CN113136347B

CN113136347B - Saccharomyces cerevisiae engineering bacterium for high yield of coniferyl alcohol and construction and application thereof

Info

Publication number: CN113136347B
Application number: CN202010062695.1A
Authority: CN
Inventors: 訾佳辰; 杨加增
Original assignee: Jinan University
Current assignee: Zhejiang Kaiman Biotechnology Co ltd
Priority date: 2020-01-20
Filing date: 2020-01-20
Publication date: 2022-06-03
Anticipated expiration: 2040-01-20
Also published as: CN113136347A

Abstract

The invention discloses a saccharomyces cerevisiae engineering bacterium for high yield of coniferyl alcohol and construction and application thereof. The engineering bacteria use saccharomyces cerevisiae as an original strain and overexpress ZWF1 and ARO4^K229L、ARO7^G229S、ARO8、TYR1、BDH1^{E221S/I222R/A223S}COMT1, 4CL5, CAD, CCR, HpaB, TAL and PhaC genes; wherein: ARO4^K229LLysine at position 229 of ARO4 was mutated to leucine; ARO7^G229SGlycine at position 229 of ARO7 was mutated to serine; BDH1^{E221S/I222R/A223S}BDH1 has a mutation of glutamic acid 221 to serine, isoleucine 222 to arginine and alanine 223 to serine. The yeast engineering bacteria can obtain coniferyl alcohol through fermentation.

Description

Saccharomyces cerevisiae engineering bacterium for high yield of coniferyl alcohol and construction and application thereof

Technical Field

The invention relates to the technical field of metabolic engineering and fermentation, in particular to a saccharomyces cerevisiae engineering bacterium for high yield of coniferyl alcohol and construction and application thereof.

Background

At present, coniferyl alcohol has no stable commercial source. Although there are reports of chemical synthesis, such as using acetyl ferulic acid as raw material, preparing acyl chloride, then esterifying, and finally reducing, the total yield is only 24.4%; or the ferulic acid is used as a raw material, and the pinosylvic alcohol is obtained by esterification, acetylation and reduction, and the total yield is 70.2%. However, both methods require the use of highly toxic organic reagents and consume expensive lithium aluminum hydride, limiting the usefulness of these methods. In addition, attempts have been made to obtain recombinant E.coli capable of producing coniferyl alcohol by heterologous expression of the biosynthetic pathway of coniferyl alcohol in E.coli. However, the use of escherichia coli heterologous expression requires the use of a large amount of antibiotics, increases the risk of drug-resistant genes entering the ecosystem, and easily causes the production of superbacteria.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provide the saccharomyces cerevisiae engineering bacteria for high yield of coniferyl alcohol.

The invention also aims to provide a construction method of the saccharomyces cerevisiae engineering bacteria for high yield of coniferyl alcohol.

The invention also aims to provide application of the saccharomyces cerevisiae engineering bacteria for high yield of coniferyl alcohol.

The purpose of the invention is realized by the following technical scheme: a Saccharomyces cerevisiae engineering bacterium for high yield of coniferyl alcohol takes Saccharomyces cerevisiae as an original strain, and overexpresses ZWF1 and ARO4^K229L、ARO7^G229S、ARO8、TYR1、BDH1^{E221S/I222R/A223S}COMT1, 4CL5, CAD, CCR, HpaB, TAL and PhaC genes; wherein the content of the first and second substances,

the ARO4^K229LLysine at position 229 of ARO4 gene was mutated to leucine;

the ARO7^G229SGlycine at position 229 of ARO7 gene was mutated to serine;

the BDH1^{E221S/I222R/A223S}The BDH1 gene has the mutation of 221 th glutamic acid to serine, 222 th isoleucine to arginine and 223 th alanine to serine.

The saccharomyces cerevisiae is preferably saccharomyces cerevisiae BY 4741.

The ZWF1, ARO4, ARO7, ARO8, TYR1 and BDH1 genes can be cloned from a saccharomyces cerevisiae BY4741 genome; wherein, the nucleotide sequence of the ZWF1 is shown as GenBank NM-001183079.1; the nucleotide sequence of ARO4 is shown in GenBank: NM-001178597.1; the nucleotide sequence of ARO7 is shown in GenBank: NM-001184157.1; the nucleotide sequence of ARO8 is shown in GenBank: NM-001181067.1; the nucleotide sequence of TYR1 is shown in GenBank: NM-001178514.1; the nucleotide sequence of BDH1 is shown in GenBank: NM-001178202.2.

The COMT1, 4CL5, CAD and CCR genes can be obtained by cloning with Arabidopsis thaliana cDNA as a template, wherein the nucleotide sequence of COMT1 is shown in GenBank: NM-124796.4; the nucleotide sequence of 4CL5 is shown in GenBank: AY 376732; the nucleotide sequence of CAD (namely CAD5) is shown in GenBank: AY 302082; the nucleotide sequence of CCR (i.e., CCR1) is shown in GenBank: NM-001332191.1.

The nucleotide sequence of HpaB is shown in GenBank: WP-003137596.1.

The nucleotide sequence of TAL is shown in GenBank: KR 095308.1.

The nucleotide sequence of PhaC is shown in GenBank: WP-001175451.1.

The construction method of the saccharomyces cerevisiae engineering bacteria for high yield of coniferyl alcohol comprises the following steps:

(1) construction of the following modules and Gene fragments Using overlapping PCR

(a) Will P_TDH3TAL and T_TDH2Sequentially connecting to construct expression module P_TDH3-TAL-T_TDH2；

(b) Will P_PGK1、ARO4^K229LAnd T_ADH1Sequentially connecting to construct expression module P_PGK1-ARO4^K229L-T_ADH1；

(c) Will P_TEF1、ARO7^G229SAnd T_CYC1Sequentially connecting to construct expression module P_TEF1-ARO7^G229S-T_CYC1；

(d) Will express the module P_TDH3-TAL-T_TDH2、P_PGK1-ARO4^K229L-T_ADH1And P_TEF1-ARO7^G229S-T_CYC1Connecting to obtain a gene fragment TAA;

(e) will P_TDH3COMT1 and T_TDH2Sequentially connecting to construct expression module P_TDH3-COMT1-T_TDH2；

(f) Will P_PGK1PhaC and T_ADH1Sequentially connecting to construct expression module P_PGK1-PhaC-T_ADH1；

(g) Will P_TEF1HpaB and T_CYC1Sequentially connecting to construct expression module P_TEF1-HpaB-T_CYC1；

(h) Will express the module P_TDH3-COMT1-T_TDH2、P_PGK1-PhaC-T_ADH1And P_TEF1-HpaB-T_CYC1Connecting to obtain a gene fragment OCB;

(i) will P_TDH34CL5 and T_TDH2Sequentially connecting to construct expression module P_TDH3-4CL5-T_TDH2；

(j) Will P_PGK1CAD and T_ADH1Sequentially connecting to construct expression module P_PGK1-CAD-T_ADH1；

(k) Will P_TEF1CCR and T_CYC1Sequentially connecting to construct expression module P_TEF1-CCR-T_CYC1；

(l) Will express the module P_TDH3-4CL5-T_TDH2、P_PGK1-CAD-T_ADH1And P_TEF1-CCR-T_CYC1Connecting to obtain a gene fragment FFA;

(m) adding P_TDH3、BDH1^{E221S/I222R/A223S}And T_TDH2Sequentially connecting to construct expression module P_TDH3-BDH1^E221S ^/I222R/A223S-T_TDH2；

(n) adding P_PGK1TYR1 and T_ADH1Sequentially connecting to construct expression module P_PGK1-TYR1-T_ADH1；

(o) adding P_TEF1ARO8 and T_CYC1Sequentially connecting to construct expression module P_TEF1-ARO8-T_CYC1；

(P) expression of the Module P_TDH3-BDH1^{E221S/I222R/A223S}-T_TDH2、P_PGK1-TYR1-T_ADH1And P_TEF1-ARO8-T_CYC1Connecting to obtain a gene segment BTA;

(q) adding P_TEF1ZWF1 and T_CYC1Sequentially connecting to construct expression module P_TEF1-ZWF1-T_CYC1；

(2) Construction of vectors

(I) The pCfB2797 vector is cut by restriction enzymes HindIII and NheI to obtain a linearized pCfB2797 vector; then connecting the gene fragment TAA obtained in the step (d) to a linearized pCfB2797 vector to obtain a plasmid pCfB2797 TAA;

(II) digesting the vector pCfB2798 by using restriction enzymes HindIII and NheI to obtain a linearized pCfB2798 vector; then connecting the gene fragment OCB obtained in the step (h) to a linearized pCfB2798 vector to obtain a plasmid pCfB2798 OCB;

(III) cutting the vector pCfB2989 by using a restriction enzyme Pst 1; obtaining a linearized pCfB2798 vector, and then connecting the MET screening label gene fragment to the linearized pCfB2798 vector to obtain a plasmid pCfB2798 m; then the vector pCfB2989m is cut by restriction enzymes HindIII and NheI to obtain a linearized pCfB2989m vector; then connecting the gene fragment FFA obtained in the step (l) to a linearized pCfB2989m vector to obtain a plasmid pCfB2989 mFFA;

(IV) connecting the His screening label gene fragment to a pEASY-Blunt vector to obtain a plasmid p-YJZ-His; then, the plasmid P-YJZ-His is cut by restriction enzyme AvaI to obtain a linearized P-YJZ-His vector; then connecting the gene segment BTA obtained in the step (P) to a linearized P-YJZ-His vector to obtain a plasmid P-HBTA;

(V) connecting the Trp screening tag gene fragment to a pEASY-Blunt vector to obtain a plasmid p-YJZ-Trp, and then utilizing a restriction enzyme AvaI to enzyme-cut the plasmid p-YJZ-Trp to obtain a linearized p-YJZ-Trp vector; then, the expression module P obtained in the step (q) is used_TEF1-ZWF1-T_CYC1Connecting to a linearized p-YJZ-Trp vector to obtain a plasmid p-TZWF 1;

(3) construction of the Strain YC1061

(A) After the plasmid pCfB2797TAA is linearized BY using a restriction enzyme NotI, Saccharomyces cerevisiae BY4741 is transformed (the linearized vector is integrated to a Ty2 site of a yeast chromosome), and Saccharomyces cerevisiae YT1003 is obtained BY screening;

(B) after the plasmid pCfB2798OCB is linearized by using a restriction enzyme NotI, Saccharomyces cerevisiae YT1003 is transformed (the linearized vector is integrated into a Ty4 site of a yeast chromosome), and Saccharomyces cerevisiae YC1031 is obtained by screening;

(C) after the plasmid pCfB2989mFFA is linearized by a restriction enzyme NotI, saccharomyces cerevisiae YC1031 is transformed (the linearized vector is integrated into a Ty1 site of a yeast chromosome), and saccharomyces cerevisiae YC1048 is obtained by screening;

(D) carrying out PCR amplification on the plasmid p-HBTA obtained in the step (IV) by using primers p-HBTA-F and p-HBTA-R to obtain a gene integration fragment HBTA; then, the gene integration fragment HBTA is transformed into saccharomyces cerevisiae YC1048 (the linearized vector is integrated to YKL211C locus of yeast chromosome), and saccharomyces cerevisiae YC1053 is obtained; wherein the nucleotide sequences of the primers p-HBTA-F and p-HBTA-R are shown as follows:

p-HBTA-F：5’-TTTCTTAGCATTTTTGACGAAATTTGCTATTTTGTTAGAGTCTTTTACACATAACAATTTCACACAGGAAACAGCTATGAC-3’(SEQ ID NO.7)；

p-HBTA-R：5’-ATGTCTGTTATTAATTTCACAGGTAGTTCTGGTCCATTGGTGAAAGTTTGTCACGACGTTGTAAAACGACG-3’(SEQ ID NO.8)；

(E) performing PCR amplification on the plasmid p-TZWF1 obtained in the step (V) by using primers p-TZWF1-F1, p-TZWF1-R1, p-TZWF1-F2 and p-TZWF1-R2 respectively to obtain gene integration fragments TZWF1-PDC5 and TZWF1-ARO 10; then simultaneously converting the strain into saccharomyces cerevisiae YC1053 (simultaneously integrating the linearized vector into the YLR134W and YDR380W loci of the yeast chromosome) to obtain saccharomyces cerevisiae YC1061, namely the saccharomyces cerevisiae engineering strain for high yield of coniferyl alcohol; wherein the nucleotide sequences of the primers p-TZWF1-F1, p-TZWF1-R1, p-TZWF1-F2 and p-TZWF1-R2 are as follows:

p-TZWF1-F1：5’-TGTCTGAAATAACCTTAGGTAAATATTTATTTGAAAGATTGAGCCAAGTCACACAGGAAACAGCTATGACCATG-3’(SEQ ID NO.9)；

p-TZWF1-R1：5’-TGTTTAGCGTTAGTAGCGGCAGTCAATTGAGCTTGTTTAACCAAGTTTTGACGACGTTGTAAAACGACGGC-3’(SEQ ID NO.10)；

p-TZWF1-F2：5’-GGCACCTGTTACAATTGAAAAGTTCGTAAATCAAGAAGAACGACACCTTGACACAGGAAACAGCTATGACCATG-3’(SEQ ID NO.11)；

p-TZWF1-R2：5’-ACGACGTTGTAAAACGACGGCTATTTTTTATTTCTTTTAAGTGCCGCTGCTTCAACCATGCACTTTAGCTG-3’(SEQ ID NO.12)。

p as described in steps (a), (e), (i) and (m)_TDH3Can be obtained BY cloning a saccharomyces cerevisiae BY4741 genome, and the nucleotide sequence of the genome is shown as SEQ ID NO. 3.

The nucleotide sequence of TAL in the step (a) is shown in GenBank: KR 095308.1.

T described in steps (a), (e), (i) and (m)_TDH2Can be obtained BY cloning a saccharomyces cerevisiae BY4741 genome, and the nucleotide sequence of the genome is shown as SEQ ID NO. 5.

P as described in steps (b), (f), (j) and (n)_PGK1Can be obtained BY cloning a saccharomyces cerevisiae BY4741 genome, and the nucleotide sequence of the genome is shown as SEQ ID NO. 1.

ARO4 described in step (b)^K229LLysine at position 229 of ARO4 gene was mutated to leucine; wherein, the nucleotide sequence of ARO4 is shown in GenBank: NM-001178597.1.

T as described in steps (b) (f), (j) and (n)_ADH1Can be obtained BY cloning a saccharomyces cerevisiae BY4741 genome, and the nucleotide sequence of the genome is shown as SEQ ID NO. 6.

P as described in steps (c), (g), (k) and (o)_TEF1Can be obtained BY cloning a saccharomyces cerevisiae BY4741 genome, and the nucleotide sequence of the genome is shown as SEQ ID NO. 2.

ARO7 described in step (c)^G229SGlycine at position 229 of ARO7 gene was mutated to serine; wherein, the nucleotide sequence of ARO7 is shown in GenBank: NM-001184157.1.

T described in steps (c), (g), (k) and (o)_CYC1Can be obtained BY cloning a saccharomyces cerevisiae BY4741 genome, and the nucleotide sequence of the genome is shown as SEQ ID NO. 4.

The COMT1 in step (e) can be obtained by cloning with Arabidopsis cDNA as a template, and the nucleotide sequence of the nucleotide sequence is shown in GenBank: NM-124796.4.

The nucleotide sequence GenBank of PhaC is shown as WP-001175451.1 in the step (f).

The nucleotide sequence of HpaB in step (g) is shown in GenBank: WP-003137596.1.

The 4CL5 described in step (i) can be obtained by cloning using Arabidopsis cDNA as a template, and the nucleotide sequence is shown in GenBank: AY 376732.

The CAD described in step (j) can be obtained by cloning using Arabidopsis cDNA as a template, and the nucleotide sequence of the CAD is shown in GenBank: AY 302082.

The CCR in the step (k) can be obtained by cloning with an Arabidopsis cDNA as a template, and the nucleotide sequence of the CCR is shown in GenBank: NM-001332191.1.

BDH1 described in step (m)^{E221S/I222R/A223S}The glutamic acid at the 221 th site of BDH1 is mutated into serine, the isoleucine at the 222 th site is mutated into arginine, and the alanine at the 223 th site is mutated into serine; wherein, the nucleotide sequence of BDH1 is shown in GenBank: NM-001178202.2.

The TYR1 described in step (n) can be obtained from the Saccharomyces cerevisiae BY4741 genome clone, and the nucleotide sequence thereof is shown in GenBank: NM-001178514.1.

The ARO8 described in step (o) can be obtained from a Saccharomyces cerevisiae BY4741 genomic clone, and the nucleotide sequence thereof is shown in GenBank: NM-001181067.1.

The ZWF1 described in step (q) can be obtained from a Saccharomyces cerevisiae BY4741 genomic clone, and the nucleotide sequence thereof is shown in GenBank: NM-001183079.1.

The nucleotide sequence of the MET screening tag gene fragment in the step (III) is shown in GenBank: CP 006432.1.

The nucleotide sequence of the His screening tag gene fragment in the step (IV) is shown in GenBank: AAA 67141.1.

The nucleotide sequence of the Trp screening tag gene fragment in the step (IV) is shown in GenBank: NP-010290.3.

The conversion in the steps (A), (B) and (C) adopts a chemical conversion method or an electric conversion method for conversion, and the integrated fragment is integrated on the chromosome of the saccharomyces cerevisiae.

The screening in the step (A) is performed by adopting SD-URA culture medium.

The SD-URA culture medium comprises the following components: YNB medium 6.7g/L, URA (uracil) defective amino acid (100X)10mL/L, glucose 20g/L (20 g/L agar powder was added when preparing solid medium).

The screening in the step (B) is performed by adopting SD-LEU culture medium.

The SD-MET culture medium comprises the following components: YNB medium 6.7g/L, MET (methionine) deficient amino acid (100X)10mL/L, glucose 20g/L (20 g/L agar powder was added when preparing solid medium).

The screening in the step (C) is carried out by adopting SD-MET culture medium.

The SD-HIS culture medium comprises the following components: YNB medium 6.7g/L, HIS (histidine) deficient amino acid (100X)10mL/L, glucose 20g/L (20 g/L agar powder was added when preparing solid medium).

And (D) screening by adopting SD-HIS and SD-MET culture media.

The SD-TRP culture medium comprises the following components: YNB medium 6.7g/L, TRP (tryptophan) deficient amino acid (100X)10mL/L, glucose 20g/L (20 g/L agar powder was added when preparing solid medium).

The screening in the step (E) is carried out by adopting SD-TRP and SD-MET culture media.

The saccharomyces cerevisiae engineering bacteria for producing coniferyl alcohol with high yield is applied to producing coniferyl alcohol.

A method for producing coniferyl alcohol, inoculate said Saccharomyces cerevisiae engineering bacterium of high yield coniferyl alcohol into the fermentation culture medium to ferment and culture, get coniferyl alcohol; the method specifically comprises the following steps:

activating the saccharomyces cerevisiae engineering bacteria for high yield of coniferyl alcohol, inoculating the activated saccharomyces cerevisiae engineering bacteria into a fermentation culture medium for fermentation culture, and supplementing a supplemented culture medium when the dissolved oxygen value is lower than 40%, so that the content of glucose in the culture medium is maintained at 5g/L, thereby obtaining the coniferyl alcohol.

The activation is multi-stage activation; the method is realized by the following steps: inoculating the saccharomyces cerevisiae engineering bacteria for high yield of coniferyl alcohol into 5mL of SD-ULMHT culture medium, and culturing at 220-250 rpm and 30 ℃ until the OD value is 2-3; then transferring the bacterial liquid into a 100mL SD-ULMHT culture medium, and culturing the bacterial liquid at 220-250 rpm and 30 ℃ until the OD value is 2-3.

The composition of the SD-ULMHT culture medium is as follows: YNB medium 6.7 g/L; URA (uracil), LEU (leucine), MET (methionine), HIS (histidine), TRP (tryptophan) co-deficient amino acids (100X)10 mL/L; 20g/L glucose (20 g/L agar powder is added when preparing solid culture medium).

The URA (uracil), LEU (leucine), MET (methionine), HIS (histidine) and TRP (tryptophan) co-deficient amino acid (100X) is the deficient amino acid mother liquor (100X) without adding URA (uracil), LEU (leucine), MET (methionine), HIS (histidine) and TRP (tryptophan); the concrete components are as follows: adenine sulfate 0.25g, arginine 0.12g, aspartic acid 0.6g, glutamic acid 0.6g, lysine 0.18g, phenylalanine 0.3g, serine 2.25g, threonine 1.2g, tyrosine 0.18g, valine 0.9g, ddH2O to 57 mL.

The fermentation medium comprises the following components: 30g/L glucose, (NH)₄)₂SO₄ 15g/L，KH₂PO₄ 8g/L，MgSO₄ 3g/L，ZnSO₄·7H₂0.72g/L of O, 12mL/L of vitamin solution and 10mL/L of trace metal salt solution; wherein:

vitamin solution: 0.05g/L of vitamin H, 1g/L of calcium pantothenate, 1g/L of nicotinic acid, 25g/L of inositol, 1g/L of thiamine hydrochloride, 1g/L of pyridoxine hydrochloride and 0.2g/L of p-aminobenzoic acid.

Trace metal salt solution: EDTA (ethylene diamine tetraacetic acid) 15g/L, ZnSO₄·7H₂O 10.2g/L，MnCl₂·4H₂O 0.5g/L，CuSO₄ 0.5g/L，CoCl₂·6H₂O 0.86g/L，Na₂MoO₄·2H₂O 0.56g/L，CaCl₂·2H₂O3.84 g/L and FeSO₄·7H₂O 5.12g/L。

The feed medium comprises the following components: grapeGlucose 585g/L, KH₂PO₄ 9g/L，MgSO₄ 2.5g/L，K₂SO₄3.5g/L，Na₂SO₄0.28g/L, 12mL/L vitamin solution and 10mL/L trace metal salt solution; wherein:

Trace metal salt solution: EDTA 15g/L, ZnSO₄·7H₂O 10.2g/L，MnCl₂·4H₂O 0.5g/L，CuSO₄0.5g/L，CoCl₂·6H₂O 0.86g/L，Na₂MoO₄·2H₂O 0.56g/L，CaCl₂·2H₂O3.84 g/L and FeSO₄·7H₂O 5.12g/L。

The inoculation amount of the saccharomyces cerevisiae engineering bacteria for high yield of coniferyl alcohol is 0.1-15% (v/v); preferably 10% (v/v).

The conditions of the fermentation culture are as follows: the temperature is 25-35 ℃, the pH value is 3-7, the dissolved oxygen value is more than 30%, the stirring speed is 300-800 rpm, the ventilation volume is 3-20L/min, and the fermentation time is 0-96 h (excluding 0); preferably: the temperature is 30 ℃, the rotating speed is 300-800 rpm, the pH value is 5.5 (adjusted by ammonia water), the dissolved oxygen value is 30%, the ventilation volume is 3-20L/min, the initial concentration of glucose is 30g/L, and the fermentation time is 12-96 h.

The speed of supplementing the feed medium is 5 ml/h.

The fermentation time is preferably 72 h.

The method for producing coniferyl alcohol further comprises the step of further purifying the obtained coniferyl alcohol; the method specifically comprises the following steps: centrifugally separating fermentation liquor obtained after fermentation to obtain thalli and supernatant; then ultrasonic extracting the thalli with ethanol water solution, mixing the extracted ethanol solvent with the supernatant, and removing ethanol in the mixed solution to obtain a coniferyl alcohol crude extract; and adsorbing the crude extract of coniferyl alcohol by using macroporous adsorption resin, eluting and drying to obtain purified coniferyl alcohol.

The rotation speed of the cells is preferably 6000 rpm.

The concentration of the ethanol water solution is preferably 50 percent by volume.

The ultrasonic extraction conditions are as follows: ultrasonic extracting at 20-40 KHz for more than 1 hr.

The macroporous adsorption resin is preferably macroporous adsorption resin D101.

The elution is performed by adopting an ethanol-water solution with the volume fraction of 30%.

Compared with the prior art, the invention has the following advantages and effects: coniferyl alcohol is a chemical raw material with high economic value, and has wide application in paper production, drug synthesis and scientific research. However, the current mode of production of coniferyl alcohol is single and unstable, which results in higher price and prevents the wide application of coniferyl alcohol. The invention constructs a saccharomyces cerevisiae engineering bacterium capable of producing coniferyl alcohol, and can obtain a coniferyl alcohol product with the purity of more than 90 percent by fermenting the saccharomyces cerevisiae strain and simply purifying. Thereby providing an environment-friendly, stable, reliable, simple and convenient coniferyl alcohol source.

Drawings

FIG. 1 is a diagram of the biosynthetic pathway reconstituted in Saccharomyces cerevisiae.

FIG. 2 is an HPLC-MS total ion flow diagram and mass spectrum of yeast strain fermentation product; wherein a is an HPLC (high Performance liquid chromatography) spectrum of an ethyl acetate extract of YC1061 fermentation liquor; b is coniferyl alcohol mass spectrum; c is the HPLC chromatogram of coniferyl alcohol after macroporous resin purification; d is coniferyl alcohol DAD scanning spectrum.

FIG. 3 is a graph showing the variation of yield and production of coniferyl alcohol by fermentation of YC1061 in a fermenter.

Detailed Description

The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited thereto. Reagents, methods and apparatus used in the present invention are conventional in the art unless otherwise indicated. The test methods in the following examples, in which specific experimental conditions are not specified, are generally performed according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. Unless otherwise specified, reagents and starting materials for use in the present invention are commercially available.

The coniferyl alcohol biosynthesis pathway designed by the invention starts from glucose, and generates tyrosine through shikimic acid pathway in yeast; tyrosine then produces p-coumaric acid under the catalysis of TAL. Generating caffeic acid under the catalysis of HpaB and HpaC, and then catalyzing with COMT1 to generate ferulic acid; ferulic acid is further reacted under the action of 4CL5, CAD5 and CCR to form coniferyl alcohol. TYR1 and BDH1^{E221S/I222R/A223S}Can increase the supply of NADPH, thereby promoting the expression of CAD and CCR; in addition, ARO8 and TYR1 are the last two key enzymes in tyrosine biosynthesis, and overexpression of the two genes can also simultaneously improve the yield of tyrosine, thereby promoting the biosynthesis of coniferyl alcohol. The ZWF1 can improve the influence of Saccharomyces cerevisiae on coniferyl alcohol as toxic metabolic intermediate, and increase thallus density, thereby increasing coniferyl alcohol yield. Furthermore, ZWF1 can increase the supply of NADPH and promote the production of coniferyl alcohol. Further, ARO7^G229SAnd ARO4^K229LCan relieve negative feedback inhibition of high concentration tyrosine, thereby further promoting tyrosine generation and further improving coniferyl alcohol yield.

The strain used in the present invention was Saccharomyces cerevisiae BY4741, purchased from ATCC.

The cloning vector (p-Blunt), E.coli DH5 alpha competence and E.coli DH10B competence used in the present invention were purchased from Kyoto Kogyo gold. The single copy integrative vectors used in the present invention were all engineered from p-Blunt. Multicopy integrative vectors such as pCfB2797, pCfB2798, pCfB2989A and the like used in the present invention are commercially available from ddgene (http:// www.addgene.org). Cloning vector P-P_TDH3、p-TAL、p-T_TDH2、p-P_PGK1、p-T_TDH2、p-P_TEF1、p-ARO4^K229L、p-T_TDH2、p-ARO7^G229S、p-COMT1、p-PhaC、p-HpaB、p-T_ADH1、p-T_CYC1p-CAD, p-CCR, p-TYR1, p-ARO8, p-4CL5, p-ZWF1 and the like are all obtained by modifying p-Blunt, namely inserting the corresponding gene segments into a p-Blunt vector.

The formulation of the culture medium involved in the examples of the present invention is as follows:

(1) YPD medium: peptone 20g/L, yeast extract 10g/L, and glucose 20g/L (20 g/L agar powder was added to the solid YPD medium at the time of preparation).

(2) SD-URA culture medium: YNB medium 6.7g/L, URA (uracil) defective amino acid (100X)10mL/L, glucose 20g/L (20 g/L agar powder was added when preparing solid medium).

(3) SD-MET Medium: YNB medium 6.7g/L, MET (methionine) deficient amino acid (100X)10mL/L, glucose 20g/L (20 g/L agar powder was added when preparing solid medium).

(4) SD-LEU medium: YNB medium 6.7g/L, LEU (leucine) defective amino acid (100X)10mL/L, glucose 20g/L (20 g/L agar powder was added when preparing solid medium).

(5) SD-HIS medium: YNB medium 6.7g/L, HIS (histidine) deficient amino acid (100X)10mL/L, glucose 20g/L (20 g/L agar powder was added when preparing solid medium).

(6) SD-TRP medium: YNB medium 6.7g/L, TRP (tryptophan) deficient amino acid (100X)10mL/L, glucose 20g/L (20 g/L agar powder was added when preparing solid medium).

(7) SD-ULMHT medium: YNB medium 6.7 g/L; URA (uracil), LEU (leucine), MET (methionine), HIS (histidine), TRP (tryptophan) co-deficient amino acid (100X) (the deficient amino acid mother liquor (100X) is not added with URA (uracil), LEU (leucine), MET (methionine), HIS (histidine), TRP (tryptophan)) 10 mL/L; 20g/L glucose (20 g/L agar powder is added when preparing solid culture medium).

Defective amino acid mother liquor (100X): 0.25g of adenine sulfate, 0.12g of arginine, 0.6g of aspartic acid, 0.6g of glutamic acid, 0.12g of histidine, 0.36g of leucine, 0.18g of lysine, 0.12g of methionine, 0.3g of phenylalanine, 2.25g of serine, 1.2g of threonine, 0.24g of tryptophan, 0.18g of tyrosine, 0.9g of valine and 0.12g of uracil, wherein the volume is fixed to 57mL by ddH2O, and a defect amino acid mother liquor (100X) can be prepared without adding any amino acid according to needs. All the above starting materials were purchased from Sigma-Aldrich.

(9) Fermentation medium: 30g/L glucose, (NH)₄)₂SO₄ 15g/L，KH₂PO₄ 8g/L，MgSO₄ 3g/L，ZnSO₄·7H₂0.72g/L of O, 12mL/L of vitamin solution and 10mL/L of trace metal salt solution;

a supplemented medium: 585g/L glucose, KH₂PO₄ 9g/L，MgSO₄ 2.5g/L，K₂SO₄ 3.5g/L，Na₂SO₄0.28g/L, 12mL/L vitamin solution and 10mL/L trace metal salt solution; wherein, the first and the second end of the pipe are connected with each other,

EXAMPLE 1 cloning of Yeast endogenous Gene, promoter, terminator

1. Extraction of Yeast genome

(1) A single clone of Saccharomyces cerevisiae BY4741 (purchased from ATCC) is picked up and cultured in 5mL YPD medium for 20-24 h at 30 ℃, and then centrifuged for 5min at 4000rpm, and the collected strain is placed in a mortar.

(2) Quick-freezing with liquid nitrogen, grinding, volatilizing the liquid nitrogen, adding 1mL of DNAiso Reagent (Baori doctor technology Co., Ltd.), and mixing.

(3) The lysate was transferred to a centrifuge tube and centrifuged at 12,000rpm at 4 ℃ or room temperature for 10 min.

(4) The supernatant was transferred to a new centrifuge tube, 1/2 volumes of absolute ethanol were added, mixed well, centrifuged at 4000rpm at room temperature, and the supernatant was removed.

(5) Washing the precipitate with 75% (v/v) ethanol for 2 times, volatilizing the residual ethanol, adding 50 μ L ddH₂O dissolved and used as PCR cloning template.

2. Cloning of Yeast endogenous genes and expression elements

(1) The yeast genome obtained above was used as a template to clone the following 6 genes, 3 promoters and 3 terminators, respectively (see Table 1 for amplification primers):

6 genes: ZWF1 (primers: ZWF1-F and ZWF1-R), ARO4 (primers ARO4-F and ARO4-R), ARO7 (primers ARO7-F and ARO7-R), ARO8(ARO8-F and ARO8-R), TYR1(TYR1-F and TYR1-R) and BDH1(BDH1-F and BDH 1-R); wherein, the nucleotide sequence of the ZWF1 is shown in GenBank NM-001183079.1; the nucleotide sequence of ARO4 is shown in GenBank: NM-001178597.1; the nucleotide sequence of ARO7 is shown in GenBank: NM-001184157.1; the nucleotide sequence of ARO8 is shown in GenBank: NM-001181067.1; the nucleotide sequence of TYR1 is shown in GenBank: NM-001178514.1; the nucleotide sequence of BDH1 is shown in GenBank: NM-001178202.2;

3 promoters: p is_PGK1(P_PGK1-F and P_PGK1-R)、P_TEF1(P_TEF1-F and P_TEF1-R) and P_TDH3(P_TDH3-F and P_TDH3-R), the nucleotide sequences of which are respectively shown in SEQ ID NO. 1-3;

3 terminators: t is_CYC1(T_CYC1-F and T_CYC1-R)、T_TDH2(T_TDH2-F and T_TDH2-R) and T_ADH1(T_ADH1-F and T_ADH1-R), the nucleotide sequences of which are respectively shown in SEQ ID NO. 4-6.

And (3) PCR reaction system: phanta Max high Fidelity enzyme (Phanta Max Super-Fidelity DNA Polymerase) 0.5. mu.L, dNTP (10mM) 0.5. mu.L, 2 x Phanta Max Buffer 10. mu.L, upstream and downstream specific primers 0.5. mu.L each, ddH₂O7. mu.l, template 1. mu.l, total reaction 20. mu.l.

The PCR amplification reaction conditions are as follows: 1min at 95 ℃; 30 cycles of 30s at 95 ℃, 30s at 50-60 ℃ and 1-2min at 72 ℃; 7min at 72 ℃.

And (3) DNA fragment purification: after the PCR reaction, detecting the strip by agarose gel electrophoresis, and recovering the gel by using a common agarose gel DNA recovery kit (Tiangen Biochemical technology Co., Ltd.), wherein the specific operation process is described in the specification.

(2) DNA fragment ligation pEASY-Blunt vector

The DNA fragment was ligated to Blunt-ended pEASY-Blunt (all-grass Biotechnology Co., Ltd.) to transform the strain DH 5. alpha. according to the product instructions.

(3) After culturing for 14-16 h at 37 ℃, selecting a single colony for colony PCR.

And (3) PCR reaction system: easy Taq polymerase 0.2. mu.L, dNTPs (2.5mM) 0.8. mu.L, 10 × Easy Taq Buffer 1. mu.L, Universal primers M13F and M13-R (Table 1) (10. mu.M) 0.3. mu.L, M13R (Table 1) (10. mu.M) 0.3. mu.L, DMSO 1. mu.L, ddH₂O 6.4μL。

The PCR amplification conditions were: 5min at 95 ℃; 30 cycles of 95 ℃ for 30s, 55 ℃ for 30s and 72 ℃ for 1-3 min; 7min at 72 ℃.

After the reaction is finished, agarose gel electrophoresis is carried out to detect positive transformants, and the transformants are sent for sequencing.

3. The ARO4 is obtained by point mutation of ARO4, ARO7 and BDH1 genes respectively^K229L、ARO7^G229S、BDH1^{E221S/I222R/A223S}Gene fragment:

ARO4 and ARO7 are rate-limiting enzymes of the tyrosine biosynthetic pathway, respectively, and are inhibited by negative feedback of the reaction product tyrosine. This negative feedback inhibition can be released by mutating lysine to leucine at position 229 of ARO4 and glycine to serine at position 229 of ARO 7. BDH1 itself generates NAPH using NAD + as a substrate, and if glutamic acid at position 221 is mutated to serine, isoleucine at position 222 is mutated to arginine, and alanine at position 223 is mutated to serine, it can be made to change the catalytic substrate NAD + to NADP + and generate NADPH. NADPH can provide reducing power to the produced cytochrome P450 oxidase CCR, thereby increasing the yield, i.e.:

using p-Blunt-ARO4 plasmid as a template (p-Blunt-ARO4 plasmid was used to ligate the ARO4 gene to pEASY-Blunt vector as described above), primer ARO4 was used^K229L-F and ARO4^K229LIn situ site directed mutagenesis of R (Table 1) to give ARO4^K229LA gene fragment;

using p-Blunt-ARO7 plasmid (construction method as above: ARO7 gene was ligated to pEASY-Blunt vector) as template, primer ARO7^G229S-F and ARO7^G229SIn situ site directed mutagenesis of R (Table 1) to give ARO7^G229SA gene fragment;

BDH1 using p-Blunt-BDH1 plasmid (construction method: joining BDH1 gene to pEASY-Blunt vector as above) as template^{E221S/I222R/A223S}-F and BDH1^{E221S/I222R/A223S}In situ site directed mutagenesis of R (Table 1) to give BDH1^E221S ^/I222R/A223SA gene fragment;

the method comprises the following specific steps:

(1) the PCR reaction system comprises: phanta Max Super-Fidelity DNA Polymerase 0.5. mu.L, dNTP 0.5. mu.L, 2 x Phanta Max buffer 10. mu.L, forward and reverse primers 1. mu.L each, template 1. mu.L, total reaction system 50. mu.L.

(2) And (3) PCR reaction conditions: 3min at 95 ℃; 30s at 95 ℃, 30s at 62 ℃, 2min at 72 ℃ and 18 cycles; 7min at 72 ℃.

(3) Adding 0.5 mu L restriction enzyme Dpn I into the PCR product, and digesting for 1h at 37 ℃.

(4) And (3) transforming all the digestion systems into escherichia coli DH5 alpha, and performing inverted culture at 37 ℃ for 14-18 h.

(5) Positive transformants were selected by colony PCR and sequenced.

Example 2 cloning of plant-derived Gene

1. Extraction of plant tissue RNA

Total RNA in leaves of Arabidopsis thaliana (all Arabidopsis thaliana can be obtained by conventional commercial purchase) is extracted by using a rapid general RNA extraction kit (Beijing Huayue Biotechnology Co., Ltd.), and the specific operation process is shown in the specification.

2. Reverse transcription of mRNA into cDNA

The Hiscript II Reverse Transcriptase kit (Nanjing Novovisan Biotechnology Co., Ltd.) is adopted to carry out Reverse transcription of RNA into cDNA, and the specific operation is described in the kit instruction book.

3. Cloning of the Gene of interest

Taking arabidopsis cDNA as a template to clone COMT1, 4CL5, CAD and CCR genes, wherein primers are shown in a table 1, and the specific implementation method can refer to an example 1; wherein, the nucleotide sequence of COMT1 is shown in GenBank NM-124796.4; the nucleotide sequence of 4CL5 is shown in GenBank: AY 376732; the nucleotide sequence of CAD (i.e., CAD5) is shown in GenBank: AY 302082; the nucleotide sequence of CCR (i.e., CCR1) is found in GenBank: NM-001332191.1.

4. Genes HpaB, TAL and PhaC were synthesized by hong Xun Bio Inc; wherein the nucleotide sequence of HpaB is shown in GenBank as WP-003137596.1; TAL nucleotide sequence is shown in GenBank: KR 095308.1; PhaC nucleotide sequence is shown in GenBank: WP-001175451.1.

TABLE 1 primer sequences for cloned genes related to the present invention

Example 3 construction of Each expression Module and construction of the relevant plasmids

1. Construction of Gene expression modules Using overlapping PCRs

(1) The construction of each module is carried out by an overlapping PCR technology, each connected fragment in the module is cloned by PCR, an overlapping region of 40-50 bp is added, and the base annealing temperature of the overlapping region is between 60-70 ℃.

(2) The first round of reaction system: 0.5 μ L of Phanta Max Super-Fidelity DNA Polymerase, 0.5 μ L of dNTP (10mM), 10 μ L of 2 x Phanta Max Buffer, 1-5 μ L of DNA fragment, 0.5 μ L of each of upstream and downstream primers, and ddH₂O to 20 μ L; the primer sequences are shown in Table 2.

First round PCR reaction conditions: 3min at 95 ℃; 30s at 95 ℃, 30s at 60-70 ℃, 1-4 min at 72 ℃ and 15 cycles; 7min at 72 ℃.

(3) Second round PCR: taking 1 mu L of the first round PCR reaction solution as a second round PCR template, wherein the second round PCR system comprises: phanta Max Super-Fidelity DNA Polymerase 0.5. mu.L, dNTP (10mM) 0.5. mu.L, 2 x Phanta Max Buffer 10. mu.L, upstream and downstream primers 0.5. mu.L each (primers for ligation into complete DNA fragments upstream and downstream primers, see Table 2), ddH₂O7. mu.L, template 1. mu.L, total reaction 20. mu.L.

Second round PCR reaction conditions: 3min at 95 ℃; 30 cycles of 30 seconds at 95 ℃, 30 seconds at 50-60 ℃ and 1-4 min at 72 ℃; 5min at 72 ℃.

(4) The gel was recovered and ligated into pEASY-Blunt vector (all-gold Biotechnology Co., Ltd.) for sequencing.

The following modules and gene segments are constructed according to the steps:

(a) promoter P Using overlapping PCR_TDH3TAL gene and T terminator_TDH2Joined to give an expression Module P_TDH3-TAL-T_TDH2(ii) a Wherein the first round is performed with P-P_TDH3As a template, AP_TDH3-F and P_TDH3TAL-R is used as a primer, and a gene segment 1 is obtained by cloning; using P-TAL as template, P_TDH3-TAL-T_TDH2-F and P_TDH3-TAL-T_TDH2R is a primer, and a gene fragment 2 is obtained by cloning; with p-T_TDH2As a template, TAL-T_TDH2-F and AT_TDH2R is a primer, and a gene fragment 3 is obtained by cloning; the second round cloning primer is AP_TDH3-F and AT_TDH2-R。

(b) Promoter P Using overlapping PCR_PGK1Gene ARO4^K229LA terminator T_ADH1Joined to give an expression Module P_PGK1-ARO4^K229L-T_ADH1(ii) a Wherein the first round is performed with P-P_PGK1As a template, AP_PGK1-F and P_PGK1-ARO4^K229Lthe-R is a primer, and a gene fragment 4 is obtained by cloning; with p-ARO4^K229LAs a template, P_PGK1-ARO4^K229L-T_ADH1-F and P_PGK1-ARO4^K229L-T_ADH1the-R is a primer, and a gene fragment 5 is obtained by cloning; with p-T_ADH1As a template, ARO4^K229L-T_ADH1-F and AT_ADH1-R is a primer, and a gene fragment 6 is obtained by cloning; the second round cloning primer is AP_PGK1-F and AT_ADH1-R。

(c) Promoter P Using overlapping PCR_TEF1Gene ARO7^G229SA terminator T_CYC1Joined to give an expression Module P_TEF1-ARO7^G229S-T_CYC1(ii) a Wherein the first round is performed with P-P_TEF1As a template, AP_TEF1-F and P_TEF1-ARO7^G229SR is a primer, and a gene fragment 7 is obtained by cloning; with p-ARO7^G229SIs a diePlate, P_TEF1-ARO7^G229S-T_CYC1-F and P_TEF1-ARO7^G229S-T_CYC1R is a primer, and a gene fragment 8 is obtained by cloning; with p-T_CYC1As a template, ARO7^G229S-T_CYC1-F and AT_CYC1the-R is a primer, and a gene fragment 9 is obtained by cloning; the second round cloning primer is AP_TEF1-F and AT_CYC1-R。

(d) Using overlapping PCRs to put Module P_TDH3-TAL-T_TDH2、P_PGK1-ARO4^K229L-T_ADH1、P_TEF1-ARO7^G229S-T_CYC1Ligated with the primer AP_TDH3-F and AT_CYC1-R, obtaining the gene fragment TAA (P)_TDH3-TAL-T_TDH2→P_PGK1-ARO4^K229L-T_ADH1→P_TEF1-ARO7^G229S-T_CYC1)。

(e) Promoter P Using overlapping PCR_TDH3Gene COMT1, terminator T_TDH2Joined to give an expression Module P_TDH3-COMT1-T_TDH2(ii) a Wherein the first round is performed with P-P_TDH3As a template, BP_TDH3-F and P_TDH3-COMT1-R as primer, cloning to obtain gene fragment 10; using P-COMT1 as a template, P_TDH3-COMT1-T_TDH2-F and P_TDH3-COMT1-T_TDH2R is a primer, and a gene fragment 11 is obtained by cloning; with p-T_TDH2As a template, COMT1-T_TDH2-F and AT_TDH2R is a primer, and a gene fragment 12 is obtained by cloning; the second round cloning primer is BP_TDH3-F and AT_TDH2-R。

(f) Promoter P Using overlapping PCR_PGK1PhaC, gene, terminator T_ADH1Joined to give an expression Module P_PGK1-PhaC-T_ADH1(ii) a Wherein the first round is performed with P-P_PGK1As a template, AP_PGK1-F and P_PGK1PhaC-R is used as a primer, and a gene fragment 13 is obtained by cloning; using P-PhaC as template, P_PGK1-PhaC-T_ADH1-F and P_PGK1-PhaC-T_ADH1R is a primer, and a gene fragment 14 is obtained by cloning; with p-T_ADH1As a template, PhaC-T_ADH1-F and AT_ADH1R is a primer, and a gene fragment 15 is obtained by cloning; the second round cloning primer is AP_PGK1-F and AT_ADH1R。

(g) Promoter P Using overlapping PCR_TEF1HpaB gene and T terminator_CYC1Are linked to give an expression module P_TEF1-HpaB-T_CYC1(ii) a Wherein the first round is performed with P-P_TEF1As a template, AP_TEF1-F and P_TEF1The HpaB-R is used as a primer, and a gene fragment 16 is obtained by cloning; using P-HpaB as template, P_TEF1-HpaB-T_CYC1-F and P_TEF1-HpaB-T_CYC1R is a primer, and a gene fragment 17 is obtained by cloning; with p-T_CYC1As a template, HpaB-T_CYC1-F and AT_CYC1-R is a primer, and a gene fragment 18 is obtained by cloning; the second round cloning primer is AP_TEF1-F and AT_CYC1-R。

(h) Using overlapping PCRs to assign modules P_TDH3-COMT1-T_TDH2、P_PGK1-PhaC-T_ADH1、P_TEF1-HpaB-T_CYC1Ligated together, using the primer BP_TDH3-F and AT_CYC1-R, obtaining the gene fragment OCB (P)_TDH3-COMT1-T_TDH2→P_PGK1-PhaC-T_ADH1→P_TEF1-HpaB-T_CYC1)。

(i) Using overlapping PCR for the promoter P_TDH3Gene 4CL5, terminator T_TDH2Are linked to give an expression module P_TDH3-4CL5-T_TDH2(ii) a Wherein the first round is performed with P-P_TDH3As a template, CP_TDH3-F and P_TDH34CL5-R is used as a primer, and a gene fragment 19 is obtained by cloning; taking P-4CL5 as a template, P_TDH3-4CL5-T_TDH2-F and P_TDH3-4CL5-T_TDH2R is a primer, and a gene segment 20 is obtained by cloning; with p-T_TDH2As a template, 4CL5-T_TDH2-F and AT_TDH2R is a primer, and a gene fragment 21 is obtained by cloning; the second round cloning primer is CP_TDH3-F and AT_TDH2-R。

(j) Promoter P Using overlapping PCR_PGK1Gene CAD, terminator T_ADH1Joined to give an expression Module P_PGK1-CAD-T_ADH1(ii) a Wherein the first round is performed with P-P_PGK1As a template, AP_PGK1-F and P_PGK1The CAD-R is used as a primer, and the gene segment 22 is obtained by cloning; using P-CAD as a template, P_PGK1-CAD-T_ADH1-F and P_PGK1-CAD-T_ADH1R is a primer, and a gene fragment 23 is obtained by cloning; with p-T_ADH1As a template, CAD-T_ADH1-F and AT_ADH1R is a primer, and a gene fragment 24 is obtained by cloning; the second round cloning primer is AP_PGK1-F and AT_ADH1R。

(k) Promoter P Using overlapping PCR_TEF1CCR of gene, terminator T_CYC1Joined to give an expression Module P_TEF1-CCR-T_CYC1(ii) a Wherein the first round is performed with P-P_TEF1As a template, AP_TEF1-F and P_TEF1the-CCR-R is used as a primer, and a gene fragment 25 is obtained by cloning; using P-CCR as template, P_TEF1-CCR-T_CYC1-F and P_TEF1-CCR-T_CYC1R is a primer, and a gene fragment 26 is obtained by cloning; with p-T_CYC1As a template, CCR-T_CYC1-F and AT_CYC1R is a primer, and a gene fragment 27 is obtained by cloning; the second round cloning primer is AP_TEF1-F and AT_CYC1-R。

(l) Using overlapping PCRs to put Module P_TDH3-4CL5-T_TDH2、P_PGK1-CAD-T_ADH1、P_TEF1-CCR-T_CYC1Ligated with CP as primer_TDH3-F and AT_CYC1-R, obtaining the gene fragment FFA (P)_TDH3-4CL5-T_TDH2→P_PGK1-CAD-T_ADH1→P_TEF1-CCR-T_CYC1)。

(m) Using overlap PCR the promoter P_TDH3BDH1 gene^{E221S/I222R/A223S}A terminator T_TDH2Joined to give an expression Module P_TDH3-BDH1^{E221S/I222R/A223S}-T_TDH2(ii) a Wherein the first round is performed with P-P_TDH3As a template, DP_TDH3-F and P_TDH3-BDH1^{E221S/I222R/A223S}R is a primer, and a gene fragment 28 is obtained by cloning; with p-BDH1^{E221S/I222R/A223S}As a template, P_TDH3-BDH1^{E221S/I222R/A223S}-T_TDH2-F and P_TDH3-BDH1^{E221S/I222R/A223S}-T_TDH2R is a primer, and a gene fragment 29 is obtained by cloning; with p-T_TDH2As a template, BDH1^{E221S/I222R/A223S}-T_TDH2-F and AT_TDH2R is a primer, and a gene fragment 30 is obtained by cloning; the second round cloning primer was DP_TDH3-F and AT_TDH2-R。

(n) Using overlap PCR for the promoter P_PGK1Gene TYR1, terminator T_ADH1Joined to give an expression Module P_PGK1-TYR1-T_ADH1(ii) a Wherein the first round is performed with P-P_PGK1As a template, AP_PGK1-F and P_PGK1-TYR1-R as primer, cloning to obtain gene fragment 31; using P-TYR1 as template, P_PGK1-TYR1-T_ADH1-F and P_PGK1-TYR1-T_ADH1R is a primer, and a gene fragment 32 is obtained by cloning; with p-T_ADH1As a template, TYR1-T_ADH1-F and AT_ADH1R is a primer, and a gene fragment 33 is obtained by cloning; the second round cloning primer is AP_PGK1-F and AT_ADH1-R。

(o) use of overlapping PCR to convert promoter P_TEF1Gene ARO8, terminator T_CYC1Joined to give an expression Module P_TEF1-ARO8-T_CYC1(ii) a Wherein the first round is performed with P-P_TEF1As a template, AP_TEF1-F and P_TEF1ARO8-R is used as a primer, and a gene fragment 34 is obtained by cloning; using P-ARO8 as template, P_TEF1-ARO8-T_CYC1-F and P_TEF1-ARO8-T_CYC1the-R is a primer, and a gene fragment 35 is obtained by cloning; with p-T_CYC1As a template, ARO8-T_CYC1-F and BT_CYC1R is a primer, and a gene fragment 36 is obtained by cloning; the second round cloning primer is AP_TEF1-F and BT_CYC1R。

(P) Using overlapping PCRs with Module P_TDH3-BDH1^{E221S/I222R/A223S}-T_TDH2、P_PGK1-TYR1-T_ADH1、P_TEF1-ARO8-T_CYC1Ligated together, using DP as primer_TDH3-F and AT_CYC1-R, obtaining the gene fragment BTA (P)_TDH3-BDH1^E221S ^/I222R/A223S-T_TDH2→P_PGK1-TYR1-T_ADH1→P_TEF1-ARO8-T_CYC1)。

(q) Using overlap PCR for the promoter P_TEF1Gene ZWF1 and terminator T_CYC1Are linked to give an expression module P_TEF1-ZWF1-T_CYC1(ii) a Wherein the first round is performed with P-P_TEF1As a template, EP_TEF1-F and EP_TEF1R is a primer, and a gene fragment 37 is obtained by cloning; using P-ZWF1 as a template, P_TEF1-ZWF1-T_CYC1-F and P_TEF1-ZWF1-T_CYC1R is a primer, and a gene fragment 38 is obtained by cloning; with p-T_CYC1As a template, ZWF1-T_CYC1-F and ET_CYC1R is a primer, and a gene fragment 39 is obtained by cloning; two rounds of cloning primers are EP_TEF1-F and ET_CYC1-R。

TABLE 2 primer sequences for construction of Gene expression modules

2. The DNA fragment is ligated to the vector.

(a) Construction of vector pCfB2797 TAA: vector pCfB2797 (Addge) was digested with restriction enzymes HindIII and NheI, and the gene fragment TAA obtained in the above method was ligated thereto using a homologous recombinase (Cloneexpress II One Step Cloning Kit, Vazyme); the connecting body is: 100ng of linearized pCfB2797 vector, 120ng of purified gene fragment TAA, 2 ul of Exnase II and 4 ul of 5 XCE Buffer; reaction conditions are as follows: 30min at 37 ℃. And (3) taking the connected vector for transforming the competence of escherichia coli DH10B, culturing for 14-16 h at 37 ℃, and screening and extracting plasmid sequencing through colony PCR to obtain the connected vector pCfB2797 TAA.

(b) Construction of vector pCfB2798 OCB: the vector pCfB2798 (Addge) was digested with restriction enzymes HindIII and NheI, and the gene fragment OCB obtained in the above-described manner was ligated thereto using a homologous recombinase (Clonexpress II One Step Cloning Kit, Vazyme). The connecting system is as follows: 100ng of linearized pCfB2798 vector, 120ng of purified gene fragment OCB, 2 ul of Exnase IIP and 4 ul of 5 × CE Buffer. The reaction conditions are as follows: 30min at 37 ℃; and (3) taking the connected vector for transforming the competence of escherichia coli DH10B, culturing for 14-16 h at 37 ℃, and screening and extracting plasmid sequencing through colony PCR to obtain the connected vector pCfB2797 OCB.

(c) Construction of the vector pCfB2989 m: the MET selection tag gene fragment (GenBank: CP006432.1) was cloned using Saccharomyces cerevisiae BY4741 genome as template and MET-F and MET-R as primers (Table 2). Taking a pCfB2989 vector (Addgene), carrying out enzyme digestion for 30min by using a restriction enzyme Pst1, and purifying the linearized vector; the MET screening tag gene fragment obtained in the above method was ligated thereto using a homologous recombinase (Cloneexpress II One Step Cloning Kit, Vazyme); the connecting system is as follows: 100ng of a linearized pCfB2989 vector, 120ng of a purified MET screening label gene fragment, 2 ul of Exnase II and 4 ul of 5 × CE Buffer; reaction conditions are as follows: 30min at 37 ℃; and (3) taking the connected vector for transforming the competence of escherichia coli DH10B, culturing for 14-16 h at 37 ℃, and screening and extracting plasmid sequencing by colony PCR to obtain the vector pCfB2989 m.

(d) Construction of the vector pCfB2989 mFFA: the vector pCfB2989m was digested with restriction enzymes HindIII and NheI, and the gene fragment FFA obtained in the above-mentioned manner was ligated thereto using a homologous recombinase (Cloneexpress II One Step Cloning Kit, Vazyme). The connecting system is as follows: 100ng of linearized pCfB-2989m vector, 120ng of purified gene fragment FFA, 2 ul of Exnase II and 4 ul of 5 × CE Buffer; the reaction conditions are as follows: 30min at 37 ℃. And (3) taking the connected vector for transforming the competence of escherichia coli DH10B, culturing for 14-16 h at 37 ℃, and obtaining the connected vector pCfB2989mFFA by colony PCR screening and extracted plasmid sequencing.

(e) Construction of a single copy integration vector p-YJZ-His: cloning BY using a saccharomyces cerevisiae BY4741 genome as a template and using primers His-F and His-R (the primers are shown in a table 2) to obtain a His screening tag gene fragment (GenBank: AAA 67141.1); after the gene fragment is purified, a Blunt-end pEASY-Blunt (all-type gold biotechnology, Inc.) is connected, a DH5 alpha strain is transformed, specific operation is shown in a product instruction, and a single-copy integration vector p-YJZ-His is obtained.

(f) Constructing a single copy integration vector p-YJZ-Trp: cloning was performed using the Saccharomyces cerevisiae BY4741 genome as a template and the primers Trp-F and Trp-R (see Table 2 for primers), to obtain a Trp selection tag gene fragment (GenBank: NP-010290.3). After the gene fragment is purified, a Blunt-end pEASY-Blunt (all-type gold biotechnology, Inc.) is connected, a DH5 alpha strain is transformed, specific operation is shown in a product instruction, and a single-copy integration vector p-YJZ-Trp is obtained.

(g) Constructing a single copy integration vector p-HBTA: digesting the plasmid p-YJZ-His with restriction enzyme AvaI for 30min, and purifying the linearized vector; the gene fragment BTA obtained in the above method was ligated with a homologous recombinase (Cloneexpress II One Step Cloning Kit, Vazyme); the connecting system is as follows: 100ng of linearized P-YJZ-His vector, 120ng of purified gene fragment BTA, 2 ul of Exnase II and 4 ul of 5 × CE Buffer; reaction conditions are as follows: 30min at 37 ℃; and (3) taking the connected vector for transforming the competence of escherichia coli DH10B, culturing for 14-16 h at 37 ℃, and screening and extracting plasmid sequencing through colony PCR to obtain the connected vector p-HBTA.

(h) Constructing a single-copy integrated vector p-TZWF 1: taking the plasmid p-YJZ-Trp, digesting for 30min by using restriction enzyme AvaI and restriction enzyme AvaI, and purifying the linearized vector; the expression module P obtained in the above method_TEF1-ZWF1-T_CYC1Ligation was performed using homologous recombinase (Clonexpress II One Step Cloning Kit, Vazyme); the connecting system is as follows: linearized P-YJZ-Trp vector 100ng, purified expression module P_TEF1-ZWF1-T _CYC1120 ng、Ex2. mu.l of nase II and 4. mu.l of 5 XCE Buffer; reaction conditions are as follows: 30min at 37 ℃. And (3) taking the connected vector for transforming the competence of escherichia coli DH10B, culturing for 14-16 h at 37 ℃, and obtaining the connected vector p-TZWF1 by colony PCR screening and extracted plasmid sequencing.

Example 4 construction of engineered Saccharomyces cerevisiae.

1. And (3) constructing a saccharomyces cerevisiae engineering bacterium YT 1003.

(a) Plasmid pCfB2797TAA was digested with restriction enzyme NotI. Enzyme digestion system: 3-5 μ g of vector pCfB2797TAA, 1-2 μ L of Not I restriction enzyme, 5 μ L of 10 x Fast Digest Buffer, adding water to 50 μ L, and performing enzyme digestion at 37 ℃ for 1-2 h. Detecting the enzyme digestion effect by running electrophoresis, and purifying.

(b) Yeast Transformation was carried out using Zymo Research FROzen-EZ Yeast Transformation II kit (Tokyo diligent Bio-Tech Co., Ltd.), Saccharomyces cerevisiae BY4741 was transformed with the linearized vector pCfB2797TAA, and the linearized vector was integrated into the Ty2 locus of the Yeast chromosome, and cultured at 30 ℃ for 4-5 days (days) according to the product manual.

(c) Positive transformants were screened using the auxotrophic medium SD-URA and colony PCR.

(d) And inoculating the positive transformant into 4ml of SD-URA culture medium, and culturing for 2 days at 30 ℃ and 220rpm to obtain Saccharomyces cerevisiae YT1003 bacterial liquid.

(e) Mu.l of the YT1003 bacterial liquid is taken, 500. mu.l of sterilized 50% glycerol (v/v) is added, and the mixture is stored at-80 ℃.

2. And (3) constructing a saccharomyces cerevisiae engineering bacterium YC 1031.

(a) Plasmid pCfB2798OCB was digested with restriction enzyme NotI. Enzyme digestion system: 3-5 μ g of vector pCfB2797OCB, 1-2 μ L of Not I restriction enzyme, 5 μ L of 10 x Fast Digest Buffer, adding water to 50 μ L, and performing enzyme digestion at 37 ℃ for 1-2 h. Detecting the enzyme digestion effect by running electrophoresis, and purifying.

(b) Yeast Transformation was carried out using Zymo Research FROzen-EZ Yeast Transformation II KitTM Yeast Transformation kit (Shanghai diligent kang Biotech Co., Ltd.), Saccharomyces cerevisiae YT1003 was transformed with linearized vector pCfB2798OCB, and the linearized vector was integrated into Ty4 locus of Yeast chromosome, and cultured at 30 ℃ for 4-5 days (days) according to the product manual.

(c) Positive transformants were screened using the auxotrophic medium SD-LEU and colony PCR.

(d) And inoculating the positive transformant into 4ml of SD-LEU culture medium, and culturing for 2d at 30 ℃ and 220rpm to obtain Saccharomyces cerevisiae YC1031 bacterial liquid.

(e) Mu.l of the YC1031 bacteria solution was taken, 500. mu.l of sterilized 50% glycerol (v/v) was added thereto, and the mixture was stored at-80 ℃.

3. And (3) construction of saccharomyces cerevisiae engineering bacteria YC 1048.

(a) Plasmid pCfB2989m FFA was digested with the restriction enzyme NotI. Enzyme digestion system: 3-5 mu g of vector pCfB29 2989m FFA, 1-2 mu L of Not I restriction enzyme, 5 mu L of 10 x Fast Digest Buffer, and water is supplemented to 50 mu L, and enzyme digestion is carried out at 37 ℃ for 1-2 h. Detecting the enzyme digestion effect by running electrophoresis, and purifying.

(b) Yeast Transformation was carried out using Zymo Research FROZEN-EZ Yeast Transformation II kit (Tokyo diligent Bio-Tech Co., Ltd.), Saccharomyces cerevisiae YC1031 was transformed with the linearized vector pCfB2989m4CC, and the linearized vector was integrated into the Ty1 site of the Yeast chromosome, and cultured at 30 ℃ for 4-5 days (days) according to the product manual.

(c) Positive transformants were screened using the auxotrophic medium SD-MET and colony PCR.

(d) And inoculating the positive transformant into 4ml of SD-MET culture medium, and culturing for 2d at 30 ℃ and 220rpm to obtain saccharomyces cerevisiae YC1048 bacterial liquid.

(e) 500. mu.l of the YC1048 bacterial solution was taken, and 500. mu.l of sterilized 50% glycerol (v/v) was added thereto, followed by storage at-80 ℃.

4. And (3) construction of saccharomyces cerevisiae engineering bacteria YC 1053.

(a) The plasmid p-HBTA was taken and subjected to PCR amplification using primers p-HBTA-F and p-HBTA-R (primers shown in Table 2) to obtain a gene integration fragment HBTA.

(b) Yeast Transformation was carried out using Zymo Research FROZEN-EZ Yeast Transformation II KitTM Yeast Transformation kit (Shanghai diligent kang Biotech Co., Ltd.), Saccharomyces cerevisiae YC1048 was transformed with the linearized vector gene integration fragment HBTA, and the linearized vector was integrated into the Yeast chromosome at YKL211C site, and cultured at 30 ℃ for 4-5 days (days) according to the product instructions.

(c) Positive transformants were screened using the auxotrophic medium SD-HIS and colony PCR.

(d) And inoculating the positive transformant into 4ml of SD-MET culture medium, and culturing for 2d at 30 ℃ and 220rpm to obtain Saccharomyces cerevisiae YC1053 bacterial liquid.

(e) 500. mu.l of the YC1053 bacterial solution was added with 500. mu.l of sterilized 50% glycerol (v/v), and the mixture was stored at-80 ℃.

5. And (3) construction of a saccharomyces cerevisiae engineering bacterium YC 1061.

(a) Taking the plasmid p-TZWF1, and respectively carrying out PCR (primer sequences are shown in Table 2) amplification by using primers p-TZWF1-F1, p-TZWF1-R1, p-TZWF1-F2 and p-TZWF1-R2 to obtain gene integration fragments TZWF1-PDC5 and TZWF1-ARO 10.

(b) Yeast Transformation was carried out using Zymo Research FROZEN-EZ Yeast Transformation II KitTM Yeast Transformation kit (Shanghai diligent kang Biotech Co., Ltd.), Saccharomyces cerevisiae YC1053 was transformed with linearized vector gene integration fragments TZWF1-PDC5 and TZWF1-ARO10, and the linearized vector was simultaneously integrated into the Yeast chromosome at the YLR134W and YDR380W sites, and cultured at 30 ℃ for 4-5 days (days) according to the product instructions.

(c) Positive transformants were screened using the auxotrophic medium SD-TRP and colony PCR.

(d) And inoculating the positive transformant into 4ml of SD-MET culture medium, and culturing for 2d at 30 ℃ and 220rpm to obtain saccharomyces cerevisiae YC1061 bacterial liquid.

(e) 500. mu.l of the YC1061 bacterial liquid was taken, and 500. mu.l of sterilized 50% glycerol (v/v) was added thereto, followed by storage at-80 ℃.

EXAMPLE 5 production of coniferyl alcohol by Strain YC1061

1. Fermentation of strain YC1061

(1) Inoculating a single colony of the strain YC1061 into 5mL of SD-ULMHT culture medium, and culturing at 30 ℃ and 220rpm until the OD value is 2-3.

(2) Inoculating the bacterial liquid into 3 bottles of 100mL SD-ULMHT culture medium, inoculating 5mL of the bacterial liquid into each bottle, and culturing the bacterial liquid at 30 ℃ and 220rpm until the OD value is 2-3.

(3) The 3 bottles of 100mL of the bacterial suspension were combined, and both were inoculated into a fermenter (5L in volume) containing 3L of fermentation medium to perform fermentation. The fermentation conditions were: the temperature is 30 ℃, the rotating speed is 300-800 rpm, the pH value is 5.5 (adjusted by ammonia water), the dissolved oxygen value is more than 30%, the ventilation volume is 3-20L/min, and the initial concentration of glucose is 30 g/L; when the dissolved oxygen value is lower than 40%, starting a feeding system to feed (adding a feeding culture medium or feeding a 500g/L glucose solution), wherein the feeding rate is 5ml/h, so that the content of glucose in the culture medium is maintained at 5 g/L; the fermentation time was 96 h.

3. Extraction and detection of fermentation products

(1) And adding the fermentation liquor into ethyl acetate with the same volume for ultrasonic extraction for 60min, standing for 24h, taking 25mL of organic layer, concentrating under reduced pressure until the organic layer is dry, dissolving the organic layer by using 1mL of methanol, then passing through a membrane, and performing LC-MS detection by using an Agilent liquid mass spectrometer 1260 and 6130. The detection method comprises the following steps: carrying out analysis by using a YMC-Pack Pro C18 RS chromatographic column; the sample volume is 1 mu L; the column temperature is 28 ℃; the detection wavelength was 290 nm.

Mobile phase conditions: a (acetonitrile), B (0.1% trifluoroacetic acid in water)

Time (min)	A(％)	B(％)
			0	15	85
6	15	85
			15	20	80
18	25	75
			22	25	75
26	40	60
			26.1	15	85
29.1	15	85

The ion detection was set to negative ion selection mode and m/z 179 ions were detected.

As a result: the biosynthetic pathway reconstructed in Saccharomyces cerevisiae is shown in FIG. 1; the LC-MS ultraviolet absorption diagram and the ion flow diagram of the yeast strain fermentation product are shown in figure 2; YC1061 yields are shown in FIG. 3.

Example 6 purification of coniferyl alcohol

(1) 3 liters of the fermentation broth of YC1061 cultured in example 5 was taken and centrifuged at 6000rpm to obtain cells and a supernatant fraction.

(2) The thallus fraction is extracted with 50% (v/v) ethanol solution under ultrasound (20KHZ-40KHZ) for 1 hr, centrifuged, and the ethanol extract is retained.

(3) And (2) mixing the thalli and the supernatant obtained in the step (1), removing ethanol in liquid by using a rotary evaporator, and adjusting the pH to 7 by using 1M sodium hydroxide to obtain a coniferyl alcohol crude extract.

(4) Enabling the coniferyl alcohol crude extract obtained in the step (3) to pass through macroporous adsorption resin D101 (Guangzhou Haoyao Biotechnology Co., Ltd.) with the column volume of 3L, and enabling the macroporous resin to fully adsorb coniferyl alcohol in the crude extract; followed by gradient elution using water, 15% (volume fraction) ethanol-water, 30% (volume fraction) ethanol-water and 100% ethanol in that order.

(5) And (4) enriching the 30% ethanol-water part eluted in the step (4), and evaporating water and ethanol by using a rotary evaporator to obtain a coniferyl alcohol crude product with the purity of over 90%.

The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.

Sequence listing

<110> river-south university

<120> saccharomyces cerevisiae engineering bacteria for high yield of coniferyl alcohol and construction and application thereof

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catctgcata ataggcattt gcaagaatta ctcgtgagta aggaaagagt gaggaactat 60

cgcatacctg catttaaaga tgccgatttg ggcgcgaatc ctttattttg gcttcaccct 120

catactatta tcagggccag aaaaaggaag tgtttccctc cttcttgaat tgatgttacc 180

ctcataaagc acgtggcctc ttatcgagaa agaaattacc gtcgctcgtg atttgtttgc 240

aaaaagaaca aaactgaaaa aacccagaca cgctcgactt cctgtcttcc tattgattgc 300

agcttccaat ttcgtcacac aacaaggtcc tagcgacggc tcacaggttt tgtaacaagc 360

aatcgaaggt tctggaatgg cgggaaaggg tttagtacca catgctatga tgcccactgt 420

gatctccaga gcaaagttcg ttcgatcgta ctgttactct ctctctttca aacagaattg 480

tccgaatcgt gtgacaacaa cagcctgttc tcacacactc ttttcttcta accaaggggg 540

tggtttagtt tagtagaacc tcgtgaaact tacatttaca tatatataaa cttgcataaa 600

ttggtcaatg caagaaatac atatttggtc ttttctaatt cgtagttttt caagttctta 660

gatgctttct ttttctcttt tttacagatc atcaaggaag taattatcta ctttttacaa 720

caaatataaa acaa 734

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PTEF1

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cacacaccat agcttcaaaa tgtttctact ccttttttac tcttccagat tttctcggac 60

tccgcgcatc gccgtaccac ttcaaaacac ccaagcacag catactaaat ttcccctctt 120

tcttcctcta gggtgtcgtt aattacccgt actaaaggtt tggaaaagaa aaaagagacc 180

gcctcgtttc tttttcttcg tcgaaaaagg caataaaaat ttttatcacg tttctttttc 240

ttgaaaattt ttttttttga tttttttctc tttcgatgac ctcccattga tatttaagtt 300

aataaacggt cttcaatttc tcaagtttca gtttcatttt tcttgttcta ttacaacttt 360

ttttacttct tgctcattag aaagaaagca tagcaatcta atctaagttt taattacaa 419

<210> 3

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<213> Artificial Sequence (Artificial Sequence)

<220>

<223> PTDH3

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tcgagtttat cattatcaat actgccattt caaagaatac gtaaataatt aatagtagtg 60

attttcctaa ctttatttag tcaaaaaatt agccttttaa ttctgctgta acccgtacat 120

gcccaaaata gggggcgggt tacacagaat atataacatc gtaggtgtct gggtgaacag 180

tttattcctg gcatccacta aatataatgg agcccgcttt ttaagctggc atccagaaaa 240

aaaaagaatc ccagcaccaa aatattgttt tcttcaccaa ccatcagttc ataggtccat 300

tctcttagcg caactacaga gaacaggggc acaaacaggc aaaaaacggg cacaacctca 360

atggagtgat gcaacctgcc tggagtaaat gatgacacaa ggcaattgac ccacgcatgt 420

atctatctca ttttcttaca ccttctatta ccttctgctc tctctgattt ggaaaaagct 480

gaaaaaaaag gttgaaacca gttccctgaa attattcccc tacttgacta ataagtatat 540

aaagacggta ggtattgatt gtaattctgt aaatctattt cttaaacttc ttaaattcta 600

cttttatagt tagtcttttt tttagtttta aaacaccaag aacttagttt cgaataaaca 660

cacataaaca aacaaa 676

<210> 4

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<213> Artificial Sequence (Artificial Sequence)

<220>

<223> TCYC1

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atccgctcta accgaaaagg aaggagttag acaacctgaa gtctaggtcc ctatttattt 60

ttttatagtt atgttagtat taagaacgtt atttatattt caaatttttc ttttttttct 120

gtacagacgc gtgtacgcat gtaacattat actgaaaacc ttgcttgaga aggttttggg 180

acgctcgaag 190

<210> 5

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<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

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atttaactcc ttaagttact ttaatgattt agtttttatt attaataatt catgctcatg 60

acatctcata tacacgttta taaaacttaa atagattgaa aatgtattaa agattcctca 120

gggattcgat ttttttggaa gtttttgttt ttttttcctt gagatgctgt agtatttggg 180

aacaattata caatcgaaag atatatgctt acattcgacc gttttagccg tgatcattat 240

cctatagtaa cataacctga agcataactg acactactat catcaatact tgtcacatga 300

gaactctgtg aataattagg ccactgaaat ttgatgcctg aaggaccggc atcacggatt 360

ttcgataaag cacttagtat cacactaatt ggcttttcgc 400

<210> 6

<211> 195

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> TADH1

<400> 6

gtagatacgt tgttgacact tctaaataag cgaatttctt atgatttatg atttttatta 60

ttaaataagt tataaaaaaa ataagtgtat acaaatttta aagtgactct taggttttaa 120

aacgaaaatt cttattcttg agtaactctt tcctgtaggt caggttgctt tctcaggtat 180

agcatgaggt cgctc 195

<210> 7

<211> 81

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<213> Artificial Sequence (Artificial Sequence)

<220>

<223> p-HBTA-F

<400> 7

tttcttagca tttttgacga aatttgctat tttgttagag tcttttacac ataacaattt 60

cacacaggaa acagctatga c 81

<210> 8

<211> 71

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> p-HBTA-R

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atgtctgtta ttaatttcac aggtagttct ggtccattgg tgaaagtttg tcacgacgtt 60

gtaaaacgac g 71

<210> 9

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> p-TZWF1-F1

<400> 9

tgtctgaaat aaccttaggt aaatatttat ttgaaagatt gagccaagtc acacaggaaa 60

cagctatgac catg 74

<210> 10

<211> 71

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> p-TZWF1-R1

<400> 10

tgtttagcgt tagtagcggc agtcaattga gcttgtttaa ccaagttttg acgacgttgt 60

aaaacgacgg c 71

<210> 11

<211> 74

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> p-TZWF1-F2

<400> 11

ggcacctgtt acaattgaaa agttcgtaaa tcaagaagaa cgacaccttg acacaggaaa 60

cagctatgac catg 74

<210> 12

<211> 71

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>

<223> p-TZWF1-R2

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acgacgttgt aaaacgacgg ctatttttta tttcttttaa gtgccgctgc ttcaaccatg 60

cactttagct g 71

Claims

1. The saccharomyces cerevisiae engineering bacteria for high yield of coniferyl alcohol is characterized by comprising the following components in percentage by weight: uses saccharomyces cerevisiae as an original strain to perform overexpressionZWF1、ARO4 ^K229L、ARO7^G229S、ARO8、TYR1、BDH1 ^{E221S/I222R/A223S}COMT1, 4CL5, CAD, CCR, HpaB, TAL and PhaC genes; wherein:

saidARO4 ^K229LLysine at position 229 of ARO4 gene was mutated to leucine;

the ARO7^G229SGlycine at the 229 th site of the ARO7 gene is mutated to serine;

saidBDH1 ^{E221S/I222R/A223S}Is composed ofBDH1The 221 th glutamic acid of the gene is mutated into serine, the 222 th isoleucine is mutated into arginine, and the 223 th alanine is mutated into serine;

the nucleotide sequence of the ZWF1 is shown as GenBank NM-001183079.1;

the nucleotide sequence of the ARO4 is shown as GenBank NM-001178597.1;

the nucleotide sequence of the ARO7 is shown as GenBank NM-001184157.1;

the nucleotide sequence of the ARO8 is shown as GenBank NM-001181067.1;

the nucleotide sequence of the TYR1 is shown as GenBank NM-001178514.1;

the nucleotide sequence of BDH1 is shown as GenBank NM-001178202.2;

the nucleotide sequence of COMT1 is shown in GenBank: NM-124796.4;

the nucleotide sequence of the 4CL5 is shown in GenBank: AY 376732;

the nucleotide sequence of the CAD is shown in GenBank: AY 302082;

the nucleotide sequence of the CCR is shown in GenBank NM-001332191.1;

the nucleotide sequence of HpaB is shown in GenBank: WP-003137596.1;

the nucleotide sequence of TAL is shown in GenBank: KR 095308.1;

the nucleotide sequence of PhaC is shown in GenBank: WP-001175451.1.

2. The saccharomyces cerevisiae engineering bacteria for high yield of coniferyl alcohol of claim 1, characterized in that: the saccharomyces cerevisiae is saccharomyces cerevisiae BY 4741.

3. The method for constructing the saccharomyces cerevisiae engineering bacteria for high yield of coniferyl alcohol in claim 1, which is characterized by comprising the following steps:

(k) Will P_TEF1CCR and T_CYC1Sequentially connecting to construct expression module P_TEF1- CCR-T_CYC1；

(l) Will express the module P_TDH3-4CL5-T_TDH2、P_PGK1-CAD-T_ADH1And P_TEF1- CCR-T_CYC1Connecting to obtain a gene fragment FFA;

(m) adding P_TDH3、BDH1^{E221S/I222R/A223S}And T_TDH2Sequentially connecting to construct expression module P_TDH3-BDH1^{E221S/I222R/A223S}-T_TDH2；

(n) adding P_PGK1TYR1 and T_ADH1Sequentially connecting to construct expression module P_PGK1- TYR1-T_ADH1；

(o) adding P_TEF1ARO8 and T_CYC1Sequentially connecting to construct expression module P_TEF1- ARO8-T_CYC1；

(P) expression Module P_TDH3-BDH1^{E221S/I222R/A223S}-T_TDH2、P_PGK1- TYR1-T_ADH1And P_TEF1- ARO8-T_CYC1Connecting to obtain a gene segment BTA;

(q) adding P_TEF1ZWF1 and T_CYC1Sequentially connecting to construct expression module P_TEF1- ZWF1-T_CYC1；

(2) Construction of vectors

(V) connecting the Trp screening tag gene fragment to a pEASY-Blunt vector to obtain a plasmid p-YJZ-Trp, and then utilizing a restriction enzyme AvaI to enzyme-cut the plasmid p-YJZ-Trp to obtain a linearized p-YJZ-Trp vector; then, the expression module P obtained in the step (q) is used_TEF1- ZWF1-T_CYC1Connecting to a linearized p-YJZ-Trp vector to obtain a plasmid p-TZWF 1;

(3) construction of the Strain YC1061

(A) After the plasmid pCfB2797TAA is linearized BY using a restriction enzyme NotI, saccharomyces cerevisiae BY4741 is transformed, and saccharomyces cerevisiae YT1003 is obtained BY screening;

(B) after the plasmid pCfB2798OCB is linearized by a restriction enzyme NotI, saccharomyces cerevisiae YT1003 is transformed, and saccharomyces cerevisiae YC1031 is obtained by screening;

(C) linearizing the plasmid pCfB2989mFFA by using a restriction enzyme NotI, then transforming saccharomyces cerevisiae YC1031, and screening to obtain saccharomyces cerevisiae YC 1048;

(D) carrying out PCR amplification on the plasmid p-HBTA obtained in the step (IV) by using primers p-HBTA-F and p-HBTA-R to obtain a gene integration fragment HBTA; then, the gene integration fragment HBTA is transformed into saccharomyces cerevisiae YC1048 to obtain saccharomyces cerevisiae YC 1053; wherein the nucleotide sequences of the primers p-HBTA-F and p-HBTA-R are respectively shown as SEQ ID NO. 7-8;

(E) performing PCR amplification on the plasmid p-TZWF1 obtained in the step (V) by using primers p-TZWF1-F1, p-TZWF1-R1, p-TZWF1-F2 and p-TZWF1-R2 respectively to obtain gene integration fragments TZWF1-PDC5 and TZWF1-ARO 10; then simultaneously converting the strain into saccharomyces cerevisiae YC1053 to obtain saccharomyces cerevisiae YC1061, namely the saccharomyces cerevisiae engineering strain for high yield of coniferyl alcohol; wherein the nucleotide sequences of the primers p-TZWF1-F1, p-TZWF1-R1, p-TZWF1-F2 and p-TZWF1-R2 are respectively shown as SEQ ID NO. 9-12.

4. The construction method of the saccharomyces cerevisiae engineering bacteria for high yield of coniferyl alcohol according to claim 3, characterized in that:

p as described in steps (a), (e), (i) and (m)_TDH3The nucleotide sequence of (A) is shown as SEQ ID number 3;

t as defined in steps (a), (e), (i) and (m)_TDH2The nucleotide sequence of (A) is shown as SEQ ID number 5;

p as described in steps (b), (f), (j) and (n)_PGK1The nucleotide sequence of (A) is shown as SEQ ID number 1;

ARO4 described in step (b)^K229LLysine at position 229 of ARO4 gene is mutated to leucine;

t as described in steps (b) (f), (j) and (n)_ADH1The nucleotide sequence of (A) is shown as SEQ ID number 6;

p as described in steps (c), (g), (k) and (o)_TEF1The nucleotide sequence of (A) is shown as SEQ ID number 2;

ARO7 described in step (c)^G229SGlycine at position 229 of ARO7 gene was mutated to serine;

t described in steps (c), (g), (k) and (o)_CYC1The nucleotide sequence of (A) is shown as SEQ ID NO. 4;

BDH1 described in step (m)^{E221S/I222R/A223S}Is composed ofBDH1The glutamic acid at position 221 is mutated to serine, the isoleucine at position 222 is mutated to arginine, and the alanine at position 223 is mutated to serine.

5. The method for constructing saccharomyces cerevisiae engineering bacteria for high yield of coniferyl alcohol as claimed in claim 3, characterized in that:

the screening in the step (A) is carried out by adopting an SD-URA culture medium;

the screening in the step (B) is carried out by adopting an SD-LEU culture medium;

the screening in the step (C) is to adopt SD-MET culture medium for screening;

the screening in the step (D) is carried out by adopting SD-HIS and SD-MET culture media;

6. The use of the engineered saccharomyces cerevisiae producing coniferyl alcohol with high yield as claimed in claim 1 or 2 in the production of coniferyl alcohol.

7. A method for producing coniferyl alcohol is characterized in that: inoculating the saccharomyces cerevisiae engineering bacteria for high yield of coniferyl alcohol described in claim 1 or 2 into a fermentation culture medium for fermentation culture to obtain coniferyl alcohol.

8. The method for producing coniferyl alcohol according to claim 7, comprising the following steps: activating the saccharomyces cerevisiae engineering bacteria with high coniferyl alcohol yield, inoculating the activated saccharomyces cerevisiae engineering bacteria into a fermentation culture medium for fermentation culture, and supplementing a supplemented culture medium when the dissolved oxygen value is lower than 40% to maintain the content of glucose in the culture medium at 5g/L to obtain coniferyl alcohol;

the fermentation medium comprises the following components: 30g/L glucose, (NH)₄)₂SO₄ 15 g/L，KH₂PO₄ 8 g/L，MgSO₄3 g/L，ZnSO₄•7H₂0.72g/L of O, 12mL/L of vitamin solution and 10mL/L of trace metal salt solution;

the feed medium comprises the following components: 585g/L glucose, KH₂PO₄ 9 g/L，MgSO₄ 2.5 g/L，K₂SO₄ 3.5 g/L，Na₂SO₄0.28g/L, 12mL/L vitamin solution and 10mL/L trace metal salt solution;

the vitamin solution comprises the following components: 0.05g/L of vitamin H, 1g/L of calcium pantothenate, 1g/L of nicotinic acid, 25g/L of inositol, 1g/L of thiamine hydrochloride, 1g/L of pyridoxine hydrochloride and 0.2g/L of p-aminobenzoic acid;

the trace metal salt solution comprises the following components: EDTA 15g/L, ZnSO₄•7H₂O 10.2 g/L，MnCl₂•4H₂O 0.5 g/L，CuSO₄ 0.5 g/L，CoCl₂•6H₂O 0.86 g/L，Na₂MoO₄•2H₂O 0.56 g/L，CaCl₂•2H₂O3.84 g/L and FeSO₄•7H₂O 5.12 g/L。

9. The method of producing coniferyl alcohol of claim 8, wherein:

the inoculation amount of the saccharomyces cerevisiae engineering bacteria for high yield of coniferyl alcohol is 0.1-15% by volume percentage;

the conditions of the fermentation culture are as follows: the temperature is 25-35 ℃, the pH value is 3-7, the dissolved oxygen value is more than 30%, the stirring speed is 300-800 rpm, the ventilation volume is 3-20L/min, the fermentation time is 0-96 h, and 0 is not included.

10. The method of producing coniferyl alcohol of claim 8, wherein:

the activation is multistage activation, and is realized by the following steps: inoculating the saccharomyces cerevisiae engineering bacteria with high coniferyl alcohol yield into 5mL of SD-ULMHT culture medium, and culturing at 220-250 rpm and 30 ℃ until the OD value is 2-3; then transferring the bacterial liquid into a 100mL SD-ULMHT culture medium, and culturing at 220-250 rpm and 30 ℃ until the OD value is 2-3;

the composition of the SD-ULMHT culture medium is as follows: YNB medium 6.7 g/L; URA, LEU, MET, HIS and TRP co-deficiency amino acid 100X 10 mL/L; 20g/L of glucose;

the URA, LEU, MET, HIS and TRP co-deficiency amino acid 100X comprises the following components: adenine sulfate 0.25g, arginine 0.12g, aspartic acid 0.6g, glutamic acid 0.6g, lysine 0.18g, phenylalanine 0.3g, serine 2.25g, threonine 1.2g, tyrosine 0.18g, valine 0.9g, and ddH₂The volume of O is 57 mL.