RECOMBINANT YEAST AND USE THEREOF

Abstract

Provided is a recombinant yeast expressing germacrene A synthetase or a fusion protein thereof, wherein the fusion protein is germacrene A synthetase and farnesyl pyrophosphate synthase. The recombinant yeast improves the yield of germacrene A, and is suitable for the industrialized production of β-elemene and/or germacrene A.

Claims

1. A recombinant strain, which is a yeast comprising or expressing germacrene A synthetase or a fusion protein of germacrene A synthetase m vivo; the fusion protein of germacrene A synthetase comprises the germacrene A synthetase and farnesyl pyrophosphate synthase.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0081] FIG. 1 shows the germacrene A biosynthetic pathway.

[0082] FIG. 2 is a GC-MS test chromatomap.

DETAILED DESCRIPTION OF THE INVENTION

[0083] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0084] Unless otherwise specified, the materials, reagents and the like used in the following examples are commercially available.

[0085] FIG. 1 shows the germacrene A biosynthetic pathway.

Example 1: Preparation of Target Genes and Plasmids Used

[0086] 1. Preparation of Target Genes

[0087] (1) Acquisition of ADH2, ALD6, ASC1, MF1, TEF1 and CYC1

[0088] Genomic DNA of yeast NK2-SQ (China Journal of Chinese Materia Medica, Lin Tingting, Wang Dong, Dai Zhubo, Zhang Xueli, Huang Luqi, 2016, 41(6): 1008-1015) was extracted as a template, and was amplified by using the primers required in the gene amplification in Table 1 to obtain ADH2, ALD6, ASC1 gene fragments with the expected size, promoter MF1, TEF1 and terminator CYCL.

[0089] PCR amplification kit TAKARA PrimeSTAR®HS DNApolymerase was used to formulate an amplification system (TAKARA). The amplification system included: 5×PS Buffer 10 μL, dNTPMix 4 μL, primers 1 μL for each, genomic DNA template 1 μL, PrimeSTAR®HS polymerase (2.5 U/μL) 0.5 μL, distilled water supplemented to a total volume of 50 μL. The amplification conditions were: pre-denaturation at 98° C. for 3 minutes (1 cycle); denaturation at 98° C. for 10 sec, annealing at 55° C. for 15 sec, extension at 72° C. for 2.5 min (30 cycles); and extension at 72° C. for 10 min (1 cycle).

TABLE-US-00003 TABLE 1 Primer sequences Gene Primer Primer sequence fragment name □ 5′.fwdarw.3′□ ADH2 SexA1-ADH2 GCGACCWGGTATGTCTATTCCAGAA ACTCAAAAAGC (SEQ ID NO: 22) ADH2-Asc1 GCGGCGCGCCTTATTTAGAAGTGT CAACAACGTATC (SEQ ID NO: 23) ALD6 SexA1-ALD6 TCGCGACCWGGTAAAACAATGACT AAGCTACACTTTGAC (SEQ ID NO: 24) ALD6-Asc1 TCGCGGCGCGCCTTACAACTTA ATTCTGACAGCT (SEQ ID NO: 25) ACS 1 SexA1-ACS 1 TCGCGACCWGGTAAAACAATGTCGC CCTCTGCCGTACAATC (SEQ ID NO: 26) ACS 1-Asc 1 TCGCGGCGCGCCTTACAACTTGAC CGAATCAATTAG (SEQ ID NO: 27) TEF1 Sac11-TEF 1 GCGCCGCGGAGTGATCCCCCAC ACACCATAGCTT (SEQ ID NO: 28) TEFl-SexA1 TGGCGACCWGGTTTTGTAATTAAA ACTTAGATTAGA (SEQ ID NO: 29) MF1 BamH1- GCGGGATCCGGGAAGACATGCT pMF1 TAACAAGAAGAT (SEQ ID NO: 30) pMF1-SexA1 GCGACCTGGTTCTTTTAATCGTT TATATTGTGTAT (SEQ ID NO: 31) CYC1 Asc1-CYC1 GCGGCGCGCCCCGCTGATCC TAGAGGGCCGCATCA (SEQ ID NO: 32) CYC1-Sac 11 GCGCCGCGGGCGCGTTGGCC GATTCATTAATGCA (SEQ ID NO: 33)

[0090] (2) Acquisition of Farnesyl Pyrophosphate Synthase Gene SynSmFPS from Salvia miltiorrhiza and Germacrene A Synthetase Gene STpGMA from Tanacetum parthenium

[0091] Nanjing GenScript Biotechnology Co., Ltd. designed full-length primers according to the sequences of SynSmFPS (SEQ ID NO: 2, derived from Salvia miltiorrhiza) and STpGMAS (SEQ ID NO: 3, derived from Tanacetum parthenium) genes, and the template DNA was formed by using OVERLAP method. The double-stranded DNAs of SynSmFPS (SEQ ID NO: 2) and STpGMAS (SEQ ID NO: 3) were obtained by PCR amplification method, and then the PCR products were transformed and cloned into a cloning vector pUC57 (Nanjing GenScript Biotechnology Co., Ltd.), and cloning plasmids of pUC57-SynSmFPS and pUC57-STpGMAS containing SynSmFPS gene and STpGMAS gene were constructed, respectively.

[0092] (3) Acquisition of Farnesyl Pyrophosphate Synthase Gene ERG20-GGGS (SEQ ID NO: 15) from Yeast and Germacrene A Synthetase Gene GGGS (SEQ ID NO: 15)-LsLTC2 from Lettuce

[0093] 200 mg of lettuce leaves was taken and ground with liquid nitrogen, and then total RNA thereof was extracted by CTAB method (Cetyltrimethylammonium Bromide method): 1 ml of 2*CTAB extract (2% CTAB, 100 mM of Tris-HCl PH 8.0, 20 mM of EDTA solution (ethylenediamine tetraacetic acid), and 1.4M NaCl solution) was added into a 1.5 ml centrifuge tube. After being pre-heated at 65° C., 20 μL of 2-mercaptoethanol was added, and a small amount of lettuce leaf powder (about 50 mg) was added thereto, and then they were mixed well and kept at 65° C. for 10 min, shaken 5 times, centrifuged at 12,000 rpm for 10 min under 4° C.; the resulted supernatant was removed, extracted with an equal volume of chloroform/isoamyl alcohol, centrifuged at 12,000 rpm for 10 min under 4° C.; the obtained supernatant was removed, extracted with an equal volume of chloroform/isoamyl alcohol, centrifuged at 12,000 rpm for 10 min under 4° C.; the resulted supernatant was removed, extracted with 1/6 volume of chloroform/isoamyl alcohol, centrifuged at 15,000 rpm for 30 min under 4° C.; the obtained supernatant was removed, to which 1/4 volume of 10 mol/L LiCl was added, kept at 4° C. overnight, centrifuged at 15,000 rpm for 30 min under 4° C.; the supernatant was discarded, and the obtained precipitate was washed twice with 75% ethanol and washed once with absolute ethanol, and placed on the super-clean bench for 15 min (room temperature); it was dissolved in 20 μL of milliQ DEPC-treated water (the solvent was milliQ pure water and the solute was diethyl pyrocarbonate, and the volume ratio diethyl pyrocarbonate:water was 1:1000), to which 1/10 volume of 2 mol/L NaAC (pH 4.0) and 2 volumes of absolute ethanol were added, kept at −20° C. for 2 h, and centrifuged at 12,000 rpm for 10 min under 4° C.; the resulted supernatant was discarded, and the obtained precipitate was washed twice with 75% ethanol and washed once with absolute ethanol, placed on a super-clean bench for 15 min (room temperature), to which 15 μL of milliQ DEPC-treated water was added to fully dissolve the precipitate, and stored at −70° C.

[0094] First-strand reverse transcription-PCR: a RNase-free PCR tube was taken, and the system was formulated according to a first strand reverse transcription kit (TaKaRa Biotechnology (Dalian) Co., Ltd.): Radom 6 Mers 2 μL, dNTP 1 μL, total RNA 1 μL (200 ng), H.sub.2O 6 μL, Total 10 μL; a transient centrifugation was performed; PCR was carried out at 65° C. for 5 min; quenching it on ice and then adding the same into the following system for reaction (coming with the first chain reverse transcription kit): 5*primer Buffer 4 μL, RNAs Inhibiter 0.5 μL, R-Transcription 1 μL, H.sub.2O 4.5 μL; transient centrifugation was performed, and a reaction was performed in a PCR instrument: 30° C. for 10 min, 42° C. for 60 min, 70° C. for 15 min, and kept at 4° C.

[0095] NK2-SQ genomic DNA and lettuce cDNA were used as templates, respectively, and amplified by using the primers in Table 2 to obtain about 1068 bp of ERG20-GGGS (SEQ ID NO: 15) (the one of positions 13-1686 in SEQ ID NO: 11 was ERG20) and 1688 bp of GGGS (SEQ ID NO: 15)-LsLTC2 (the one of positions 1-1056 in SEQ. ID NO. 12 was LsLTC2).

[0096] The system was formulated according to the PCR amplification kit Phusion High-Fidelity PCR Master Mix with HF Buffer (purchased from NEB (Beijing) Co., Ltd.). The amplification system included: 5× Phusion HF Buffer 10 μL, dNTP (10 mM each dNTP) 1 μL, DNA template 20 ng, primers (10 μM) 1.5 μL for each, Phusion High-Fidelity DNA Polymerase (2.5 U/μL) 0.5 μL, and distilled water supplemented to a total volume of 50 μL. Amplification conditions: pre-denaturation at 98° C. for 3 min (1 cycle); denaturation at 98° C. for 10 sec, annealing at 58° C. for 10 sec, extension at 72° C. for imin 30 cycles); and extension at 72° C. for 10 min 1 cycle).

TABLE-US-00004 TABLE 2 Primer sequences Gene Primer Primer fragment name sequence (5′.fwdarw.3′) ERG20-GGGS SEXA1-ERG20 GCGACCWGGTAAAACAAT (SEQ ID GGCTTCAGAAAAAGAAATT NO: 15) AGGAGD □ SEQ ID NO: 34□ ERG20-GGGS CTTTCCCATAGAACCACCA (SEQ ID CCCTATTTGCTTCTCTTGT NO: 15) AAACTTTG □ SEQ ID NO: 35□ GGGS GGGS (SEQ ID GGTGGTGGTTCTATGGCA (SEQ ID NO: 15)- GCAGTTGACACTAA NO: 15)- LSLTC2 □ SEQ ID NO: 36□ LSLTC2 LSLTC2-ASC1 GCGGGCGCGCCTTACATGGA TACAGAACCAACAAAT □ SEQ ID NO: 37□

[0097] 2. Construction of Recombinant Plasmids

[0098] (1) Plasmid pM2-ADH2

[0099] ADH2 obtained through amplification in the above “1. Preparation of target genes” and plasmid pM2-tHMG1 (described in Chinese patent ZL201310399947.X) were double enzyme digested by using SexA1 (purchased from NEB (Beijing) Co., Ltd.) and Asc1 (purchased from NEB (Beijing) Co., Ltd.) to obtain 1052 bp of ADH2 enzyme-digested product and 4738 bp of enzyme-digested plasmid pM2-tHMG1 backbone; the ADH2 enzyme-digested product was then ligated with the enzyme-digested plasmid pM2-tHMG1 backbone to obtain the recombinant plasmid pM2-ADH2.

[0100] (2) Plasmid pM4-ACS1

[0101] ACS1 obtained through amplification in the above “1. Preparation of target genes” and plasmid pM4-AtCPR1 (described in Chinese patent ZL201310399947.X) were double enzyme digested by using SexA1 and Asc1 to obtain 2201 bp of ACS1 enzyme-digested product and 5061 bp of enzyme-digested plasmid pM4-AtCPR1 backbone; the ACS1 enzyme-digested product was then ligated with the enzyme-digested plasmid pM4-AtCPR1 backbone to obtain the recombinant plasmid pM4-ACS1.

[0102] (3) Plasmid pM3-ALD6

[0103] ALD6 obtained through amplification in the above “1. Preparation of target genes” and plasmid pM3-ERG9 (described in Chinese patent ZL201310399947.X) were double enzyme digested by using SexA1 and Asc1 to obtain 1511 bp of ALD6 enzyme-digested product and 4598 bp of enzyme-digested plasmid pM3-ERG9 backbone; the ALD6 enzyme-digested product was then ligated with the enzyme-digested plasmid pM3-ERG9 backbone to obtain the recombinant plasmid pM3-ALD6.

[0104] (4) Construction of Plasmids pRS313-LEU2-P.sub.TEF1-STpGMAS-T.sub.CYC1 and pRS425-LEU2-P.sub.TEF1-STpGMAS-T.sub.CYC1

[0105] TEF1 obtained through amplification in the above “1. Preparation of target genes” was enzyme digested by using SexA1, and 440 bp of TEF1 enzyme-digested product was obtained;

[0106] CYC1 obtained through amplification in the above “1. Preparation of target genes” was enzyme digested by using Asc1, and 322 bp of CYC1 enzyme-digested product was obtained;

[0107] pUC57-STpGMAS was enzyme digested by using SexA1 and Asc1, and 1694 bp of STpGMAS was recovered.

[0108] 50 ng of each of the enzyme-digested products TEF1, CYC1 and STpGMAS was added into a ligation system including: 2 μL of 10×T4 DNA Ligase Reaction Buffer (NEB), 1 μL of T4 DNA Ligase (NEB, 400,000 cohesive end units/ml), distilled water supplemented to 20 μL; they reacted at room temperature for 2 hours to obtain a ligation product.

[0109] 1 μL of the ligation product was added into a PCR system (Phusion High-Fidelity PCR Master Mix with HF Buffer kit, NEB) including: 5× Phusion HF Buffer 10 μL, dNTP (10 mM each dNTP) 1 μL, DNA template 20 ng, and primers Sac11-TEF1 and CYC1-Sac11 (10 μM) in Table 3, 1.5 μL for each, Phusion High-Fidelity DNA Polymerase (2.5 U/μL) 0.5 μL, and distilled water supplemented to a total volume of 50 μL. The amplification conditions were: pre-denaturation at 98° C. for 3 min (1 cycle); denaturation at 98° C. for 10 sec, annealing at 58° C. for 10 sec, extension at 72° C. for 1.5 min (30 cycles); and extension at 72° C. for 10 min (1 cycle). 2456 bp of PCR amplification product was obtained.

[0110] The amplification product was purified, and then enzyme digested by using SacII. The target fragment SacII-TEF1-STpGMAS-CYC1-Sac was recovered from gel, and prepared to use.

[0111] Plasmids pRS313 (Sikorski, R. S. and Hieter, P. 1989, Genetics 122 (1): 19-27) and pRS425 (Sikorski, R. S. and Hieter, P. 1989, Genetics 122 (1): 19-27) were enzyme digested with SacII, respectively, and 4967 bp of pRS313 vector fragment and 6849 bp of pRS425 vector fragment were obtained; 4 μL of NEB buffer and 1 μL of CIP dephosphorylation enzyme (NEB) were then added, and distilled water was supplemented to 40 μL; it was treated at 37° C. for 1 h, and EDTA with the final concentration of 10 μmol was added; it was kept at 65° C. for 30 min to terminate the reaction, and pRS313-SacII vector fragment and pRS425-SacII vector fragment were recovered from gel.

[0112] 50 ng of each of the vector fragments pRS313-SacII, pRS425-SacII and SacII-TEF1-STpGMAS-CYC1-SacII obtained in the above step “1. Preparation of target genes” were respectively added into a ligation system including: 2 μL 10×T4 DNA Ligase Reaction Buffer (NEB)), 1 μL T4 DNA Ligase (NEB, 400,000 cohesive end units/ml), distilled water supplemented to 20 μL; they reacted at room temperature for 2 hours to obtain the ligation product, which was transferred into Trans10 competent cells and verified by sequencing, and thus plasmids pRS313-HIS3-P.sub.TEF1-STpGMAS-T.sub.CYC1 and pRS425-LEU2-P.sub.TEF1-STpGMAS-T.sub.CYC1 were obtained.

[0113] Using plasmid pRS313-HIS3-P.sub.TEF1-STpGMAS-T.sub.CYC1 as a template, 6692 bp of plasmid pRS313-TEF1-STpGMAS-CYC1 backbone was amplified by using the primers in Table 3.

[0114] Using pRS425 as a template, LEU2 (1808 bp) was amplified by using the primers in Table 3.

[0115] The amplification system included: 5× Phusion HF Buffer 10 μL, dNTP (10 mM each dNTP) 1 μL, DNA template 20 ng, primers (10 μM) 1.5 μL for each, Phusion High-Fidelity DNA Polymerase (2.5 U/μL) 0.5 μL, and distilled water supplemented to a total volume of 50 μL. The amplification conditions were: pre-denaturation at 98° C. for 3 min (1 cycle); denaturation at 98° C. for 10 sec, annealing at 58° C. for 10 sec, extension at 72° C. for 4 min (30 cycles); and extension at 72° C. for 10 min (1 cycle). The target fragment was purified from gel. 2 μL of 10×T4 DNA Ligase Reaction Buffer (NEB) and 1 μL of T4 Polynucleotide kinase (NEB) were added into the product of LEU2 fragment, and distilled water was supplemented to a total volume of 20 μL. A phosphorylation was performed at 37° C. for 1 h, and it was ligated to pRS313-P.sub.TEF1-STpGMAS-T.sub.CYC1 by T4 DNA ligase (NEB) after being recovered from gel, transformed, and verified by sequencing to obtain plasmid pRS313-LEU2-P.sub.TEF1-STpGMAS-T.sub.CYC1.

TABLE-US-00005 TABLE 3 Primer sequences Primer Gene Primer sequence fragment name □ 5′.fwdarw.3′□ TEF1-STpGMAS- Sac11-TEF1 GCGCCGCGGAGTGATCC CYC1 CCCACACACCATAGCTT (SEQ ID NO: 28) CYC1-Sac11 GCGCCGCGGGCGCGTTG GCCGATTCATTAATGCA (SEQ ID NO: 33) pRS313-TEF1- V313-to-R CTTTGCCTTCGTTTATC STpGMAS- TTGC CYC1 (SEQ ID NO: 38) V313-to-F TATATGTATACCTA TGAATGTCAG (SEQ ID NO: 39) LEU2 Bsp-Leu-F TGGcgTCCGGATTAAGC AAGGATTTTCTTAACTT CTTC (SEQ ID NO: 40) Bsp-Leu-R TGGcgTCCGGAGATGC GGTATTTTCTCCTTAC GCA (SEQ ID NO: 41)

[0116] (5) Construction of Plasmid pRS425-LEU2-P.sub.TEF1-SynSmFPS-GGGS (SEQ ID NO: 15)-STpGMAS-T.sub.CYC1

[0117] Using pUC57-SynSmFPS and pUC57-STpGMAS as templates, 1080 bp of SynSmFPS-GGGS (SEQ ID NO: 15) and 1704 bp of GGGS (SEQ ID NO: 15)-STpGMAS were obtained by amplification using the primers in Table 4.

[0118] The amplification system included: 5× Phusion HF Buffer 10 μL, dNTP (10 mM each dNTP) 1 μL, DNA template 20 ng, primers (10 μM) 1.5 μL for each, Phusion High-Fidelity DNA Polymerase (2.5 U/μL) 0.5 μL, and distilled water supplemented to a total volume of 50 μL. The amplification conditions were: pre-denaturation at 98° C. for 3 min (1 cycle); denaturation at 98° C. for 10 sec, annealing at 58° C. for 10 sec, extension at 72° C. for 1 min (30 cycles); extension at 72° C. for 10 min (1 cycle). SynSmFPS-GGGS (SEQ ID NO: 15) and GGGS (SEQ ID NO: 15)-STpGMAS were used together as templates, and 2767 bp of SynSmFPS-GGGS (SEQ ID NO: 15)-STpGMAS fragment was obtained by amplification using the primers in Table 4 (SexA1-SynSmFPS and STpGMAS-Asc1).

[0119] The amplification system included: 5× Phusion HF Buffer 10 μL, dNTP (10 mM each dNTP) 1 μL, DNA templates SynSmFPS-GGGS (SEQ ID NO: 15) and GGGS (SEQ ID NO: 15)-STpGMAS 20 ng for each, primers (10 μM) 1.5 μL for each, Phusion High-Fidelity DNA Polymerase (2.5 U/μL) 0.5 μL, and distilled water supplemented to a total volume of 50 μL. The amplification conditions were: pre-denaturation at 98° C. for 3 min (1 cycle); denaturation at 98° C. for 10 sec, annealing at 58° C. for 10 sec, extension at 72° C. for 2 min (30 cycles); extension at 72° C. for 10 min (1 cycle).

[0120] The amplification product was purified, and then enzyme digested with SexA1 and Asc1, and the target fragment SexA1-SynSmFPS-GGGS (SEQ ID NO: 15)-STpGMAS-Asc1 (2760 bp) was recovered from gel, and prepared to use.

[0121] The plasmid pRS425-LEU2-P.sub.TEF1-STpGMAS-T.sub.CYC1 constructed in the above item “(4)” was enzyme digested with SexA1 and Asc1, and the 7602 bp large fragment was recovered from gel, so as to obtain the vector pRS425-LEU2-P.sub.TEF1- . . . -T.sub.CYC1; 50 ng of each of the vectors pRS425-LEU2-P.sub.TEF1- . . . -T.sub.CYC1 and SexA1-SynSmFPS-GGGS (SEQ ID NO: 15)-STpGMAS-Asc1 was added into the ligation system including: 2 μL 10×T4 DNA Ligase Reaction Buffer (NEB), 1 μL T4 DNA Ligase (NEB, 400,000 cohesive end units/ml), and distilled water supplemented to 20 μL; they reacted at room temperature for 2 hours to obtain a ligation product which was transferred into Trans10 competent cells, the plasmid was extracted and verified by sequencing, and plasmid pRS425-LEU2-P.sub.TEF1-SynSmFPS-GGGS (SEQ ID NO: 15)-STpGMAS-T.sub.CYC1 was obtained.

TABLE-US-00006 TABLE 4 Primer sequences Primer Gene Primer sequence fragment name □ 5′.fwdarw.3′□ SynSmFPS-GGGS SexA1- ACCTGGTAAAACAATGGC (SEQ ID SynSmFPS TAATTTGAATGGTGAATC NO: 15) (SEQ ID NO: 42) SynSmFPS-GGGS TGCTGCCATAGAACCACC SEQ ID NO: 15) ACCTTTTTGTCTTTTATAG ATTTTACC (SEQ ID NO: 43) GGGS GGGS GGTGGTGGTTCTATGGCA (SEQ ID (SEQ ID NO: 5) GCAGTACAAGCAACCAC NO: 15)- -STpGMAS (SEQ ID NO: 44) STpGMAS STpGMAS-Asc1 GGCGCGCCTCAGACTGG CAAGGAATCTA (SEQ ID NO: 45) SynSmFPS- SexA1- ACCTGGTAAAACAATGGC GGGS SynSmFPS TAATTTGAATGGTGAATC (SEQ ID (SEQ ID NO: 42) NO: 15)- STpGMAS STpGMAS-Asc1 GGCGCGCCTCAGACTGG CAAGGAATCTA (SEQ ID NO: 45)

[0122] (6) Construction of Plasmid pRS425-LEU2-P.sub.MF1-SynSmFPS-GGGS (SEQ ID NO: 15)-STpGMAS-T.sub.CYC1

[0123] MF1 obtained in the above “1. Preparation of target genes” and plasmid pRS425-LEU2-P.sub.TEF1-SynSmFPS-GGGS (SEQ ID NO: 15)-STpGMAS-T.sub.CYC1 constructed in the above item “(5)” were double enzyme digested by using BamHI (purchased from TaKaRa) and SexA1, respectively. 814 bp target promoter gene MF1 and 9898 bp vector fragment pRS425-LEU2- . . . -SynSmFPS-GGGS (SEQ ID NO: 15)-STpGMAS-T.sub.CYC1 were purified from gel and the two (50 ng for each) were added into a ligation system including: 2 μL 10×T4 DNA Ligase Reaction Buffer (NEB), 1 μL T4 DNA Ligase (NEB, 400,000 cohesive end units/ml), and distilled water supplemented to 20 μL; they reacted at room temperature for 2 hours to obtain the ligation product which was transformed into Trans10 competent cells, and the plasmid was extracted and verified by sequencing. The plasmid obtained accordant with the correct sequence was named as pRS425-LEU2-P.sub.MF1-SynSmFPS-GGGS (SEQ ID NO: 15)-STpGMAS-T.sub.CYC1.

[0124] (7) Construction of Plasmid pM2-ERG20-GGGS (SEQ ID NO: 15)-LsLTC2

[0125] Using ERG20-GGGS (SEQ ID NO: 15) and GGGS (SEQ ID NO: 15)-LsLTC2 together as templates, an ERG20-GGGS (SEQ ID NO: 15)-LsLTC2 fragment of about 2744 bp was obtained by amplification using the primers (SexA1-ERG20 and LsLTC2-Asc1) in Table 5.

[0126] The amplification system included: 5× Phusion HF Buffer 10 μL, dNTP (10 mM each dNTP) 1 μL, DNA templates ERG20-GGGS (SEQ ID NO: 15) and GGGS (SEQ ID NO: 15)-LsLTC2 20 ng for each, primers (10 μM) 1.5 μL for each, Phusion High-Fidelity DNA Polymerase (2.5 U/μL) 0.5 μL, and distilled water supplemented to a total volume of 50 μL. The amplification conditions were: pre-denaturation at 98° C. for 3 min (1 cycle); denaturation at 98° C. for 10 sec, annealing at 58° C. for 10 sec, extension at 72° C. for 2 min (30 cycles); and extension at 72° C. for 10 min (1 cycle). The amplification product was purified, and then enzyme digested with SexA1 and Asc1, and the target fragment SexA1-ERG20-GGGS (SEQ ID NO: 15)-LsLTC2-Asc1 (about 2744 bp) was recovered from gel, and then ligated with the enzyme-digested plasmid vector pM2-tHMG1 backbone, so as to obtain the recombinant plasmid pM2-ERG20-GGGS (SEQ ID NO: 15)-LsLTC2.

TABLE-US-00007 TABLE 5 Primer sequences Primer Primer sequence Gene fragment name □ 5′.fwdarw.3′□ ERG20-GGGS SEXA1- GCGACCWGGTAAAACAATGGCT (SEQ ID ERG20 TCAGAAAAAGAAATTAGGAG NO: 15) (SEQ ID NO: 34) ERG20- CTTTCCCATAGAACCACCACCC GGGS TATTTGCTTCTCTTGTAAACTT (SEQ ID TG NO: 15) (SEQ ID NO: 35) GGGS (SEQ ID NO: GGGS GGTGGTGGTTCTATGGCAGCAG 15)-LSLTC2 (SEQ ID TTGACACTAA LSLTC2 (SEQ ID NO: 36) NO: 15) LSLTC2- GCGGGCGCGCCTTACATGGATA ASC1 CAGAACCAACAAAT (SEQ ID NO: 37) ERG20-GGGS SEXA1- GCGACCWGGTAAAACAATGGCT (SEQ ID ERG20 TCAGAAAAAGAAATTAGGAG NO: 15)-STPGMAS (SEQ ID NO: 34) LSLTC2- GCGGGCGCGCCTTACATGGAT ASC1 ACAGAACCAACAAAT (SEQ ID NO: 37)

[0127] (8) Construction of Plasmid pEASY-NDT80-HIS3

[0128] Using NK2-SQ genomic DNA and pRS313 as templates, 1252 bp of NDT80 (SEQ ID NO: 13) and 1168 bp of HIS3 (SEQ ID NO: 14) were obtained by amplification using the primers in Table 6.

[0129] The amplification system included: 5× Phusion HF Buffer 10 μL, dNTP (10 mM each dNTP) 1 μL, DNA template 20 ng, primers (10 μM) 1.5 μL for each, Phusion High-Fidelity DNA Polymerase (2.5 U/μL) 0.5 μL, and distilled water supplemented to a total volume of 50 μL. The amplification conditions were: pre-denaturation at 98° C. for 3 min (1 cycle); denaturation at 98° C. for 10 sec, annealing at 58° C. for 10 sec, extension at 72° C. for 1 min (30 cycles); and extension at 72° C. for 10 min (1 cycle).

[0130] The amplification product NDT80 was cloned into pEASY-Blunt Simple cloning vector (pEASY cloning vector, Beijing TransGen Biotech Co., Ltd.), transformed into Trans10 competent cells, and the plasmid was extracted and verified by sequencing, and thus plasmid pEASY-NDT80 was obtained.

TABLE-US-00008 TABLE 6 Primers Gene Primer Primer sequence fragment name Template □ 5′.fwdarw.3′□ NDT80 NDT80-up- Genomic GCGGTTTAAACGTTCGAC PmeI DNA CATATTGATGAAGAGTGGG TAGG(SEQ ID NO: 46) NDT80- NK2-SQ CTGTTCCATTGATTTCTTC down TCTATTGTTATATC (SEQ ID NO: 47) HIS3 Bsp-HIS-F pRS313 TGGCGTCCGGATCGCGCGT TTCGGTGATGACGG (SEQ ID NO: 48) Pme1-HIS-R GCGGTTTAAACGTGTCACT ACATAAGAACACCT (SEQ ID NO: 49)

[0131] pEASY-NDT80 was enzyme digested by using PmeI (purchased from NEB (Beijing) Co., Ltd.), and 5122 bp target fragment (30 ng) was purified from gel, 4 μL NEB buffer (reaction buffer, purchased from NEB (Beijing) Co., Ltd.) and 1 μL CIP dephosphorylation enzyme (NEB) were added, and distilled water was supplemented to 40 μL; it was treated at 37° C. for 1 h, to which EDTA at a final concentration of 10 μmol was added, and it was kept at 65° C. for 30 min to terminate the reaction. 5122 bp target fragment pEASY-NDT80 was recovered from gel, and prepared to use.

[0132] HIS3 (30 ng) was purified from gel, 4 μL of 10×T4 DNA Ligase Reaction Buffer (NEB) and 1 μL of T4 Polynucleotide kinase (NEB) were added, and distilled water was supplemented to 40 μL, and it was phosphorylated at 37° C. for 1 h. After being recovered from gel, it was ligated with pEASY-NDT80 by using T4 DNA ligase (NEB), transformed into Trans10 competent cells, and verified by sequencing to obtain plasmid pEASY-NDT80-HIS3.

[0133] The information of plasmids constructed above was shown in Table 7 below:

TABLE-US-00009 TABLE 7 Plasmid Information Plasmid name Basic information pM2-ADH2 Containing P.sub.PGK1-ADH2-T.sub.ADH1 cassette pM4-ACS1 Containing P.sub.TDH3-ACS1-T.sub.TPI1 cassette pM3-ALD6 Containing P.sub.TEF1-ALD6-T.sub.CYC1 cassette pRS313-LEU2-P.sub.TEF1-STpGMAS-T.sub.CYC1 Containing P.sub.TEF1-SynSmFPS-T.sub.CYC1 cassette, LEU2, low-copy plasmid pRS425-LEU2-P.sub.TEF1-STpGMAS-T.sub.CYC1 Containing P.sub.TEF1-SynSmFPS-T.sub.CYC1 cassette, LEU2, high-copy plasmid pRS425-LEU2-P.sub.TEF1-SynSmFPS-GGGS Containing P.sub.TEF1-SynSmFPS-GGGS (SEQ (SEQ ID NO: 15)-STpGMAS-T.sub.CYC1 ID NO: 15)-STpGMAS-T.sub.CYC1 cassette, LEU2, high-copy plasmid pRS425-LEU2-P.sub.MF1-SynSmFPS-GGGS Containing P.sub.MF1-SynSmFPS-GGGS (SEQ ID (SEQ ID NO: 15)-STpGMAS-T.sub.CYC1 NO: 15)-STpGMAS-T.sub.CYC1 cassette, LEU2, high-copy plasmid pEASY-NDT80-HIS3 NDT80, HIS3

[0134] (9) Construction of Plasmid pEASY-rDNA-TRP1

[0135] Using NK2-SQ genomic DNA and pRS314 (Sikorski, R. S. and Hieter, P. 1989, Genetics 122(1): 19-27) as templates, respectively, rDNA (SEQ ID NO: 9) and TRP1 (SEQ ID NO: 10) were obtained by amplification using the primers in Table 8.

[0136] The amplification system included: 5× Phusion HF Buffer 10 μL, dNTP (10 mM each dNTP) 1 μL, DNA template 20 ng, primers (10 μM) 1.5 μL for each, Phusion High-Fidelity DNA Polymerase (2.51 U/μL) 0.5 μL, and distilled water supplemented to a total volume of 50 μL. The amplification conditions were: pre-denaturation at 98° C. for 3 min (1 cycle); denaturation at 98° C. for 10 sec, annealing at 58° C. for 10 sec, extension at 72° C. for Imin (30 cycles); and extension at 72° C. for 10 min (1 cycle) The amplification product rDNA was cloned into pEASY-Blunt Simple cloning vector and transformed into Trans10 competent cells, and the plasmid was extracted and verified by sequencing, so as to obtain plasmid pEASY-rDNA.

TABLE-US-00010 TABLE 8 Primers Gene Primer Primer sequence fragment name Template □ 5′.fwdarw.3′□ rDNA rDNA-up-F Genomic ATGAGAGTAGCAAACGT DNA of AAGTCT NK2-SQ (SEQ ID NO: 50) rDNA-R-PmeI GCGGTTTAAACTTTCCT CTAATCAGGTTCCACCA (SEQ ID NO: 51) TRP1 BSP-TRP1-F pRS314 TGGCGTCCGGATACAAT CTTGATCCGGAGCT (SEQ ID NO: 52) BSP-TRP1-R TGGCGTCCGGACACAAA CAATACTTAAATAAATA C(SEQ ID NO: 53)

[0137] pEASY-rDNA was enzyme digested by using PmeI, and 5122 bp target fragment (30 ng) was purified from gel, 4 μL NEB buffer and 1 μL CIP dephosphorylation enzyme (NEB) was added, and distilled water supplemented to a total volume of 40 μL; it was treated at 37° C. for 1 h, to which EDTA at a final concentration of 10 μmol was added, and it was kept at 65° C. for 30 min to terminate the reaction. 5122 bp target fragment pEASY-rDNA was recovered from gel, and prepared to use. TRP1 (30 ng) was purified from gel, 4 μL of 10×T4 DNA Ligase Reaction Buffer (NEB) and 1 μL of T4 Polynucleotide kinase (NEB) were added, and distilled water was supplemented to a total volume of 40 μL, and it was phosphorylated at 37° C. for 1 h. After being recovered from gel, it was ligated with pEASY-rDNA by using T4 DNA ligase (NEB), transformed into Trans10 competent cells, and verified by sequencing, and thus plasmid pEASY-rDNA-TRP1 was obtained.

Example 2: Construction of Recombinant Strains

[0138] 1. Preparation of Yeast Competent Cells

[0139] The original strains were respectively cultured in the corresponding medium (Table 13) at 30° C., 250 rpm overnight. 1 mL of the culture suspension (with OD around 0.6-10) was added into a 1.5 mL EP tube, centrifuged at 10,000 g for imin under 4° C.; the resulted supernatant was discarded, the precipitate was washed with sterile water (4° C.) and centrifuged under the same conditions; and the resulted supernatant was discarded. 1 mL of a treatment solution (10 mM LiAc (lithium acetate); 10 mM DTT (dithiothreitol); 0.6M sorbitol; 10 mM Tris-HCl (tris(hydroxymethyl)aminomethane hydrochloride buffer, pH7.5), DTT was added immediately before using the treatment solution) was added into the yeast, and it was kept at 25° C. for 20 min. After centrifugation, the supernatant was discarded, and 1 mL of 1M sorbitol (filtered and sterilized through a 0.22 m aqueous membrane) was added to re-suspend the yeast, then it was centrifuged, and the supernatant was discarded (re-suspended twice with 1M sorbitol) until the final volume became about 90 μL.

[0140] 2. Construction of Strain FPP-001

[0141] 1) Preparation of NDT80-HIS3-Up, P.sub.PGK1-ADH2-T.sub.ADH1, P.sub.TDH3-ACS1-T.sub.TPI1, P.sub.TEF1-ALD6-T.sub.CYC1 and NDT80-HIS3-Down

[0142] P.sub.PGK1-ADH2-T.sub.ADH1, P.sub.TDH3-ACS1-T.sub.TPI1, and P.sub.TEF1-ALD6-T.sub.CYC1 were expression cassettes carrying alcohol dehydrogenase 2, acetyl-CoA synthetase 1, and acetaldehyde dehydrogenase 6, respectively; NDT80-HIS3-up and NDT80-HIS3-down were the upstream and downstream homology arms of HIS3, respectively; the fragments were respectively amplified according to the following methods:

[0143] The functional modules were obtained by PCR using the templates and primers of PCR described in Table 9, respectively: 698 bp M1 (NDT80-HIS3-up), 2081 bp M2 (P.sub.PGK1-ADH2-T.sub.ADH1), 3519 bp M3 (P.sub.TDH3-ACS1-T.sub.TPI1), 2376 bp M4 (P.sub.TEF1-ALD6-T.sub.CYC1), 1835 bp M5 (NDT80-HIS3-down).

[0144] The amplification system included: 5× Phusion HF Buffer 10 μL, dNTP (10 mM each dNTP) 1 μL, DNA template 20 ng, primers (10 μM) 1.5 μL for each, Phusion High-Fidelity DNA Polymerase (2.5 U/μL) 0.5 μL, and distilled water supplemented to a total volume of 50 μL. The amplification conditions were: pre-denaturation at 98° C. for 3 min (1 cycle); denaturation at 98° C. for 10 sec, annealing at 58° C. for 10 sec, extension at 72° C. for 2 min (30 cycles); extension at 72° C. for 10 min (1 Cycle). The product was recovered from gel and stored.

TABLE-US-00011 TABLE 9 Primers Amplifi- PCR cation tem- fragment Primer Primer sequence Module plate name name □ 5′.fwdarw.3′□ M1 pEASY- NDT80- X1-M- CTTGCAAATGCCTATTGT NDT80- HIS3- pEASY- GCAGATGTTATAATATCT HIS3 UP r-t-F GTGCGTTTAATTAAGGCT CGTATGTTGTGTGGAATT GT(SEQ ID NO: 54) NDT80- CTGGCTTTAAAAAATGGA interg-2 TAAAAAGGGATG(SEQ ID NO: 55) M2 PM2- P.sub.PGK1- 1-M- CTGTTTCCTGTGTGAAAT ADH2 ADH2- pEASY- TGTTATCCGCTCACAATT T.sub.ADH1 PGK1-F CCACACAACATACGAGCC TTAATTAAACGCACAGAT ATTATAAC(SEQ ID NO: 56) 3G-1-M- CCTCCGCGTCATTAAACT ADHt- TCTTGTTGTTGACGCTAA TDH3-R CATTCAACGCTAGTATTC GGCATGCCGGTAGAGGTG TGG (SEQ ID NO: 57) M3 PM4- P.sub.TDH3- 3G-3-M- CAGGTATAGCATGAGGTC ACS1 ACS1- ADHt- GCTCTTATTGACCACACC T.sub.TPI1 TDH3-F TCTACCGGCATGCCGAAT ACTAGCGTTGAATGTTAG CGTC (SEQ ID NO: 58) 3G-3-M- AGGAGTAGAAACATTTTG TPI1t- AAGCTATGGTGTGTGGGG TEF1-R GATCACTTTAATTAATCT ATATAACAGTTGAAATTT GGA(SEQ ID NO: 59) M4 PM3- P.sub.TEF1- 3G-2-M- GTCATTTTCGCGTTGAGA ALD6 ALD6- TPI1t- AGATGTTCTTATCCAAAT T.sub.CYC1 TEF1-F TTCAACTGTTATATAGAT TAATTAAAGTGATCCCCC ACAC(SEQ ID NO: 60) M-CYC1- CGTATTACAATTCACTGG pEASY-R CCGTCGTTTTACAACGTC GTGACTGGGAAAACCCTG GCGCGTTGGCCGATTCAT TAATGC(SEQID NO: 61) M5 PEASY- NDT80- NDT80- CATCATAAGGAATTCCGG NDT80- HIS3- interg- GATTCTCCCCAT (SEQ HIS3 down 1 ID NO: 62) X2-M- CGAAGGCTTTAATTTGCA pEASY- AGCTGCGGCCCTGCATTA r-t-R ATGAATCGGCCAACGCGC CAGGGTTTTCCCAGTCAC GACGTTG(SEQID NO: 63)

[0145] 2) Construction of Strain FPP-001

[0146] Original strain Saccharomyces cerevisiae NK2-SQ was cultured in a SD-Ura liquid medium (0.8% yeast selective medium SD-Ura-Trp-His (Beijing FunGenome Technology Co., Ltd.), 2% glucose, 0.005% His, 0.01% Trp) overnight, followed by being prepared into competent cells. Then, the transformation fragments M1, M2, M3, M4 and M5 in Table 9 were added in a total amount of 5 μg (molar ratio=1:1:1:1:1), mixed well and transferred to an electric shock cup, electrically shocked at 2.7 kv for 5.7 ms, to which 1 mL of 1M sorbitol was added, and it was resuscitated at 30° C. for 1 h, and spread onto a SD-Ura-His medium and cultured at 30° C. for 36 h or more. The ingredients in the screening medium composition were: 0.8% yeast selective medium SD-Ura-Trp-His (Beijing FunGenome Technology Co., Ltd.), 2% glucose, and 0.01% Trp. The true positive clone was identified by PCR, and named as strain FPP-001.

[0147] 3 Construction of Strains ELE-001 and ELE-002

[0148] Original strain Saccharomyces cerevisiae FPP-001 was cultured in a SD-Ura-His liquid medium overnight, followed by being prepared into competent cells. Then, plasmids pRS313-LEU2-P.sub.TEF1-STpGMAS-T.sub.CYC1 and pRS425-LEU2-P.sub.TEF1-STpGMAS-T.sub.CYC1 were respectively added, mixed well and transferred into an electric shock cup, electrically shocked at 2.7 kv for 5.7 ms, to which 1 mL of 1M sorbitol was added, and it was resuscitated at 30° C. for 1 h, and spread onto a SD-Ura-His-Leu medium and cultured at 30° C. for 36 h or more. The ingredients in the screening medium composition were: 0.8% yeast selective medium SD-Ura-Trp-His (Beijing FunGenome Technology Co., Ltd.), 2% glucose, and 0.01% Trp. The true positive clone was identified by PCR, and named as strains ELE-001 (into which plasmid pRS313-LEU2-P.sub.TEF1-STpGMAS-T.sub.CYC1 was transferred) and ELE-002 (into which plasmid pRS425-LEU2-P.sub.TEF1-STpGMAS-T.sub.CYC1 was transferred), respectively.

[0149] 4 Construction of Strain ELE-011

[0150] FPP-001 competent cells were prepared according to the steps in the above item 3. Then, plasmid pRS425-LEU2-P.sub.TEF1-SynSmFPS-GGGS (SEQ ID NO: 15)-STpGMAS-T.sub.CYC1 was added thereto, mixed well and transferred into an electric shock cup, electrically shocked at 2.7 kv for 5.7 ms, to which 1 mL of 1M sorbitol was added, and it was resuscitated at 30° C. for 1 h, and spread onto a SD-Ura-His-Leu medium and cultured at 30° C. for 36 h or more. The true positive clone was identified by PCR, and named as strain ELE-011.

[0151] Construction of Strains ELE-012 to ELE-019

[0152] Using plasmid pRS425-LEU2-P.sub.TEF1-SynSmFPS-GGGS (SEQ ID NO: 15)-STpGMAS-T.sub.CYC1 as a template, PCR amplification was performed by using the primers of Table 11 to obtain the amplification products corresponding to different primers. Then, the amplification products corresponding to different primers were respectively transferred into yeast FPP-001 for carrying out its own homologous recombination, and recombinant strains ELE-012 to ELE-018 were obtained, respectively. The linker peptide GGGS (SEQ ID NO: 15) of the fusion protein SynSmFPS-GGGS (SEQ ID NO: 15)-STpGMAS in the vector were replaced with 3A001, 4A001, 5A002, 6A005, 6B004, 8A005, 12A003, respectively (as shown in Table 10).

[0153] Using plasmid pRS425-LEU2-P.sub.MF1-SynSmFPS-GGGS (SEQ ID NO: 15)-STpGMAS-T.sub.CYC1 as a template, PCR amplification was performed by using the primers with the linker peptide of 8A005 in Table 10 (Table 11) to obtain the amplification products corresponding to different primers. Then, the amplification products corresponding to the different primers were respectively transferred into yeast FPP-001 for carrying out its own homologous recombination, and recombinant strain ELE-019 was obtained. The linker peptide GGGS (SEQ ID NO: 15) of the fusion protein SynSmFPS-GGGS (SEQ ID NO: 15)-STpGMAS in the vector was replaced with 8A005.

[0154] Table 10 Showing the nucleotide sequences and amino acid sequences of linker peptide

TABLE-US-00012 Linker Nucleotide Amino acid peptide sequence sequence of name (5′.fwdarw.3′) linker peptide 3A001 TACGGTCAG YGQ 4A001 CCGGGGGGACAC PGGH (SEQ ID NO: 64) (SEQ ID NO: 16) 5A002 TATAGAAGTCAAATC YRSQI (SEQ ID NO: 65) (SEQ ID NO: 17) 6A005 GTGATACCTTTTATTTCA VIPFIS (SEQ ID NO: 66) (SEQ ID NO: 18) 6B004 TTTTTGTATCTTAAGTTT FLYLKF (SEQ ID NO: 67) (SEQ ID NO: 19) 8A005 TGGCGGTTCTCGCCGA WRFSPKLQ AGCTTCAG (SEQ ID NO: 20) (SEQ ID NO: 68) 12A003 CACCACGTGCAGGAGTCA HHVQESQCISTV CAATGTATTTCCACAG TG(SEQ ID NO: 69) (SEQ ID NO: 21)

[0155] The specific reaction conditions were as follows:

[0156] The above amplification system included: 5× Phusion HF Buffer 10 μL, dNTP (10 mM each dNTP) 1 μL, DNA template 20 ng, primers (as shown in Table 11) (10 μM) 1.5 μL for each, Phusion High-Fidelity DNA Polymerase (2.5 U/μL) 0.5 μL, and distilled water supplemented to a total volume of 50 μL. The amplification conditions were: pre-denaturation at 98° C. for 3 min (1 cycle); denaturation at 98° C. for 10 sec, annealing at 58° C. for 10 sec, extension at 72° C. for 5.5 min (30 cycles); extension at 72° C. for 10 min (1 cycle).

[0157] The amplification product was digested by using DpnI enzyme from Fermentas Company after being purified. The system thereof included: 5× Fast Digest Green Buffer 4 μL, purified product 34 μL, DpnI 2 μL. The enzyme digestion temperature and reaction time were 37° C. and 1 h, respectively. Finally, it was recovered from gel and stored.

TABLE-US-00013 TABLE 11 Primers Primer Linker Primer sequence peptide name (5′.fwdarw.3′) 3A001 50 bp- CAAGCAGTTTTGAAATCATTTTTG 3A001- GGTAAAATCTATAAAAGACAAAAA STpGmA TACGGTCAGATGGCAGCAGTACAA GCAACCAC (SEQ ID NO: 70) SynSmFPS- TTTTTGTCTTTTATAGATTTTACC Linker-R (SEQ ID NO: 71) 4A001 50 bp- CAAGCAGTTTTGAAATCATTTTTG 4A001- GGTAAAATCTATAAAAGACAAAAA STpGmA CCGGGGGGACACATGGCAGCAGTA CAAGCAACCAC (SEQ ID NO: 72) SynSmFPS- TTTTTGTCTTTTATAGATTTTACC Linker-R (SEQ ID NO: 71) 5A002 50 bp- CAAGCAGTTTTGAAATCATTTTTG 5A002- GGTAAAATCTATAAAAGACAAAAA STpGmA TATAGAAGTCAAATCATGGCAGCA GTACAAGCAACCAC (SEQ ID NO: 73) SynSmFPS- TTTTTGTCTTTTATAGATTTTACC Linker-R (SEQ ID NO: 71) 6A005 50 bp- CAAGCAGTTTTGAAATCATTTTTG 6A005- GGTAAAATCTATAAAAGACAAAAA STpGmA GTGATACCTTTTATTTCAATGGCA GCAGTACAAGCAACCAC (SEQ ID NO: 74) SynSmFPS- TTTTTGTCTTTTATAGATTTTACC Linker-R (SEQ ID NO: 71) 6B004 50 bp- CAAGCAGTTTTGAAATCATTTTTGG 6B004- GTAAAATCTATAAAAGACAAAAATT STpGmA TTTGTATCTTAAGTTTATGGCAGCA GTACAAGCAACCAC (SEQ ID NO: 75) SynSmFPS- TTTTTGTCTTTTATAGATTTTACC Linker-R (SEQ ID NO: 71) 8A005 50 bp- CAAGCAGTTTTGAAATCATTTTTG 8A005- GGTAAAATCTATAAAAGACAAAAA STpGmA TGGCGGTTCTCGCCGAAGCTTCAG ATGGCAGCAGTACAAGCAACCAC (SEQ ID NO: 76) SynSmFPS- TTTTTGTCTTTTATAGATTTTACC Linker-R (SEQ ID NO: 71) 12A003 50 bp- CAAGCAGTTTTGAAATCATTTTTGG 12A003- GTAAAATCTATAAAAGACAAAAACA STpGmA CCACGTGCAGGAGTCACAATGTATT TCCACAGTGATGGCAGCAGTACAAG CAACCAC (SEQ ID NO: 77) SynSmFPS- TTTTTGTCTTTTATAGATTTTACC Linker-R (SEQ ID NO: 71)

[0158] FPP-001 competent cells were prepared according to the steps in above item 3. Then, the products recovered from gel obtained in the previous step were respectively added thereto, mixed well and transferred into an electric shock cup, electrically shocked at 2.7 kv for 5.7 ms, to which 1 mL of 1M sorbitol was added, and it was resuscitated at 30° C. for 1 h, and respectively spread onto SD-Ura-His-Leu medium and cultured at 30° C. for 36 h or more. The true positive clone was identified by PCR, and named as strains ELE-012 to ELE-019, respectively.

[0159] 6 Construction of Recombinant Strain ELE-020

[0160] 1) Preparation of P.sub.PGK1-ERG20-GGGS (SEQ ID NO: 15)-LsLTC2-T.sub.ADH1, P.sub.TEF1-SynSmFPS-GGGS (SEQ ID NO: 15)-STpGMAS-T.sub.CYC1, rDNA-TRP1-Up, and rDNA-TRP1-Down

[0161] P.sub.PGK1-ERG20-GGGS (SEQ ID NO: 15)-LsLTC2-T.sub.ADH1 and P.sub.TEF1-SynSmFPS-GGGS (SEQ ID NO: 15)-STpGMAS-T.sub.CYC1 were expression cassette carrying a fusion protein of yeast farnesyl pyrophosphate synthase and lettuce-derived germacrene A synthetase, and a fusion protein of codon-optimized Salvia miltiorrhiza-derived farnesyl pyrophosphate synthase and codon-optimized Tanacetum parthenium-derived germacrene A synthetase, respectively; and rDNA-TRP1-up and rDNA-TRP1-down were the upstream and downstream homologous arms of rDNA, respectively; the fragments were amplified according to the following methods: The functional modules were obtained by PCR using templates and primers described in Table 12, respectively:

TABLE-US-00014 M1 (rDNA-TRP1-up)□ M2 (P.sub.PGK1-ERG20-GGGS (SEQ ID NO: 15)- LsLTC2-T.sub.ADH1)□ M3 (P.sub.TEF1-SynSmFPS-GGGS (SEQ ID NO: 15)-STpGMAS-T.sub.CYC1)□ M4 (rDNA-TRP1-down).

[0162] The amplification system included: 5× Phusion HF Buffer 10 μL, dNTP (10 mM each dNTP) 1 μL, DNA template 20 ng, primers (10 μM) 1.5 μL for each, Phusion High-Fidelity DNA Polymerase (2.5 U/μL) 0.5 μL, and distilled water supplemented to a total volume of 50 μL. The amplification conditions were: pre-denaturation at 98° C. for 3 min (1 cycle); denaturation at 98° C. for 10 sec, annealing at 58° C. for 10 sec, extension at 72° C. for 2 min (30 cycles); and extension at 72° C. for 10 min (1 cycle). The product was recovered from gel and stored.

TABLE-US-00015 TABLE 12 Primers Amplifi- PCR cation tem- fragment Primer Primer sequence Module plate name name □ 5′.fwdarw.3′□ M1 pEASY- rDNA- X1-M- CTTGCAAATGCCTATTGTG rDNA- TRP1- pEASY- CAGATGTTATAATATCTGT TRP1 up r-t-F GCGTTTAATTAAGGCTCGT ATGTTGTGTGG AATTGT (SEEQ ID NO: 54) X1-r-t- CTCACTATTTTTTACTGCG R-rDNA GAAGCGG (SEEQ ID NO: 78) M2 pM2-ERG20- PPGK1- 1-M-pEASY- CTGTTTCCTGTGTGAAATT GGGS ERG20- PGK1-F GTTATCCGCTCACAATTCC (SEQ ID GGGS ACACAACATACGAGCCTT NO: 15)- (SEQ ID AATTAAACGCACAGATATT LsLTC2 NO: 15)- ATAAC LTC2-T.sub.ADH1 (SEEQ ID NO: 56) 1-M-ADHt- GGAGTAGAAACATTTTGAA TEF1-R GCTATGGTGTGTGGGGGA TCACTTTAATTAATCGGCA TGCCGGTAGAGGTG (SEEQ ID NO: 79) M3 pRS425- PTEF1- 2-M-ADHt- GGTATAGCATGAGGTCGC LEU2- SynSm FPS- TEF1-F TCTTATTGACCACACCTCT PTEF1- GGGS ACCGGCATGCCGATTAATT SynSm (SEQ ID AAAGTGATCCCCCA FPS- NO: 15)- (SEEQ ID NO: 80) GGGS STpGMAS- (SEQ T.sub.CYC1 ID NO: 15)- STpGMAS- T.sub.CYC1 M-CYC1- CGTATTACAATTCACTGGC pEASY-R CGTCGTTTTACAACGTCGT GACTGGGAAAACCCTGGC GCGTTGGCCGATTCATTAA TGC (SEEQ ID NO: 61) M4 pEASY- rDNA- X2-r-t-F- GAACTGGGTTACCCGGGG rDNA- TRP1- rDNA CACCTGTC TRP1 down (SEEQ ID NO: 81) X2-M-pEASY- CGAAGGCTTTAATTTGCAA r-1-R GCTGCGGCCCTGCATTAA TGAATCGGCCAACGCGCC AGGGTTTTCCCAGTCACG ACGTTG (SEEQ ID NO: 63)

[0163] Original strain Saccharomyces cerevisiae ELE-019 was cultured in a SD-Ura-His-Leu liquid medium overnight, followed by being prepared into competent cells. Then, the transformation fragments M1, M2, M3, and M4 in Table 12 were added in a total amount of 4 μg (molar ratio=1:1:1:1), mixed well and transferred into an electric shock cup, electrically shocked at 2.7 kv for 5.7 ms, to which 1 mL of 1M sorbitol was added, and it was resuscitated at 30° C. for 1 h, and spread onto SD-Ura-His-Leu-Trp medium and cultured at 30° C. for 36 h or more. The ingredients in the screening medium composition were: 0.8% yeast selective medium SD-Ura-His-Leu-Trp (Beijing FunGenome Technology Co., Ltd.), 2% glucose. The true positive clone was identified by PCR, and named as strain ELE-020.

[0164] This ELE-020 recombinant strain was deposited on Oct. 20, 2017 at the China General Microbiological Culture Collection Center, CGMCC. The deposition address was Building 3, No. 1 West Beichen Road, Chaoyang District, Beijing. The strain name was: Saccharomyces cerevisiae, the latin name thereof is: Saccharomyces cerevisiae; and the deposition number thereof was: CGMCC No. 14829.

[0165] The information of all the above engineering strains was shown in Table 13.

TABLE-US-00016 TABLE 13 Information of engineering strains Strain name Basic information Medium NK2-SQ P.sub.PGK1-tHMG1-T.sub.ADH1, P.sub.PDC1-ERG12-T.sub.ADH2, P.sub.ENO2-IDI1-T-.sub.PDC1, SD-Ura P.sub.PYK1-ERG19-T.sub.PGI1, P.sub.FBA1-ERG13- T.sub.TDH2, P.sub.TDH3-ERG8-T.sub.TPI1 and P.sub.TEF1-ERG10-T.sub.CYC1 and the screening marker of URA3 were integrated into GAL7 site of the chromosome of strain CEN. PK2-1D (MATaura3-52; trp1-289; leu2-3, 112; his3Δ1; MAL2-8C; SUC2) FPP-001 P.sub.PGK1-ADH2-T.sub.ADH1, P.sub.TEF1-ALD6-T.sub.CYC1, P.sub.TDH3-ACS1- SD-Ura-His T.sub.TPL1 and the screening marker of HIS3 were integrated into NDT80 site of the chromosome of strain NK2-SQ ELE-001 FPP-001 transferred with pRS313-LEU2-P.sub.TEF1-STpGMAS-T.sub.CYC1 SD-Ura-His-Leu ELE-002 FPP-001 transferred with pRS425-LEU2-P.sub.TEF1-STpGMAS-T.sub.CYC1 SD-Ura-His-Leu ELE-011 FPP-001 transferred with pRS425-LEU2-P.sub.TEF1- SD-Ura-His-Leu SynSmFPS-GGGS (SEQ ID NO: 15)-STpGMAS-T.sub.CYC1 ELE-012 FPP-001 transferred with pRS425-LEU2-P.sub.TEF1-SynSmFPS-3A001-STpGMAS-T.sub.CYC1 SD-Ura-His-Leu ELE-013 FPP-001 transferred with pRS425-LEU2-P.sub.TEF1-SynSmFPS-4A001-STpGMAS-T.sub.CYC1 SD-Ura-His-Leu ELE-014 FPP-001 transferred with pRS425-LEU2-P.sub.TEF1-SynSmFPS-5A002-STpGMAS-T.sub.CYC1 SD-Ura-His-Leu ELE-015 FPP-001 transferred with pRS425-LEU2-P.sub.TEF1-SynSmFPS-6A005-STpGMAS-T.sub.CYC1 SD-Ura-His-Leu ELE-016 FPP-001 transferred with pRS425-LEU2-P.sub.TEF1-SynSmFPS-6B004-STpGMAS-T.sub.CYC1 SD-Ura-His-Leu ELE-017 FPP-001 transferred with pRS425-LEU2-P.sub.TEF1-SynSmFPS-8A005-STpGMAS-T.sub.CYC1 SD-Ura-His-Leu ELE-018 FPP-001 transferred with pRS425-LEU2-P.sub.TEF1-SynSmFPS-12A003-STpGMAS-T.sub.CYC1 SD-Ura-His-Leu ELE-019 FPP-001 transferred with pRS425-LEU2-P.sub.MF1-SynSmFPS-8A005-STpGMAS-T.sub.CYC1 SD-Ura-His-Leu ELE-020 P.sub.PGK1-ERG20-GGGS (SEQ ID NO: 15)-LsLTC2-T.sub.ADH1, SD-Ura-His-Leu-Trp P.sub.TEF1-SynSmFPS-GGGS (SEQ ID NO: 15)- STpGMAS-T.sub.CYC1 and the screening marker of TRP1 were integrated into the rDNA site of the chromosome of strain ELE-019

Example 3: Application of Recombinant Strain in Producing β-Elemene

[0166] 1. Engineering Strain Culture and Product Extraction

[0167] All engineering yeast strains prepared in Example 2 were activated in the corresponding solid selective medium SD-Ura-His-Leu, and seed solutions were prepared in the corresponding liquid selective medium SD-Ura-His-Leu (30° C., 250 rpm, 16 h), inoculated in an amount of 1% into a 100 mL trigonal flask containing 15 mL of the corresponding liquid selective medium, shaken at 250 rpm and cultured at 30° C. for id. Then, 1.5 mL of n-dodecane was added thereto, and continued to be shaken and cultured for 5 d. Finally, the liquid in the trigonal flask was transferred to a 50 mL centrifuge tube, centrifuged at 5,000 rpm for 5 min, and the organic phase was collected for use.

[0168] 2. β-Elemene Conversion and its Qualitative and Quantitative Analyses

[0169] 1) β-Elemene Conversion

[0170] The above organic phase sample was heated in an oil bath at 100-380° C. (180° C.) within a fuming cupboard for 1 h to obtain a converted material.

[0171] 2) Detection

[0172] The converted material was diluted 10 times with n-hexane, filtered through an organic nylon membrane (0.22 μm), and detected by using GC-MS. Testing equipment: Agilent GCMSD Agilent 7890A/5975C; GC-MS measurement conditions: inlet temperature 250° C., injection volume 1 μL, splitless, solvent delay 3 min; column: HP-5 ms (30 m*0.25 mm); Chromatographic conditions: 45° C. for 1 min, warming up to 300° C. at 10° C./min and keeping for 5 min; MS conditions: Full Scan: 50-750 amu. Qualitative and quantitative analyses were carried out by using the standard of β-elemene, which was purchased from the China National Institutes for Food and Drug Control (Cat. No. 100268). FIG. 2 is a GC-MS test chromatomap of 3-elemene produced by all engineering yeast strains prepared in Example 2.

[0173] As a result, the yield of each engineering strain after fermentation for 6 days was as follows:

[0174] Engineering strains ELE-001 and ELE-002 were obtained by introducing low and high copy number of STpGMAS based on FPP-001. Wherein, the yield of β-elemene of ELE-001 reached 9.3 mg/L, and the yield of β-elemene of ELE-002 reached 22.1 mg/L;

[0175] Engineering strain ELE-011 was obtained by introducing high copy number of fusion protein gene SynSmFPS-GGGS (SEQ ID NO: 15)-STpGMAS based on FPP-001, and the yield of β-elemene reached 101.1 mg/L.

[0176] Engineering strains ELE-012 to ELE-019 (the promoters and linkers thereof were TEF1 and 3A001, TEF1 and 4A001, TEF1 and 5A002, TEF1 and 6A005, TEF1 and 6B004, TEF1 and 8A005, TEF1 and 12A003, MF1 and 8A005, respectively) were obtained by introducing high copy number of fusion protein gene SynSmFPS-Linker-STpGMAS based on FPP-001.

[0177] Engineering strain ELE-020 was obtained by the recombination and introduction of fusion protein genes P.sub.PGK1-ERG20-GGGS (SEQ ID NO: 15)-LsLTC2-T.sub.ADH1 and P.sub.TEF1-SynSmFPS-GGGS (SEQ ID NO: 15)-STpGMAS-T.sub.CYC1 based on ELE-019.

[0178] The yields of β-elemene produced by using strains ELE-012 to ELE-020 were 2.2 mg/L (relative to the culture solution), 35.5 mg/L, 110.4 mg/L, 108.6 mg/L, 73.6 mg/L, 109.7 mg/L, 48.3 mg/L, 158.1 mg/L and 469 mg/L, respectively.

[0179] 3. Bioreactor Fermentation Culture

[0180] 1) Medium Formulation

[0181] The calcium chloride mother liquid: 19.2 g/L aqueous solution of calcium chloride dihydrate.

[0182] The trace metal salt mother liquid: 19.1 g/L of disodium ethylenediamine tetraacetate, 10.2 g/L of zinc sulfate heptahydrate, 0.5 g/L of manganese chloride tetrahydrate, 0.86 g/L of cobalt chloride hexahydrate, 0.78 g/L of copper sulfate pentahydrate, 0.56 g/L of sodium molybdate dehydrate, and 5.12 g/L of iron sulphite heptahydrate.

[0183] The vitamin mother liquid: 0.05 g/L of biotin, 0.2 g/L of sodium p-aminobenzoate, 1 g/L of niacin, 1 g/L of calcium pantothenate, 1 g/L pyridoxine hydrochloride, 1 g/L of thiamine hydrochloride, and 25 g/L of inositol.

[0184] The seed medium and the fermentation medium: 25 g/L of glucose, 15 g/L of ammonium sulfate, 6.15 g/L of magnesium sulfate heptahydrate, 0.72 g/L of zinc sulfate heptahydrate, 8 g/L of potassium dihydrogen phosphate, 2 mL/L of calcium chloride mother liquid, 10 mL/L of trace metal salt mother liquid; 12 mL/L of vitamin mother liquid, 1 g/L of tryptophan, and the balance of water.

[0185] The fed-batch medium: 800 g/L of glucose, 5.125 g/L of magnesium sulfate heptahydrate, 3.5 g/L of potassium sulfate, 0.28 g/L of sodium sulfate, 9 g/L of potassium dihydrogen phosphate, 1 g/L of tryptophan, and the balance of water.

[0186] 2) Fermentation of Engineering Strain ELE-019

[0187] The engineering strain ELE-019 was activated according to the methods in item 1. The monoclonal colony on the plate was picked up and inoculated into a test tube containing SD-Ura-His-Leu medium, and shaken at 250 rpm and cultured at 30° C. overnight; 500 μL of the strain culture was pipetted into a 250 mL trigonal flask containing 50 mL of SD-Ura-His-Leu medium, and shaken at 250 rpm and cultured at 30° C. for 24 h.

[0188] 2 mL of the strain culture was respectively pipetted into three 1 L trigonal flasks containing 100 mL of seed medium, shaken at 250 rpm and cultured at 30° C. for 48 h; finally, the seed solution was inoculated into a 7 L fermentation tank containing 3 L of the fermentation medium via a flame inoculation loop (Eppendorf Company, Germany, model no.: BioFlo®320).

[0189] The parameters set in the fermentation process were: temperature 30° C., pH 5.0, dissolved oxygen 30%, air flow rate 3-20 L/min, stirring speed 300-1000 rpm; and dissolved oxygen were cascading with stirring speed and air flowing. When the dissolved oxygen value was greater than 60%, the fed-batch medium was added into the fermentation tank until the glucose concentration in the fermentation liquid was 5 g/L.

[0190] Three hours before the end of the fermentation, 10% (relative to the volume of the culture solution) of n-dodecane was added, and after the end of the fermentation, the organic phase was separated.

[0191] After the treatment carried out according to the conversion and detection methods in item 2, qualitative and quantitative analyses were performed. After high-density fermentation of the engineering strain ELE-019 for 96 hours, 2 g/L (relative to the culture solution) of β-elemene may be obtained. The recombinant strains complying with the object of the present invention, including but not limited to the specific experimental examples described in Table 13, may be subjected to a fermentation culture according to the fermentation methods described in item “3” to obtain germacrene A.

INDUSTRIAL APPLICATION

[0192] The experiments of the present invention verified that a recombinant strain can be obtained by expressing germacrene A synthetase gene or fusion protein gene thereof in a host yeast in the present invention, which can greatly improve the yield of germacrene A. It is suitable for industrial production of β-elemene and/or germacrene A, and provides a potent strain and research basis for the biosynthesis of anti-cancer raw material β-elemene.