WO2023208037A1 - 一种橙花叔醇合成酶及应用 - Google Patents

一种橙花叔醇合成酶及应用 Download PDF

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WO2023208037A1
WO2023208037A1 PCT/CN2023/090827 CN2023090827W WO2023208037A1 WO 2023208037 A1 WO2023208037 A1 WO 2023208037A1 CN 2023090827 W CN2023090827 W CN 2023090827W WO 2023208037 A1 WO2023208037 A1 WO 2023208037A1
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synthase
seq
nerolidol
nucleic acid
plasmid
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French (fr)
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叶紫玲
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武汉合生科技有限公司
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/02—Preparation of oxygen-containing organic compounds containing a hydroxy group
    • C12P7/04—Preparation of oxygen-containing organic compounds containing a hydroxy group acyclic
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • This application belongs to the field of nerolidol biosynthesis, and specifically relates to a nerolidol synthase and its application.
  • Nerol is a sesquiterpene compound that was originally discovered from orange flowers, so it was named nerolidol. It was later found to be present in aromatic plants such as citronella, lavender, lemongrass and ginger. Nerol has a wide range of biological functions, such as acaricidal, anti-herbivore, antibacterial, antioxidant, anti-inflammatory and anti-anxiety activities. It is a promising phytochemical drug. At the same time, it is also a synthetic intermediate for the herbivore-induced volatile substance DMNT, which can protect plants from damage by herbivores. In addition, nerolol is approved by the U.S. Food and Drug Administration (FDA) for use as a food flavoring.
  • FDA U.S. Food and Drug Administration
  • nerolid alcohol There are three main ways to synthesize nerolid alcohol, namely plant extraction, chemical synthesis and biosynthesis. Plant extraction and chemical synthesis are subject to various limitations, such as complex technological processes, low yields affected by seasons, and high extraction costs. In addition, plant extraction and chemical synthesis often involve toxic reagents in the reaction, which have a great impact on the environment, and the products may contain relatively large amounts of chemicals. There are many solvent residues and toxic reagent residues, and product safety needs to be improved. Metabolic engineering and synthetic biology provide another approach to engineering microbial cell factories for sustainable and efficient production of nerolidol.
  • the purpose of this application is to provide a nerolidol synthase and its application in the biosynthesis of nerolidol.
  • the first aspect of the present application provides a nerolidol synthase, which contains domains with Pfam numbers PF01397 and PF03936 and has nerolidol synthase activity.
  • the second aspect of the present application provides a polynucleotide molecule, which contains at least one of the nucleotide sequence encoding the nerolidol synthase provided in the first aspect of the present application or its complementary sequence.
  • the third aspect of the present application provides a nucleic acid construct comprising at least one of the polynucleotide molecules provided in the second aspect of the present application.
  • the fourth aspect of the present application provides a recombinant bacterium, which contains the polynucleotide molecule provided by the second aspect of the present application, or the nucleic acid construct provided by the third aspect of the present application.
  • the fifth aspect of the present application provides the recombinant bacteria using the nerol synthase of the first aspect of the present application, the polynucleotide molecule of the second aspect of the present application, the nucleic acid construct of the third aspect of the present application or the fourth aspect of the present application.
  • the sixth aspect of the present application provides a method for preparing nerolidol, which includes biosynthesizing nerolidol using the recombinant bacteria of the fourth aspect of the present application.
  • This application identifies a new type of nerolithyl alcohol synthase and its encoding gene.
  • the encoding gene of the nerolithyl alcohol synthase is integrated into a nucleic acid construct and introduced into a host cell to obtain a recombinant bacterium, thereby making the encoding gene Genes expressed in recombinant bacteria to achieve the biosynthesis of nerolidol.
  • the production of nerolid alcohol is significantly improved.
  • Figure 1 shows a schematic diagram of the construction of plasmid pZY900
  • Figure 2 shows a schematic diagram of the construction of plasmids pYR013, pYR007, pArar-TPS27, pArar-TPS28, pYR006, pYR010, pCaNES2, pCaNES1, pTwNES, pAcNES1, pFaNES1, pCsNES2 and pLpNES1;
  • Figure 4 is the mass spectrum of nerolidol in the fermentation products of strains CCJ-1 and S900;
  • Figure 6 shows the mass spectra of nerolidol in the fermentation products of strains LXF-1, LXF-1-1, LXF-1-2, and S900;
  • Figure 8 shows the mass spectra of nerolidol in the fermentation products of strains AH-1, AH-2 and S900;
  • Figure 9 shows a schematic diagram of the construction of plasmids pYR020, pYR021, pYR017, and pYR018;
  • Figure 10A shows a schematic diagram of the construction of plasmid pYH395
  • Figure 10B shows a schematic diagram of the construction of the knockout element of the activating cis-element (-220 to -175) upstream of the ERG9 promoter;
  • Figure 11 is a schematic diagram of the construction of pZY521 knockout cassette
  • Figure 12 shows the shake flask fermentation yield of the strain containing nerol synthase CCJ_TPS23;
  • Figure 13 shows the shake flask fermentation yield of the strain containing nerolithyl alcohol synthase ACH_TPS07.
  • the terms "about” and “similar to” mean within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which error range may depend in part on how the value is measured or determined, or Depends on the limitations of the measurement system.
  • nucleic acid refers to deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) and their polymers in single- or double-stranded form.
  • nucleic acid or “polynucleotide” also includes nucleic acids containing known analogs of natural nucleotides that have similar binding properties to the reference nucleic acid and in a manner similar to those found in nature. nucleotides are metabolized in a similar manner (see, U.S. Patent No.
  • Construct refers to any recombinant polynucleotide molecule (e.g., plasmid, cosmid, virus, autonomously replicating polynucleotide molecule, bacteriophage, linear or circular single- or double-stranded DNA or RNA polynucleotide molecule) that can Derived from any source, capable of integrating into the genome or replicating autonomously, it can be operably linked to one or more polynucleotide molecules.
  • a construct generally includes a polynucleotide molecule of the present application operably linked to transcription initiation regulatory sequences that direct the transcription of the polynucleotide molecule of the present application in a host cell.
  • Heterologous promoters or endogenous promoters may be used to direct expression of the nucleic acids of the present application.
  • Vector refers to any recombinant nucleic acid construct that can be used for the purpose of transformation (ie, the introduction of heterologous DNA into a host cell).
  • the vector may contain a resistance gene for growth in the organism and a promoter for expression of the protein of interest in the organism.
  • Certain vectors are capable of autonomous replication in the host cell into which they are introduced (eg, vectors with an origin of replication functioning in the host cell).
  • Other vectors can be introduced into a host cell, integrated into the host cell's genome, and thus replicated with the host genome.
  • certain preferred vectors are capable of directing the expression of foreign genes to which they are linked.
  • Plasmid generally refers to a circular double-stranded DNA circle into which additional DNA segments (foreign genes) can be ligated, but may also include linear double-stranded molecules, such as those derived from polymerase chains. Amplification by reaction (PCR) or treatment of circular plasmid with restriction enzymes yields linear double-stranded molecules.
  • Plasmid vectors include vector backbone (i.e. empty vector) and expression framework.
  • expression framework refers to a sequence with the potential to encode a protein.
  • host cell refers to a cell that can introduce a gene of interest and provide conditions for cloning and/or expression of the gene of interest, such as a microorganism, specifically bacteria (such as Escherichia coli), yeast (such as Saccharomyces cerevisiae), Actinomycetes etc.
  • mutant bacteria refers to bacteria that have been genetically modified (such as bacteria, yeast, actinomycetes, etc.), which means that foreign gene fragments have been introduced into their bacteria.
  • One way of modification includes bacteria. The bacterial genome is changed after new DNA fragments are introduced. Another way includes the introduction of artificially constructed or modified plasmids into the bacterial cells, so that the bacterial cells gain the ability to express the target genes.
  • the first aspect of the present application provides a nerolidol synthase, which contains domains with Pfam numbers PF01397 and PF03936 and has nerolidol synthase activity.
  • the nerol synthase is from pyrethrum, astilbe, or mugwort.
  • the nerol synthase has the same properties as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.11 or SEQ ID NO.
  • the amino acid sequence shown in .12 is an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
  • the nerolidol synthase has the following amino acid sequence (amino terminus to carboxyl terminus):
  • the second aspect of the present application provides a polynucleotide molecule, which contains at least one of the nucleotide sequence encoding the nerolidol synthase provided in the first aspect of the present application or its complementary sequence.
  • the polynucleotide molecule comprises the same sequence as SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, SEQ ID NO.8, SEQ ID NO.9 or SEQ ID NO.10.
  • the nucleotide sequences shown are nucleotide sequences having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity.
  • the polynucleotide molecule comprises a nucleotide sequence (5' end to 3' end) as shown below:
  • the third aspect of the present application provides a nucleic acid construct comprising at least one of the polynucleotide molecules provided in the second aspect of the present application.
  • the polynucleotide molecule connected to the nucleic acid construct is called the target gene, and the enzyme encoded by the polynucleotide molecule is called the target protein.
  • the nucleic acid construct also contains regulatory elements that regulate the expression of the gene of interest, such as promoters, terminators, etc.
  • the promoter can be a constitutive promoter such as PTEF1 , PTDH3 , PGPM1 , PTPI1 , etc., inducible promoters such as PHXT1 (high concentration glucose induction), PCUP1 (copper ion induction), PGAL1 , PGAL2 , PGAL7 , PGAL10 (galactose induction), etc., in this field
  • PHXT1 high concentration glucose induction
  • PCUP1 copper ion induction
  • PGAL1 , PGAL2 , PGAL7 , PGAL10 galactose induction
  • the nucleic acid construct also contains a marker gene for screening recombinant bacteria containing the target gene or target protein, such as a leucine screening marker, a histidine screening marker, a tryptophan screening marker, a urine screening marker, and a leucine screening marker.
  • a marker gene for screening recombinant bacteria containing the target gene or target protein such as a leucine screening marker, a histidine screening marker, a tryptophan screening marker, a urine screening marker, and a leucine screening marker.
  • Pyrimidine screening markers, etc. can be specifically selected by those skilled in the art according to needs, and are not limited in this application.
  • the nucleotide sequence is located between two insertion elements used to integrate the nucleotide sequence into the genome of the host cell.
  • a nucleotide sequence with insertion elements connected at both ends is connected to a nucleic acid construct, such as a plasmid backbone of a plasmid vector.
  • a nucleic acid construct such as a plasmid backbone of a plasmid vector.
  • the nucleic acid construct can be restricted by restriction. Endonuclease and other tools are used to digest the nucleic acid construct to obtain a linearized target gene fragment with insertion elements connected at both ends.
  • the linearized target gene fragment is introduced into the host cell and allowed to pass through both ends.
  • the end insertion element is inserted into the corresponding position of the host cell genome, thereby obtaining the recombinant bacterium of the present application.
  • the two insertion elements appear in pairs, for example, they can be the left and right homology arms of leu2, the left and right homology arms of Ura3, the left and right homology arms of YPRCdelta15, etc.
  • the homology arms of different genes can combine the target genes. Integrate into For different positions in the host cell genome, those skilled in the art can specifically select the type of homology arm according to the position where they wish to be integrated into the host cell genome. This application is not limited here.
  • regulatory elements such as promoters and terminators for regulating the expression of the target gene are also included between the two inserted elements. This application does not limit the types of promoters and terminators.
  • the nucleic acid construct further comprises encoding acetoacetyl-CoA thiolase (ERG10), hydroxymethylglutaryl-CoA synthase (ERG13), hydroxymethylglutaryl-CoA reductase ( HMG1), mevalonate kinase (ERG12), mevalonate-5-phosphate kinase (ERG8), mevalonate pyrophosphate decarboxylase (MVD1), isoprene pyrophosphate isomerase (IDI1), At least one nucleotide sequence of farnesene pyrophosphate synthase (ERG20); the names of genes encoding these enzymes are shown in brackets.
  • the hydroxymethylglutaryl-CoA reductase is a truncated hydroxymethylglutaryl-CoA reductase (tHMG1), which truncates the endoplasmic reticulum localization sequence, thereby enhancing the enzyme's Stability in the cytoplasm.
  • tHMG1 truncated hydroxymethylglutaryl-CoA reductase
  • ERG10 Accession/GENE ID:856079
  • ERG13 Accession/GENE ID:854913
  • tHMG1 Accession/GENE ID:854900, truncated 4-1659bp
  • ERG12 Accession/GENE ID:NM_001182715.1
  • ERG8 Accession/GENE ID: CP046093.1, 689693..691048
  • MVD1 Accession/GENE ID: NM_001183220.1
  • IDI1 Accession/GENE ID: NM_001183931.1
  • ERG20 Accession/GENE ID: 853272.
  • acetoacetyl-CoA thiolase, hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase, mevalonate kinase, mevalonate-5-phosphate kinase, Mevalonate pyrophosphate decarboxylase and isoprene pyrophosphate isomerase are enzymes in the mevalonate pathway, which can synthesize isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), both of which can be used as precursors to synthesize farnesene pyrophosphate (FPP) under the catalysis of farnesene pyrophosphate synthase, and FPP is the substrate for the biosynthesis of nerol. Therefore, When the nucleic acid construct contains at least one of an enzyme in the mevalonate pathway and farnesene pyrophosphate synthase, it is beneficial to the synthesis of FPP and further to the
  • the nucleic acid construct is a plasmid vector; preferably, the plasmid vector is a eukaryotic expression vector.
  • the nucleic acid construct includes a pRS426 plasmid backbone.
  • the inventor found that the pRS426 plasmid backbone contains the AmpR screening marker suitable for E. coli, the URA3 screening marker suitable for S. cerevisiae, and the replicon suitable for E. coli and the multi-copy replicon of S. cerevisiae.
  • pRS426 plasmid backbone It is beneficial to maintain a high copy of the plasmid containing the target gene after being introduced into Saccharomyces cerevisiae.
  • the mutation present in the pRS426 plasmid backbone eliminates the restriction site BsaI in the pRS426 plasmid backbone, so that BsaI can be used as a restriction endonuclease when constructing the vector using the Goldengate method. .
  • the nucleic acid construct is at least one of plasmid vectors pYR006, pYR007, pYR010, pYR013, pAra-TPS27, pAra-TPS28, pYR017, pYR018, pYR020, and pYR021; a schematic diagram of the construction of the plasmid vector As shown in Figure 2 or Figure 9.
  • the plasmid vector can be directly introduced into the host cell, or the plasmid vector can be digested by enzymes.
  • the vector is used to obtain the target gene fragment containing the insertion element, and the gene fragment is further integrated into the genome of the host cell.
  • the fourth aspect of the present application provides a recombinant bacterium, which contains the polynucleotide molecule provided by the second aspect of the present application, or the nucleic acid construct provided by the third aspect of the present application.
  • the polynucleotide molecule is integrated into the genome of the host cell; preferably, the host cell is a eukaryotic cell; more preferably, it is Saccharomyces cerevisiae.
  • the recombinant bacterium can directly contain a nucleic acid construct containing a nucleotide sequence encoding the nerolithyl alcohol synthase.
  • the nucleic acid construct exists alone in the host cell in the form of a plasmid. , expressing the nerol synthase.
  • the polynucleotide molecule is integrated into the genome of the host cell.
  • the polynucleotide molecule is integrated into the genome of the host cell, which is beneficial to the long-term stable expression of the target gene, thereby obtaining a recombinant bacterium capable of stable inheritance.
  • the target gene can be connected between two insertion elements, and the insertion element can be used to integrate the polynucleotide molecules into the genome of the host cell.
  • the target gene is inserted into the genome of the host cell.
  • the insertion element can be the left and right homology arms of leu2, the left and right homology arms of Ura3, and the left and right homology arms of YPRCdelta15.
  • the homology arms of different genes are used to The target gene is inserted into different positions of the host cell genome.
  • the inventor found that the recombinant bacteria of the present application can be obtained by inserting the target gene into a site that does not interfere with the normal physiological metabolism of the host cell.
  • the copy number of the polynucleotide molecule in the genome of the recombinant bacterium is 1-3.
  • the recombinant bacteria can endogenously and/or exogenously express acetoacetyl-CoA thiolase, hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase, At least one of mevalonate kinase, mevalonate-5-phosphate kinase, mevalonate pyrophosphate decarboxylase, isoprene pyrophosphate isomerase, and farnesene pyrophosphate synthase.
  • the copy numbers of phosphokinase, mevalonate pyrophosphate decarboxylase, isoprene pyrophosphate isomerase and farnesene pyrophosphate synthase in the genome of the recombinant bacteria are each independently 2, 2, 4, 2, 2, 2, 2, 2, 2.
  • Saccharomyces cerevisiae can synthesize FPP endogenously. Therefore, in some preferred embodiments, using Saccharomyces cerevisiae as a host cell is beneficial to obtain a recombinant bacterium that can efficiently synthesize nerolidol.
  • the recombinant bacterium further includes knockout or down-regulation of at least one of the genes encoding FPP hydrolase DPP1, FPP hydrolase LPP1, citrate synthase, malate synthase or squalene synthase.
  • FPP hydrolase DPP1 diacylglycerol pyrophosphate phosphatase 1
  • FPP hydrolase LPP1 lipid phosphate phosphatase 1
  • MLS1 malate synthase
  • FPP hydrolase DPP1 diacylglycerol pyrophosphate phosphatase 1
  • FPP hydrolase LPP1 lipid phosphate phosphatase 1
  • FPP hydrolase LPP1 lipid phosphate phosphatase 1
  • FPP hydrolase LPP1 lipid phosphate phosphatase 1
  • MLS1 malate synthase
  • EMG9 uses FPP as a substrate to synthesize squalene.
  • These enzymes competitively consume the substrates required for nerolidol biosynthesis. Therefore, knocking out or down-regulating these enzymes
  • the coding genes reduce the expression of these enzymes in the recombinant bacteria, which is beneficial to improving the efficient synthesis of nerolol in the recombin
  • the down-regulation described in this application has its general meaning. In this application, it can be understood that the expression of the gene is inhibited, resulting in a reduction in the expression of the protein regulated by the gene.
  • the knockout described in this application has its general meaning, which refers to inactivating or deleting a specific gene through a certain pathway, thereby reducing or not expressing the protein it encodes.
  • the fifth aspect of the present application provides the recombinant bacteria using the nerol synthase of the first aspect of the present application, the polynucleotide molecule of the second aspect of the present application, the nucleic acid construct of the third aspect of the present application or the fourth aspect of the present application.
  • the sixth aspect of the present application provides a method for preparing nerolidol, which includes biosynthesizing nerolidol using the recombinant bacteria of the fourth aspect of the present application.
  • the recombinant bacterium is capable of endogenously and/or exogenously expressing acetoacetyl-CoA thiolase, hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase, At least one of valonate kinase, mevalonate-5-phosphate kinase, mevalonate pyrophosphate decarboxylase, isoprene pyrophosphate isomerase, and farnesene pyrophosphate synthase.
  • the nucleic acid construct can also be connected to the nucleic acid construct encoding acetoacetyl-CoA thiolase, hydroxymethylglutaryl-CoA synthase, hydroxymethylglutaryl-CoA reductase, mevalonate At least one of the nucleotide sequences of acid kinase, mevalonate-5-phosphate kinase, mevalonate pyrophosphate decarboxylase, isoprene pyrophosphate isomerase, and farnesene pyrophosphate synthase, so that The recombinant bacterium can synthesize FPP efficiently, which is beneficial to further increasing the production of nerol.
  • the copy numbers of phosphokinase, mevalonate pyrophosphate decarboxylase, isoprene pyrophosphate isomerase and farnesene pyrophosphate synthase in the genome of the recombinant bacteria are each independently 2, 2, 4, 2, 2, 2, 2, 2, 2.
  • knockout or down-regulation of at least one of the genes encoding FPP hydrolase DPP1, FPP hydrolase LPP1, citrate synthase, malate synthase or squalene synthase is also included.
  • the nucleic acid construct used when the nucleic acid construct used contains an inducible promoter, it may also include knockout of the recombinant bacterial transcription repressor.
  • the transcription repressor GAL80 when using the GAL (galactose-inducible) promoter, the transcription repressor GAL80 may be Knocking out the transcription inhibitor can enable the recombinant bacteria to express the target gene independently without the need for inducers, thus reducing fermentation costs.
  • the nerolidol synthase and its application of the present application will be described below through specific examples.
  • the following examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention.
  • the plasmids involved in the following examples are all plasmids well known to those skilled in the art. If specific techniques or conditions are not specified in the examples, the techniques or conditions described in literature in the field or product instructions will be followed. If the manufacturer of the reagents or instruments used is not indicated, they are all conventional products that can be purchased commercially.
  • Tissues from different stages and parts of pyrethrum were sampled, RNA was extracted from the samples, and second- and third-generation transcriptome sequencing was performed. From the second- and third-generation transcriptome data, two terpene synthases, PF01397 and PF03936, were found.
  • a total of 166 potential protein sequences were obtained from the pyrethrum transcriptome protein sequences of Pfam domains; CD-Hit was used to perform de-redundant clustering of the found protein sequences, and sequences with more than 90% sequence similarity were defined as In the same category, a total of 33 categories were obtained; in each category, according to the sequence integrity, protein sequence lengths greater than 500 were selected as candidate genes, and the genes with the highest expression levels were further selected as test genes, and a total of 24 were obtained.
  • the genes to be verified are named CCJ-TPS01 to CCJ-TPS24.
  • plasmid pZY900 use Saccharomyces cerevisiae S288c genome (extraction method, see: Li Xiaowei. Engineering acetyl-CoA pathway to construct an efficient synthesis platform of Saccharomyces cerevisiae [D].
  • a gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae was used as a template to amplify fragment 9004 (nonsense gene lacZ, used for replacement of the target gene); pRS426 was used as a template, and primers 900-8F/8R, 900-9F/9R, and 900-10F were used. /10R amplification to obtain the plasmid backbone (introducing MssI restriction site and screening markers (AmpR, URA3, etc.)).
  • FIG. 1 The schematic diagram of the construction of plasmid pZY900 is shown in Figure 1, in which fragments 9001(HA), 9002(T), 9003(T), 9004, 9005, 9006, and 9007(HA) are connected in sequence from left to right, and the remaining parts are from the plasmid backbone of pRS426 .
  • Design primers to connect the gene to be verified on a universal vector is the verification process of the gene CCJ_TPS23 in pyrethrum that was finally verified to have nerol synthase activity.
  • Design specific gene primer pair CCJ_TPS23-F/R use pyrethrum cDNA (using TIANGEN company's RNAprep Pure Plant Plus Kit (Cat. No. DP441) to extract pyrethrum ovule tissue RNA, use Vazyme's HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper ) kit (Cat. No. R212) to reverse transcribe RNA to obtain cDNA) as a template, use Novozant's Phanta high-fidelity enzyme to obtain the CCJ_TPS23 gene fragment through PCR amplification, and use the Tiangen glue recovery kit to recover the gel.
  • pyrethrum cDNA using TIANGEN company's RNAprep Pure Plant Plus Kit (Cat. No. DP441) to extract pyrethrum ovule tissue RNA
  • Vazyme's HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper ) kit Cat. No. R212
  • the homologous recombination method was used to connect it to the BsaI-cut yeast expression vector pZY900. After sequencing and confirmation, the yeast expression vector containing the CCJ_TPS23 gene was obtained, named pYR013, and its plasmid construction The schematic diagram is shown in pYR013 in Figure 2, in which the lacZ gene in pZY900 is replaced by the CCJ_TPS23 gene.
  • the CCJ_TPS23-F/R primer sequence is shown in Table 2 below:
  • Plasmid pYR013 was introduced into strain JCR27 through the lithium acetate method (Li Xiaowei. Engineering acetyl-CoA pathway to construct an efficient synthesis platform for Saccharomyces cerevisiae [D]. Wuhan University, 2015.2.3.14 LiAc transformation of Saccharomyces cerevisiae) (for the construction of yeast strain JCR27, see the literature Siemon, Thomas et al.
  • plasmid pZY900 was introduced into strain JCR27, which was used as a control strain and was named S900.
  • Strains CCJ-1 and S900 were inoculated into SC-URA liquid culture medium (Li Xiaowei. Engineering the acetyl-CoA pathway to construct an efficient synthesis platform for Saccharomyces cerevisiae [D]. Wuhan University, 2015, uracil-deficient medium), at 30°C.
  • the chromatographic column is TR-5MS column (30m ⁇ 0.25mm ⁇ 0.25um).
  • the carrier gas is high-purity helium with a flow rate of 1mL/min.
  • Acetone is used as a needle cleanser.
  • the injection volume is 1uL, and the split ratio is 50.
  • the injection port temperature is 240°C, and the ion transfer tube temperature is 270°C.
  • Detection procedure The initial column temperature is 50°C and maintained for 1 min; the temperature is increased to 280°C at 15°C/min and maintained for 1 min; the temperature is increased to 300°C at 20°C/min and maintained for 2 minutes.
  • the mass spectrum of nerolid alcohol is shown in Figure 4. It passed and nerolid alcohol standard By comparing the chromatogram retention time and mass spectrum fragments of the product, it can be determined that strain CCJ-1 can synthesize nerolidol, but strain S900 cannot. This result shows that the protein encoded by the CCJ_TPS23 gene is nerol synthase.
  • CD-Hit was used to perform de-redundant clustering of the found protein sequences, and those with sequence similarity greater than 90 were % of the sequences are defined as the same category, so a total of 25 categories are obtained; in each category, according to the sequence integrity, the protein sequence length greater than 500 is selected as the candidate gene, and the gene with the highest expression is further selected as the test gene. , a total of 17 genes to be verified were obtained, and the genes were named ACH-TPS01 to ACH-TPS17.
  • Design primers to construct the gene to be verified on a universal vector Shown here is the verification process of the genes ACH_TPS07, ACH_TPS08, and ACH_TPS09 that were finally verified to have nerol synthase activity in astilbe.
  • ACH_TPS07, ACH_TPS08, and ACH_TPS09 genes are named pYR007, pYR006, and pYR010.
  • FIG. 1 The schematic diagram of the construction of plasmids pYR007, pYR006, and pYR010 is shown in pYR007, pYR006, and pYR010 in Figure 2, which replace the lacZ gene in pZY900 with the genes ACH_TPS07, ACH_TPS08, and ACH_TPS09 respectively.
  • the primer sequences are shown in Table 3 below.
  • Plasmid pYR007 was introduced into strain JCR27, and the transformed strain was named LXF-1; plasmid pYR006 was introduced into strain JCR27, and the transformed strain was named LXF-1-1; Plasmid pYR010 was introduced into strain JCR27, and the transformed strain was named LXF-1-2. Plasmid pZY900 was introduced into strain JCR27 as a control strain and named S900.
  • Inoculate strains LXF-1, LXF-1-1, LXF-1-2, and S900 into SC-URA liquid culture medium respectively, and culture on a shaking table at 30°C and 200 rpm overnight; transfer to 45 the next day according to the initial OD600 0.1 5 ml of isopropyl myristate was added to ml of YPDHG liquid culture medium, and the mixture was cultured on a shaking table at 30° C. and 200 rpm for 72 hours. The oil layer was collected and the product was detected using GC-MS. The detection conditions were the same as 1.2.4 of Example 1.
  • the mass spectrum of nerolid alcohol As shown in Figure 6, by comparing the chromatographic retention time and mass spectrum fragments with the standard substance of nerolid alcohol, it can be determined that strains LXF-1, LXF-1-1, and LXF-1-2 can synthesize nerolid alcohol. alcohol, but strain S900 cannot synthesize it. This result shows that the proteins encoded by the ACH_TPS07, ACH_TPS08, and ACH_TPS09 genes are nerolidol synthase.
  • Plasmids pYR007, pYR006, and pYR010 were digested with MssI respectively (refer to the MssI enzyme instructions for enzyme digestion conditions) to obtain linearized fragments.
  • the linearized fragments were integrated into strain JCR27 through the lithium acetate method to obtain strains LXF-2, LXF-2-6, LXF-2-10.
  • Inoculate the bacteria into YPD liquid culture medium and culture overnight on a shaker at 30°C and 200rpm; transfer it to 45 ml of YPDHG liquid culture medium according to the initial OD600 0.1 the next day, add 5 ml of isopropyl myristate, and shake at 30°C and 200rpm.
  • the shake flask fermentation yields of nerolol from strains LXF-2, LXF-2-6, and LXF-2-10 were 269 mg/L and 240 mg/L respectively. L, 221mg/L.
  • Design primers to construct the gene to be verified on a universal vector Shown here is the verification process of the genes Arar-TPS27 and Arar-TPS28 that were finally verified to have nerol synthase activity in mugwort.
  • the genes Arar-TPS27 and Arar-TPS28 derived from mugwort are directly optimized and synthesized according to the codons of Saccharomyces cerevisiae, and the sequences are shown in SEQ ID NO.7 and SEQ ID NO.8.
  • FIG. 1 The schematic diagram of the construction of plasmids pArar-TPS27 and pArar-TPS28 is shown in pArar-TPS27 and pArar-TPS28 in Figure 2, which replace the lacZ gene in pZY900 with the genes pArar-TPS27 and pArar-TPS28 respectively.
  • the primer sequences are shown in Table 4 below.
  • Plasmid pZY900 was introduced into strain JCR27 as a control strain and named S900.
  • Inoculate strains AH-1, AH-2, and S900 into SC-URA liquid medium respectively, and cultivate overnight on a shaking table at 30°C and 200 rpm; transfer them to 45 ml of YPDHG liquid medium according to the initial OD600 0.1 the next day, and add 5 ml of isopropyl myristate, 30°C, 200rpm shaker incubation for 72 hours, collect the oil layer, use GC-MS to detect the product, detection conditions Same as 1.2.4 of Example 1.
  • Plasmids pArar-TPS27 and pArar-TPS28 were digested with MssI respectively (refer to MssI enzyme instructions for enzyme digestion conditions) to obtain linearized fragments.
  • the shake flask fermentation yields of nerolol from strains AH-27 and AH-28 were 162 mg/L and 158 mg/L respectively.
  • 1.1 Contains nerolithyl alcohol derived from Celastrus angulatus (CaNES2), Celastrus angulatus (CaNES1), Tripterygium wilfordii (TwNES), Actinidia chinensis (AcNES1), Fragaria x ananassa (FaNES1), Camellia sinensis (CsNES2), Laggera pterodonta (LpNES1) Plasmid construction of synthase gene
  • the nerolidol synthases from different sources used in this comparative example are nerolidol synthases that have been widely studied in this field and have a recognized high yield.
  • the nucleotide sequence of nerol synthase derived from Celastrus angulatus is as follows (5'-3') after codon optimization in Saccharomyces cerevisiae.
  • the gene is named CaNES2
  • the nucleotide sequence of nerol synthase derived from Celastrus angulatus is as follows (5'-3') after codon optimization in Saccharomyces cerevisiae.
  • the gene is named CaNES1
  • the nucleotide sequence of nerol synthase derived from Tripterygium wilfordii is as follows (5'-3') after codon optimization in Saccharomyces cerevisiae.
  • the gene is named TwNES.
  • nucleotide sequence of nerol synthase derived from Actinidia chinensis is as follows (5'-3') after codon optimization in Saccharomyces cerevisiae.
  • the gene is named AcNES1
  • the nucleotide sequence of nerol synthase derived from Fragaria x ananassa is as follows (5'-3') after codon optimization in S. cerevisiae.
  • the gene is named FaNES1
  • the nucleotide sequence of nerol synthase derived from Camellia sinensis is as follows (5'-3') after codon optimization in Saccharomyces cerevisiae.
  • the gene is named CsNES2
  • the nucleotide sequence of nerol synthase derived from Laggera pterodonta is as follows (5'-3') after codon optimization in Saccharomyces cerevisiae.
  • the gene is named LpNES1
  • FIG. 2 The schematic diagram of the construction of plasmids pCaNES2, pCaNES1, pTwNES, pAcNES1, pFaNES1, pCsNES2 and pLpNES1 is shown in Figure 2, which replaces the lacZ gene in pZY900 with the genes CaNES2, CaNES1, TwNES, AcNES1, FaNES1, CsNES2 and LpNES1 respectively.
  • pCaNES2, pCaNES1, pTwNES, pAcNES1, pFaNES1, pCsNES2 and pLpNES1 were linearized with MssI respectively, the fragments containing the target genes were recovered using a gel recovery kit and introduced into the strain JCR27 by the lithium acetate method.
  • the transformed strains were named JCaNES2, JCaNES1, JTwNES, JAcNES1, JFaNES1, JCsNES2 and JLpNES1.
  • the shake flask fermentation yields were 33.7 mg/L, 8.3 mg/L, 69 mg/L, 111 mg/L, 86.5 mg/L, 21 mg/L, and 5.3 mg/L respectively.
  • plasmid pYR020 The schematic diagram of the construction of plasmid pYR020 is shown in pYR020 in Figure 9, which also contains the MssI restriction site, and the rest comes from the pRS426 plasmid backbone.
  • the primer sequences are shown in Table 5 below.
  • plasmid pYR021 The schematic diagram of the construction of plasmid pYR021 is shown in Figure 9 pYR021, which also contains the Not I restriction site, and the remaining parts are from the pRS426 plasmid backbone.
  • the primer sequences are shown in Table 6 below.
  • the primer sequences are shown in Table 7 below.
  • ERG9-1F/R and ERG9-2F/R Use primers ERG9-1F/R and ERG9-2F/R to amplify the whole genome of Saccharomyces cerevisiae CEN.PK2-1D as a template to obtain two fragments, and then use primers ERG9-1F and ERG9-2R to obtain two fragments.
  • overlap extension PCR was performed to obtain the ERG9 knockout cassette.
  • the schematic diagram of constructing the knockout element of the activating cis-element (-220 to -175) upstream of the ERG9 promoter is shown in Figure 10B.
  • the two HAs represent the homology arm on the left side of the sequence (-220 to -175) and the sequence (- 220 to - 175) Homology arm on the right.
  • the primer sequences are shown in Table 8 below.
  • Primers 5211-F/R, 5212-F/R, and 5213-F/R were used to amplify the whole genome of Saccharomyces cerevisiae CEN.PK2-1D, pRS426 plasmid, and the whole genome of Saccharomyces cerevisiae CEN.PK2-1D as templates respectively. Three fragments were obtained, and then using the three fragments obtained with primers 5211-F and 5213-R as templates, overlap extension PCR was performed to obtain the pZY521 knockout cassette.
  • FIG. 11 The schematic diagram of pZY521 knockout cassette construction is shown in Figure 11, in which the sequences are GAL80 left homology arm (HA), URA3 selection marker (promoter P URA3 , URA3 gene, terminator T), and GAL80 right homology arm (HA).
  • HA GAL80 left homology arm
  • URA3 selection marker promoter P URA3
  • HA GAL80 right homology arm
  • the primer sequences are shown in Table 9 below.
  • step 2) After linearizing plasmid pYR020 using MssI enzyme, use the same method as step 1) to integrate it into strain CCJ-2 to obtain strain CCJ-3.
  • step 3 After linearizing plasmid pYR021 using NotI enzyme, use the same method as step 1) to integrate it into strain CCJ-3 to obtain strain CCJ-4.
  • Plasmid pYH395 and ERG9 knockout cassette were co-transferred into strain CCJ-4 through the lithium acetate method.
  • the colony was verified by PCR to verify the correctness of the knockout, and then YPD liquid culture medium (20g/L tryptone, 10g/L Yeast extract, 20g/L glucose) culture, shaker at 220rpm, incubate at 30 degrees for 8 hours, wash with water, and draw the bacteria on a 5-FOA plate (Li Xiaowei. Engineering acetyl-CoA pathway to construct an efficient synthesis platform for Saccharomyces cerevisiae [D]. Wuhan University , 2015.), cultured in a 30°C incubator for 3 days, picked bacteria from the plate, and verified the correctness of the knockout by PCR.
  • the correct strain was named CCJ-5.
  • This step is used to knock out the activating cis-elements (-220 to -175) upstream of the ERG9 promoter.
  • the squalene synthase encoded by ERG9 uses FPP as the substrate to synthesize squalene, which is a competitive pathway for the nerolithyl alcohol synthesis pathway.
  • knocking out the activating cis-element upstream of the ERG9 promoter can down-regulate the squalene synthesis pathway and reduce the competition of the squalene synthesis pathway to consume the substrate required for the synthesis of nerolidol. material, which is conducive to further increasing the yield of nerol.
  • the pZY521 knockout cassette was transferred into strain CCJ-5 using the lithium acetate method to obtain strain CCJ-6.
  • the function of this step is to knock out the transcription repressor GAL80, so that the target gene transferred into the bacteria can be expressed independently without the need for inducers, which is beneficial to reducing the fermentation product and further increasing the nerolithyl alcohol. output.
  • plasmid pYR017 The schematic diagram of the construction of plasmid pYR017 is shown in pYR017 in Figure 9, which also contains the MssI restriction site, and the rest comes from the pRS426 plasmid backbone.
  • the primer sequences are shown in Table 10 below.
  • plasmid pYR018 The schematic diagram of the construction of plasmid pYR018 is shown in pYR018 in Figure 9, which also contains the Not I restriction site, and the remaining parts are from the pRS426 plasmid backbone.
  • the primer sequences are shown in Table 11 below.
  • plasmid pYR007 was linearized with MssI and then integrated into strain JCR27 to obtain strain LXF-2; plasmid pYR017 was linearized with MssI and then integrated into strain LXF-2 to obtain strain LXF-3. ; Plasmid pYR018 was linearized with NotI and integrated into strain LXF-3 to obtain strain LXF-4; plasmid pYH395 and ERG9 knockout cassette were jointly transferred into strain LXF-4, and the colony was verified by PCR to verify the correctness of the knockout.
  • the correct strain was named LXF-5; the pZY521 knockout cassette was transferred into the strain LXF -5, strain LXF-6 was obtained.
  • the dissolved oxygen is controlled to be above 20%, the pH is 5, the glucose concentration is 1-2g/L, and the ethanol concentration is below 5g/L. Finally, in the 1L fermentation tank, the production of nerolol reached 55.7g/L, which is the highest production level reported so far.

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Abstract

提供一种包含Pfam编号为PF01397和PF03936的结构域的橙花叔醇合成酶及其应用，所述橙花叔醇合成酶的氨基酸序列如SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.11或SEQ ID NO.12所示，编码核酸分子的核苷酸序列如SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9或SEQ ID NO.10所示。所述应用包括将橙花叔醇合成酶的编码基因整合入核酸构建体中导入宿主细胞，获得重组菌，从而使所述编码基因在重组菌中表达，进而实现橙花叔醇的生物合成，且橙花叔醇的产量显著提高。

Description

一种橙花叔醇合成酶及应用 技术领域
本申请属于橙花叔醇生物合成领域，具体涉及一种橙花叔醇合成酶及应用。
背景技术
橙花叔醇是一种倍半萜化合物，最初是从橙花中发现的，所以被命名为橙花叔醇，后来它被发现存在于香茅、薰衣草、柠檬草和生姜等芳香植物中。橙花叔醇具有广泛的生物功能，如杀螨、抗草食动物、抗菌、抗氧化、抗炎和抗焦虑等活性，是一种很有发展前景的植物化学药物。同时它也是草食动物诱导的挥发性物质DMNT的合成中间体，可以保护植物免受草食动物的伤害。另外，橙花叔醇被美国食品和药物管理局(FDA)允许作为食品调味剂使用。
橙花叔醇的合成方式主要有三种，植物提取、化学合成以及生物合成。植物提取与化学合成受到种种限制如工艺过程复杂，受季节影响产率低，提取成本高；此外，植物提取和化学合成往往有有毒试剂参与反应，对坏境影响大、同时产物中可能有较多的溶剂残留以及有毒试剂残留，产品安全性有待提高。代谢工程和合成生物学为工程微生物细胞工厂可持续高效地生产橙花叔醇提供了另一种途径。
发明内容
本申请的目的在于提供一种橙花叔醇合成酶，以及其在生物合成橙花叔醇中的应用。
本申请为解决上述技术问题，提出了如下技术方案：
本申请第一方面提供了一种橙花叔醇合成酶，其包含Pfam编号为PF01397和PF03936的结构域，且具有橙花叔醇合成酶活性。
本申请第二方面提供了一种多核苷酸分子，其包含编码本申请第一方面所提供的橙花叔醇合成酶的核苷酸序列或其互补序列的至少一种。
本申请第三方面提供了一种核酸构建体，其包含本申请第二方面所提供的多核苷酸分子的至少一种。
本申请第四方面提供了一种重组菌，其包含本申请第二方面所提供的多核苷酸分子，或本申请第三方面所提供的核酸构建体。
本申请第五方面提供了采用本申请第一方面的橙花叔醇合成酶、本申请第二方面的多核苷酸分子、本申请第三方面的核酸构建体或本申请第四方面的重组菌生产橙花叔醇的用途。
本申请第六方面提供了一种橙花叔醇的制备方法，其包括采用本申请第四方面的重组菌生物合成橙花叔醇。
本申请鉴定出一种新型的橙花叔醇合成酶以及其编码基因，将所述橙花叔醇合成酶的编码基因整合入核酸构建体中导入宿主细胞，获得重组菌，从而使所述编码基因在重组菌中表 达，进而实现橙花叔醇的生物合成。采用本申请的重组菌和生物合成橙花叔醇的方法，橙花叔醇的产量显著提高。
附图说明
图1显示了质粒pZY900构建示意图；
图2显示了质粒pYR013、pYR007、pArar-TPS27、pArar-TPS28、pYR006、pYR010、pCaNES2、pCaNES1、pTwNES、pAcNES1、pFaNES1、pCsNES2和pLpNES1构建示意图；
图3为菌株CCJ-1、S900发酵产物中橙花叔醇特征离子m/z＝93提取离子流色谱图；
图4为菌株CCJ-1、S900发酵产物中橙花叔醇的质谱图；
图5为菌株LXF-1、LXF-1-1、LXF-1-2、S900发酵产物中橙花叔醇特征离子m/z＝93提取离子流色谱图；
图6为菌株LXF-1、LXF-1-1、LXF-1-2、S900发酵产物中橙花叔醇的质谱图；
图7为菌株AH-1、AH-2、S900发酵产物中橙花叔醇特征离子m/z＝93提取离子流色谱图；
图8为菌株AH-1、AH-2、S900发酵产物中橙花叔醇的质谱图；
图9显示了质粒pYR020、pYR021、pYR017、pYR018构建示意图；
图10A显示了质粒pYH395构建示意图；
图10B显示了ERG9启动子上游激活顺式元件(-220至-175)敲除元件构建示意图；
图11为pZY521敲除盒构建示意图；
图12显示了含橙花叔醇合酶CCJ_TPS23的菌株摇瓶发酵产量；
图13显示了含橙花叔醇合酶ACH_TPS07的菌株摇瓶发酵产量。
具体实施方式
本文使用的术语和说明仅仅是为了描述特定的实施方案，而不意在限制本申请。除非另有定义，本文所用的所有技术和科学术语具有与本公开所属领域的普通技术人员通常理解的相同含义。此外，除非上下文另有要求，否则单数术语应包括复数，并且复数术语应包括单数。
定义
如本文所用，术语“一个”和“一种”以及“所述”和类似的指代物指示单数和复数，除非本文另外指明或上下文明显矛盾。
如本文所用，术语“约”和“类似于”是指在本领域普通技术人员所确定的特定值的可接受误差范围内，所述误差范围可部分取决于该值的测量或确定方式，或取决于测量系统的局限性。
术语“核酸”或“多核苷酸”是指脱氧核糖核酸(DNA)或核糖核酸(RNA)及其呈单链或双链形式的聚合物。除非明确地限制，否则术语“核酸”或“多核苷酸”还包括含有已知的天然核苷酸的类似物的核酸，其具有与参照核酸相似的结合性质，并且以与天然存在 的核苷酸相似的方式被代谢(参见，属于Kariko等人的美国专利No.8278036，其公开了尿苷被假尿苷替代的mRNA分子，合成所述mRNA分子的方法以及用于在体内递送治疗性蛋白的方法)。除非另有所指，否则特定核酸序列还隐含地包括其保守修饰的变体(例如，简并密码子取代)、等位基因、直系同源物、单核苷酸多态性(SNP)和互补序列以及明确指出的序列。
“构建体”是指任何重组多核苷酸分子(例如质粒、粘粒、病毒、自主复制多核苷酸分子、噬菌体、线性或环状单链或双链DNA或RNA多核苷酸分子)，其可衍生自任何来源，能够与基因组整合或自主复制，其可以以可操作的方式连接一个或多个多核苷酸分子。本申请中，构建体通常包含本申请的多核苷酸分子，其可操作地连接至转录起始调节序列，这些序列会导引本申请的多核苷酸分子在宿主细胞中的转录。可使用异源启动子或内源启动子导引本申请的核酸的表达。
“载体”是指任何重组核酸构建体，该构建体可用于转化的目的(即将异源DNA引入到宿主细胞中)。载体可以包含用于在生物体中生长的抗性基因和用于在生物体中表达目的蛋白质的启动子。某些载体能够在引入它们的宿主细胞中自主复制(例如，具有在宿主细胞中起作用的复制起点的载体)。其他载体可以引入宿主细胞后整合到宿主细胞的基因组中，并因此与宿主基因组一起复制。此外，某些优选的载体能够指导与它们连接的外源基因的表达。一种类型的载体是“质粒”，其通常是指可以连接入另外的DNA区段(外源基因)的环状双链DNA环，也可以包括线性双链分子，诸如从通过聚合酶链式反应(PCR)的扩增或用限制酶处理环状质粒得到线性双链分子。
质粒载体包括载体骨架(即空载体)与表达框架。
术语“表达框架”是指具有编码蛋白质潜能的序列。
术语"宿主细胞"指的是能够将目的基因导入，并为目的基因克隆和/或表达提供条件的细胞，诸如微生物，具体地可以为细菌(诸如大肠杆菌)、酵母菌(诸如酿酒酵母)、放线菌等。
术语“重组菌”指的经过基因工程改造的菌(如细菌、酵母菌、放线菌等)，这意味着它们的菌体中引入了外源基因片段，其中，改造的一种方式包括菌体基因组被引入新的DNA片段后发生了改变，另一种方式包括菌体中引入了经过人工构建或改造过的质粒，从而使菌体获得表达目的基因的能力。
本申请第一方面提供了一种橙花叔醇合成酶，其包含Pfam编号为PF01397和PF03936的结构域，且具有橙花叔醇合成酶活性。
在一些实施方式中，所述橙花叔醇合成酶来自除虫菊、落新妇或艾蒿。
在一些实施方式中，所述的橙花叔醇合成酶具有与SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.11或SEQ ID NO.12所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列。
在一些实施方式中，所述橙花叔醇合成酶具有以下氨基酸序列(氨基末端至羧基末端)：

本申请第二方面提供了一种多核苷酸分子，其包含编码本申请第一方面所提供的橙花叔醇合成酶的核苷酸序列或其互补序列的至少一种。
在一些实施方式中，所述多核苷酸分子包含与SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9或SEQ ID NO.10所示的核苷酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的核苷酸序列。
在一些实施方式中，所述多核苷酸分子包含如下所示的核苷酸序列(5’末端至3’末端)：



本申请第三方面提供了一种核酸构建体，其包含本申请第二方面所提供的多核苷酸分子的至少一种。本申请中，将连接入所述核酸构建体的多核苷酸分子称为目的基因，将所述多核苷酸分子编码的酶称为目标蛋白。
在一些实施方式中，所述核酸构建体中还包含调控目的基因表达的调控元件，例如启动子、终止子等，示例性的，所述启动子可以为组成型启动子如PTEF1、PTDH3、PGPM1、PTPI1等，诱导型启动子如PHXT1(高浓度葡萄糖诱导)、PCUP1(铜离子诱导)、PGAL1、PGAL2、PGAL7、PGAL10(半乳糖诱导)等，本领域技术人员可根据需要选择，本申请在此不做限定。
在一些实施方式中，所述核酸构建体中还包含用于筛选包含目的基因或目标蛋白的重组菌的标记基因，例如亮氨酸筛选标记、组氨酸筛选标记、色氨酸筛选标记、尿嘧啶筛选标记等，本领域技术人员可根据需要具体选择，本申请在此不做限定。
在一些实施方式中，所述核苷酸序列位于两个插入元件之间，所述插入元件用于将所述核苷酸序列整合入宿主细胞的基因组中。
在一些实施方式中，两端连接有插入元件的核苷酸序列连接于核酸构建体，例如质粒载体的质粒骨架中，所述核酸构建体用于向宿主细胞导入目的基因时，可以通过限制性内切酶等工具，将所述核酸构建体酶切，从而获得两端连接有插入元件的线性化的目的基因片段，通过将所述线性化的目的基因片段导入宿主细胞中，使其通过两端的插入元件插入宿主细胞基因组的相应位置，从而获得本申请的重组菌。
本领域技术人员可采用常规的方法将线性化的目的基因片段导入宿主细胞中，例如对于酵母菌，可采用醋酸锂法，对于大肠杆菌，可采用钙转法等，此为本领域的常规操作，本申请在此不做限定。
在一些实施方式中，所述两个插入元件成对出现，例如可以是leu2的左右同源臂、Ura3的左右同源臂、YPRCdelta15左右同源臂等，不同基因的同源臂可以将目的基因整合入 宿主细胞基因组的不同位置，本领域技术人员可根据希望整合入宿主细胞基因组的位置具体选择同源臂的种类，本申请在此不做限定。
在一些实施方式中，所述两个插入元件之间还包括用于调控目的基因表达的启动子、终止子等调控元件。本申请对所述启动子和终止子的种类不做限定。
在一些实施方式中，所述核酸构建体还包含编码乙酰乙酰辅酶A硫解酶(ERG10)、羟甲基戊二酰辅酶A合酶(ERG13)、羟甲基戊二酰辅酶A还原酶(HMG1)、甲羟戊酸激酶(ERG12)、甲羟戊酸-5-磷酸激酶(ERG8)、甲羟戊酸焦磷酸脱羧酶(MVD1)、异戊二烯焦磷酸异构酶(IDI1)、法尼烯焦磷酸合酶(ERG20)的核苷酸序列的至少一种；其中括号中显示了编码这些酶的基因名称。
在一些实施方式中，所述羟甲基戊二酰辅酶A还原酶为截短的羟甲基戊二酰辅酶A还原酶(tHMG1)，tHMG1截去了内质网定位序列，增强了酶在细胞质中的稳定性。
编码以上酶的基因的示例性而非限制性的公开如下：
ERG10(Accession/GENE ID:856079)、ERG13(Accession/GENE ID:854913)、tHMG1(Accession/GENE ID:854900，截去4-1659bp)、ERG12(Accession/GENE ID:NM_001182715.1)、ERG8(Accession/GENE ID:CP046093.1，689693..691048)、MVD1(Accession/GENE ID:NM_001183220.1)、IDI1(Accession/GENE ID:NM_001183931.1)、ERG20(Accession/GENE ID:853272)。
发明人发现，乙酰乙酰辅酶A硫解酶、羟甲基戊二酰辅酶A合酶、羟甲基戊二酰辅酶A还原酶、甲羟戊酸激酶、甲羟戊酸-5-磷酸激酶、甲羟戊酸焦磷酸脱羧酶、异戊二烯焦磷酸异构酶属于甲羟戊酸途径中的酶，甲羟戊酸途径可以合成异戊烯基二磷酸(IPP)和二甲基烯丙基二磷酸(DMAPP)，二者可以作为前体，在法尼烯焦磷酸合酶的催化下合成法尼烯焦磷酸(FPP)，而FPP是生物合成橙花叔醇的底物，因此，当所述核酸构建体中包含甲羟戊酸途径中的酶和法尼烯焦磷酸合酶的至少一种时，有利于FPP的合成，进而有利于橙花叔醇的生物合成。
在一些实施方式中，所述的核酸构建体为质粒载体；优选地，所述质粒载体为真核表达载体。
在一些实施方式中，所述的核酸构建体包括pRS426质粒骨架。发明人发现，pRS426质粒骨架中包含适用于大肠杆菌的AmpR筛选标记，适用于酿酒酵母的URA3筛选标记，以及适用于大肠杆菌的复制子和酿酒酵母的多拷贝复制子，采用所述pRS426质粒骨架有利于含有目的基因的质粒在导入酿酒酵母中后维持质粒的高拷贝。
在一些实施方式中，所述pRS426质粒骨架中存在的突变，所述突变消除了pRS426质粒骨架中的酶切位点BsaI，从而可以在使用Goldengate方法构建载体时，以BsaI作为限制性内切酶。
在一些实施方式中，所述的核酸构建体为质粒载体pYR006、pYR007、pYR010、pYR013、pAra-TPS27、pAra-TPS28、pYR017、pYR018、pYR020、pYR021的至少一种；所述质粒载体的构建示意图如图2或图9所示。
在一些实施方式中，可以将所述质粒载体直接导入宿主细胞中，也可以通过酶切所述质 粒载体，获得包含插入元件的目的基因片段，进一步将所述基因片段整合入所述宿主细胞的基因组中。
本申请第四方面提供了一种重组菌，其包含本申请第二方面所提供的多核苷酸分子，或本申请第三方面所提供的核酸构建体。
在一些实施方式中，所述多核苷酸分子整合入所述宿主细胞的基因组中；优选地，所述宿主细胞为真核细胞；更优选为酿酒酵母。
在一些实施方式中，所述重组菌中可以直接包含含有编码所述橙花叔醇合成酶的核苷酸序列的核酸构建体，例如，所述核酸构建体以质粒的形式单独存在于宿主细胞中，表达所述橙花叔醇合成酶。
在另一些实施方式中，所述多核苷酸分子整合入所述宿主细胞的基因组中。所述多核苷酸分子整合入所述宿主细胞的基因组中，有利于所述目的基因的长期稳定表达，从而获得能够稳定遗传的重组菌。
本领域技术人员可采用常规的方法将多核苷酸分子整合入所述宿主细胞的基因组中，本申请在此不做限定，例如可以将目的基因连接于两个插入元件之间，通过插入元件将所述目的基因插入宿主细胞的基因组中，示例性的，所述插入元件可以为leu2的左右同源臂、Ura3的左右同源臂、YPRCdelta15左右同源臂，不同基因的同源臂用于将目的基因插入宿主细胞基因组的不同位置，发明人发现，将目的基因插入不干扰宿主细胞正常生理代谢的位点，均能够获得本申请的重组菌。
在一些实施方式中，所述多核苷酸分子在所述重组菌的基因组中的拷贝数为1-3个。
在一些实施方式中，所述的重组菌能够内源和/或外源表达乙酰乙酰辅酶A硫解酶、羟甲基戊二酰辅酶A合酶、羟甲基戊二酰辅酶A还原酶、甲羟戊酸激酶、甲羟戊酸-5-磷酸激酶、甲羟戊酸焦磷酸脱羧酶、异戊二烯焦磷酸异构酶、法尼烯焦磷酸合酶的至少一种。
在一些实施方式中，所述乙酰乙酰辅酶A硫解酶、羟甲基戊二酰辅酶A合酶、羟甲基戊二酰辅酶A还原酶、甲羟戊酸激酶、甲羟戊酸-5-磷酸激酶、甲羟戊酸焦磷酸脱羧酶、异戊二烯焦磷酸异构酶和法尼烯焦磷酸合酶在所述重组菌基因组中的拷贝数各自独立地为2、2、4、2、2、2、2、2个。
发明人发现，酿酒酵母可以内源合成FPP，因此在一些优选地实施方式中，采用酿酒酵母作为宿主细胞，有利于获得高效合成橙花叔醇的重组菌。
在一些实施方式中，所述重组菌还包括FPP水解酶DPP1、FPP水解酶LPP1、柠檬酸合酶、苹果酸合酶或角鲨烯合成酶的编码基因的至少一种的敲除或下调。发明人发现，FPP水解酶DPP1(diacylglycerol pyrophosphate phosphatase 1)和FPP水解酶LPP1(lipid phosphate phosphatase 1)具有水解FPP生成法尼醇的能力，柠檬酸合酶(CIT2)和苹果酸合酶(MLS1)能够消耗乙酰辅酶A；角鲨烯合成酶(ERG9)以FPP为底物合成角鲨烯，这些酶均竞争性消耗橙花叔醇生物合成所需要的底物，因此，敲除或下调这些酶的编码基因，减少重组菌中这些酶的表达，有利于提高重组菌中橙花叔醇的高效合成。
本申请中所述的下调具有其一般含义，在本申请中可以理解为基因的表达受到抑制，导致基因调控的蛋白表达量减少。
本申请中所述的敲除具有其一般含义，是指通过一定的途径使特定的基因失活或缺失，从而使其所编码的蛋白减少表达或不表达。
本申请第五方面提供了采用本申请第一方面的橙花叔醇合成酶、本申请第二方面的多核苷酸分子、本申请第三方面的核酸构建体或本申请第四方面的重组菌生产橙花叔醇的用途。
本申请第六方面提供了一种橙花叔醇的制备方法，其包括采用本申请第四方面的重组菌生物合成橙花叔醇。
在一些实施方式中，所述重组菌能够内源和/或外源表达乙酰乙酰辅酶A硫解酶、羟甲基戊二酰辅酶A合酶、羟甲基戊二酰辅酶A还原酶、甲羟戊酸激酶、甲羟戊酸-5-磷酸激酶、甲羟戊酸焦磷酸脱羧酶、异戊二烯焦磷酸异构酶、法尼烯焦磷酸合酶的至少一种。
发明人发现，采用能够内源表达甲羟戊酸途径中的酶和法尼烯焦磷酸合酶的至少一种的宿主细胞中导入所述目的基因，有利于进一步提高橙花叔醇的产量。
在一些实施方式中，还可以在所述核酸构建体中连接编码乙酰乙酰辅酶A硫解酶、羟甲基戊二酰辅酶A合酶、羟甲基戊二酰辅酶A还原酶、甲羟戊酸激酶、甲羟戊酸-5-磷酸激酶、甲羟戊酸焦磷酸脱羧酶、异戊二烯焦磷酸异构酶、法尼烯焦磷酸合酶的核苷酸序列的至少一种，使所述重组菌能够高效合成FPP，从而有利于橙花叔醇产量的进一步提高。
在一些实施方式中，所述乙酰乙酰辅酶A硫解酶、羟甲基戊二酰辅酶A合酶、羟甲基戊二酰辅酶A还原酶、甲羟戊酸激酶、甲羟戊酸-5-磷酸激酶、甲羟戊酸焦磷酸脱羧酶、异戊二烯焦磷酸异构酶和法尼烯焦磷酸合酶在所述重组菌基因组中的拷贝数各自独立地为2、2、4、2、2、2、2、2个。
在一些实施方式中，还包括FPP水解酶DPP1、FPP水解酶LPP1、柠檬酸合酶、苹果酸合酶或角鲨烯合成酶的编码基因的至少一种的敲除或下调。
在一些实施方式中，当采用的核酸构建体中包含诱导型启动子时，还可以包括重组菌转录抑制因子的敲除，例如采用GAL(半乳糖诱导)启动子时，可以对转录抑制因子GAL80进行敲除，转录抑制因子的敲除可以使所述重组菌在不需要诱导剂的情况下即可自主表达目的基因，降低了发酵成本。
下面通过具体实施例来说明本申请的橙花叔醇合成酶及其应用。下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。以下实施例中所涉及的质粒均为本领域技术人员公知质粒。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1 除虫菊中橙花叔醇合成酶的功能鉴定
1.1除虫菊来源的潜在橙花叔醇合成基因筛选
对除虫菊不同时期和不同部位的组织进行取样，对样品进行RNA提取，并进行了二代、三代转录组测序；从二代和三代转录组数据中，寻找同时含有萜类合酶PF01397和PF03936两个Pfam结构域的除虫菊转录组蛋白质序列，共获得166条潜在的蛋白质序列；使用CD-Hit对所找到的蛋白质序列进行去冗余聚类，将序列相似的大于90％的序列定义为 同一类，如此共获得了33类；在每一类中，按照序列完整度，选择出蛋白质序列长度大于500的作为候选基因，进一步从中选择出表达量最高的基因作为测试基因，共获得了24条待验证基因，基因命名为CCJ-TPS01至CCJ-TPS24。
1.2除虫菊中橙花叔醇合成酶及基因的鉴定
1.2.1酵母表达通用载体的构建
质粒pZY900具体构建过程：以酿酒酵母S288c基因组(提取方法见：李晓伟.工程乙酰辅酶A通路构建酿酒酵母高效合成平台[D].武汉大学,2015.2.3.6酵母基因组DNA提取方法)为模板，用引物900-1F/1R、900-2F/2R、900-6F/6R、900-7F/7R分别扩增获得片段9001(Leu2的左同源臂)、9002(终止子tTDH2)、9006(基因ERG20与终止子tERG20)、9007(Leu2右同源臂)；以酿酒酵母CEN.PK2-1D的基因组(提取方法见：李晓伟.工程乙酰辅酶A通路构建酿酒酵母高效合成平台[D].武汉大学,2015.2.3.6酵母基因组DNA提取方法)为模板，用引物900-3F/3R、900-5F/5R分别扩增获得片段9003(终止子tCYC1)和9005(启动子pGAL1和pGAL10)；用引物900-4F/4R以pCAS(见文献Zhang,Yueping et al.“A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae.”Nature communications vol.10,1 1053.5 Mar.2019,doi:10.1038/s41467-019-09005-3)为模板扩增获得片段9004(无义基因lacZ，用于目的基因的替换)；以pRS426为模板，用引物900-8F/8R、900-9F/9R、900-10F/10R扩增获得质粒骨架(引入MssI酶切位点，筛选标记(AmpR、URA3等))。通过DNA assemble(又称酵母组装，李晓伟.工程乙酰辅酶A通路构建酿酒酵母高效合成平台[D].武汉大学,2015.)的方法将以上片段在酿酒酵母体内重组构建pZY900，然后在大肠杆菌内扩增，酶切验证以及测序正确后，得到pZY900。质粒pZY900构建示意图见图1，其中，片段9001(HA)、9002(T)、9003(T)、9004、9005、9006、9007(HA)从左至右依次连接，其余部分来自pRS426的质粒骨架。
构建质粒pZY900所用引物的序列见下表1。
表1

1.2.2酵母表达载体的构建
设计引物将待验证基因连接在通用载体上。此处展示了除虫菊中最终验证有橙花叔醇合成酶活性的基因CCJ_TPS23的验证过程。
设计特异基因引物对CCJ_TPS23-F/R，以除虫菊的cDNA(采用TIANGEN公司RNAprep Pure Plant Plus Kit试剂盒(货号DP441)提取除虫菊胚珠组织RNA，使用Vazyme的HiScript II 1st Strand cDNA Synthesis Kit(+gDNA wiper)试剂盒(货号R212)对RNA进行反转录获得cDNA)为模板，利用诺唯赞公司的Phanta高保真酶通过PCR扩增获得CCJ_TPS23基因片段，利用天根胶回收试剂盒进行胶回收后，通过翊圣公司同源重组试剂盒，采用同源重组的方法连接到BsaI切后的酵母表达载体pZY900中，经过测序确认无误后，获得含有CCJ_TPS23基因的酵母表达载体，命名为pYR013，其质粒构建示意图见图2中的pYR013，其中，以CCJ_TPS23基因替代了pZY900中的lacZ基因。
CCJ_TPS23-F/R引物序列如下表2所示：
表2
1.2.3菌株构建
将质粒pYR013通过醋酸锂法(李晓伟.工程乙酰辅酶A通路构建酿酒酵母高效合成平台[D].武汉大学,2015.2.3.14酿酒酵母LiAc转化)导入菌株JCR27中(酵母菌株JCR27的构建见文献Siemon,Thomas et al.“Semisynthesis of Plant-Derived Englerin A Enabled by Microbe Engineering of Guaia-6,10(14)-diene as Building Block.”Journal of the American Chemical Society vol.142,6(2020):2760-2765.doi:10.1021/jacs.9b12940)，将改造菌株命名为CCJ-1。
以同样的方法，将质粒pZY900导入菌株JCR27中，作为对照菌株，命名为S900。
1.2.4菌株发酵和产物鉴定
将菌株CCJ-1、S900分别接菌至SC-URA液体培养基(李晓伟.工程乙酰辅酶A通路构建酿酒酵母高效合成平台[D].武汉大学,2015，缺尿嘧啶培养基)，30℃，200rpm摇床培养过夜；次日按照初始OD600＝0.1转接至45毫升YPDHG液体培养基(20g/L蛋白胨，10g/L酵母粉，10g/L葡萄糖，10g/L半乳糖)中，加入5毫升肉豆蔻酸异丙酯，30℃，200rpm摇床培养72小时，收集油层，使用正己烷稀释至合适的浓度，使用GC-MS检测产物。
Thermo Fisher Scientific配备AS 3000自动进样，分流/不分流进样器的TRACE GC ULTRA气相色谱，以及配备三重四极杆检测器的TSQ QUANTUM XLS MS。
色谱柱为TR-5MS column(30m×0.25mm×0.25um)。载气为高纯氦气，流速1mL/min。丙酮作为洗针液。进样量1uL，分流比50。进样口温度240℃，离子传输管温度270℃。
检测程序：起始柱温为50℃，保持1min；按15℃/min升温至280℃，保持1min；按20℃/min升温至300℃，保持2min。
CCJ-1、S900发酵产物中橙花叔醇特征离子m/z＝93提取离子流色谱图如图3所示，橙花叔醇的质谱图如图4所示，通过和橙花叔醇标准品进行色谱图保留时间，质谱图碎片比对，可以确定菌株CCJ-1中可以合成橙花叔醇，而菌株S900不能合成。此结果表明了CCJ_TPS23基因所编码的蛋白为橙花叔醇合成酶。
将质粒pYR013使用MssI酶切(酶切体条件参见MssI酶说明书)，获得线性化片段，将所述线性化片段通过醋酸锂法整合至菌株JCR27中，获得菌株CCJ-2。接菌至YPD液体培养基中，30℃，200rpm摇床培养过夜；次日按照初始OD600＝0.1转接至45毫升YPDHG液体培养基中，加入5毫升肉豆蔻酸异丙酯，30℃，200rpm摇床培养72小时，收集油层，使用GC-MS检测产物，菌株CCJ-2的橙花叔醇的摇瓶发酵产量为271mg/L。
实施例2 落新妇中橙花叔醇合成酶的功能鉴定
2.1落新妇来源的潜在橙花叔醇合成基因筛选
按照同样的方式，对落新妇不同时期和不同部位的组织进行取样，对样品进行RNA提取，并进行了二代、三代转录组测序；从二代和三代转录组数据中，寻找同时含有萜类合酶PF01397和PF03936两个Pfam结构域的落新妇转录组蛋白质序列，共获得89条潜在的蛋白质序列；使用CD-Hit对所找到的蛋白质序列进行去冗余聚类，将序列相似的大于90％的序列定义为同一类，如此共获得了25类；在每一类中，按照序列完整度，选择出蛋白质序列长度大于500的作为候选基因，进一步从中选择出表达量最高的基因作为测试基因，共获得了17条待验证基因，基因命名为ACH-TPS01至ACH-TPS17。
2.2落新妇中橙花叔醇合成酶及基因的鉴定
2.2.1酵母表达载体的构建
设计引物将待验证基因构建在通用载体上。此处展示了落新妇中最终验证有橙花叔醇合成酶活性的基因ACH_TPS07、ACH_TPS08、ACH_TPS09的验证过程。
设计特异基因引物对ACH_TPS07-F/R，ACH_TPS08-F/R，ACH_TPS09-F/R，以落新妇的cDNA(采用TIANGEN公司RNAprep Pure Plant Plus Kit试剂盒(货号DP441)提取落新妇叶片，使用Vazyme的HiScript II 1st Strand cDNA Synthesis Kit(+gDNA wiper)试剂盒(货号R212)对RNA进行反转录获得cDNA)为模板，利用诺唯赞公司的Phanta高保真酶通过PCR扩增获得ACH_TPS07、ACH_TPS08、ACH_TPS09基因片段，利用天根胶回收试剂盒进行胶回收后，通过翊圣公司同源重组试剂盒，采用同源重组的方法分别将获得的基因片段连接到BsaI切后的酵母表达载体pZY900中，经过测序确认无误后，获得分别含有 ACH_TPS07、ACH_TPS08、ACH_TPS09基因的酵母表达载体，命名为pYR007、pYR006、pYR010。质粒pYR007、pYR006、pYR010构建示意图见图2中的pYR007、pYR006、pYR010，其分别以基因ACH_TPS07、ACH_TPS08、ACH_TPS09替代pZY900中的lacZ基因。
引物序列如下表3所示。
表3
2.2.2菌株构建
采用与实施例1的1.2.3相同的方法，将质粒pYR007导入菌株JCR27中，将改造菌株命名为LXF-1；将质粒pYR006导入菌株JCR27中，将改造菌株命名为LXF-1-1；将质粒pYR010导入菌株JCR27中，将改造菌株命名为LXF-1-2。将质粒pZY900导入菌株JCR27中，作为对照菌株，命名为S900。
2.2.3菌株发酵和产物鉴定
将菌株LXF-1、LXF-1-1、LXF-1-2、S900分别接菌至SC-URA液体培养基，30℃，200rpm摇床培养过夜；次日按照初始OD600＝0.1转接至45毫升YPDHG液体培养基中，加入5毫升肉豆蔻酸异丙酯，30℃，200rpm摇床培养72小时，收集油层，使用GC-MS检测产物，检测条件与实施例1的1.2.4相同。
LXF-1、LXF-1-1、LXF-1-2、S900发酵产物中橙花叔醇的特征离子m/z＝93提取离子流色谱图如图5所示，橙花叔醇的质谱图如图6所示，通过和橙花叔醇标准品进行色谱图保留时间，质谱图碎片比对，可以确定菌株LXF-1、LXF-1-1、LXF-1-2中可以合成橙花叔醇，而菌株S900不能合成。此结果表明了ACH_TPS07、ACH_TPS08、ACH_TPS09基因所编码的蛋白为橙花叔醇合成酶。
将质粒pYR007、pYR006、pYR010分别使用MssI酶切(酶切体条件参见MssI酶说明书)，获得线性化片段，将所述线性化片段通过醋酸锂法整合至菌株JCR27中，获得菌株LXF-2、LXF-2-6、LXF-2-10。接菌至YPD液体培养基中，30℃，200rpm摇床培养过夜；次日按照初始OD600＝0.1转接至45毫升YPDHG液体培养基中，加入5毫升肉豆蔻酸异丙酯，30℃，200rpm摇床培养72小时，收集油层，使用GC-MS检测产物，菌株LXF-2、LXF-2-6、LXF-2-10的橙花叔醇的摇瓶发酵产量分别为269mg/L、240mg/L、221mg/L。
实施例3 艾蒿中橙花叔醇合成酶的功能鉴定
3.1艾蒿来源的潜在橙花叔醇合成基因筛选
从艾蒿的转录组数据(https://www.ncbi.nlm.nih.gov/bioproject/PRJNA722539)中，寻找同时含有萜类合酶PF01397和PF03936两个Pfam结构域的艾蒿转录组蛋白质序列，共获得167条潜在的蛋白质序列；使用CD-Hit对所找到的蛋白质序列进行去冗余聚类，将序列相似的大于90％的序列定义为同一类，如此共获得了47类；在每一类中，按照序列完整度，选择出蛋白质序列长度大于500的作为候选基因，进一步从中选择出表达量最高的基因作为测试基因，共获得了29条待验证基因，基因命名为Arar-TPS01至Arar-TPS29。
3.2艾蒿中橙花叔醇合成酶及基因的鉴定
3.2.1酵母表达载体的构建
设计引物将待验证基因构建在通用载体上。此处展示了艾蒿中最终验证有橙花叔醇合成酶活性的基因Arar-TPS27、Arar-TPS28的验证过程。艾蒿来源的基因Arar-TPS27、Arar-TPS28直接按照酿酒酵母密码子优化合成，序列如SEQ ID NO.7、SEQ ID NO.8所示。
设计特异基因引物对Arar-TPS27-F/R，Arar-TPS28-F/R以合成的基因为模板，利用诺唯赞公司的Phanta高保真酶通过PCR扩增获得Arar-TPS27、Arar-TPS28基因片段，利用天根胶回收试剂盒进行胶回收后，通过翊圣公司同源重组试剂盒，采用同源重组的方法分别连接到BsaI切后的酵母表达载体pZY900中，经过测序确认无误后，获得分别含有Arar-TPS27、Arar-TPS28的酵母表达载体，命名为pArar-TPS27、pArar-TPS28。质粒pArar-TPS27、pArar-TPS28构建示意图见图2中的pArar-TPS27、pArar-TPS28，其分别以基因pArar-TPS27、pArar-TPS28替代pZY900中的lacZ基因。
引物序列如下表4所示。
表4
3.2.2菌株构建
采用与实施例1的1.2.3相同的方法，将质粒pArar-TPS27导入菌株JCR27中，将改造菌株命名为AH-1；将质粒pArar-TPS28导入菌株JCR27中，将改造菌株命名为AH-2；将质粒pZY900导入菌株JCR27中，作为对照菌株，命名为S900。
3.2.3菌株发酵和产物鉴定
将菌株AH-1、AH-2、S900分别接菌至SC-URA液体培养基，30℃，200rpm摇床培养过夜；次日按照初始OD600＝0.1转接至45毫升YPDHG液体培养基中，加入5毫升肉豆蔻酸异丙酯，30℃，200rpm摇床培养72小时，收集油层，使用GC-MS检测产物，检测条件 与实施例1的1.2.4相同。
AH-1、AH-2、S900发酵产物中橙花叔醇的特征离子m/z＝93提取离子流色谱图如图7所示，橙花叔醇的质谱图如图8所示，通过和橙花叔醇标准品进行色谱图保留时间，质谱图碎片比对，可以确定菌株AH-1、AH-2均可以合成橙花叔醇，而菌株S900不能合成。此结果表明了Arar-TPS27、Arar-TPS28基因所编码的蛋白为橙花叔醇合成酶。
将质粒pArar-TPS27、pArar-TPS28分别使用MssI酶切(酶切体条件参见MssI酶说明书)，获得线性化片段，将所述线性化片段通过醋酸锂法整合至菌株JCR27中，获得菌株AH-27、AH-28。接菌至YPD液体培养基中，30℃，200rpm摇床培养过夜；次日按照初始OD600＝0.1转接至45毫升YPDHG液体培养基中，加入5毫升肉豆蔻酸异丙酯，30℃，200rpm摇床培养72小时，收集油层，使用GC-MS检测产物，菌株AH-27、AH-28的橙花叔醇的摇瓶发酵产量分别为162mg/L、158mg/L。
对比例1 现有橙花叔醇合成酶基因的表达
1.1含Celastrus angulatus(CaNES2)、Celastrus angulatus(CaNES1)、Tripterygium wilfordii(TwNES)、Actinidia chinensis(AcNES1)、Fragaria x ananassa(FaNES1)、Camellia sinensis(CsNES2)、Laggera pterodonta(LpNES1)来源的橙花叔醇合酶基因的质粒构建
本对比例中采用的不同来源的橙花叔醇合成酶是目前本领域研究较广泛，且产量公认较高的橙花叔醇合成酶。
Celastrus angulatus来源的橙花叔醇合成酶按照酿酒酵母密码子优化后的核苷酸序列如下(5’-3’)，基因命名为CaNES2

Celastrus angulatus来源的橙花叔醇合成酶按照酿酒酵母密码子优化后的核苷酸序列如下(5’-3’)，基因命名为CaNES1

Tripterygium wilfordii来源的橙花叔醇合成酶按照酿酒酵母密码子优化后的核苷酸序列如下(5’-3’)，基因命名为TwNES
Actinidia chinensis来源的橙花叔醇合成酶按照酿酒酵母密码子优化后的核苷酸序列如下(5’-3’)，基因命名为AcNES1

Fragaria x ananassa来源的橙花叔醇合成酶按照酿酒酵母密码子优化后的核苷酸序列如下(5’-3’)，基因命名为FaNES1

Camellia sinensis来源的橙花叔醇合成酶按照酿酒酵母密码子优化后的核苷酸序列如下(5’-3’)，基因命名为CsNES2

Laggera pterodonta来源的橙花叔醇合成酶按照酿酒酵母密码子优化后的核苷酸序列如下(5’-3’)，基因命名为LpNES1
设计引物将上述密码子优化后的橙花叔醇合酶编码基因构建在通用载体pZY900上。针对各基因设计的引物序列如下表所示，以上述合成的基因为模板，采用与实施例1的1.2.2相同的方法，分别构建含有CaNES2、CaNES1、TwNES、AcNES1、FaNES1、CsNES2和LpNES1基因的酵母表达载体，命名为pCaNES2、pCaNES1、pTwNES、pAcNES1、pFaNES1、pCsNES2和pLpNES1。质粒pCaNES2、pCaNES1、pTwNES、pAcNES1、pFaNES1、pCsNES2和pLpNES1构建示意图见图2，其分别以基因CaNES2、CaNES1、TwNES、AcNES1、FaNES1、CsNES2和LpNES1替代pZY900中的lacZ基因。

1.2含Celastrus angulatus(CaNES2)、Celastrus angulatus(CaNES1)、Tripterygium wilfordii TwNES(KU588405)、Actinidia chinensis(AcNES1)、Fragaria x ananassa(FaNES1)、Camellia sinensis(CsNES2)、Laggera pterodonta(LpNES1)来源的橙花叔醇合酶基因的菌株构建与摇瓶发酵
将质粒pCaNES2、pCaNES1、pTwNES、pAcNES1、pFaNES1、pCsNES2和pLpNES1分别使用MssI线性化后，将含有目标基因的片段使用胶回收试剂盒回收后通过醋酸锂法导入菌株JCR27中，将改造菌株分别命名为JCaNES2、JCaNES1、JTwNES、JAcNES1、JFaNES1、JCsNES2和JLpNES1。将菌株分别接菌至YPD液体培养基中，30℃，200rpm摇床培养过夜；次日按照初始OD600＝0.1转接至45毫升YPDHG液体培养基中，加入5毫升肉豆蔻酸异丙酯，30℃，200rpm摇床培养72小时，收集油层，使用GC-MS检测产物，检测条件与实施例1的1.2.4相同，菌株JCaNES2、JCaNES1、JTwNES、JAcNES1、JFaNES1、JCsNES2和JLpNES1的橙花叔醇的摇瓶发酵产量分别为33.7mg/L、8.3mg/L、69mg/L、111mg/L、86.5mg/L、21mg/L、5.3mg/L。
通过实施例1、2、3与对比例1的比较可见，本申请的除虫菊、落新妇、艾蒿来源的橙花叔醇合成酶相比于现有橙花叔醇合成酶具有更优的性能。
实施例4 含有除虫菊来源的橙花叔醇合成酶CCJ_TPS23的高产菌株的构建
4.1高产质粒构建
使用引物020-1F/R、020-2F/R、020-3F/R、020-4F/R、020-5F/R、020-6F/R、020-7F/R、020-8F/R、020-9F/R，分别以酿酒酵母CEN.PK2-1D的全基因组、pRS423质粒、酿酒酵母CEN.PK2-1D的全基因组、酿酒酵母S288C全基因组、酿酒酵母CEN.PK2-1D的全基因组、除虫菊cDNA、CEN.PK2-1D的全基因组、CEN.PK2-1D的全基因组、pRS426质粒为模板扩增获得片段Ura3左同源臂(HA)、组氨酸筛选标记(HIS3)、CYC1终止子(T)、tHMG1基因、GAL1-GAL10启动子(PGAL10和PGAL1)、CCJ_TPS23、PGK1终止子(T)、Ura3右同源臂(HA)、质粒骨架。随后采用与实施例1相同酵母组装的方法，在酿酒酵母体内重组构建，将以上片段顺序连接，获得质粒pYR020。质粒pYR020构建示意图见图9中的pYR020，其中还包含MssI酶切位点，其余部分来自pRS426质粒骨架。
引物序列见下表5。
表5
使用引物021-1F/R、021-2F/R、021-3F/R、021-4F/R、021-5F/R、021-6F/R、021-7F/R、021-8F/R，分别以酿酒酵母CEN.PK2-1D的全基因组、pRS424质粒、酿酒酵母S288C全基因组、酿酒酵母CEN.PK2-1D的全基因组、除虫菊cDNA、酿酒酵母CEN.PK2-1D的全基因组、酿酒酵母CEN.PK2-1D的全基因组、pRS426质粒为模板扩增获得片段YPRCdelta15左同源臂(HA)、色氨酸筛选标记(TRP1)、GPM1终止子(T)、GAL1-GAL10启动子(PGAL10和PGAL1)、CCJ_TPS23、PGK1终止子(T)、YPRCdelta15右同源臂(HA)、质粒骨架。随后使用与前述相同的酵母组装的方法，将以上片段在酿酒酵母体内重组构建，将以 上片段顺序连接，获得质粒pYR021。质粒pYR021构建示意图见图9中的pYR021，其中还包含Not I酶切位点，其余部分来自pRS426质粒骨架。
引物序列见下表6。
表6
4.2基因编辑质粒以及敲除盒构建
使用引物3951-F/R以质粒pKlURA100(见文献Zhang,Yueping et al.“A gRNA-tRNA array for CRISPR-Cas9 based rapid multiplexed genome editing in Saccharomyces cerevisiae.”Nature communications vol.10,1 1053.5 Mar.2019,doi:10.1038/s41467-019-09005-3)为模板扩增获得片段。随后使用Goldengate(Zhang,Yueping et al.“A gRNA-tRNA array for CRISPR-Cas9based rapid multiplexed genome editing in Saccharomyces cerevisiae.”Nature communications vol.10,1 1053.5 Mar.2019,doi:10.1038/s41467-019-09005-3)的方法，将以上片段和pCas进行组装，构建获得质粒pYH395，质粒构建示意图如图10A所示。
引物序列见下表7。
表7
使用引物ERG9-1F/R、ERG9-2F/R以酿酒酵母CEN.PK2-1D的全基因组为模板扩增获得两个片段，随后使用引物ERG9-1F、ERG9-2R，以获得的两个片段为模板，进行重叠延伸PCR，获得ERG9敲除盒。ERG9启动子上游激活顺式元件(-220至-175)敲除元件构建示意图见图10B，其中，两个HA为分别代表序列(-220至-175)左侧的同源臂和序列(-220至- 175)右侧的同源臂。
引物序列见下表8。
表8
使用引物5211-F/R、5212-F/R、5213-F/R，分别以酿酒酵母CEN.PK2-1D的全基因组、pRS426质粒、酿酒酵母CEN.PK2-1D的全基因组为模板扩增获得三个片段，随后使用引物5211-F、5213-R以获得的三个片段为模板，进行重叠延伸PCR，获得pZY521敲除盒。pZY521敲除盒构建示意图见图11，其中序列依次为GAL80左同源臂(HA)、URA3选择标记(启动子PURA3、URA3基因、终止子T)、GAL80右同源臂(HA)。
引物序列见下表9。
表9
4.3菌株构建
1)将质粒pYR013使用MssI酶切(酶切体条件参见MssI酶说明书)，获得线性化片段，将所述线性化片段通过醋酸锂法整合至菌株JCR27中，获得菌株CCJ-2。
2)将质粒pYR020使用MssI酶线性化后，采用与步骤1)相同的方法整合至菌株CCJ-2中，获得菌株CCJ-3。
3)将质粒pYR021使用NotI酶线性化后，采用与步骤1)相同的方法整合至菌株CCJ-3中，获得菌株CCJ-4。
4)将质粒pYH395和ERG9敲除盒通过醋酸锂法共同转入至菌株CCJ-4中，菌落经PCR验证敲除的正确性，随后使用YPD液体培养基(20g/L胰蛋白胨、10g/L酵母提取物、20g/L葡萄糖)培养，220rpm摇床，30度培养8小时后水洗，将菌划5-FOA平板(李晓伟.工程乙酰辅酶A通路构建酿酒酵母高效合成平台[D].武汉大学,2015.)，30℃培养箱培养3天，从平板上挑菌，菌落经PCR验证敲除的正确性，正确的菌株命名为CCJ-5。
本步骤用于敲除ERG9启动子上游激活顺式元件(-220至-175)，ERG9编码的角鲨烯合成酶以FPP为底物合成角鲨烯，为橙花叔醇合成途径的竞争途径，ERG9启动子上游激活顺式元件的敲除可下调角鲨烯合成途径，减少角鲨烯合成途径竞争消耗橙花叔醇合成所需的底 物，有利于进一步提高橙花叔醇的产量。
5)将pZY521敲除盒使用醋酸锂法转入菌株CCJ-5中，获得菌株CCJ-6。本步骤的作用是敲除转录抑制因子GAL80，从而可以使转入菌中的目的基因可以在不需要诱导剂的情况下即可自主表达，有利于降低发酵成品，还可进一步提高橙花叔醇的产量。
4.4菌株摇瓶发酵
将菌株CCJ-2、CCJ-3、CCJ-4、CCJ-5、CCJ-6分别接菌至YPD液体培养基中，30℃，200rpm摇床培养过夜；次日按照初始OD600＝0.1转接至45毫升YPDHG液体培养基(CCJ-2、CCJ-3、CCJ-4、CCJ-5)或YPD液体培养基(CCJ-6)中，加入5毫升肉豆蔻酸异丙酯，30℃，200rpm摇床培养72小时，收集油层，使用GC-MS检测产物，检测条件与实施例1的1.2.4相同。不同菌株橙花叔醇产量数据如图12所示。从结果中可以看出，随着目的基因CCJ_TPS23拷贝数的增多，橙花叔醇的产量逐渐升高，CCJ-2为271mg/L，CCJ-3为557mg/L，CCJ-3为627mg/L；下调角鲨烯合成途径后(CCJ-5)，橙花叔醇产量进一步明显提高，产量达到1140mg/L；更进一步敲除半乳糖诱导的主要转录抑制因子GAL80(CCJ-6)，橙花叔醇产量进一步明显提高，产量可达到1942mg/L。
实施例5 含有落新妇来源的橙花叔醇合成酶ACH_TPS07的高产菌株的构建
5.1高产质粒构建
使用引物020-1F/R、020-2F/R、020-3F/R、020-4F/R、020-5F/017-5R、017-6F/R、017-7F/020-7R、020-8F/R、020-9F/R，分别以酿酒酵母CEN.PK2-1D的全基因组、pRS423质粒、酿酒酵母CEN.PK2-1D的全基因组、酿酒酵母S288C的全基因组、酿酒酵母CEN.PK2-1D的全基因组、落新妇cDNA、酿酒酵母CEN.PK2-1D的全基因组、酿酒酵母CEN.PK2-1D的全基因组、pRS426质粒为模板扩增获得片段Ura3左同源臂(HA)、组氨酸筛选标记(HIS3)、CYC1终止子(T)、tHMG1基因、GAL1-GAL10启动子(PGAL10和PGAL1)、ACH_TPS07、PGK1终止子(T)、Ura3右同源臂(HA)、质粒骨架。随后采用与实施例1相同酵母组装的方法，在酿酒酵母体内重组构建，将以上片段顺序连接，获得质粒pYR017。质粒pYR017构建示意图见图9中的pYR017，其中还包含MssI酶切位点，其余部分来自pRS426质粒骨架。
引物序列见下表10。
表10
使用引物021-1F/R、021-2F/R、021-3F/R、021-4F/018-4R、018-5F/R、018-6F/021-6R、021-7F/R、021-8F/R，分别以酿酒酵母CEN.PK2-1D的全基因组、pRS424质粒、酿酒酵母 S288C全基因组、酿酒酵母CEN.PK2-1D的全基因组、落新妇cDNA、酿酒酵母CEN.PK2-1D的全基因组、酿酒酵母CEN.PK2-1D的全基因组、pRS426质粒为模板扩增获得片段YPRCdelta15左同源臂(HA)、色氨酸筛选标记(TRP1)、GPM1终止子(T)、GAL1-GAL10启动子(PGAL10和PGAL1)、ACH_TPS07、PGK1终止子(T)、YPRCdelta15右同源臂(HA)、质粒骨架。随后使用与前述相同的酵母组装的方法，将以上片段在酿酒酵母体内重组构建，将以上片段顺序连接，获得质粒pYR018。质粒pYR018构建示意图见图9中的pYR018，其中还包含Not I酶切位点，其余部分来自pRS426质粒骨架。
引物序列见下表11。
表11
5.2菌株构建
采用与实施例4相同的方法，将质粒pYR007使用MssI线性化后整合至菌株JCR27中，获得菌株LXF-2；将质粒pYR017使用MssI线性化后整合至菌株LXF-2中，获得菌株LXF-3；将质粒pYR018使用NotI线性化后整合至菌株LXF-3中，获得菌株LXF-4；将质粒pYH395和ERG9敲除盒共同转入至菌株LXF-4中，菌落经PCR验证敲除的正确性，随后使用YPD培养后水洗，将菌划5-FOA平板，从平板上挑菌，菌落经PCR验证敲除的正确性，正确的菌株命名为LXF-5；将pZY521敲除盒转入菌株LXF-5中，获得菌株LXF-6。
5.3菌株摇瓶发酵
将菌株LXF-2、LXF-3、LXF-4、LXF-5、LXF-6分别接菌至YPD液体培养基，30℃，200rpm摇床培养过夜；次日按照初始OD600＝0.1转接至45毫升YPDHG液体培养基(LXF-2、LXF-3、LXF-4、LXF-5)或YPD液体培养基(LXF-6)中，加入5毫升肉豆蔻酸异丙酯，30℃，200rpm摇床培养72小时，收集油层，使用GC-MS检测产物，检测条件与实施例1的1.2.4相同。产量数据如图13所示。不同菌株橙花叔醇产量数据如图13所示。从结果中可以看出，随着目的基因ACH_TPS07拷贝数的增多，橙花叔醇的产量逐渐升高，LXF-2约269mg/L，LXF-3约436mg/L，LXF-4约556mg/L；下调角鲨烯合成途径后(LXF-5)，橙花叔醇产量进一步明显提高，产量达到1112mg/L；更进一步敲除转录抑制因子GAL80后(LXF-6)，橙花叔醇产量进一步明显提高，产量可达到2105mg/L。
实施例6 橙花叔醇高产菌株的发酵罐发酵
参照文献(Ye Z,Huang Y,Shi B,et al.Coupling cell growth and biochemical pathway induction in Saccharomyces cerevisiae for production of(+)-valencene and its chemical conversion to(+)-nootkatone[published online ahead of print,2022 Mar 13].Metab Eng.2022；72:107-115. doi:10.1016/j.ymben.2022.03.005)中所记载的发酵罐培养基和发酵方法，对所构建的菌株LXF-6进行分批补料发酵，在发酵过程中添加覆盖剂以实现原位萃取，覆盖剂为肉豆蔻酸异丙酯。发酵过程控制溶氧在20％以上，pH为5，葡萄糖浓度为1-2g/L，乙醇浓度为5g/L以下。最终在1L发酵罐上，橙花叔醇产量达到了55.7g/L，是目前报道的最高产量水平。

Claims (19)

  1. 一种橙花叔醇合成酶，其包含Pfam编号为PF01397和PF03936的结构域，且具有橙花叔醇合成酶活性。
  2. 根据权利要求1所述的橙花叔醇合成酶，其来自除虫菊、落新妇或艾蒿。
  3. 根据权利要求1所述的橙花叔醇合成酶，其具有与SEQ ID NO.1、SEQ ID NO.2、SEQ ID NO.3、SEQ ID NO.4、SEQ ID NO.11或SEQ ID NO.12所示的氨基酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的氨基酸序列。
  4. 一种多核苷酸分子，其包含编码权利要求1-3中任一项所述的橙花叔醇合成酶的核苷酸序列或其互补序列的至少一种。
  5. 根据权利要求4所述的多核苷酸分子，其包含与SEQ ID NO.5、SEQ ID NO.6、SEQ ID NO.7、SEQ ID NO.8、SEQ ID NO.9或SEQ ID NO.10所示的核苷酸序列具有至少90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或100％序列同一性的核苷酸序列。
  6. 一种核酸构建体，其包含权利要求4或5所述的多核苷酸分子的至少一种。
  7. 根据权利要求6所述的核酸构建体，其还包含编码乙酰乙酰辅酶A硫解酶、羟甲基戊二酰辅酶A合酶、羟甲基戊二酰辅酶A还原酶、甲羟戊酸激酶、甲羟戊酸-5-磷酸激酶、甲羟戊酸焦磷酸脱羧酶、异戊二烯焦磷酸异构酶、法尼烯焦磷酸合酶的核苷酸序列的至少一种。
  8. 根据权利要求6或7所述的核酸构建体，所述核苷酸序列位于两个插入元件之间，所述插入元件用于将所述核苷酸序列整合入宿主细胞的基因组中。
  9. 根据权利要求6所述的核酸构建体，其为质粒载体；优选地，所述质粒载体为真核表达载体。
  10. 根据权利要求9所述的核酸构建体，其包括pRS426质粒骨架。
  11. 根据权利要求10所述的核酸构建体，其为质粒载体pYR006、pYR007、pYR010、pYR013、pAra-TPS27、pAra-TPS28、pYR017、pYR018、pYR020、pYR021的至少一种；所述质粒载体的构建示意图如图2或图9所示。
  12. 一种重组菌，其包含根据权利要求4或5所述的多核苷酸分子，或权利要求6-11中任一项所述的核酸构建体。
  13. 根据权利要求12所述的重组菌，其中，所述多核苷酸分子整合入宿主细胞的基因组中；优选地，所述宿主细胞为真核细胞；更优选为酿酒酵母。
  14. 根据权利要求13所述的重组菌，其中，所述多核苷酸分子在所述重组菌的基因组中的拷贝数为1-3个。
  15. 根据权利要求12所述的重组菌，其能够内源和/或外源表达乙酰乙酰辅酶A硫解酶、羟甲基戊二酰辅酶A合酶、羟甲基戊二酰辅酶A还原酶、甲羟戊酸激酶、甲羟戊酸-5-磷酸激酶、甲羟戊酸焦磷酸脱羧酶、异戊二烯焦磷酸异构酶、法尼烯焦磷酸合酶的至少一 种。
  16. 根据权利要求15所述的重组菌，其中，编码乙酰乙酰辅酶A硫解酶、羟甲基戊二酰辅酶A合酶、羟甲基戊二酰辅酶A还原酶、甲羟戊酸激酶、甲羟戊酸-5-磷酸激酶、甲羟戊酸焦磷酸脱羧酶、异戊二烯焦磷酸异构酶和法尼烯焦磷酸合酶的核苷酸序列在所述重组菌基因组中的拷贝数各自独立地为2、2、4、2、2、2、2、2个。
  17. 根据权利要求12所述的重组菌，其还包括FPP水解酶DPP1、FPP水解酶LPP1、柠檬酸合酶、苹果酸合酶或角鲨烯合成酶的编码基因的至少一种的下调或敲除。
  18. 采用权利要求1-3中任一项所述的橙花叔醇合成酶、权利要求4或5所述的多核苷酸分子、权利要求6-11中任一项所述的核酸构建体或权利要求12-17中任一项所述的重组菌生产橙花叔醇的用途。
  19. 一种橙花叔醇的制备方法，其包括采用权利要求12-17中任一项所述的重组菌生物合成橙花叔醇。
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DATABASE Protein 8 May 2018 (2018-05-08), ANONYMOUS : "(3S,6E)-nerolidol synthase 2 [Artemisia annua] ", XP093104385, retrieved from NCBI Database accession no. PWA37709.1 *
DATABASE Protein 8 May 2018 (2018-05-08), ANONYMOUS : "hypothetical protein CTI12_AA004750 [Artemisia annua]", XP093104393, retrieved from NCBI Database accession no. PWA96917.1 *
ZHOU HAN-CHEN, SHAMALA LUBOBI FERDINAND, YI XING-KAI, YAN ZHEN, WEI SHU: "Analysis of Terpene Synthase Family Genes in Camellia sinensis with an Emphasis on Abiotic Stress Conditions", SCIENTIFIC REPORTS, vol. 10, no. 1, XP093104382, DOI: 10.1038/s41598-020-57805-1 *

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