CN113265391B

CN113265391B - Linalool synthase CcLS and coding gene and application thereof

Info

Publication number: CN113265391B
Application number: CN202110587075.4A
Authority: CN
Inventors: 杨孝廉
Original assignee: Shenzhen Tianxiong Biotechnology Co ltd
Current assignee: Shenzhen Tianxiong Biotechnology Co ltd
Priority date: 2021-05-27
Filing date: 2021-05-27
Publication date: 2022-11-04
Anticipated expiration: 2041-05-27
Also published as: CN113265391A

Abstract

The invention provides linalool synthase CcLS and a coding gene and application thereof, and relates to the technical field of plant genetic engineering. The amino acid sequence of the linalool synthase CcLS is shown in SEQ ID No. 2. The invention obtains a novel linalool synthase CcLS participating in the synthesis of linalool, and proves that the novel linalool synthase CcLS can be used for synthesizing or preparing linalool.

Description

Linalool synthase CcLS and coding gene and application thereof

Technical Field

The invention relates to the technical field of plant genetic engineering, in particular to linalool synthase CcLS and a coding gene and application thereof.

Background

Camphor trees (Cinnamomum camphora (l.) presl) are also called Cinnamomum camphora trees, and have the name: cinnamomum camphora, camphorwood, yao people firewood, lao, cinnamomum camphora and Cinnamomum camphora; the camphor tree is a tree species of evergreen trees of cinnamomum of Lauraceae, a national II-level protective plant, and is an economic plant which integrates multiple purposes of medicine, essence, spice, oil, landscape, garden and the like. The camphor trees have camphor fragrance and can extract camphor oil, and the camphor trees can be divided into at least 5 different chemical types (chemotype) according to the main components contained in the camphor oil, namely linalool type (the main component is linalool), borneol type (the main component is D-borneol), camphor type (the main component is camphor), 1.8-eucalyptus type (the main component is 1, 8-cineole), nerolidol type (the main component is nerolidol), etc. (Caihui C, yongjie Z, yongda Z, et al. Transcripto biology and identification of genes related to specific biosyntheses in Cinnamomum camphora [ J ]. BMC genetics, 2018,19 (1): 550). Wherein the main component of the linalool type camphor trees is linalool which is an ideal raw material for extracting the linalool.

Linalool (linalool) is widely distributed in secretory tissues such as glands, oil chambers and resin tracts of higher plants and is a main component of plant volatile oil. The product is mainly used as perfume or synthetic intermediate in industry, and is an important raw material in medicine, cosmetics and other industries. Recent studies have shown that Linalool has a variety of biological activities, including Anti-inflammatory analgesic Effects (Huo M, cui X, xue J et AL. Anti-inflammatory Effects of linear in RAW 264.7 macrogels and lipopolysaccharides-induced lung in surgery model [ J ]. J Surg Res,2013,180 (1): E47-E54; evidence for the innovative elements of ionic glutamatergic receptors on the antigenic effect of (-) -linolool in the micro [ J ]. Neurosci Lett,2008,440 (3): 299-303.), tumour chemosensitisation (Miyashita M, sadzuka Y. Effect of linear as a component of Humulus luculus on doxorubicin-induced activity [ J ]. Food Chem Toxicol,2013, 53-179.), sedative anxiolytic (Linck VM, da-Silva AL, figueim et AL. Effects of inert linear in excitation, sodium interaction and affinity in microorganism [ J ]. Phytodimecine, 2010,17 (8/9): 679-683. Sesamum et AL., repellents (de-Oliviania-Souza-alcohol-T, zeringia V, aralia-oligna-C.sub.112. Reactive and 103. Lactic acid-C.sub.112. Reactive and 3. Pacific acids of biological sample J.), repellent insecticidal (de-oligna-cholesterol-G. T, zeyla V, simple-alidium-C.sub.sub.112, molecular and 103. Environmental sample of biological in biological and biological sample J. (3. Environmental sample J.) (reaction J.sub.sub.sub..

Linalool (linalool) belongs to the enols of chain-like oxygen-containing monoterpene derivatives. In plants, isopentenyl pyrophosphate (IPP), a universal substrate for terpenes, and dimethylpropylene pyrophosphate (DMAPP), an isomer thereof, are produced via the cytoplasmic mevalonate pathway (MVA) pathway and the plastid 2-methyl-D-erythritol-4-phosphate (MEP) pathway. And from this, monoterpenes, sesquiterpenes, diterpenes, triterpenes, geranyl diphosphates (GPP), farnesyl diphosphates (FPP) and Geranylgeranyl diphosphates (GGPP) which are substrates for triterpenes are produced. Monoterpene synthase (also known as monoterpene cyclase), which catalyzes The formation of various monoterpene skeletons by GPP, is considered to be a key enzyme in The synthesis of terpene secondary metabolites (Chen F, tholl D, bohlmann J et al, the family of family synthases in plants: a mid-size family of genes for specialized metabolism is high-purity transformed family of The genes [ J ] The Plant Journal,2011,66 (1): 212-229 Trap SC, croteau R.general organization of family synthases and molecular evaluation instruments J ]. Genes, 2001-811.

Therefore, it is necessary to research a key enzyme gene having the capability of synthesizing linalool, an oxygen-containing monoterpene derivative.

In view of this, the invention is particularly proposed.

Disclosure of Invention

The invention aims to provide a key enzyme for synthesizing oxygen-containing monoterpene derivative linalool, a coding gene thereof and application thereof in synthesizing linalool.

In order to solve the above problems, the technical scheme provided by the invention is as follows:

in one aspect, the invention provides a linalool synthase CcLS having an amino acid sequence as set forth in SEQ ID No. 2. SEQ ID No.2 consists of 582 amino acid residues.

Linalool synthase CcLS of the present invention, derived from camphor tree (Cinnamomum camphora (l.) presl), is named linalool synthase CcLS of camphor tree.

Furthermore, the invention comprises a fusion protein obtained by connecting protein labels at the N terminal or/and the C terminal of the amino acid shown in the sequence SEQ ID No. 2.

Further, the present invention also includes a protein having a derivative sequence in which one to several amino acids are deleted, substituted, inserted or added based on the amino acid sequence shown in SEQ ID No.2 and having linalool synthase activity. For example, conservative variants, biologically active fragments or derivatives of the protein consisting of the amino acid sequence are included in the scope of the present invention, as long as the homology between the fragment or protein variant of the protein and the amino acid sequence is above 90%. Preferably, the amino acid sequence of the conservative variant, biologically active fragment or derivative of said protein has more than 95% identity, e.g. 96%, 97%, 98%, 99% or 100%, with the amino acid sequence shown in SEQ ID No. 2.

In another aspect, the invention provides a coding gene of the linalool synthase CcLS, and the nucleotide sequence of the coding gene is shown as SEQ ID No. 1.

Further, the nucleotides may comprise variants of conservative substitutions thereof (e.g., substitutions of degenerate codons) and complementary sequences. For example, a nucleotide that hybridizes to the nucleotide sequence under stringent conditions. The protein sequence coded by the gene of the invention can be used for designing and artificially synthesizing a nucleotide sequence which is optimized by codons and is favorable for expression in plants. The stringent conditions are hybridization and washing at 68 ℃ for 2 times, 5min each time, in a solution of 2 XSSC, 0.1% SDS, and hybridization and washing at 68 ℃ for 2 times, 15min each time, in a solution of 0.5 XSSC, 0.1% SDS.

In another aspect, the present invention also provides a recombinant vector comprising the gene encoding the linalool synthase CcLS. The vector may be a plasmid vector or a viral vector.

In another aspect, the invention provides a recombinant cell or transgenic plant cell line comprising said recombinant vector or expressing said linalool synthase CcLS.

In one embodiment, the recombinant cell may be a yeast, bacterium or fungus.

The expression vector, the transgenic cell and the host bacterium containing the gene belong to the protection scope of the invention.

In another aspect, the present invention also provides the use of the linalool synthase CcLS, the gene encoding the linalool synthase CcLS, the recombinant vector or the recombinant cell for the preparation of linalool.

In another aspect, the present invention provides a method for producing linalool synthase CcLS, comprising introducing a gene encoding the linalool synthase CcLS into a recipient microorganism to obtain a recombinant microorganism expressing the linalool synthase CcLS, culturing the recombinant microorganism, and expressing to obtain linalool synthase CcLS.

In one embodiment, the expression vector containing the encoding gene may be introduced into a recipient microorganism by a calcium chloride method or an electroporation transformation method.

In one embodiment, the recipient microorganism is E.coli. More specifically, the Escherichia coli is Escherichia coli expression strain Transetta (DE 3).

In a specific embodiment, the gene encoding CcLS can be introduced into the E.coli expression strain Transetta (DE 3) via recombinant plasmid pET32 a:; the recombinant plasmid pET32a comprises a CcLS which is a recombinant expression vector obtained by constructing a Ccls gene shown in a sequence SEQ ID No.1 to a BamHI enzyme cutting site of a pET32a (+) vector and keeping other sequences of the pET32a (+) vector unchanged.

A method of producing linalool, comprising the steps of: and (3) introducing the coding gene of the linalool synthase CcLS into saccharomyces cerevisiae to obtain recombinant saccharomyces cerevisiae, and fermenting the recombinant saccharomyces cerevisiae to obtain linalool.

Further, in the above method, the saccharomyces cerevisiae is preferably a BY4741 yeast strain.

In one embodiment, the fermentation comprises inoculating the recombinant Saccharomyces cerevisiae to a fermentation medium and culturing at 30 ℃ for 48-96h; preferably 60-72h. Preferably, the culture is a shaking culture.

Furthermore, in the method, the coding gene of the CcLS can be introduced into GPP-producing yeast through a recombinant plasmid pESC-Leu:; the recombinant plasmid pESC-Leu is characterized in that CcLS is a recombinant expression vector which is constructed by constructing a CcLS gene shown in a sequence SEQ ID No.1 to a BamHI enzyme cutting site of a pESC-Leu vector and keeping other sequences of the pESC-Leu vector unchanged.

In another aspect, the present invention provides a method for synthesizing linalool using the linalool synthase CcLS, comprising the steps of: the linalool synthase CcLS is utilized to catalyze and synthesize linalool under neutral conditions by taking geranyl pyrophosphate (GPP) as a substrate.

Further, in the process of catalyzing and synthesizing linalool by utilizing the linalool synthase CcLS, an enzymatic buffer is added, wherein the enzymatic buffer comprises HEPES (with the concentration of 20-25 mM), mgCl2 (with the concentration of 5-8 mM) and DTT (with the concentration of 5-8 mM).

Has the advantages that:

the linalool synthase gene Ccls is obtained by cloning, and the linalool synthase Ccls with high expression level and high activity is successfully obtained after the gene is expressed. The invention can improve the linalool content in plants by genetic engineering technology.

The linalool synthase can catalyze geranyl pyrophosphate to synthesize linalool (linalool), and has high catalytic activity. The invention also provides a method for synthesizing linalool, 15mg of linalool can be obtained in each liter of fermentation liquor, a foundation is laid for biosynthesis of linalool, and the method has wide industrial application prospects.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is the agarose gel electrophoresis picture of Ccls gene clone of camphor tree; m represents Trans2K DNA Marker (nucleic acid molecular weight standard, the bands are 2000, 1000, 750, 500, 250 and 100bp from top to bottom respectively), cccls represents Cccls gene;

FIG. 2 is CcLS structural domain prediction analysis (from NCBI database);

FIG. 3 is a CcLS phylogenetic tree;

FIG. 4 is a polyacrylamide gel electrophoresis (SDS-PAGE) analysis of CcLS protein expressed in E.coli; m is a PageRuler non-prestained broad-range protein molecular weight standard (the bands are 250, 150, 100, 70 and 50KDa from top to bottom respectively), 1 is a protein expressed by an empty vector pET32a, 2 is a recombinant plasmid pET32a:: a protein expressed by CcLS, and an arrow indicates a target protein (recombinant protein CcLS);

FIG. 5 is a GC-MS analysis of the CcLS enzymatic reaction product; wherein, a in A is an extracted ion flow diagram of a target compound of a supernatant of a reference bacterium, b is a pET32a:: an extracted ion flow diagram of a target compound of a supernatant of a CcLS recombinant bacterium, and c is an extracted ion flow diagram of a standard linalool; b is pET32a, wherein the mass spectrogram of the target compound of the CcLS recombinant strain supernatant is shown in the specification; c is a mass spectrogram of a standard linalool;

FIG. 6 is a GC-MS analysis of the fermentation production of linalool by the introduction of CcLS into a yeast strain (GPP-producing); in the figure, a is an extracted ion flow diagram of a fermentation product of an empty vector pESC-Leu introduced yeast strain (GPP-reducing), b is an extracted ion flow diagram of a fermentation product of a CcLS introduced yeast strain (GPP-reducing) for producing linalool, and c is an extracted ion flow diagram of a standard linalool.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the following embodiments, and it should be apparent that the described embodiments are some, but not all, embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

In the following examples

High-Fidelity DNA Polymerase, bamHI restriction enzyme is a product of New England Biolabs;

the rapid general plant RNA extraction kit is a product of Beijing Huayuyo Biotechnology Limited;

TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix, trans2K DNA Marker, pEASY-Uni Sensless Cloning and Assembly Kit, escherichia coli competent cell Transetta (DE 3) is a product of Beijing Quanjin Biotechnology Limited;

HEPES，MgCl ₂ DTT is a product of Sigma-Aldrich company;

PageRuler non-prestained broad range protein molecular weight standards are products of Thermo Fisher Scientific, inc.;

the pET32a (+) vector is a product of Novagen corporation;

the pESC-Leu vector is a product of Agilent;

SD-Ura and SD-Ura-Leu are products of Beijing pan-Kino science and technology Limited;

ZYMO RESEARCH FRORCON-EZ Yeast Transformation II kit Zymo RESEARCH;

BY4741 Yeast Strain (genotype: MATa his 3. DELTA.1 leu 2. DELTA.0 met15. DELTA.0 ura 3. DELTA.0) is a product of Biotech, inc., of Beijing Huayuyang;

geranyl pyrophosphate (GPP) is a product of Sigma, with a product catalog number of G6772, a CAS number of 763-10-0;

linalool (Linalool) is a product from Sigma company under catalog number CRM40437 and CAS number 78-70-6.

Example 1 cloning of full-Length cDNA sequence of CcLS Gene of Cinnamomum camphora

1. Extraction of Total RNA

The total RNA of the camphor tree leaves is extracted by referring to the specification of the rapid universal plant RNA extraction kit of Beijing Huayuanyang biotechnology limited company.

2. Synthesis of first Strand cDNA

Referring to the specifications of TransScript One-Step gDNA Removal and cDNA Synthesis Supermix of the first strand cDNA Synthesis kit of Beijing all-purpose gold Biotechnology Co., ltd, the following reverse transcription reaction systems are prepared, the reverse transcription steps are sequentially completed, and finally the first strand cDNA is obtained.

The reverse transcription reaction system is as follows:

the reverse transcription procedure was as follows:

(1) To obtain higher synthesis efficiency, total RNA and absorbed Oligo (dT) are sequentially extracted ₁₈ Placing Primer and RNase-free Water in a PCR tube, gently mixing uniformly, slightly centrifuging, and placing in a PCR instrument for reaction at 65 ℃ for 5min;

(2) After the Reaction, the PCR tube was placed on ice, and 10.0. Mu.L of 2 XTS Reaction Mix and 1.0. Mu.L of

RT/RI Enzyme Mix, 1.0. Mu.L gDNA Remover, gently mixing, and slightly centrifuging;

(3) Placing in a PCR instrument, and performing reverse transcription reaction at 42 deg.C 30min and 85 deg.C for 5s to obtain first strand cDNA;

(4) First strand cDNA was stored at-20 ℃.

3. Primer 5.0 design Primer

Deeply digging camphor tree leaf transcriptome data, extracting a camphor tree Ccls gene Open Reading Frame (ORF) sequence from the data, and designing and cloning primers CcLS-F1 and CcLS-R1 based on the sequence, wherein the primer sequences are as follows:

CcLS-F1：5’-ATGAGTTTGATCATTCAGTATCTTCCTCAC-3’(SEQ ID No.3)；

CcLS-R1：5’-TCAAGGCCACTGGCTTGGAAC-3’(SEQ ID No.4)。

4. PCR amplification

Taking the first strand cDNA obtained in step 2 as a template, and adopting high fidelity enzyme

Carrying out PCR amplification reaction on the High-Fidelity DNA Polymerase, the CcLS-F1 and the CcLS-R1 primers, cutting the gel, purifying and recovering to obtain a PCR amplification product, wherein the result is shown in figure 1, and sequencing and analyzing the PCR amplification product.

The PCR amplification procedure is as follows:

performing pre-denaturation at 98 ℃ for 3min; 2 s at 98 ℃,2 s at 55 ℃,10 min at 72 ℃ and 35 cycles; extension at 72 ℃ for 5min.

The sequencing result shows that: the sequence of the PCR amplification product was identical to that shown in sequence No.1, and the gene shown in sequence No.1 was designated Ccls, and encoded a protein consisting of 582 amino acid residues, which was designated CcLS, and the amino acid sequence of the protein was SEQ ID No. 2.

Cccls sequence (SEQ ID No. 1):

ATGAGTTTGATCATTCAGTATCTTCCTCATTGGTCTAGAATTCCACCTAGACCTCCTCAGCTCTCTCAATTTCAAAACTCATCCAGGCCCAAACCTGTAATTCAGGCAGGCCAAGTACAACGCGACGTGCTTCAAATCGCCCGTCGATCAGCAAATTACCACCCAAGCATTTGGGACCCCCAATATATTGAGTCGCTAGCAAGTCCATATGGTGATGAGTGCTTTGGGACTCGGCTTGAGGAGTTGAAATTCGAAGCCAAACGGCTGCTCGAAGCTACCGTAGAGCCATTGTCTTGTCTGGAGCTTGTCGACTCGATCCAACGGCTAGGGGTGGCATACCACTTTGAGGATGAGATCAAAAACAGCCTTGATGGTGTTTATGGGGTTAACGCCCACGTCGGCGATGATCTTTACACTGCAGCATTACGGTTTCGGCTTCTTCGACAACACGGTTATGGTGTTACTCCAGATATATTCAGCAAGTTTTTGGAGAAGGAAAGAACATTCAAGCCATGCACAAGCCTAGATGCAAAAGGCCTTCTGAGCCTATATGAAGCATCACATACTATGATACATGGAGAGCAAGTGTTGGAAGACGCCAAAGAATTCAGTGTCAAGCATCTTAACTACTTGATGGGGAACTTACAGAGCAATCTAAGAGAGCAAGTGCAACATGCCCTAGAAATGCCCTTGCATTGGAGGATGCCAAGGCTAGAAGCAAAGCATTATATAGACGTGAATGGGAGGTCAGATGAGAGGAATATGGTTTTACTAGAGCTGGCAAGGTTGGATTTCAATTTCGTGCAATCCAAGCACCAAGAAGAACTGAAGGAGGTGTCAAGATGGTGGAAAGACTTGGGTCTTGCAAAGAAGCTGGATTTTTCTAGGGATCGATTGGTTGAAAATTACTTATGGGCCGTGGGAATCGCTCCCGAGCCCAAGTTCTCCAACTGCAGGAAAGGGCTCACCAAACTCATCTCCATTTTAACAGTGATCGATGACATCTACGATGTATATGGATCACTTGATGAACTTGAACTCTTTACAGAAGCTATAAAGAGATGGGACATTGAGGCTTTGGAGACTCTACCAGAGTACATGAAGATATGTTACTTGGCACTATTTAACTTTGTTCATGAAGTATCCTATGACACACTTAAGGATTATGGGTGGAACATCTTACCCTTTATCAGGAAAGAGTGGGAAAGGCTATGCATGTCATATCTGGTAGAAGCAAAATGGTTTGGCAATGACAATAAGCCAGTCCTTGATGAGTATTTGAGAAATGGTTGGATCTCAGTGGGTGGCCCAGTGGCGATGGTTCACGCTTATTTTCTTCAAGGGCAACCAATCAGGAAGGATTCAATCAACTTTTTAGACAACGGATCAGAGCTCATTTATTGGTCATCGGTTGCTACTCGACTCAATGATGACTTGGGCACTTCTAAGGCTGAGATGAAGCGAGGAGATGTGCCGAAAGCAGTCGAGTGCCACATGATCCAGACAGGTAGGTCCCATGAAGATGCAAGAGAGTACATAAAGGGTCTAGCAAGAGATTGTTGGAAGAAAATGAATGAGGAATGCTTGAAATGTAGTCTTCCTAACAGTTATGCAGAAACAGTTTTGAACATGGTTCGTACAGCCCAATGCATCTACCAGCATGGAGATGGTATTGGAACTTCGACTGGAGTGACCCAAGATAGAGTCATCTCATTGATCTGTGAGCCCGTTCCAAGCCAGTGGCCTTGA。

CcLS sequence (SEQ ID No. 2):

MSLIIQYLPHWSRIPPRPPQLSQFQNSSRPKPVIQAGQVQRDVLQIARRSANYHPSIWDPQYIESLASPYGDECFGTRLEELKFEAKRLLEATVEPLSCLELVDSIQRLGVAYHFEDEIKNSLDGVYGVNAHVGDDLYTAALRFRLLRQHGYGVTPDIFSKFLEKERTFKPCTSLDAKGLLSLYEASHTMIHGEQVLEDAKEFSVKHLNYLMGNLQSNLREQVQHALEMPLHWRMPRLEAKHYIDVNGRSDERNMVLLELARLDFNFVQSKHQEELKEVSRWWKDLGLAKKLDFSRDRLVENYLWAVGIAPEPKFSNCRKGLTKLISILTVIDDIYDVYGSLDELELFTEAIKRWDIEALETLPEYMKICYLALFNFVHEVSYDTLKDYGWNILPFIRKEWERLCMSYLVEAKWFGNDNKPVLDEYLRNGWISVGGPVAMVHAYFLQGQPIRKDSINFLDNGSELIYWSSVATRLNDDLGTSKAEMKRGDVPKAVECHMIQTGRSHEDAREYIKGLARDCWKKMNEECLKCSLPNSYAETVLNMVRTAQCIYQHGDGIGTSTGVTQDRVISLICEPVPSQWP。

example 2 analysis of Ccls Gene bioinformatics of Cinnamomum camphora

The full-length cDNA of the camphor tree Ccls gene consists of 1749 nucleotides, the detailed sequence is shown as sequence 1 (SEQ ID No. 1), and the protein shown as coding sequence 2 (SEQ ID No. 2). Nucleotide homology search was performed on the camphor tree Ccls gene sequence in the Non-redundant GenBank + EMBL + DDBJ + PDB and Non-redundant GenBank CDS translation + PDB + Swissprot + Superdate + PIR databases using the BLAST program in the NCBI database, which has high homology at the amino acid level with monoterpene synthases in other species, as well as a typical Isoprenoid _ Biosyn _ C1 superfamily domain, as shown in fig. 2 and 3; wherein fig. 2 is the CcLS structural functional domain prediction analysis (from NCBI database), and fig. 3 is the CcLS phylogenetic tree (adjacency).

Example 3 obtaining of CcLS protein of Cinnamomum camphora and functional analysis thereof

1. Obtaining CcLS protein of camphor tree

1. Construction of recombinant vectors

By performing the operation according to the specification of the Kit pEASY-Uni SEAmless Cloning and Assembly Kit of Beijing all-purpose gold Biotechnology, inc., the CcLS gene shown in the sequence 1 is constructed to the BamHI enzyme cutting site of pET32a (+) vector (Novagen corporation), and other sequences of pET32a (+) vector are kept unchanged, so as to obtain recombinant plasmids pET32a:: ccLS.

The method comprises the following specific steps:

1) PCR amplification reaction was performed using the PCR amplification product obtained in example 1 as a template and primers CcLS-F2 and CcLS-R2, and a purified PCR product was obtained by cutting and recovering the gel. Wherein, the primer sequences are as follows (sequences shown by underlining are vector homologous regions):

the primer sequences are as follows (sequences shown underlined are regions of vector homology):

CcLS-F2(SEQ ID No.5)：

5’-CCATGGCTGATATCGGAATGAGTTTGATCATTCAGTATCTTCCTCAC-3’；

CcLS-R2(SEQ ID No.6)：

5’-ACGGAGCTCGAATTCGGTCAAGGCCACTGGCTTGGAAC-3’。

2) The pET32a (+) vector (Novagen) was digested with BamHI, and the linearized vector fragment was recovered by gel-cutting and purification.

3) The purified PCR product obtained in the step 1) is cloned to the linearized vector fragment obtained in the step 2) according to the operation of the specification of pEASY-Uni Seamless Cloning and Assembly Kit of Beijing Omegand Biotechnology Limited, so as to obtain a recombinant plasmid pET32a:: ccLS.

2. Obtaining of recombinant bacteria

Transforming the recombinant plasmid pET32a: ccLS into an Escherichia coli expression strain Transetta (DE 3) (purchased from Beijing all-type gold biotechnology, inc.) to obtain pET32a: ccLS recombinant bacteria; meanwhile, pET32a (+) empty vector without target gene is used to transform Escherichia coli expression strain Transetta (DE 3) as a control bacterium.

3. Obtaining of recombinant protein CcLS

Monoclonal colonies of the recombinant CcLS strain and the control strain, which are pET32a, are picked and inoculated into a fresh LB liquid medium (containing ampicillin 100 mg/L) respectively, and are subjected to shaking culture in a shaking table at 37 ℃ overnight. The following day is as follows: diluting at 100 ratio, inoculating into 200mL LB liquid medium (containing ampicillin 100 mg/L), shaking culturing at 37 deg.C in shaking table to OD ₆₀₀ When the concentration is 0.6-0.8 ℃, transferring the mixture into a shaker at 18 ℃ for shaking for 1 hour, adding IPTG (isopropyl-beta-D-thiogalactoside) to the final concentration of 0.5mM, and continuing shaking culture at 18 ℃ for 24 hours to induce the expression of the target protein. After the induction is finished, centrifuging the bacterial liquid for 5min by 8000g, removing the supernatant, collecting pET32a, namely CcLS recombinant bacteria and thalli of control bacteria, and storing the bacterial liquid in a refrigerator at the temperature of 80 ℃ below zero for later use.

Extracting proteins in the strains of the CcLS recombinant strain and the control strain, wherein the steps are as follows: with 5mL of HEPES buffer (25mM HEPES,5mM MgCl) pre-chilled ₂ 5mM DTT, pH 7.0) resuspending pET32a, ccLS recombinant bacteria and control bacteria; ultrasonically breaking the bacteria in ice bath (30% power, 5s ultrasonic, 5s interval, 10min continuous), after ultrasonically breaking, centrifuging at 12000g and 4 DEG CAnd (3) obtaining pET32a, namely CcLS recombinant strain supernatant and control strain supernatant which are protein solutions respectively after 30 min.

The supernatant of the recombinant CcLS strain pET32a and the supernatant of the control strain were subjected to polyacrylamide gel electrophoresis (SDS-PAGE), and the results are shown in FIG. 4. As indicated by the black arrow in the figure, pET32a:: ccLS recombinant strain supernatant has recombinant plasmid pET32a:: ccLS expressed recombinant protein CcLS, which is about 85kDa in size and is in agreement with the expected size. Control supernatants were free of the corresponding proteins.

2. Detection of enzymatic Activity of recombinant protein CcLS

1. Enzymatic Activity

And (3) taking pET32a as a reference, carrying out enzymatic reaction on the supernatant of the CcLS recombinant strain, and extracting by using normal hexane to obtain an enzymatic reaction product. Wherein the enzymatic reaction comprises the following specific steps:

the total enzymatic reaction system is 0.2mL, and 190. Mu.L of supernatant of recombinant CcLS strain pET32a, i.e. HEPES buffer (25mM HEPES,5mM MgCl. RTM ₂ 5mM DTT, pH 7.0), resuspending the thalli and carrying out ultrasonic disruption to obtain a protein solution, adding 10 mu L of substrate geranyl pyrophosphate (GPP), mixing uniformly, slightly centrifuging, slowly adding 200 mu L of n-hexane to cover the whole system of the enzymatic reaction to form a liquid seal, standing in an incubator at 30 ℃ for reaction for 2 hours, carrying out vortex oscillation, 12000g, centrifuging for 5min, and taking a supernatant n-hexane layer to obtain pET32a:: ccLS recombinant bacteria supernatant enzymatic reaction product.

2. GC-MS detection

The enzymatic reaction products were detected by GC-MS using gas chromatography-Mass spectrometer: the GC-MS analysis system is Thermo TRACE 1310/TSQ 8000gas chromatography, the sample introduction amount is 1 muL, the split mode is adopted, the gas chromatographic column is Thermo Scientific TG-5MS (30 m multiplied by 0.25mm multiplied by 0.25μm), the helium gas flow rate is 1.0mL/min, the injection port temperature is 220 ℃, the ion source temperature is 200 ℃, the temperature raising program is 50 ℃ for 2min, and the temperature raising program is 5 ℃ and min ^-1 Heating to 150 deg.C and maintaining at 2min,30 deg.C/min ^-1 To 300 ℃, electron energy 70eV, and a 50-500m/z range scan was performed on the sample.

And replacing 190 mu L of CcLS recombinant bacterium supernatant in the reaction with 190 mu L of control bacterium supernatant, and repeating the test to obtain the target compound of the control bacterium supernatant.

The results of GC-MS analysis are shown in FIG. 5: linalool (linalool) is not detected in the target compound of the supernatant of the control strain, pET32a, linalool (linalool) is detected in the target compound of the supernatant of the CcLS recombinant strain, which indicates that the CcLS recombinant protein can catalyze GPP to form linalool (linalool), and indicates that the recombinant protein CcLS is linalool synthase.

Example 4 introduction of CcLS from Cinnamomum camphora into Yeast Strain for fermentation production of linalol

1. Construction of eukaryotic expression vectors

By performing the operation according to the specification of pEASY-Uni SEAmless Cloning and Assembly Kit of Beijing Omegal Biotechnology, inc., the CcLS gene of sequence 1 is constructed to the BamHI enzyme cutting site of pESC-Leu vector (Agilent), and other sequences of pESC-Leu vector are kept unchanged, so as to obtain the recombinant plasmid pESC-Leu:: ccLS.

The method comprises the following specific steps:

1) PCR amplification reaction was performed using the PCR amplification product obtained in example 1 as a template and primers CcLS-F3 and CcLS-R3, and a purified PCR product was obtained by cutting and recovering the gel. Wherein, the primer sequences are as follows (sequences shown by underlining are vector homologous regions):

CcLS-F3(SEQ ID No.7)：

5’-GGAGAAAAAACCCCGATGAGTTTGATCATTCAGTATCTTCCTCAC-3’；

CcLS-R3(SEQ ID No.8)：

5’-GTGAGTCGTATTACGGTCAAGGCCACTGGCTTGGAAC-3’。

2) The pESC-Leu vector (Agilent) was digested with BamHI, and the linearized vector fragment was recovered by gel-cutting purification.

3) Taking the purified PCR product obtained in the step 1), and constructing the purified PCR product on the linearized vector fragment obtained in the step 2) according to the instruction of the Beijing Quanyu gold biotechnology Co., ltd, so as to obtain a recombinant plasmid pESC-Leu:: ccLS.

2. Construction of GPP-producing Yeast Strain

YPD solid plate: 1% yeast extract +2% peptone +2% glucose +1.5% agar; preparing corresponding liquid culture medium (YPD liquid culture medium) without adding agar; YPL induction medium: 1% yeast extract +2% peptone +2% galactose; SD-Ura solid plate: SD-Ura +2% glucose +2% agar; the corresponding liquid culture medium (SD-Ura liquid culture medium) is obtained without adding agar; SD-Ura-Leu solid plate: SD-Ura-Leu +2% glucose +2% agar; the corresponding liquid medium (SD-Ura-Leu liquid medium) was prepared without adding agar.

The BY4741 yeast strain (genotype: MATa his 3. DELTA.1 leu 2. DELTA.0 met15. DELTA.0 ura 3. DELTA.0) was spread on YPD solid plates and cultured in an inverted state at 30 ℃ for 48 to 72 hours to obtain a freshly activated BY4741 yeast colony. According to the equimolar ratio, ura3 marker, yeast-derived tHMGR1 (containing a promoter sequence P) _TDH3 And a terminator sequence T _TPI1 I.e. P _TDH3 -tHMGR1-T _TPI1 ) IDI1 derived from yeast (containing promoter sequence P) _ADH1 And a terminator sequence T _PGI I.e. P _ADH1 -IDI1-T _PGI ) Yeast-derived tHMGR1 (containing promoter sequence P) _PGK1 And a terminator sequence T _ADH1 I.e. P _PGK1 -tHMGR1-T _ADH1 ) Yeast-derived ERG20 ^F96W-N127W (containing promoter sequence P _TEF2 And a terminator sequence T _CYC1 I.e. P _TEF2 -ERG20 ^F96W-N127W -T _CYC1 ) After mixing, electrotransformation integrated into BY4741 yeast strain at chromosome 15 YPRC Δ 15 (chromosome XVI long _ terminal _ repeat and Autonomously Replicating Sequence, YPRC Δ 15).

1) Picking a single colony of freshly activated BY4741 yeast in 5mL YPD liquid culture medium, carrying out shake culture at 30 ℃ and 200rpm overnight until OD600=0.6-1.0;

2) Taking an electric rotating cup (inner diameter of 0.2 cm) soaked in ethanol, cleaning with ultrapure water, standing upside down, placing on water-absorbing filter paper, air drying, and sterilizing in a super clean bench for 30min;

3) In an ultra-clean bench sterile environment, sucking 1-2mL of bacterial liquid, placing the bacterial liquid in a sterile 1.5mL EP tube, centrifuging at normal temperature for 1min at 10000g, and removing supernatant;

4) Adding 1mL of precooled sterile water, resuspending the thalli, 10000g, centrifuging for 1min at normal temperature, and discarding the supernatant;

5) Repeating step 4) once, adding 1mL of pre-cooled buffer (10mM LiAc,10mM DTT,0.6M sorbitol,10mM pH7.5 Tris-HCl), and culturing in a water bath at 25 ℃ for 20min;

6) 10000g, centrifuging for 1min at normal temperature, and discarding the supernatant;

7) Adding precooled 1mL of sorbitol (1M) solution to resuspend the thalli, 10000g of thalli, centrifuging for 1min at normal temperature, and discarding supernatant;

8) Repeating the step 7) once, adding pre-cooled 100 mu L of sorbitol (1M) solution to resuspend the thalli, and preparing electroporation competent cells;

9) Mixing Ura3 marker and P _TDH3 -tHMGR1-T _TPI1 、P _ADH1 -IDI1-T _PGI 、P _PGK1 -tHMGR1-T _ADH1 、P _TEF2 -ERG20 ^F96W-N127W -T _CYC1 Mixing 5 DNA fragments at equal molar ratio, adding into the competent cells with total mass of 500ng (total volume not more than 1/10 of the volume of the competent cells), gently mixing, centrifuging slightly, transferring to electric rotating cup (0.2 cm), and ice-cooling for 2-5min; performing electrotransformation under the conditions of 2.7kV, 25 muF and 200 omega (Bio-Rad, hercules, CA), adding 1mL of sorbitol (1M) solution into a super clean bench after electric shock to resuspend electrotransformation solution, transferring the electrotransformation solution into a sterile 1.5mL of EP tube, culturing for 1-2h in an incubator at 30 ℃, turning the EP tube up and down during the period, and uniformly mixing for 2-3 times;

10 10000g, centrifuging for 1min at normal temperature, discarding the supernatant, suspending the thallus by 100 mu L of solution, dripping the thallus in the center of a defective SD-Ura solid plate, uniformly coating the thallus by using a coater until all the coated bacteria liquid is completely absorbed, placing the thallus in a 30 ℃ incubator for inverted culture for 2-3d, and naming the obtained strain as GPP-producing yeast strain.

3. Competent preparation of GPP-degrading Yeast

GPP-degrading yeast (genotype: MATa his 3. DELTA.1 leu2. DELTA.0 met15. DELTA.0 Ura3. DELTA.0, YPRC. DELTA.15 Ura3-P _TDH3 -tHMGR1-T _TPI1 -P _ADH1 -IDI1-T _PGI -P _PGK1 -tHMGR1-T _ADH1 -P _TEF2 -ERG20 ^F96W-N127W -T _CYC1 )。

Yeast competent cells were prepared using ZYMO RESEARCH FROZEN-EZ Yeast Transformation II kit:

(1) Selecting newly activated GPP-producing yeast single colony from SD-Ura solid plate, inoculating in 10mL SD-Ura liquid culture medium, shake culturing in 30 deg.C shaker to OD ₆₀₀ About = 0.8-1.0;

(2) Centrifuging at room temperature at 500g for 4min, and removing supernatant;

(3) Adding 10mL of Frozen-EZ Solution 1 for resuspending thalli, centrifuging at room temperature for 4min at 500g, and removing supernatant;

(4) Adding 1mL of Frozen-EZ Solution 2 heavy suspension thalli to obtain GPP-producing yeast competent cells, and subpackaging the cells into sterile 1.5mL of EP tubes with each tube being 50 mu L;

(5) Slowly cooling and storing at-70 deg.C (4 deg.C, 1h; 20 deg.C, 1h; 40 deg.C, 1h; 70 deg.C), and inhibiting liquid nitrogen from quick freezing the competent cells to reduce their activity.

4. Recombinant plasmid pESC-Leu:: ccLS is transformed into GPP-producing yeast competent cell

(1) Taking a 50 mu L GPP-reducing yeast competent cell, unfreezing on ice, sucking 0.2-1 mu g of recombinant plasmid pESC-Leu:: ccLS (less than 5 mu L) to be added into the yeast competent cell, and gently mixing;

(2) Adding 500 mu L of Frozen-EZ Solution 3, and violently and uniformly mixing;

(3) Incubating in an incubator at 30 ℃ for 1-2h, turning the EP tube up and down during the incubation period, and mixing uniformly for 2-3 times;

(4) Taking 50-150 mu L of incubated bacterial liquid, coating the incubated bacterial liquid on an SD-Ura-Leu solid plate, airing, placing the plate at 30 ℃ for inverted culture for 48-96h to obtain the recombinant yeast which is transferred into the recombinant yeast pESC-Leu:: ccLS, and naming the recombinant yeast as GPP-producing/pESC-Leu:: ccLS.

5. Fermentation of

(1) In an ultra-clean bench sterile environment, selecting a single colony of CcLS (GPP-producing/pESC-Leu) growing on the SD-Ura-Leu solid plate in the step 4, placing the single colony in 10mL SD-Ura-Leu liquid culture medium, and carrying out shaking culture at 30 ℃ and 200rpm for 48h;

(2) The cells were collected by centrifugation at 5000g for 5min at room temperature, resuspended in 2mL of YPL induction medium, transferred to 20mL of YPL induction medium, and subjected to induction culture at 30 ℃ and 200rpm for 72 hours.

6. Fermentation product extraction

The target component is a monoterpene compound which is fat-soluble and easily soluble in ethyl acetate, so that the ethyl acetate is selected as a solvent to extract the target monoterpene compound. The extraction steps are as follows:

(1) Collecting the fermented bacteria liquid, and adding ethyl acetate with the same volume;

(2) Carrying out ultrasonic bacteria breaking for 1h, and oscillating and mixing for many times;

(3) At room temperature of 5000g for 5min, carefully absorbing the upper organic phase, adding appropriate amount of anhydrous sodium sulfate (drying at 120 ℃ for 30 min), shaking while adding, and removing water in the extract;

(4) Concentrating on a rotary evaporator to be nearly dry;

(5) Sucking the concentrated solution, filtering with 0.22 μm PTFE needle filter, storing the filtrate in liquid phase vial, sealing with sealing membrane, and storing in refrigerator at 4 deg.C.

7. GC-MS detection of fermentation product

Detecting a target compound by GC-MS (gas chromatography-mass spectrometry): the GC-MS analysis system is Thermo TRACE 1310/TSQ 8000gas chromatography, the sample introduction amount is 1 muL, the split mode is adopted, the gas chromatographic column is Thermo Scientific TG-5MS (30 m multiplied by 0.25mm multiplied by 0.25μm), the helium gas flow rate is 1.0mL/min, the injection port temperature is 220 ℃, the ion source temperature is 200 ℃, the temperature raising program is 50 ℃ for 2min, and the temperature raising program is 5 ℃ and min ^-1 Heating to 150 deg.C, maintaining at 2min, and cooling at 30 deg.C/min ^-1 To 300 ℃, electron energy 70eV, and a 50-500m/z range scan was performed on the sample.

The results of GC-MS analysis are shown in FIG. 6: the yeast strain GPP-reduction/pESC-Leu containing pESC-Leu recombinant plasmid CcLS can synthesize linalool (linalool). 15.0mg linalool per liter of fermentation broth can be obtained.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

SEQUENCE LISTING

<110> Shenzhen Tianxiong Biotech Limited

<120> linalool synthase CcLS and coding gene and application thereof

<130> PA21004854

<160> 8

<170> PatentIn version 3.3

<210> 1

<211> 1749

<212> DNA

<213> Ccls sequence

<400> 1

atgagtttga tcattcagta tcttcctcat tggtctagaa ttccacctag acctcctcag 60

ctctctcaat ttcaaaactc atccaggccc aaacctgtaa ttcaggcagg ccaagtacaa 120

cgcgacgtgc ttcaaatcgc ccgtcgatca gcaaattacc acccaagcat ttgggacccc 180

caatatattg agtcgctagc aagtccatat ggtgatgagt gctttgggac tcggcttgag 240

gagttgaaat tcgaagccaa acggctgctc gaagctaccg tagagccatt gtcttgtctg 300

gagcttgtcg actcgatcca acggctaggg gtggcatacc actttgagga tgagatcaaa 360

aacagccttg atggtgttta tggggttaac gcccacgtcg gcgatgatct ttacactgca 420

gcattacggt ttcggcttct tcgacaacac ggttatggtg ttactccaga tatattcagc 480

aagtttttgg agaaggaaag aacattcaag ccatgcacaa gcctagatgc aaaaggcctt 540

ctgagcctat atgaagcatc acatactatg atacatggag agcaagtgtt ggaagacgcc 600

aaagaattca gtgtcaagca tcttaactac ttgatgggga acttacagag caatctaaga 660

gagcaagtgc aacatgccct agaaatgccc ttgcattgga ggatgccaag gctagaagca 720

aagcattata tagacgtgaa tgggaggtca gatgagagga atatggtttt actagagctg 780

gcaaggttgg atttcaattt cgtgcaatcc aagcaccaag aagaactgaa ggaggtgtca 840

agatggtgga aagacttggg tcttgcaaag aagctggatt tttctaggga tcgattggtt 900

gaaaattact tatgggccgt gggaatcgct cccgagccca agttctccaa ctgcaggaaa 960

gggctcacca aactcatctc cattttaaca gtgatcgatg acatctacga tgtatatgga 1020

tcacttgatg aacttgaact ctttacagaa gctataaaga gatgggacat tgaggctttg 1080

gagactctac cagagtacat gaagatatgt tacttggcac tatttaactt tgttcatgaa 1140

gtatcctatg acacacttaa ggattatggg tggaacatct taccctttat caggaaagag 1200

tgggaaaggc tatgcatgtc atatctggta gaagcaaaat ggtttggcaa tgacaataag 1260

ccagtccttg atgagtattt gagaaatggt tggatctcag tgggtggccc agtggcgatg 1320

gttcacgctt attttcttca agggcaacca atcaggaagg attcaatcaa ctttttagac 1380

aacggatcag agctcattta ttggtcatcg gttgctactc gactcaatga tgacttgggc 1440

acttctaagg ctgagatgaa gcgaggagat gtgccgaaag cagtcgagtg ccacatgatc 1500

cagacaggta ggtcccatga agatgcaaga gagtacataa agggtctagc aagagattgt 1560

tggaagaaaa tgaatgagga atgcttgaaa tgtagtcttc ctaacagtta tgcagaaaca 1620

gttttgaaca tggttcgtac agcccaatgc atctaccagc atggagatgg tattggaact 1680

tcgactggag tgacccaaga tagagtcatc tcattgatct gtgagcccgt tccaagccag 1740

tggccttga 1749

<210> 2

<211> 582

<212> PRT

<213> CcLS sequence

<400> 2

Met Ser Leu Ile Ile Gln Tyr Leu Pro His Trp Ser Arg Ile Pro Pro

1 5 10 15

Arg Pro Pro Gln Leu Ser Gln Phe Gln Asn Ser Ser Arg Pro Lys Pro

20 25 30

Val Ile Gln Ala Gly Gln Val Gln Arg Asp Val Leu Gln Ile Ala Arg

35 40 45

Arg Ser Ala Asn Tyr His Pro Ser Ile Trp Asp Pro Gln Tyr Ile Glu

50 55 60

Ser Leu Ala Ser Pro Tyr Gly Asp Glu Cys Phe Gly Thr Arg Leu Glu

65 70 75 80

Glu Leu Lys Phe Glu Ala Lys Arg Leu Leu Glu Ala Thr Val Glu Pro

85 90 95

Leu Ser Cys Leu Glu Leu Val Asp Ser Ile Gln Arg Leu Gly Val Ala

100 105 110

Tyr His Phe Glu Asp Glu Ile Lys Asn Ser Leu Asp Gly Val Tyr Gly

115 120 125

Val Asn Ala His Val Gly Asp Asp Leu Tyr Thr Ala Ala Leu Arg Phe

130 135 140

Arg Leu Leu Arg Gln His Gly Tyr Gly Val Thr Pro Asp Ile Phe Ser

145 150 155 160

Lys Phe Leu Glu Lys Glu Arg Thr Phe Lys Pro Cys Thr Ser Leu Asp

165 170 175

Ala Lys Gly Leu Leu Ser Leu Tyr Glu Ala Ser His Thr Met Ile His

180 185 190

Gly Glu Gln Val Leu Glu Asp Ala Lys Glu Phe Ser Val Lys His Leu

195 200 205

Asn Tyr Leu Met Gly Asn Leu Gln Ser Asn Leu Arg Glu Gln Val Gln

210 215 220

His Ala Leu Glu Met Pro Leu His Trp Arg Met Pro Arg Leu Glu Ala

225 230 235 240

Lys His Tyr Ile Asp Val Asn Gly Arg Ser Asp Glu Arg Asn Met Val

245 250 255

Leu Leu Glu Leu Ala Arg Leu Asp Phe Asn Phe Val Gln Ser Lys His

260 265 270

Gln Glu Glu Leu Lys Glu Val Ser Arg Trp Trp Lys Asp Leu Gly Leu

275 280 285

Ala Lys Lys Leu Asp Phe Ser Arg Asp Arg Leu Val Glu Asn Tyr Leu

290 295 300

Trp Ala Val Gly Ile Ala Pro Glu Pro Lys Phe Ser Asn Cys Arg Lys

305 310 315 320

Gly Leu Thr Lys Leu Ile Ser Ile Leu Thr Val Ile Asp Asp Ile Tyr

325 330 335

Asp Val Tyr Gly Ser Leu Asp Glu Leu Glu Leu Phe Thr Glu Ala Ile

340 345 350

Lys Arg Trp Asp Ile Glu Ala Leu Glu Thr Leu Pro Glu Tyr Met Lys

355 360 365

Ile Cys Tyr Leu Ala Leu Phe Asn Phe Val His Glu Val Ser Tyr Asp

370 375 380

Thr Leu Lys Asp Tyr Gly Trp Asn Ile Leu Pro Phe Ile Arg Lys Glu

385 390 395 400

Trp Glu Arg Leu Cys Met Ser Tyr Leu Val Glu Ala Lys Trp Phe Gly

405 410 415

Asn Asp Asn Lys Pro Val Leu Asp Glu Tyr Leu Arg Asn Gly Trp Ile

420 425 430

Ser Val Gly Gly Pro Val Ala Met Val His Ala Tyr Phe Leu Gln Gly

435 440 445

Gln Pro Ile Arg Lys Asp Ser Ile Asn Phe Leu Asp Asn Gly Ser Glu

450 455 460

Leu Ile Tyr Trp Ser Ser Val Ala Thr Arg Leu Asn Asp Asp Leu Gly

465 470 475 480

Thr Ser Lys Ala Glu Met Lys Arg Gly Asp Val Pro Lys Ala Val Glu

485 490 495

Cys His Met Ile Gln Thr Gly Arg Ser His Glu Asp Ala Arg Glu Tyr

500 505 510

Ile Lys Gly Leu Ala Arg Asp Cys Trp Lys Lys Met Asn Glu Glu Cys

515 520 525

Leu Lys Cys Ser Leu Pro Asn Ser Tyr Ala Glu Thr Val Leu Asn Met

530 535 540

Val Arg Thr Ala Gln Cys Ile Tyr Gln His Gly Asp Gly Ile Gly Thr

545 550 555 560

Ser Thr Gly Val Thr Gln Asp Arg Val Ile Ser Leu Ile Cys Glu Pro

565 570 575

Val Pro Ser Gln Trp Pro

580

<210> 3

<211> 30

<212> DNA

<213> Artificial sequence

<400> 3

atgagtttga tcattcagta tcttcctcac 30

<210> 4

<211> 21

<212> DNA

<213> Artificial sequence

<400> 4

tcaaggccac tggcttggaa c 21

<210> 5

<211> 47

<212> DNA

<213> Artificial sequence

<400> 5

ccatggctga tatcggaatg agtttgatca ttcagtatct tcctcac 47

<210> 6

<211> 38

<212> DNA

<213> Artificial sequence

<400> 6

acggagctcg aattcggtca aggccactgg cttggaac 38

<210> 7

<211> 45

<212> DNA

<213> Artificial sequence

<400> 7

ggagaaaaaa ccccgatgag tttgatcatt cagtatcttc ctcac 45

<210> 8

<211> 37

<212> DNA

<213> Artificial sequence

<400> 8

gtgagtcgta ttacggtcaa ggccactggc ttggaac 37

Claims

1. Linalool synthase CcLS, wherein the amino acid sequence of the linalool synthase CcLS is shown in SEQ ID No. 2.

2. The gene encoding linalool synthase CcLS of claim 1, characterized in that the nucleotide sequence of said encoding gene is represented by SEQ ID No. 1.

3. A recombinant vector comprising the gene encoding linalool synthase CcLS of claim 2.

4. A recombinant cell comprising the recombinant vector of claim 3 or expressing the linalool synthase CcLS of claim 1.

5. The recombinant cell of claim 4, wherein the recombinant cell is E.coli.

6. The recombinant cell according to claim 4, wherein the recombinant cell is an E.coli expression strain Transetta DE3.

7. Use of the linalool synthase CcLS of claim 1, the gene encoding the linalool synthase CcLS of claim 2, the recombinant vector of claim 3, or the recombinant cell of any one of claims 4-6 for the preparation of linalool.

8. A method for producing linalool synthase CcLS, comprising introducing the coding gene of linalool synthase CcLS according to claim 2 into a recipient microorganism to obtain a recombinant microorganism expressing linalool synthase CcLS according to claim 1, culturing the recombinant microorganism, and expressing to obtain linalool synthase CcLS.

9. A method for producing linalool, comprising the steps of: the method comprises the steps of introducing the coding gene of the linalool synthase CcLS of claim 2 into Saccharomyces cerevisiae to obtain recombinant Saccharomyces cerevisiae, and fermenting the recombinant Saccharomyces cerevisiae to obtain linalool.

10. The method of claim 9, wherein the fermentation comprises inoculating the recombinant saccharomyces cerevisiae into a fermentation medium and culturing at 30 ℃ for 48-96 h.

11. The method of claim 9, wherein the fermentation comprises inoculating the recombinant saccharomyces cerevisiae into a fermentation medium and culturing at 30 ℃ for 60-72 hours.

12. A method of synthesizing linalool using the linalool synthase CcLS of claim 1, comprising the steps of: geranyl pyrophosphate is used as a substrate, and linalool is catalytically synthesized by utilizing the linalool synthase CcLS.