-T CYC1 ) and ELE-002 (transferred into plasmid pRS425-LEU2-P TEF1 -STpGMAS-T CYC1 ).
  • the FPP-001 competent state was prepared according to the above three steps. Then, the plasmid pRS425-LEU2-P TEF1 -SynSmFPS-GGGS-STpGMAS-T CYC1 was added , mixed and transferred to an electric rotor, 2.7kv electric shock for 5.7ms, 1mL 1M sorbitol was added, resuscitated at 30 °C for 1h, and applied to SD- Ura-His-Leu medium was cultured at 30 ° C for more than 36 h. A positive positive clone was identified by PCR and designated as strain ELE-011.
  • PCR amplification was carried out with the primers of Table 11 to obtain amplification products corresponding to different primers; respectively, amplification products corresponding to different primers were respectively used.
  • Transfer to yeast FPP-001 for self-homologous recombination respectively obtain recombinant ELE-012-ELE-018, and replace the linker peptide GGGS between the fusion protein SynSmFPS-GGGS-STpGMAS in the vector to 3A001, 4A001, 5A002, 6A005, 6B004, 8A005, 12A003 (shown in Table 10).
  • PCR amplification was carried out using primers in Table 10 with a linker of 8A005 (Table 11) to obtain amplification products corresponding to different primers;
  • the amplified products corresponding to the different primers were transferred into yeast FPP-001 for self-homologous recombination to obtain recombinant ELE-019, and the linker peptide GGGS between the fusion protein SynSmFPS-GGGS-STpGMAS in the vector was replaced with 8A005.
  • Table 10 shows the nucleotide sequence and amino acid sequence of the linker peptide
  • the above amplification system was: 5 ⁇ Phusion HF Buffer 10 ⁇ L, dNTP (10 mM each dNTP) 1 ⁇ L, DNA template 20 ng, primer (Table 11) (10 ⁇ M) each 1.5 ⁇ L, Phusion High-Fidelity DNA Polymerase (2.5 U/ ⁇ L) 0.5 ⁇ L, supplemented with distilled water to a total volume of 50 ⁇ L.
  • the amplification conditions were: pre-denaturation at 98 ° C for 3 minutes (1 cycle); denaturation at 98 ° C for 10 seconds, annealing at 58 ° C for 10 seconds, extension at 72 ° C for 5.5 minutes (30 cycles); extension at 72 ° C for 10 minutes (1 cycle).
  • the amplified product was purified and digested with Ferentas DpnI enzyme, and the system was: 5 ⁇ Fast Digest Green Buffer 4 ⁇ L, purified product 34 ⁇ L, and DpnI 2 ⁇ L.
  • the digestion temperature and reaction time were 37 ° C and 1 h, respectively, and finally the tapping was recovered and stored.
  • the FPP-001 competent state was prepared according to the above three steps. Then, the recovered rubber obtained by the above step was separately added, mixed and transferred to an electric rotor, 2.7 kv electric shock for 5.7 ms, 1 mL of 1 M sorbitol was added, and resuscitated at 30 ° C for 1 h, respectively, and applied to SD-Ura-His-Leu medium 30. °C, culture for more than 36h. The correct positive clones were identified by PCR and named as strain ELE-012-ELE-019.
  • P TEF1 -SynSmFPS-GGGS-STpGMAS- T CYC1 was prepared 1, P PGK1 -ERG20-GGGS- LsLTC2-T ADH1,, rDNA-TRP1-up and rDNA-TRP1-down of
  • P PGK1 -ERG20-GGGS-LsLTC2- T ADH1 and P TEF1 -SynSmFPS-GGGS-STpGMAS- T CYC1 yeast are carried farnesyl pyrophosphate synthase fusion protein source with lettuce Zimmer alkenyl and codon synthase A Optimized fusion protein expression cassette of Danshen-derived farnesyl pyrophosphate synthase and codon-optimized white chrysanthemum-derived gemasene A synthase; rDNA-TRP1-up and rDNA-TRP1-down are rDNA The upstream homology arm and the downstream homology arm; the fragments were amplified as follows:
  • the functional modules were obtained by PCR using the PCR templates and primers described in Table 12, respectively:
  • the amplification system was: 5 ⁇ Phusion HF Buffer 10 ⁇ L, dNTP (10 mM each dNTP) 1 ⁇ L, DNA template 20 ng, primer (10 ⁇ M) 1.5 ⁇ L each, Phusion High-Fidelity DNA Polymerase (2.5 U/ ⁇ L) 0.5 ⁇ L, supplemented with distilled water To a total volume of 50 ⁇ L.
  • the amplification conditions were: pre-denaturation at 98 ° C for 3 minutes (1 cycle); denaturation at 98 ° C for 10 seconds, annealing at 58 ° C for 10 seconds, extension at 72 ° C for 2 minutes (30 cycles); extension at 72 ° C for 10 minutes (1 Cycle), the product is recovered by tapping.
  • the screening medium composition was: 0.8% yeast selection medium SD-Ura-His-Leu-Trp (Beijing Fenino (Functional Genome) Technology Co., Ltd.), 2% glucose.
  • a positive positive clone was identified by PCR and designated as strain ELE-020.
  • the ELE-020 recombinant strain was deposited on October 20, 2017 at the General Microbiology Center (CGMCC) of the China Microbial Culture Collection Management Committee.
  • CGMCC General Microbiology Center
  • the deposit address is No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing.
  • Yeast Latin name: Saccharomyces cerevisiae, accession number: CGMCC No. 14829.
  • yeast engineering strains prepared in Example 2 were activated in the corresponding solid selection medium SD-Ura-His-Leu, and seed liquids were prepared in the corresponding liquid selection medium SD-Ura-His-Leu (30 ° C, 250 rpm, 16 h)
  • the cells were inoculated with a 1% inoculum in a 100 mL flask containing 15 mL of the corresponding liquid selection medium, and cultured at 30 ° C, shaking at 250 rpm for 1 day, then 1.5 mL of n-dodecane was added, and the culture was further shaken for 5 days. Finally, the liquid in the flask was transferred to a 50 mL centrifuge tube, centrifuged at 5000 rpm for 5 min, and the organic phase was collected for use.
  • the above organic phase sample was heated in a ventilated kitchen in a 100-380 ° C (180 ° C) oil bath for 1 hour to obtain a converted material.
  • the converted material was diluted 10 times with n-hexane, passed through an organic nylon membrane (0.22 ⁇ m), and detected by GC-MS.
  • Testing equipment Agilent GC/MS Agilent 7890A/5975C.
  • GC-MS measurement conditions inlet temperature 250 ° C, injection volume 1 ⁇ L, no split, solvent delay 3 min; column: HP-5ms (30m * 0.25mm); chromatographic conditions: 45 ° C, 1 min, 10 ° C / Min to 300 ° C for 5 min; MS conditions: F ull Scan: 50-750 amu.
  • the high copy number fusion protein gene SynSmFPS-GGGS-STpGMAS was introduced on the basis of FPP-001 to obtain the engineering strain ELE-011, and the yield of ⁇ -elemene was up to 101.1 mg/L;
  • the high copy number fusion protein gene SynSmFPS-Linker-STpGMAS was introduced to obtain the engineering strain ELE-012-ELE-019 (the promoter and linker were TEF1 and 3A001, TEF1 and 4A001, TEF1 and 5A002, respectively). , TEF1 and 6A005, TEF1 and 6B004, TEF1 and 8A005, TEF1 and 12A003, MF1 and 8A005).
  • the fusion protein genes PPGK1-ERG20-GGGS-LsLTC2-TADH1 and PTEF1-SynSmFPS-GGGS-STpGMAS-TCYC1 were recombined on the basis of ELE-019 to obtain the engineering strain ELE-020.
  • Calcium chloride mother liquor 19.2 g / L calcium chloride dihydrate.
  • Trace metal salt mother liquor 19.1 g / L disodium ethylenediaminetetraacetate; 10.2 g / L zinc sulfate heptahydrate; 0.5 g / L manganese chloride tetrahydrate; 0.86 g / L cobalt chloride hexahydrate; 0.78 g / L Copper sulfate pentahydrate; 0.56 g/L sodium molybdate dihydrate; 5.12 g/L iron sulphite heptahydrate.
  • Vitamin mother liquor 0.05g/L biotin; 0.2g/L sodium p-aminobenzoate; 1g/L niacin; 1g/L calcium pantothenate; 1g/L pyridoxine hydrochloride; 1g/L thiamine hydrochloride; 25g/ L inositol.
  • Seed medium and fermentation medium 25g/L glucose, 15g/L ammonium sulfate, 6.15g/L magnesium sulfate heptahydrate, 0.72g/L zinc sulfate heptahydrate, 8g/L potassium dihydrogen phosphate, 2mL/L chlorination Calcium mother liquor, 10 mL / L trace metal salt mother liquor; 12 mL / L vitamin mother liquor, 1 g / L tryptophan, the rest is water.
  • Feeding medium 800g/L glucose, 5.125g/L magnesium sulfate heptahydrate, 3.5g/L potassium sulfate, 0.28g/L sodium sulfate, 9g/L potassium dihydrogen phosphate, 1g/L tryptophan, the rest water.
  • the engineered bacteria ELE-019 was activated by the method of 1. Pick the monoclonal on the plate to the tube containing SD-Ura-His-Leu medium, incubate overnight at 30 ° C, shaking at 250 rpm; pipet 500 ⁇ L of the bacterial solution into a 250 mL triangle containing 50 mL of SD-Ura-His-Leu medium. In a bottle, cultured at 30 ° C, shaking at 250 rpm for 24 h;
  • the parameters set in the fermentation process are: temperature 30 ° C, pH 5.0, dissolved oxygen 30%, air flow 3-20 L / min, stirring speed 300-1000 rpm, dissolved oxygen and stirring speed, ventilation cascade.
  • the feed medium is added to the fermenter until the glucose concentration in the fermentation liquid is 5 g/L.
  • the high-density fermentation of the engineering bacteria ELE-019 can obtain 2g/L (relative to the culture solution) ⁇ -elemene after 96 hours.
  • the recombinant bacteria according to the object of the present invention including but not limited to the specific experimental examples described in Table 13, can be subjected to fermentation culture according to the fermentation method described in "3" to obtain gemasene A.
  • the experiments of the present invention prove that the present invention expresses the gemasene synthase gene or the fusion protein gene thereof in the host yeast, and obtains the recombinant bacteria, which can greatly improve the yield of the gemasene A, and is suitable for industrial production of ⁇ -elemene and / or gemmene A, provides a powerful strain and research basis for the biosynthesis of anti-cancer raw material ⁇ -elemene.

Abstract

提供一种表达吉玛烯A合成酶或其融合蛋白的重组酵母菌，所述融合蛋白为吉玛烯A合成酶和法尼基焦磷酸合酶，该重组菌提高吉玛烯A的产量，适用于工业化生产β-榄香烯和/或吉玛烯A。

Description

一种重组菌及其用途 技术领域
本发明涉及生物化工领域，尤其涉及一种重组菌，以及基于重组微生物法合成β-榄香烯。
背景技术
β-榄香烯(beta-elemene)是一种具有郁金香味的挥发性倍半萜化合物，为国家一类肿瘤新药的原料药。目前主要从郁金和莪术等植物中分离提取得到，但这种方法有较多的缺点，包括含量低和差异大，产品纯化难，植物生长周期长，对生物资源尤其野生资源造成严重破坏等。
利用合成生物学的原理，设计和改造微生物菌株来生产天然产物已被国际认为是一种最有潜力的方法，如在大肠杆菌中生产紫杉醇的前体紫杉二烯已达到1000mg/L(Parayil KuMaran AjikuMar et al.,2010,Science,330:70-74)；银杏内酯类(Ginkgolides)前体左旋海松二烯(Levopimaradiene)，在改造后的大肠杆菌工程菌中达到700mg/L的产量(Effendi Leonard et al.,2010,PNAS,107(31):13654–13659)；在酵母工程菌中生产青蒿素(Artemisinin)的前体青蒿酸(Artemisinic acid)最高达到25g/L(PaddonC J et al.,2013,Nature,496(7446):528-531)；目前国内在青蒿素，紫杉醇和丹参酮等药物分子的生物合成方面有相关研究。
自然界中，法尼基焦磷酸(FPP)能被吉玛烯A合成酶(GMAS)催化合成吉玛烯A，吉玛烯A具有热不稳定性，易发生分子内热重排为β-榄香烯目前已有部分利用重组菌生产β榄香烯前体吉玛烯A的研究，但产量均较低，达不到工业应用的要求。例如：高允允等人通过在大肠杆菌中构建吉玛烯A的生物合成途径，得到的重组菌合成吉玛烯A的最高产量仅为6.32mg/L，距离工业化还有较大的差距[微生物生物合成β-榄香烯前体—吉马烯A研究，高允允，2012，杭州师范大学]。
发明公开
本发明一个目的是提供一种重组菌。
本发明提供的重组菌，为体内含有或表达吉玛烯A合成酶或吉玛烯A 合成酶融合蛋白的酵母菌；
所述吉玛烯A合成酶融合蛋白包括所述吉玛烯A合成酶和法尼基焦磷酸合酶。
上述重组菌中，根据融合蛋白基因宿主来源不同，分为1种或多种，所述融合蛋白的编码核酸包括所述吉玛烯A合成酶的编码核酸和所述法尼基焦磷酸合酶的编码核酸；
所述融合蛋白的编码核酸为1种或多种；
多种所述融合蛋白的编码核酸中，至少2种所述吉玛烯A合成酶编码核酸来源宿主不同，且至少2种所述法尼基焦磷酸合酶编码核酸来源宿主不同。
本发明中所述的基因来源宿主不同是指基因的原始来源宿主不同，本发明所述吉玛烯A合成酶(germacrene A synthase)基因可从已知含有吉玛烯A合成酶的植物或微生物中克隆得到，例如可选自向日葵(Helianthus annuus L.)、小白菊(Tanacetum parthenium)、莴苣(Lactuca sativa Linn.)、青蒿(Artemisia carvifolia)、蓝细菌等，所述法尼基焦磷酸合酶(farnesyl diphosphate synthase)基因可从已知含有吉玛烯A合成酶的植物或微生物中克隆得到，例如可选自丹参(Salvia miltiorrhiza)、酵母菌(Yeast)、刺五加(Acanthopanax senticosus(Rupr.Maxim.)Harms)、杜仲(Eucommia ulmoides Oliv.)等。
所述吉玛烯A合成酶的编码核酸包括SEQ ID NO.3所示的核酸或SEQ ID NO.12第13-1686位所示的核酸；
所述法尼基焦磷酸合酶的编码核酸包括SEQ ID NO.2所示的核酸或SEQ ID NO.11第1-1056位所示的核酸。
上述重组菌中，所述融合蛋白还包括用于连接所述吉玛烯A合成酶和所述法尼基焦磷酸合酶的连接肽；
所述连接肽选自GGGS、YGQ(3A001)、PGGH(4A001)、YRSQI(5A002)、VIPFIS(6A005)、FLYLKF(6B004)、WRFSPKLQ(8A005)或HHVQESQCISTV(12A003)。
上述重组菌中，所述体内含有或表达吉玛烯A合成酶或吉玛烯A合成酶融合蛋白为将所述吉玛烯A合成酶的编码核酸或所述融合蛋白的编码核 酸导入所述酵母菌；
和/或，所述将吉玛烯A合成酶的编码核酸导入所述酵母菌为将含有所述吉玛烯A合成酶的编码核酸表达盒导入所述酵母菌；
所述将融合蛋白的编码核酸导入所述酵母菌为将含有所述融合蛋白的编码核酸表达盒导入所述酵母菌；
和/或，所述含有吉玛烯A合成酶的编码核酸表达盒包括启动子、所述吉玛烯A合成酶的编码核酸和终止子；
和/或，所述含有融合蛋白的编码核酸表达盒包括启动子、所述融合蛋白的编码核酸和终止子；
或所述启动子选自TEF1或MF1或PGK1；所述终止子为CYC1或ADH1；
或所述启动子为TEF1，且终止子为CYC1；
或所述启动子为MF1，且终止子为CYC1；
或所述启动子为PGK1，且终止子为ADH1。
上述中，启动子TEF1包含SEQ ID NO.4所示的序列；所述启动子MF1包含SEQ ID NO.1所示的序列；所述终止子CYC1t包含SEQ ID NO.5所示的序列。
上述重组菌中，所述重组菌还表达1个或多个标记基因；和/或，所述标记基因选自his3或trp1。
上述重组菌中，所述含有吉玛烯A合成酶的编码核酸表达盒通过表达所述吉玛烯A合成酶的编码核酸表达盒的载体导入所述酵母菌；
所述含有融合蛋白的编码核酸表达盒通过表达所述含有融合蛋白的编码核酸表达盒的载体导入所述酵母菌。
上述重组菌中，所述吉玛烯A合成酶的编码核酸表达盒通过质粒形式导入所述酵母菌；
或，所述融合蛋白的编码核酸表达盒通过质粒形式和/或整合在染色体的形式导入所述酵母菌。
在本发明的实施例中，
融合蛋白选自如下中至少一种：SynSmFPS-GGGS-STpGMAS，SynSmFPS-YGQ-STpGMAS，SynSmFPS-PGGH-STpGMAS，SynSmFPS-YRSQI-STpGMAS，SynSmFPS-VIPFIS-STpGMAS， SynSmFPS-FLYLKF-STpGMAS，SynSmFPS-WRFSPKLQ-STpGMAS，SynSmFPS-HHVQESQCISTV-STpGMAS，SynSmFPS-WRFSPKLQ-STpGMAS，ERG20-GGGS-LsLTC2；
融合蛋白优选为SynSmFPS-8A005-STpGMAS；
融合蛋白尤其优选为3种融合蛋白：SynSmFPS-WRFSPKLQ(8A005)-STpGMAS、ERG20-GGGS-LsLTC2、SynSmFPS-GGGS-STpGMAS；
表达融合蛋白编码核酸的表达盒选自如下至少一种：
PTEF1-SynSmFPS-GGGS-STpGMAS-TCYC1，
PTEF1-SynSmFPS-YGQ-STpGMAS-TCYC1，
PTEF1-SynSmFPS-PGGH-STpGMAS-TCYC1，
PTEF1-SynSmFPS-YRSQI-STpGMAS-TCYC1，
PTEF1-SynSmFPS-VIPFIS-STpGMAS-TCYC1，
PTEF1-SynSmFPS-FLYLKF-STpGMAS-TCYC1，
PTEF1-SynSmFPS-WRFSPKLQ(8A005)-STpGMAS-TCYC1，
PTEF1-SynSmFPS-HHVQESQCISTV-STpGMAS-TCYC1，
或PMF1-SynSmFPS-WRFSPKLQ(8A005)-STpGMAS-TCYC1；
表达融合蛋白编码核酸的表达盒优选为：PMF1-SynSmFPS-8A005-STpGMAS-TCYC1；
表达融合蛋白编码核酸的表达盒尤其优选为3种：PMF1-SynSmFPS-8A005-STpGMAS-TCYC1、PPGK1-ERG20-GGGS-LsLTC2-TADH1和PTEF1-SynSmFPS-GGGS-STpGMAS-TCYC1。
表达吉玛烯A合成酶编码核酸表达盒的载体选自如下：
pRS313-LEU2-PTEF1-STpGMAS-TCYC1，
pRS425-LEU2-PTEF1-STpGMAS-TCYC1，
表达吉玛烯A合成酶编码核酸表达盒的载体选自如下：
pRS425-LEU2-PTEF1-SynSmFPS-GGGS-STpGMAS-TCYC1，
pRS425-LEU2-PTEF1-SynSmFPS-YGQ-STpGMAS-TCYC1，
pRS425-LEU2-PTEF1-SynSmFPS-PGGH-STpGMAS-TCYC1，
pRS425-LEU2-PTEF1-SynSmFPS-YRSQI-STpGMAS-TCYC1，
pRS425-LEU2-PTEF1-SynSmFPS-VIPFIS-STpGMAS-TCYC1，
pRS425-LEU2-PTEF1-SynSmFPS-FLYLKF-STpGMAS-TCYC1，
pRS425-LEU2-PTEF1-SynSmFPS-WRFSPKLQ-STpGMAS-TCYC1，
pRS425-LEU2-PTEF1-SynSmFPS-HHVQESQCISTV-STpGMAS-TCYC1，
或pRS425-LEU2-PMF1-SynSmFPS-WRFSPKLQ-STpGMAS-TCYC1；
上述整合在染色体上的融合蛋白基因表达盒选自PTEF1-SynSmFPS-GGGS-STpGMAS-TCYC1和PPGK1-ERG20-GGGS-LsLTC2-TADH1。
上述重组菌中，所述酵母菌为提高出发酵母菌中乙醇脱氢酶、乙醛脱氢酶和乙酰辅酶A合成酶的含量和/或活性得到的菌。
所述提高出发酵母菌中乙醇脱氢酶、乙醛脱氢酶和乙酰辅酶A合成酶的含量和/或活性得到的菌为增加所述出发酵母菌中乙醇脱氢酶的编码核酸、乙醛脱氢酶的编码核酸和乙酰辅酶A合成酶的编码核酸的拷贝数；
上述重组菌中，所述增加出发酵母菌中乙醇脱氢酶的编码核酸、乙醛脱氢酶的编码核酸和乙酰辅酶A合成酶的编码核酸的拷贝数为将乙醇脱氢酶的编码核酸表达盒、乙醛脱氢酶的编码核酸表达盒、乙酰辅酶A合成酶的编码核酸表达盒和另一个所述标记基因(his3)采用同源重组导入所述出发酵母菌。
上述重组菌中，所述出发酵母菌为酿酒酵母；和/或，所述酿酒酵母为NK2-SQ。
1个所述标记基因为TRP1；另一个所述标记基因为HIS3。
上述乙醇脱氢酶的基因ADH2包含SEQ ID NO.6所示的序列，乙醛脱氢酶的基因ALD6包含SEQ ID NO.7所示的序列，所述乙酰辅酶A合成酶的基因ACS1包含SEQ ID NO.8所示的序列。
本发明的重组菌及各个所需载体和片段的构建见实施例。
在本发明的实施例中，重组菌具体如下：
重组菌ELE-001，为将pRS313-LEU2-PTEF1-STpGMAS-TCYC1导入酵母FPP-001得到的菌；
重组菌ELE-002，为将pRS425-LEU2-PTEF1-STpGMAS-TCYC1导入酵母FPP-001得到的菌；
重组菌ELE-011，为将pRS425-LEU2-PTEF1-SynSmFPS-GGGS-STpGMAS-TCYC1导入酵母FPP-001得到的菌；
重组菌ELE-012，为将pRS425-LEU2-PTEF1-SynSmFPS-3A001-STpGMAS-TCYC1导入酵母FPP-001得到的菌；
重组菌ELE-013，为将pRS425-LEU2-PTEF1-SynSmFPS-4A001-STpGMAS-TCYC1导入酵母FPP-001得到的菌；
重组菌ELE-014，为将pRS425-LEU2-PTEF1-SynSmFPS-5A002-STpGMAS-TCYC1导入酵母FPP-001得到的菌；
重组菌ELE-015，为将pRS425-LEU2-PTEF1-SynSmFPS-6A005-STpGMAS-TCYC1导入酵母FPP-001得到的菌；
重组菌ELE-016，为将pRS425-LEU2-PTEF1-SynSmFPS-6B004-STpGMAS-TCYC1导入酵母FPP-001得到的菌；
重组菌ELE-017，为将pRS425-LEU2-PTEF1-SynSmFPS-8A005-STpGMAS-TCYC1导入酵母FPP-001得到的菌；
重组菌ELE-018，为将pRS425-LEU2-PTEF1-SynSmFPS-12A003-STpGMAS-TCYC1导入酵母FPP-001得到的菌；
重组菌ELE-019，为将pRS425-LEU2-PMF1-SynSmFPS-8A005-STpGMAS-TCYC1导入酵母FPP-001得到的菌；
重组菌ELE-020，为将pRS425-LEU2-PMF1-SynSmFPS-8A005-STpGMAS-TCYC1、导入酵母FPP-001后，再通过同源重组导入PPGK1-ERG20-GGGS-LsLTC2-TADH1，PTEF1-SynSmFPS-GGGS-STpGMAS-TCYC1,rDNA-TRP1-up和rDNA-TRP1-down得到的菌。
上述酵母FPP-001为将NDT80-HIS3-up、PPGK1-ADH2-TADH1、PTDH3-ACS1-TTPI1、 PTEF1-ALD6-TCYC1和NDT80-HIS3-down导入酿酒酵母中得到的菌。
其中重组菌ELE-020为酿酒酵母Saccharomyces cerevisiae CGMCC No.14829也是本发明保护的范围。
该ELE-020重组菌株于2017年10月20日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏地址：北京市朝阳区北辰西路1号院3号，菌种名称：酿酒酵母，拉定名：Saccharomyces cerevisiae，保藏编号：CGMCC No.14829。
上述重组菌在生产β-榄香烯和/或吉玛烯A中的应用也是本发明保护的范围。
本发明的第3个目的是提供一种生产吉玛烯A的方法。
本发明提供的方法，包括如下步骤：发酵上述的重组菌，得到吉玛烯A；
本发明的第4个目的是提供一种生产β-榄香烯的方法。
本发明提供的方法，包括如下步骤：
1)发酵上述的重组菌，得到发酵产物；
2)有机溶液萃取所述发酵产物，收集有机相；
3)将所述有机相加热，得到β-榄香烯。
上述方法中，所述发酵为先将所述重组菌在种子培养基中培养获得种子液；再将所述种子液接种到发酵培养基中发酵培养，将所述发酵培养所述产物记作发酵体系。
上述方法中，在所述发酵培养过程中，向所述发酵体系中添加补料培养基；优选地，待所述发酵体系中的溶氧值大于60％时，向所述发酵体系中加入补料培养基至所述发酵体系中葡萄糖浓度为5g/L。
上述方法中，每L体积所述种子培养基和所述发酵培养基配方包含：25g葡萄糖，15g硫酸铵，6.15g七水硫酸镁，0.72g七水硫酸锌，8g磷酸二氢钾，2mL氯化钙母液，10mL微量金属盐母液；12mL维他命母液，1g色氨酸，其余为水。
所述氯化钙母液为19.2g/L二水氯化钙水溶液；
每L体积所述微量金属盐母液的配方包含：19.1g乙二胺四乙酸二钠；10.2g七水硫酸锌；0.5g四水氯化锰；0.86g六水氯化钴；0.78g五 水硫酸铜；0.56g二水钼酸钠；5.12g七水亚硫酸铁；其余为水。
每L体积维他命母液的配方包含：0.05g生物素；0.2g对氨基苯甲酸纳；1g烟酸；1g泛酸钙；1g盐酸吡哆醇；1g盐酸硫胺素；25g肌醇；其余为水。
每L体积所述补料培养基的配方含有800g葡萄糖，5.125g七水硫酸镁，3.5g硫酸钾，0.28g硫酸钠，9g磷酸二氢钾和1g色氨酸；其余为水。
在所述发酵前，还包括如下步骤：
a)在固体选择培养基中活化重组菌；
b)在液体选择培养基中震荡培养后，转入种子培养基培养，得到种子液；
其中，所述固体或液体选择培养基为SD-Ura-His-Leu培养基；
上述步骤b)中培养条件为30℃，250rpm；所述接种步骤为火焰环接种；
上述发酵方法中所述种子液的培养方法具体为重组菌活化后，挑取平板上的单克隆至装有SD-Ura-His-Leu培养基的试管，30℃，250rpm振荡培养过夜；吸取500μL菌液至装有50mL SD-Ura-His-Leu培养基的250mL三角瓶中，30℃，250rpm振荡培养24h；分别吸取2mL菌液至3个装有100mL种子培养基的1L三角瓶中，30℃，250rpm振荡培养48h。
上述生产β-榄香烯的方法中，所述有机溶剂为正十二烷；所述加热条件为：100-380℃加热1小时。
附图说明
图1为吉玛烯A生物合成途径。
图2为GC-MS检测图。
实施发明的最佳方式
下述实施例中所使用的实验方法如无特殊说明，均为常规方法。
下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
图1为吉玛烯A生物合成途径。
实施例1、目标基因和所用质粒制备
一、目标基因的制备
1、ADH2、ALD6、ASC1、MF1、TEF1和CYC1的获得
提取酵母NK2-SQ(中国中药杂志，林庭庭，王冬，戴住波，张学礼，黄璐琦，2016，41(6)：1008-1015)的基因组DNA作为模板，用表1中基因扩增所需的引物进行扩增，得到符合预期大小的片段ADH2、ALD6、ASC1基因、启动子MF1、TEF1和终止子CYC1。
取PCR扩增试剂盒TAKARA
Figure PCTCN2017109029-appb-000001
DNApolymerase配置扩增体系(TAKARA公司)，扩增体系为：5×PS Buffer 10μL，dNTPMix 4μL，引物各1μL，基因组DNA模板1μL，
Figure PCTCN2017109029-appb-000002
聚合酶(2.5U/μL)0.5μL，补加蒸馏水至总体积50μL。扩增条件为：98℃预变性3分钟(1个循环)；98℃变性10秒、55℃退火15秒、72℃延伸2.5分钟(30个循环)；72℃延伸10分钟(1个循环)。
表1 为引物序列
Figure PCTCN2017109029-appb-000003
2、来自丹参的法尼基焦磷酸合酶基因SynSmFPS和来自小白菊的吉玛烯A合成酶基因STpGMA的获得
南京金斯瑞生物科技有限公司依照SynSmFPS(SEQ ID NO.2，来源于 丹参)和STpGMAS(SEQ ID NO.3，来源于小白菊)基因序列，设计全长引物，利用OVERLAP方法形成模版DNA，再利用PCR扩增的方法得到SynSmFPS(SEQ ID NO.2)和STpGMAS(SEQ ID NO.3)的双链DNA，然后将PCR产物转化克隆至克隆载体pUC57(南京金斯瑞生物科技有限公司)中，分别构建了含SynSmFPS基因和STpGMAS基因的克隆型质粒pUC57-SynSmFPS和pUC57-STpGMAS。
3、来自酵母的法尼基焦磷酸合酶基因ERG20-GGGS和来自莴苣的吉玛烯A合成酶基因GGGS-LsLTC2的获得
提取莴苣叶片200mg用液氮研磨后CTAB法(十六烷基三甲基溴化铵法)提取总RNA：在1.5ml离心管中加入1ml 2*CTAB提取液(2％CTAB，100mM Tris-HCl PH 8.0,20mM EDTA溶液(乙二胺四乙酸),1.4M NaCl溶液)，65℃预热之后，加入20μl 2-mercaptoethanol(2-巯基乙醇)；加入少量莴苣叶片粉末(约50mg)，混合均匀，65℃保温10min，摇匀5次；4℃，12000rpm离心10min，移出上清，用等体积的氯仿/异戊醇抽提；4℃，12000rpm离心10min，移出上清，用等体积的氯仿/异戊醇抽提；4℃，12000rpm离心10min，移出上清，用1/6体积的氯仿/异戊醇抽提；4℃，15000rpm离心30min，移出上清，加入1/4体积的10mol/LLiCl，4℃放置过夜；4℃，15000rpm离心30min，弃去上清，用75％乙醇洗涤沉淀2次，无水乙醇洗涤沉淀1次，超净台放置15min(室温)；用20μl mill iQ DEPC(溶剂为mi liQ纯水，溶质为diethyl pyrocarbonate焦碳酸二乙酯；焦碳酸二乙酯：水的体积比为1:1000)处理水溶解，加入1/10体积的2mol/L NaAC(pH 4.0)，加入2体积的无水乙醇，-20℃放置2h；4℃，12000rpm离心10min，弃上清，沉淀用75％乙醇洗涤两次，无水乙醇洗涤沉淀1次；超净台放置15min(室温)，加15μlmil liQ DEPC处理水使沉淀充分溶解，-70℃保存。
第一链反转录-PCR：取无RNA酶PCR管，按第一链反转录试剂盒(宝生物工程(大连)有限公司)配备体系：Radom 6Mers 2ul、dNTP 1ul、total RNA 1ul(200ng)、H2O 6ul、Total 10ul、瞬间离心，PCR 65℃5min，冰上急冷；再加入以下体系中反应(第一链反转录试剂盒自带)：5*primer Buffer 4ul、RNAs Inhibiter 0.5ul、R-Transcription1 ul、 H2O 4.5ul，瞬间离心，PCR仪进行反应：30℃10min、42℃60min、70℃15min、4℃保温。
分别以NK2-SQ的基因组DNA和莴苣cDNA为模板，用表2中引物分别扩增得到约1068bp ERG20-GGGS(SEQ ID NO.11中第13-1686为ERG20)和1688bp GGGS-LsLTC2(SEQ ID NO.12中第1-1056为LsLTC2)。
按PCR扩增试剂盒Phusion High-Fidelity PCR Master Mix with HF Buffer((购自NEB(北京)有限公司)配置体系。扩增体系均为：5×Phusion HF Buffer 10μL、dNTP(10mM each dNTP)1μL、DNA模板20ng、引物(10μM)各1.5μL、Phusion High-Fidelity DNA Polymerase(2.5U/μL)0.5μL、补加蒸馏水至总体积50μL。扩增条件为：98℃预变性3分钟(1个循环)；98℃变性10秒、58℃退火10秒、72℃延伸1分钟(30个循环)；72℃延伸10分钟(1个循环)。
表2 引物序列
Figure PCTCN2017109029-appb-000004
二、重组质粒的构建
1、pM2-ADH2质粒
用SexA1(购自NEB(北京)有限公司)和Asc1(购自NEB(北京)有限公司)分别对上述“一、目标基因的制备”中扩增得到的ADH2以及质粒pM2-tHMG1(记载在中国专利ZL201310399947.X中)进行双酶切，得到1052bpADH2酶切产物和4738bp质粒pM2-tHMG1酶切后骨架；再将ADH2酶切产物与质粒pM2-tHMG1酶切后骨架连接，得到重组质粒pM2-ADH2。
2、pM4-ACS1质粒
用SexA1和Asc1分别对上述“一、目标基因的制备”中扩增得到的ACS1以及质粒pM4-AtCPR1(记载在中国专利ZL201310399947.X中)进行双酶切；得到2201bp ACS1酶切产物、和5061bp质粒pM4-AtCPR1酶切后 骨架；再将ACS1酶切产物与质粒pM4-AtCPR1酶切后骨架连接，得到重组质粒pM4-ACS1。
3、pM3-ALD6质粒
用SexA1和Asc1分别上述“一、目标基因的制备”中扩增得到的ALD6以及质粒pM3-ERG9(记载在中国专利申请ZL201310399947.X中)进行双酶切；得到1511bp ALD6酶切产物和4598bp质粒pM3-ERG9酶切后骨架；再将ALD6酶切产物与质粒pM3-ERG9酶切后骨架连接，得到重组质粒pM3-ALD6。
4、pRS313-LEU2-PTEF1-STpGMAS-TCYC1和pRS425-LEU2-PTEF1-STpGMAS-TCYC1质粒的构建
SexA1酶切上述“一、目标基因的制备”中扩增得到的TEF1，得到440bpTEF1酶切产物；
Asc1酶切上述“一、目标基因的制备”中扩增得到的CYC1，得到322bpCYC1酶切产物；
SexA1和Asc1酶切pUC57-STpGMAS，回收1694bp的STpGMAS；
将酶切产物TEF1、CYC1和STpGMAS各50ng加入连接体系：2μL 10×T4 DNA Ligase Reaction Buffer(NEB公司)、1μL T4 DNA Ligase(NEB公司，400,000cohesive end units/ml)，补充蒸馏水至20μL，室温反应2小时得到连接产物；
取1μL连接产物加入PCR体系(Phusion High-Fidelity PCR Master Mix with HF Buffer试剂盒，NEB公司)：5×Phusion HF Buffer 10μL、dNTP(10mM each dNTP)1μL、DNA模板20ng、加入表3引物Sac11-TEF1和CYC1-Sac11(10μM)各1.5μL、Phusion High-Fidelity DNA Polymerase(2.5U/μL)0.5μL、补加蒸馏水至总体积50μL。扩增条件为：98℃预变性3分钟(1个循环)；98℃变性10秒、58℃退火10秒、72℃延伸1.5分钟(30个循环)；72℃延伸10分钟(1个循环)。得到2456bp的PCR扩增产物。
将扩增产物纯化，然后用SacII酶切，割胶回收目的片段SacII-TEF1-STpGMAS-CYC1-SacII，备用。
用SacII分别酶切质粒pRS313(Sikorski,R.S.and  Hieter,P.1989,Genetics 122(1):19-27)和pRS425(Sikorski,R.S.and Hieter,P.1989,Genetics 122(1):19-27)，得到4967bp的pRS313载体片段和6849bp的pRS425载体片段；再分别加入4μL NEB buffer、1μL CIP去磷酸化酶(NEB公司)，补充蒸馏水至40μL，37℃处理1h，加入终浓度为10μmol的EDTA，65℃30min终止反应，割胶回收pRS313-SacII载体片段和pRS425-SacII载体片段。
将上述“一、目标基因的制备”步骤所得载体片段pRS313-SacII、pRS425-SacII分别和SacII-TEF1-STpGMAS-CYC1-SacII各50ng加入连接体系：2μL 10×T4 DNA Ligase Reaction Buffer(NEB公司)、1μL T4 DNA Ligase(NEB公司，400,000cohesive end units/ml)，补充蒸馏水至20μL，室温反应2小时得到连接产物，转入Trans10感受态细胞中和测序验证，得到pRS313-HIS3-PTEF1-STpGMAS-TCYC1和pRS425-LEU2-PTEF1-STpGMAS-TCYC1质粒。
以pRS313-HIS3-PTEF1-STpGMAS-TCYC1质粒为模板，用引物表3中引物，扩增6692bp质粒骨架pRS313-TEF1-STpGMAS-CYC1；
用pRS425为模板，用引物表3中引物，扩增LEU2(1808bp)。
扩增体系均为：5×Phusion HF Buffer 10μL、dNTP(10mM each dNTP)1μL、DNA模板20ng、引物(10μM)各1.5μL、Phusion High-Fidelity DNA Polymerase(2.5U/μL)0.5μL、补加蒸馏水至总体积50μL。扩增条件为：98℃预变性3分钟(1个循环)；98℃变性10秒、58℃退火10秒、72℃延伸4分钟(30个循环)；72℃延伸10分钟(1个循环)。
割胶纯化目的片段。在产物LEU2片段中加入2μL 10×T4 DNA Ligase Reaction Buffer(NEB公司)、1μL T4 Polynucleotide kinase(NEB公司)，补充蒸馏水至20μL，37℃磷酸化1h，割胶回收后同pRS313-PTEF1-STpGMAS-TCYC1用T4 DNA连接酶(NEB公司)连接，转化，测序验证后得到质粒：pRS313-LEU2-PTEF1-STpGMAS-TCYC1。
表3 引物序列
Figure PCTCN2017109029-appb-000005
Figure PCTCN2017109029-appb-000006
5、pRS425-LEU2-PTEF1-SynSmFPS-GGGS-STpGMAS-TCYC1质粒的构建
以pUC57-SynSmFPS和pUC57-STpGMAS为模板，用表4中引物分别扩增得到1080bpSynSmFPS-GGGS和1704bp GGGS-STpGMAS。
扩增体系均为：5×Phusion HF Buffer 10μL、dNTP(10mM each dNTP)1μL、DNA模板20ng、引物(10μM)各1.5μL、Phusion High-Fidelity DNA Polymerase(2.5U/μL)0.5μL、补加蒸馏水至总体积50μL。扩增条件为：98℃预变性3分钟(1个循环)；98℃变性10秒、58℃退火10秒、72℃延伸1分钟(30个循环)；72℃延伸10分钟(1个循环)。
对SynSmFPS-GGGS和GGGS-STpGMAS共同作为模板，用表4中引物(SexA1-SynSmFPS和STpGMAS-Asc1)扩增得到2767bpSynSmFPS-GGGS-STpGMAS片段。
扩增体系均为：5×Phusion HF Buffer 10μL、dNTP(10mM each dNTP)1μL、DNA模板SynSmFPS-GGGS和GGGS-STpGMAS各20ng、引物(10μM)各1.5μL、Phusion High-Fidelity DNA Polymerase(2.5U/μL)0.5μL、补加蒸馏水至总体积50μL。扩增条件为：98℃预变性3分钟(1个循环)；98℃变性10秒、58℃退火10秒、72℃延伸2分钟(30个循环)；72℃延伸10分钟(1个循环)。
将扩增产物纯化，然后用SexA1和Asc1酶切，割胶回收目的片段SexA1-SynSmFPS-GGGS-STpGMAS-Asc1(2760bp)，备用。
将前述第“4”项中所构建质粒pRS425-LEU2-PTEF1-STpGMAS-TCYC1用SexA1和Asc1酶切，割胶回收7602bp大片段，得到载体pRS425-LEU2-PTEF1-...-TCYC1；将载体pRS425-LEU2-PTEF1-...-TCYC1与SexA1-SynSmFPS-GGGS-STpGMAS-Asc1各50ng加入连接体系：2μL 10×T4 DNA Ligase Reaction Buffer(NEB公司)、1μLT4 DNA Ligase(NEB公 司，400,000cohesive end units/ml)，补充蒸馏水至20μL，室温反应2小时得到连接产物，转入Trans10感受态细胞中，提取质粒测序验证，得到pRS425-LEU2-PTEF1-SynSmFPS-GGGS-STpGMAS-TCYC1质粒。
表4 引物序列
Figure PCTCN2017109029-appb-000007
6、pRS425-LEU2-PMF1-SynSmFPS-GGGS-STpGMAS-TCYC1质粒的构建
用BamH1(购自TaKaRa公司)和SexA1分别对上述“一、目标基因的制备”得到的MF1和前述第“5”项所构建的质粒pRS425-LEU2-PTEF1-SynSmFPS-GGGS-STpGMAS-TCYC1进行双酶切；割胶纯化814bp目标启动子基因MF1和9898bp载体片段pRS425-LEU2-...-SynSmFPS-GGGS-STpGMAS-TCYC1，并将二者加入连接体系(各50ng)：2μL10×T4 DNA Ligase Reaction Buffer(NEB公司)、1μL T4 DNA Ligase(NEB公司，400,000cohesive end units/ml)，补充蒸馏水至20μL，室温反应2小时得到连接产物，转化Trans10感受态细胞中，提取质粒测序验证，将得到的序列正确的质粒命名为pRS425-LEU2-PMF1-SynSmFPS-GGGS-STpGMAS-TCYC1。
7、pM2-ERG20-GGGS-LsLTC2质粒的构建
对ERG20-GGGS和GGGS-LsLTC2共同作为模板，用表5中引物(SexA1-ERG20和LsLTC2-Asc1)扩增得到约2744bp ERG20-GGGS-LsLTC2片段。
扩增体系均为：5×Phusion HF Buffer 10μL、dNTP(10mM each dNTP)1μL、DNA模板ERG20-GGGS和GGGS-LsLTC2各20ng、引物(10μM)各1.5μL、Phusion High-Fidelity DNA Polymerase(2.5U/μL)0.5μL、补加蒸馏水至总体积50μL。扩增条件为：98℃预变性3分钟(1个循环)；98℃变性10秒、58℃退火10秒、72℃延伸2分钟(30个循环)；72℃延伸10分钟(1个循环)。
将扩增产物纯化，然后用SexA1和Asc1酶切，割胶回收目的片段SexA1-ERG20-GGGS-LsLTC2-Asc1(约2744bp)，然后将其与载体与质粒pM2-tHMG1酶切后骨架连接，得到重组质粒pM2-ERG20-GGGS-LsLTC2。
表5 引物序列
Figure PCTCN2017109029-appb-000008
8、pEASY-NDT80-HIS3质粒的构建
分别以NK2-SQ的基因组DNA和pRS313模板，用引物表6中引物，扩增得到1252bpNDT80(SEQ ID NO.13)和1168bp HIS3(SEQ ID NO.14)。
扩增体系为：5×Phusion HF Buffer 10μL、dNTP(10mM each dNTP)1μL、DNA模板20ng、引物(10μM)各1.5μL、Phusion High-Fidelity DNA Polymerase(2.5U/μL)0.5μL、补加蒸馏水至总体积50μL。扩增条件为：98℃预变性3分钟(1个循环)；98℃变性10秒、58℃退火10秒、72℃延伸1分钟(30个循环)；72℃延伸10分钟(1个循环)。
将扩增产物NDT80克隆到pEASY-Blunt Simple克隆载体(pEASY克隆载体，北京全式金生物技术(TransGen Biotech)有限公司)转化Trans10感受态细胞中，提取质粒测序验证，得到质粒pEASY-NDT80。
表6 引物
Figure PCTCN2017109029-appb-000009
PmeI(购自NEB(北京)有限公司))酶切pEASY-NDT80，割胶纯化5122bp目的片段(30ng)，4μL NEB buffer(反应缓冲液，购自NEB(北京)有限公司)、1μL CIP去磷酸化酶(NEB公司)，补充蒸馏水至40μL，37℃处理1h，加入终浓度为10μmol的EDTA，65℃30min终止反应，割胶回收5122bp目的片段pEASY-NDT80，备用。
割胶纯化HIS3(30ng)，加入4μL 10×T4 DNA Ligase Reaction Buffer(NEB公司)、1μL T4 Polynucleotide kinase(NEB公司)，补充蒸馏水至40μL，37℃磷酸化1h，割胶回收后，与pEASY-NDT80用T4 DNA连接酶连接(NEB公司)，转化Trans10感受态细胞，测序验证，得到质粒pEASY-NDT80-HIS3。
上述构建的各质粒信息如下表7所示：
表7 质粒信息
Figure PCTCN2017109029-appb-000010
Figure PCTCN2017109029-appb-000011
9、pEASY-rDNA-TRP1质粒的构建
分别以NK2-SQ的基因组DNA和pRS314(Sikorski,R.S.and Hieter,P.1989,Genetics 122(1):19-27)模板，用引物表8中引物，扩增得到约rDNA(SEQ ID NO.9)和TRP1(SEQ ID NO.10)。
扩增体系为：5×Phusion HF Buffer 10μL、dNTP(10mM each dNTP)1μL、DNA模板20ng、引物(10μM)各1.5μL、Phusion High-Fidelity DNA Polymerase(2.5U/μL)0.5μL、补加蒸馏水至总体积50μL。扩增条件为：98℃预变性3分钟(1个循环)；98℃变性10秒、58℃退火10秒、72℃延伸1分钟(30个循环)；72℃延伸10分钟(1个循环)。
将扩增产物rDNA克隆到pEASY-Blunt Simple克隆载体转化Trans10感受态细胞中，提取质粒测序验证，得到质粒pEASY-rDNA。
表8 引物
Figure PCTCN2017109029-appb-000012
PmeI酶切pEASY-rDNA，割胶纯化5122bp目的片段(30ng)，4μL NEB buffer、1μL CIP去磷酸化酶(NEB公司)，补充蒸馏水至40μL，37℃处理1h，加入终浓度为10μmol的EDTA，65℃30min终止反应，割胶回收5122bp目的片段pEASY-rDNA，备用。
割胶纯化TRP1(30ng)，加入4μL 10×T4 DNA Ligase Reaction Buffer(NEB公司)、1μL T4 Polynucleotide kinase(NEB公司)，补充蒸馏水至40μL，37℃磷酸化1h，割胶回收后，与pEASY-rDNA用T4 DNA连接酶(NEB公司)连接，转化Trans10感受态细胞，测序验证，得到质粒pEASY-rDNA-TRP1。
实施例2、重组菌的构建
一、酵母感受态的制备
将出发菌分别于相应培养基(表13)中30℃，250rpm过夜培养，取1mL(OD约0.6-1.0)分装到1.5mL EP管中，4℃、10000g离心1min，弃上清，沉淀用无菌水(4℃)洗涤，同样条件下离心，弃上清。菌体加入1mL处理液(10mM LiAc(醋酸锂)；10mM DTT(二硫苏糖醇)；0.6M sorbitol(山梨糖醇)；10mM Tris-HCl(三羟甲基氨基甲烷盐酸缓冲溶液，pH 7.5),处理液使用时才加DTT)，25℃下放置20min。离心，弃上清，菌体中加入1mL 1M sorbitol(0.22μm水系膜过膜除菌)重悬，离心，弃上清(用1M sorbitol重悬二次)，到最终体积约为90μL。
二、FPP-001菌株的构建
1、NDT80-HIS3-up,PPGK1-ADH2-TADH1，PTDH3-ACS1-TTPI1，PTEF1-ALD6-TCYC1和NDT80-HIS3-down的制备
PPGK1-ADH2-TADH1，PTDH3-ACS1-TTPI1，PTEF1-ALD6-TCYC1分别为携带有乙醇脱氢酶2、乙酰辅酶A合成酶1和乙醛脱氢酶6的表达盒；NDT80-HIS3-up和NDT80-HIS3-down分别为HIS3的上游同源臂和下游同源臂；分别按照如下方法扩增片段：
分别用表9描述的PCR模板和引物进行PCR获得功能模块：698bpM1(NDT80-HIS3-up)，2081bp M2(PPGK1-ADH2-TADH1)，3519bp M3(PTDH3-ACS1-TTPI1)，2376bp M4(PTEF1-ALD6-TCYC1)，1835bp M5(NDT80-HIS3-down)。
扩增体系为：5×Phusion HF Buffer 10μL、dNTP(10mM each dNTP)1μL、DNA模板20ng、引物(10μM)各1.5μL、Phusion High-Fidelity DNA Polymerase(2.5U/μL)0.5μL、补加蒸馏水至总体积50μL。扩 增条件为：98℃预变性3分钟(1个循环)；98℃变性10秒、58℃退火10秒、72℃延伸均用2分钟(30个循环)；72℃延伸10分钟(1个循环)，产物经割胶回收保存。
表9 引物
Figure PCTCN2017109029-appb-000013
Figure PCTCN2017109029-appb-000014
2、FPP-001菌株的构建
出发菌酿酒酵母NK2-SQ于SD-Ura液体培养基(0.8％酵母选择培养基SD-Ura-Trp-His(北京泛基诺(功能基因组)科技有限公司)，2％葡萄糖，0.005％His，0.01％Trp)中过夜培养后制备感受态。然后，加入表9中转化用片段M1，M2，M3，M4和M5共5μg(摩尔比＝1:1:1:1:1)，混匀后转移至电转杯中，2.7kv电击5.7ms，加入1mL 1M sorbitol，30℃复苏1h，涂布于SD-Ura-His培养基，30℃，培养36h以上。筛选培养基成分为：0.8％酵母选择培养基SD-Ura-Trp-His(北京泛基诺(功能基因组)科技有限公司)，2％葡萄糖，0.01％Trp。PCR鉴定出正确的阳性克隆，命名为菌株FPP-001。
三、ELE-001和ELE-002菌株的构建
出发菌酿酒酵母FPP-001于SD-Ura-His液体培养基中过夜培养后制备感受态。然后，分别加入质粒pRS313-LEU2-PTEF1-STpGMAS-TCYC1和pRS425-LEU2-PTEF1-STpGMAS-TCYC1，混匀后转移至电转杯中，2.7kv电击5.7ms，加入1mL 1M sorbitol，30℃复苏1h，涂布于SD-Ura-His-Leu培养基，30℃，培养36h以上。筛选培养基成分为：0.8％酵母选择培养基SD-Ura-Trp-Leu-His(北京泛基诺(功能基因组)科技有限公司)，2％葡萄糖，0.01％Trp。PCR鉴定出正确的阳性克隆，分别命名为菌株ELE-001(转入质粒pRS313-LEU2-PTEF1-STpGMAS-TCYC1)和ELE-002(转入质粒pRS425-LEU2-PTEF1-STpGMAS-TCYC1)。
四、ELE-011菌株的构建
按照上述三中步骤制备FPP-001感受态。然后，加入质粒pRS425-LEU2-PTEF1-SynSmFPS-GGGS-STpGMAS-TCYC1，混匀后转移至电转杯中，2.7kv电击5.7ms，加入1mL 1M sorbitol，30℃复苏1h，涂布于SD-Ura-His-Leu培养基30℃，培养36h以上。PCR鉴定出正确的阳性克隆，命名为菌株ELE-011。
五、ELE-012-ELE-019菌株的构建
以pRS425-LEU2-PTEF1-SynSmFPS-GGGS-STpGMAS-TCYC1质粒为模板，用表11的引物分別进行PCR扩增，得到不同引物对应的扩增产物；再分别将不同引物对应的扩增产物转入酵母FPP-001中进行自身同源重组，分别得到重组菌ELE-012-ELE-018，使该载体中的融合蛋白SynSmFPS-GGGS-STpGMAS之间的连接肽GGGS分别替换成3A001、4A001、5A002、6A005、6B004、8A005、12A003(表10所示)。
以pRS425-LEU2-PMF1-SynSmFPS-GGGS-STpGMAS-TCYC1质粒为模板，用表10中连接肽为8A005的引物(表11)进行PCR扩增，得到不同引物对应的扩增产物；再分别将不同引物对应的扩增产物转入酵母FPP-001中进行自身同源重组，得到重组菌ELE-019，使该载体中的融合蛋白SynSmFPS-GGGS-STpGMAS之间的连接肽GGGS替换成8A005。
表10 为连接肽核苷酸序列和氨基酸序列
连接肽名称 核苷酸序列(5’→3’) 连接肽氨基酸序列
3A001 TACGGTCAG YGQ
4A001 CCGGGGGGACAC PGGH
5A002 TATAGAAGTCAAATC YRSQI
6A005 GTGATACCTTTTATTTCA VIPFIS
6B004 TTTTTGTATCTTAAGTTT FLYLKF
8A005 TGGCGGTTCTCGCCGAAGCTTCAG WRFSPKLQ
12A003 CACCACGTGCAGGAGTCACAATGTATTTCCACAGTG HHVQESQCISTV
具体反应条件如下：
上述扩增体系为：5×Phusion HF Buffer 10μL、dNTP(10mM each dNTP)1μL、DNA模板20ng、引物(表11所示)(10μM)各1.5μL、Phusion High-Fidelity DNA Polymerase(2.5U/μL)0.5μL、补加蒸馏水至总体积50μL。扩增条件为：98℃预变性3分钟(1个循环)；98℃变性10秒、58℃退火10秒、72℃延伸均用5.5分钟(30个循环)；72℃延伸10分钟(1个循环)。
扩增产物经纯化后，利用Fermentas公司的DpnI酶进行消化处理，其体系为：5×Fast Digest Green Buffer 4μL，纯化产物34μL，DpnI 2μL。酶切温度和反应时间分别为37℃和1h，最后割胶回收保存。
表11 引物
Figure PCTCN2017109029-appb-000015
Figure PCTCN2017109029-appb-000016
按照上述三中步骤制备FPP-001感受态。然后，分别加入上步所得割胶回收产物，混匀后转移至电转杯中，2.7kv电击5.7ms，加入1mL 1M sorbitol，30℃复苏1h，分别涂布于SD-Ura-His-Leu培养基30℃，培养36h以上。PCR鉴定出正确的阳性克隆，分别命名为菌株ELE-012-ELE-019。
六、ELE-020重组菌株的构建
1、PPGK1-ERG20-GGGS-LsLTC2-TADH1，PTEF1-SynSmFPS-GGGS-STpGMAS-TCYC1，rDNA-TRP1-up,和rDNA-TRP1-down的制备
PPGK1-ERG20-GGGS-LsLTC2-TADH1和PTEF1-SynSmFPS-GGGS-STpGMAS-TCYC1分别为携带有酵母法尼基焦磷酸合酶与莴苣来源吉玛烯A合酶的融合蛋白和密码子优化后的丹参来源的的法尼基焦磷酸合酶与密码子优化后的小白菊来源吉玛烯A合酶的融合蛋白表达盒；rDNA-TRP1-up和rDNA-TRP1-down分别为rDNA的上游同源臂和下游同源臂；分别按照如下方法扩增片段：
分别用表12描述的PCR模板和引物进行PCR获得功能模块：
M1(rDNA-TRP1-up)，
M2(PPGK1-ERG20-GGGS-LsLTC2-TADH1)，
M3(PTEF1-SynSmFPS-GGGS-STpGMAS-TCYC1)，
M4(rDNA-TRP1-down)。
扩增体系为：5×Phusion HF Buffer 10μL、dNTP(10mM each dNTP)1μL、DNA模板20ng、引物(10μM)各1.5μL、Phusion High-Fidelity DNA Polymerase(2.5U/μL)0.5μL、补加蒸馏水至总体积50μL。扩增条件为：98℃预变性3分钟(1个循环)；98℃变性10秒、58℃退火10秒、72℃延伸均用2分钟(30个循环)；72℃延伸10分钟(1个循环)，产物经割胶回收保存。
表12 引物
Figure PCTCN2017109029-appb-000017
Figure PCTCN2017109029-appb-000018
出发菌酿酒酵母ELE-019于SD-Ura-His-Leu液体培养基中过夜培养后制备感受态。然后，加入表12中转化用片段M1，M2，M3和M4共4μg(摩尔比＝1:1:1:1)，混匀后转移至电转杯中，2.7kv电击5.7ms，加入1mL 1M sorbitol，30℃复苏1h，涂布于SD-Ura-His-Leu-Trp培养基，30℃，培养36h以上。筛选培养基成分为：0.8％酵母选择培养基SD-Ura-His-Leu-Trp(北京泛基诺(功能基因组)科技有限公司)，2％葡萄糖。PCR鉴定出正确的阳性克隆，命名为菌株ELE-020。
该ELE-020重组菌株于2017年10月20日保藏于中国微生物菌种保藏管理委员会普通微生物中心(CGMCC)，保藏地址：北京市朝阳区北辰西路1号院3号，菌种名称：酿酒酵母，拉丁名：Saccharomyces cerevisiae，保藏编号：CGMCC No.14829。
上述所有的工程菌株信息如表13所示。
表13 工程菌株信息
Figure PCTCN2017109029-appb-000019
Figure PCTCN2017109029-appb-000020
Figure PCTCN2017109029-appb-000021
实施例3、重组菌在生产β-榄香烯中的应用
1、工程菌培养及产物提取
在相应固体选择培养基SD-Ura-His-Leu中活化实施例2制备的所有酵母工程菌株，于相应液体选择培养基SD-Ura-His-Leu中制备种子液(30℃，250rpm，16h)，以1％的接种量接种于含15mL相应液体选择培养基的100mL三角瓶中，30℃，250rpm振荡培养1天，然后加入1.5mL正十二烷，继续震荡培养5天。最后，将三角瓶中液体转移至50mL离心管，5000rpm离心5min，收集有机相备用。
2、β-榄香烯转化及其定性定量分析
1)β-榄香烯转化
将上述有机相样品在通风厨内中经100-380℃(180℃)油浴加热1小时，得到转化后物质。
2)检测
将转化后物质用正己烷稀释10倍，过有机尼龙膜(0.22μm)，用GC-MS检测。检测仪器：安捷伦气质联用仪Agilent 7890A/5975C。GC-MS测定条件：进样口温度250℃，进样体积1μL，不分流，溶剂延时3min；色谱柱：HP-5ms(30m*0.25mm)；色谱条件：45℃，1min，10℃/min到300℃保温5min；MS条件：F ull Scan:50-750amu。用β-榄香烯的标准品进行定性定量，β-榄香烯标准品在中国药品生物制品检定所购买(货号：100268)。图2为实施例2制备的所有酵母工程菌株生产β-榄香烯的GC-MS检测图。
结果各工程菌发酵6天时产量如下：
在FPP-001的基础上分别引入低、高拷贝数的STpGMAS，得到工程菌株ELE-001和ELE-002。其中，ELE-001的β-榄香烯产量达9.3mg/L，ELE-002的β-榄香烯产量达22.1mg/L；
在FPP-001的基础上引入高拷贝数的融合蛋白基因SynSmFPS-GGGS-STpGMAS，得到工程菌株ELE-011，其β-榄香烯的产量达 101.1mg/L；
在FPP-001的基础上分别引入高拷贝数的融合蛋白基因SynSmFPS-Linker-STpGMAS，得到工程菌株ELE-012-ELE-019(启动子和Linker分别为TEF1和3A001、TEF1和4A001、TEF1和5A002、TEF1和6A005、TEF1和6B004、TEF1和8A005、TEF1和12A003、MF1和8A005)。
在ELE-019的基础上重组导入融合蛋白基因PPGK1-ERG20-GGGS-LsLTC2-TADH1和PTEF1-SynSmFPS-GGGS-STpGMAS-TCYC1，得到工程菌株ELE-020。
利用ELE-012-ELE-020菌株制备β-榄香烯的产量分别达2.2mg/L(相对于培养液)、35.5mg/L、110.4mg/L、108.6mg/L、73.6mg/L、109.7mg/L、48.3mg/L、158.1mg/L和469mg/L。
3、生物反应器发酵培养
1)培养基配置
氯化钙母液：19.2g/L二水氯化钙。
微量金属盐母液：19.1g/L乙二胺四乙酸二钠；10.2g/L七水硫酸锌；0.5g/L四水氯化锰；0.86g/L六水氯化钴；0.78g/L五水硫酸铜；0.56g/L二水钼酸钠；5.12g/L七水亚硫酸铁。
维他命母液：0.05g/L生物素；0.2g/L对氨基苯甲酸纳；1g/L烟酸；1g/L泛酸钙；1g/L盐酸吡哆醇；1g/L盐酸硫胺素；25g/L肌醇。
种子培养基和发酵培养基：25g/L葡萄糖，15g/L硫酸铵，6.15g/L七水硫酸镁，0.72g/L七水硫酸锌，8g/L磷酸二氢钾，2mL/L氯化钙母液，10mL/L微量金属盐母液；12mL/L维他命母液，1g/L色氨酸，其余为水。
补料培养基：800g/L葡萄糖，5.125g/L七水硫酸镁，3.5g/L硫酸钾，0.28g/L硫酸钠，9g/L磷酸二氢钾，1g/L色氨酸，其余为水。
2)工程菌ELE-019发酵
按1方法活化工程菌ELE-019。挑取平板上的单克隆至装有SD-Ura-His-Leu培养基的试管，30℃，250rpm振荡培养过夜；吸取500μL菌液至装有50mL SD-Ura-His-Leu培养基的250mL三角瓶中，30℃，250rpm振荡培养24h；
分别吸取2mL菌液至3个装有100mL种子培养基的1L三角瓶中，30℃，250rpm振荡培养48h；最后经火焰接种环，将种子液加入含3L发酵培养基的7L发酵罐(德国Eppendorf公司，型号：
Figure PCTCN2017109029-appb-000022
)中。
发酵过程中参数设定值分别为：温度30℃，pH 5.0，溶氧30％，空气流量3-20L/min，搅拌转速300-1000rpm，溶氧与搅拌转速、通气级联。当溶氧值大于60％时，向发酵罐中加入补料培养基至发酵液中葡萄糖浓度为5g/L。
在发酵结束前3小时，添加10％(相对于培养液体积)的正十二烷，发酵结束后分离有机相。
按2中转化方法和检测方法处理，并进行定性定量分析，工程菌ELE-019高密度发酵96小时后能得到2g/L(相对于培养液)β-榄香烯。符合本发明目的的重组菌，包括但不限于表13中记载的具体实验例，均可按照“3”项下所述的发酵方法进行发酵培养，得到吉玛烯A。
工业应用
本发明的实验证明，本发明在宿主酵母中表达吉玛烯A合成酶基因或其融合蛋白基因，得到重组菌，可以大大提高吉玛烯A的产量，适用于工业化生产β-榄香烯和/或吉玛烯A，为抗癌原料β-榄香烯的生物合成方面提供有力菌株及研究基础。

Claims (20)

  1. 一种重组菌，为体内含有或表达吉玛烯A合成酶或吉玛烯A合成酶融合蛋白的酵母菌；
    所述吉玛烯A合成酶融合蛋白包括所述吉玛烯A合成酶和法尼基焦磷酸合酶。
  2. 根据权利要求1所述的重组菌，其特征在于：所述融合蛋白的编码核酸包括所述吉玛烯A合成酶的编码核酸和所述法尼基焦磷酸合酶的编码核酸；
    所述融合蛋白的编码核酸为1种或多种；
    多种所述融合蛋白的编码核酸中，至少2种所述吉玛烯A合成酶编码核酸来源宿主不同，且至少2种所述法尼基焦磷酸合酶编码核酸来源宿主不同。
  3. 根据权利要求2所述的重组菌，其特征在于：
    所述吉玛烯A合成酶的编码核酸包括SEQ ID NO.3所示的核酸或SEQ ID NO.12第13-1686位所示的核酸；
    所述法尼基焦磷酸合酶的编码核酸包括SEQ ID NO.2所示的核酸或SEQ ID NO.11第1-1056位所示的核酸。
  4. 根据权利要求1-3中任一所述的重组菌，其特征在于：所述融合蛋白还包括用于连接所述吉玛烯A合成酶和所述法尼基焦磷酸合酶的连接肽；
    所述连接肽选自GGGS、YGQ、PGGH、YRSQI、VIPFIS、FLYLKF、WRFSPKLQ或HHVQESQCISTV。
  5. 根据权利要求1-4中任一所述的重组菌，其特征在于：
    所述体内含有或表达吉玛烯A合成酶或吉玛烯A合成酶融合蛋白为为将所述吉玛烯A合成酶的编码核酸或所述融合蛋白的编码核酸导入所述酵母菌；
    和/或，所述将吉玛烯A合成酶的编码核酸导入所述酵母菌为将含有所述吉玛烯A合成酶的编码核酸表达盒导入所述酵母菌；
    所述将融合蛋白的编码核酸导入所述酵母菌为将含有所述融合蛋白的编码核酸表达盒导入所述酵母菌；
    和/或，所述含有吉玛烯A合成酶的编码核酸表达盒包括启动子、所述吉玛烯A合成酶的编码核酸和终止子；
    和/或，所述含有融合蛋白的编码核酸表达盒包括启动子、所述融合蛋白的编码核酸和终止子；
    或所述启动子选自TEF1或MF1或PGK1；所述终止子为CYC1或ADH1；
    或所述启动子为TEF1，且终止子为CYC1；
    或所述启动子为MF1，且终止子为CYC1；
    或所述启动子为PGK1，且终止子为ADH1。
  6. 根据权利要求1-5中任一所述的重组菌，其特征在于：所述重组菌还表达1个或多个标记基因；和/或，所述标记基因选自his3或trp1。
  7. 根据权利要求5或6所述的重组菌，其特征在于：
    所述含有吉玛烯A合成酶的编码核酸表达盒通过表达所述吉玛烯A合成酶的编码核酸表达盒的载体导入所述酵母菌；
    所述含有融合蛋白的编码核酸表达盒通过表达所述含有融合蛋白的编码核酸表达盒的载体导入所述酵母菌。
  8. 根据权利要求5-7中任一所述的重组菌，其特征在于：
    所述吉玛烯A合成酶的编码核酸表达盒通过质粒形式导入所述酵母菌；
    或，所述融合蛋白的编码核酸表达盒通过质粒形式和/或整合在染色体的形式导入所述酵母菌。
  9. 根据权利要求1-8中任一项所述的重组菌，其特征在于：所述酵母菌为提高出发酵母菌中乙醇脱氢酶、乙醛脱氢酶和乙酰辅酶A合成酶的含量和/或活性得到的菌。
  10. 根据权利要求9所述的重组菌，其特征在于：
    所述提高出发酵母菌中乙醇脱氢酶、乙醛脱氢酶和乙酰辅酶A合成酶的含量和/或活性得到的菌为增加所述出发酵母菌中乙醇脱氢酶的编码核酸、乙醛脱氢酶的编码核酸和乙酰辅酶A合成酶的编码核酸的拷贝数；
    和/或，所述增加出发酵母菌中乙醇脱氢酶的编码核酸、乙醛脱氢酶的编码核酸和乙酰辅酶A合成酶的编码核酸的拷贝数为将乙醇脱氢酶的编码核酸表达盒、乙醛脱氢酶的编码核酸表达盒、乙酰辅酶A合成酶的编码 核酸表达盒和另一个所述标记编码核酸采用同源重组导入所述出发酵母菌。
  11. 根据权利要求9或10所述的重组菌，其特征在于：所述出发酵母菌为酿酒酵母；和/或，所述酿酒酵母为NK2-SQ。
  12. 权利要求11中任一所述重组菌为酿酒酵母Saccharomyces cerevisiae CGMCC No.14829。
  13. 权利要求1-12中任一所述重组菌在生产β-榄香烯和/或吉玛烯A中的应用。
  14. 一种生产吉玛烯A的方法，包括如下步骤：发酵权利要求1-12中任一所述重组菌，得到吉玛烯A。
  15. 一种生产β-榄香烯的方法，包括如下步骤：
    1)发酵权利要求1-12中任一所述重组菌，得到发酵产物；
    2)有机溶液萃取所述发酵产物，收集有机相；
    3)将步骤2)中所述有机相加热，得到β-榄香烯。
  16. 根据权利要求14或15所述的方法，其特征在于：
    所述发酵的方法为：先将所述重组菌在种子培养基中培养获得种子液；再将所述种子液接种到发酵培养基中发酵培养，将所述发酵培养所述产物记作发酵体系。
  17. 根据权利要求16所述的方法，其特征在于：在所述发酵培养过程中，向所述发酵体系中添加补料培养基；优选地，待所述发酵体系中的溶氧值大于60％时，向所述发酵体系中加入补料培养基至所述发酵体系中葡萄糖浓度为5g/L。
  18. 根据权利要求16所述的方法，其特征在于：每L体积所述种子培养基和所述发酵培养基配方包含：25g葡萄糖，15g硫酸铵，6.15g七水硫酸镁，0.72g七水硫酸锌，8g磷酸二氢钾，2mL氯化钙母液，10mL微量金属盐母液；12mL维他命母液，1g色氨酸；
    所述氯化钙母液为19.2g/L二水氯化钙水溶液；
    每L体积所述微量金属盐母液的配方包含：19.1g乙二胺四乙酸二钠；10.2g七水硫酸锌；0.5g四水氯化锰；0.86g六水氯化钴；0.78g五水硫酸铜；0.56g二水钼酸钠；5.12g七水亚硫酸铁；
    每L体积维他命母液的配方包含：0.05g生物素；0.2g对氨基苯甲酸纳；1g烟酸；1g泛酸钙；1g盐酸吡哆醇；1g盐酸硫胺素；25g肌醇。
  19. 根据权利要求17或18所述的方法，其特征在于：每L体积所述补料培养基的配方含有800g葡萄糖，5.125g七水硫酸镁，3.5g硫酸钾，0.28g硫酸钠，9g磷酸二氢钾和1g色氨酸。
  20. 根据权利要求15-19中任一项所述的方法，其特征在于：
    所述有机溶剂为正十二烷；
    所述加热条件为：100-380℃加热1小时。
PCT/CN2017/109029 2016-11-04 2017-11-02 一种重组菌及其用途 WO2018082588A1 (zh)

Priority Applications (5)

Application Number Priority Date Filing Date Title
JP2019544968A JP7129990B2 (ja) 2016-11-04 2017-11-02 組換え酵母及びその使用
EP17867973.4A EP3536792A4 (en) 2016-11-04 2017-11-02 RECOMBINANT YEAST AND ASSOCIATED USE
US16/347,552 US11421199B2 (en) 2016-11-04 2017-11-02 Recombinant yeast and use thereof
US17/812,157 US20220396759A1 (en) 2016-11-04 2022-07-12 Recombinant yeast and use thereof
US18/456,333 US20240010969A1 (en) 2016-11-04 2023-08-25 Recombinant yeast and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201610961269.5 2016-11-04
CN201610961269 2016-11-04

Related Child Applications (2)

Application Number Title Priority Date Filing Date
US16/347,552 A-371-Of-International US11421199B2 (en) 2016-11-04 2017-11-02 Recombinant yeast and use thereof
US17/812,157 Continuation US20220396759A1 (en) 2016-11-04 2022-07-12 Recombinant yeast and use thereof

Publications (1)

Publication Number Publication Date
WO2018082588A1 true WO2018082588A1 (zh) 2018-05-11

Family

ID=62075401

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/109029 WO2018082588A1 (zh) 2016-11-04 2017-11-02 一种重组菌及其用途

Country Status (5)

Country Link
US (3) US11421199B2 (zh)
EP (1) EP3536792A4 (zh)
JP (1) JP7129990B2 (zh)
CN (1) CN108060092B (zh)
WO (1) WO2018082588A1 (zh)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111154665A (zh) * 2020-01-21 2020-05-15 南京工业大学 一株重组解脂耶罗维亚酵母及其构建方法和应用
CN112063540A (zh) * 2020-09-21 2020-12-11 山东大学 一种用于生产β-榄香烯或吉马烯A的重组菌株
CN115305254A (zh) * 2021-05-08 2022-11-08 中国科学院天津工业生物技术研究所 一种萜类底盘微生物与工程菌及其构建方法和应用
CN115322913A (zh) * 2021-05-10 2022-11-11 中国科学院天津工业生物技术研究所 产玫瑰精油的重组酿酒酵母及其构建方法和应用

Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111423300B (zh) * 2019-01-10 2023-02-28 四川弘合生物科技有限公司 一种β榄香烯的制备方法
CN111187786A (zh) * 2020-02-03 2020-05-22 天津大学 重组载体及其在制备比卡菌素中的应用
CN113249282B (zh) * 2021-04-23 2023-06-20 大连大学 一种产β-榄香烯重组菌及其构建方法和用途
CN115247183B (zh) * 2021-04-28 2024-03-19 中国科学院天津工业生物技术研究所 重组微生物的构建方法、相关生物材料及其应用
CN114672425B (zh) * 2022-04-11 2023-09-15 湖北工业大学 产α-古巴烯的重组酿酒酵母及其应用
CN116064266A (zh) * 2022-11-01 2023-05-05 四川大学 盐胁迫抗性增强的重组酿酒酵母菌及其构建方法与应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103820344A (zh) * 2011-12-30 2014-05-28 天津工业生物技术研究所 生产次丹参酮二烯的酿酒酵母基因工程菌及其构建方法与应用
WO2016029153A1 (en) * 2014-08-21 2016-02-25 Manus Biosynthesis, Inc. Methods for production of oxygenated terpenes

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH06261741A (ja) * 1993-03-11 1994-09-20 Nippon Sharyo Seizo Kaisha Ltd 微生物増殖方法
JP4700617B2 (ja) * 2003-11-26 2011-06-15 フイルメニツヒ ソシエテ アノニム パッチュリからのセスキテルペン合成酵素
JP5590769B2 (ja) * 2004-07-27 2014-09-17 ザ リージェンツ オブ ザ ユニバーシティ オブ カリフォルニア イソプレノイド化合物を生産するための遺伝的に修飾された宿主細胞および同宿主細胞の使用方法
CN101928716A (zh) * 2009-06-30 2010-12-29 中国中医科学院中药研究所 丹参法呢基焦磷酸合酶(SmFPS)基因及其编码的蛋白和应用
KR101137026B1 (ko) * 2009-10-30 2012-04-19 한국생명공학연구원 효모 변이주 및 이를 이용한 스쿠알렌의 생산 방법
WO2011141855A1 (en) * 2010-05-10 2011-11-17 Firmenich Sa Method for producing patchoulol and 7 - epi - alpha - slinene
EP2800818B1 (en) * 2012-01-06 2017-07-26 Firmenich SA Genetically engineered yeast cells

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103820344A (zh) * 2011-12-30 2014-05-28 天津工业生物技术研究所 生产次丹参酮二烯的酿酒酵母基因工程菌及其构建方法与应用
WO2016029153A1 (en) * 2014-08-21 2016-02-25 Manus Biosynthesis, Inc. Methods for production of oxygenated terpenes

Non-Patent Citations (8)

* Cited by examiner, † Cited by third party
Title
EFFENDI LEONARD ET AL., PNAS, vol. 107, no. 31, 2010, pages 13654 - 13659
GAO YUNYUN: "Studies on the microbial biosynthesis of the precursor of β-elemene--germacrene A", 2012, HANG-ZHOU NORMAL UNIVERSITY
LIN TINGTINGWANG DONGDAI ZHUBOZHANG XUELIHUANG LUQI, CHINA JOURNAL OF CHINESE MATERIA MEDICA, vol. 41, no. 6, 2016, pages 1008 - 1015
PADDON CJ ET AL., NATURE, vol. 496, no. 7446, 2013, pages 528 - 531
PARAYIL KUMARAN AJIKUMAR ET AL., SCIENCE, vol. 330, 2010, pages 70 - 74
See also references of EP3536792A4
SIKORSKI, R.S.HIETER, P., GENETICS, vol. 122, no. 1, 1989, pages 19 - 27
ZHANG, YAN ET AL.: "Progress of Heterologous Expression of Terpenes in Saccharomyces Cerevisiae", CHEMICAL INDUSTRY AND ENGINEERING PROGRESS, vol. 33, no. 5, 31 December 2014 (2014-12-31), pages 12665 - 1270, XP009514826, ISSN: 1000-6613 *

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111154665A (zh) * 2020-01-21 2020-05-15 南京工业大学 一株重组解脂耶罗维亚酵母及其构建方法和应用
CN112063540A (zh) * 2020-09-21 2020-12-11 山东大学 一种用于生产β-榄香烯或吉马烯A的重组菌株
CN112063540B (zh) * 2020-09-21 2022-05-17 山东大学 一种用于生产β-榄香烯或吉马烯A的重组菌株
CN115305254A (zh) * 2021-05-08 2022-11-08 中国科学院天津工业生物技术研究所 一种萜类底盘微生物与工程菌及其构建方法和应用
CN115305254B (zh) * 2021-05-08 2024-03-26 中国科学院天津工业生物技术研究所 一种萜类底盘微生物与工程菌及其构建方法和应用
CN115322913A (zh) * 2021-05-10 2022-11-11 中国科学院天津工业生物技术研究所 产玫瑰精油的重组酿酒酵母及其构建方法和应用
CN115322913B (zh) * 2021-05-10 2024-03-22 中国科学院天津工业生物技术研究所 产玫瑰精油的重组酿酒酵母及其构建方法和应用

Also Published As

Publication number Publication date
US20220396759A1 (en) 2022-12-15
EP3536792A4 (en) 2020-07-01
US20200362297A1 (en) 2020-11-19
US20240010969A1 (en) 2024-01-11
CN108060092B (zh) 2021-08-27
EP3536792A1 (en) 2019-09-11
US11421199B2 (en) 2022-08-23
JP2020503888A (ja) 2020-02-06
JP7129990B2 (ja) 2022-09-02
CN108060092A (zh) 2018-05-22

Similar Documents

Publication Publication Date Title
WO2018082588A1 (zh) 一种重组菌及其用途
CN110106209B (zh) 一种利用解脂耶氏酵母途径定位合成萜类化合物的方法
CN108085262B (zh) 对萜类或含萜精油耐受性提高或萜类产量提高的重组宿主细胞、其产生方法及其用途
CN106367361B (zh) 一种酿酒酵母工程菌株及其构建方法、应用
CN108795789A (zh) 一种高产衣康酸解脂耶氏酵母工程菌株及其构建方法、发酵工艺与应用
CN114621968A (zh) 四氢嘧啶生物合成基因簇、突变体及制备四氢嘧啶的方法
CN114058525A (zh) 一种高产角鲨烯基因工程菌及其构建方法与应用
CN107354118B (zh) 一种具有γ-松油烯合成能力的基因工程菌及其构建方法与应用
CN114107078A (zh) 一种高产瓦伦烯基因工程菌及其构建方法与应用
CN109097342A (zh) 蓝色犁头霉中甾类11β-羟化酶及其编码基因与应用
CN111154665B (zh) 一株重组解脂耶罗维亚酵母及其构建方法和应用
CN110283806B (zh) 一种紫色红曲霉酯合成酶lip05-50、编码基因及其应用
CN109055417B (zh) 一种重组微生物、其制备方法及其在生产辅酶q10中的应用
CN116396876A (zh) 一种生产人参皂苷Rd的酿酒酵母工程菌及其构建方法
CN110904062A (zh) 一株高产l-丙氨酸的菌株
CN111607546B (zh) 一种高产法尼烯的基因工程菌及其构建方法与应用
CN110551702B (zh) 重组塔宾曲霉单宁酶及其表达和应用
CN112646834A (zh) 一种羽扇豆醇衍生物及其合成方法和应用
CN107903227B (zh) 琥珀酸酐类化合物、与其相关的基因和蛋白及其制备方法
CN115322913B (zh) 产玫瑰精油的重组酿酒酵母及其构建方法和应用
WO2016106988A1 (zh) 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
CN114456964B (zh) 一种高产豆甾醇的重组解脂亚罗酵母、其构建方法、用于产豆甾醇的发酵培养基及应用
CN115305254B (zh) 一种萜类底盘微生物与工程菌及其构建方法和应用
CN116064639A (zh) 单萜合酶、基因工程菌株及其应用
CN113999783A (zh) 一种表达杂萜类化合物的重组酿酒酵母及其构建方法和应用

Legal Events

Date Code Title Description
DPE1 Request for preliminary examination filed after expiration of 19th month from priority date (pct application filed from 20040101)
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17867973

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2019544968

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2017867973

Country of ref document: EP

Effective date: 20190604