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 -elemene and/or germacrene A.

Claims

1. A recombinant yeast strain, comprising: a. germacrene A synthetase; or b. a fusion protein comprising germacrene A synthetase and farnesyl pyrophosphate synthase, wherein said recombinant yeast strain has been modified to have an increased content and/or activity of alcohol dehydrogenase, acetaldehyde dehydrogenase and acetyl-CoA synthetase as compared to original yeast prior to modification.

2-20. (canceled)

21. The recombinant yeast strain of claim 1, wherein said recombinant yeast strain comprises said fusion protein, and said fusion protein is encoded by one or more nucleic acids encoding the germacrene A synthetase and one or more nucleic acids encoding the farnesyl pyrophosphate synthase.

22. The recombinant yeast strain of claim 21, wherein said fusion protein is encoded by at least two nucleic acids encoding the germacrene A synthetase and at least two nucleic acids encoding the farnesyl pyrophosphate synthase and, wherein the at least two nucleic acids encoding the germacrene A synthetase are different or the same, and the at least two nucleic acids encoding the farnesyl pyrophosphate synthase are different or the same.

23. The recombinant yeast strain of claim 21, wherein said germacrene A synthetase is encoded by: a nucleic acid represented by SEQ ID NO:3 or a nucleic acid represented by positions 13-1686 of SEQ ID NO:12; and said farnesyl pyrophosphate synthase is encoded by: a nucleic acid represented by SEQ ID NO:2 or a nucleic acid represented by positions 1-1056 of SEQ ID NO:11.

24. The recombinant yeast strain of claim 1, wherein the fusion protein further comprises a linker peptide linking the germacrene A synthetase with the farnesyl pyrophosphate synthase.

25. The recombinant yeast strain of claim 24, wherein the linker peptide is selected from GGGS, YGQ, PGGH, YRSQI, VIPFIS, FLYLKF, WRFSPKLQ or HHVQESQCISTV.

26. The recombinant yeast strain of claim 1, wherein said yeast strain comprises a nucleic acid encoding the germacrene A synthetase or a nucleic acid encoding the fusion protein.

27. The recombinant yeast strain of claim 26, wherein the nucleic acid encoding the germacrene A synthetase or the fusion protein is contained in an expression cassette.

28. The recombinant yeast strain of claim 27, wherein the expression cassette further comprises a promoter and a terminator.

29. The recombinant yeast strain of claim 28, wherein the promoter is selected from TEF1, MF1 or PGK1 and the terminator is CYC1 or ADH1.

30. The recombinant yeast strain of claim 1, wherein the recombinant yeast strain further expresses one or more marker genes.

31. The recombinant yeast strain of claim 30, wherein the marker gene is selected from his3 or trp1.

32. The recombinant yeast strain of claim 27, wherein the expression cassette is contained in a vector.

33. The recombinant yeast strain of claim 27, wherein the expression cassette is contained in a plasmid or is integrated into a chromosome of said yeast strain.

34. The recombinant yeast strain of claim 1, wherein said yeast strain comprises an increased copy number of a nucleic acid encoding the alcohol dehydrogenase, a nucleic acid encoding the acetaldehyde dehydrogenase and a nucleic acid encoding the acetyl-CoA synthetase, as compared to the original yeast prior to modification.

35. The recombinant yeast strain of claim 34, wherein said yeast strain comprises an expression cassette configured to increase the copy number of said nucleic acid encoding the alcohol dehydrogenase, an expression cassette configured to increase the copy number of said nucleic acid encoding the acetaldehyde dehydrogenase, an expression cassette configured to increase the copy number of said nucleic acid encoding the acetyl-CoA synthetase, and a marker gene introduced by homologous recombination.

36. The recombinant yeast strain of claim 1, wherein the original yeast is Saccharomyces cerevisiae.

37. The recombinant yeast strain of claim 36, wherein said Saccharomyces cerevisiae is Saccharomyces cerevisiae NK2-SQ.

38. The recombinant yeast strain of claim 36, wherein said recombinant yeast strain is Saccharomyces cerevisiae CGMCC No.14829.

39. A method of producing germacrene A, comprising fermenting the recombinant yeast strain of claim 1 to obtain germacrene A.

40. A method of producing -elemene, comprising: a. fermenting the recombinant yeast strain of claim 1 to obtain a fermentation product; b. extracting the fermentation product with an organic solvent, and collecting the organic phase; and c. heating the organic phase of step b to obtain -elemene.

41. The method of claim 40, wherein the fermentation of step a comprises: first, culturing the recombinant strain in a seed medium to obtain a seed liquid; second, inoculating the seed liquid into a fermentation medium and conducting fermentation culture; and third, generating a product of the fermentation culture, which is named as a fermentation system.

42. The method of claim 41, wherein during the fermentation culture, a fed-batch medium is added into the fermentation system.

43. The method of claim 42, wherein when the dissolved oxygen value in the fermentation system is greater than 60%, a fed-batch medium is added into the fermentation system until glucose concentration in the fermentation system reaches 5 g/L.

44. The method of claim 41, wherein a formulation of the seed medium and the fermentation medium contains per L volume: 25 g of glucose, 15 g of ammonium sulfate, 6.15 g of magnesium sulfate heptahydrate, 0.72 g of zinc sulfate heptahydrate, 8 g of potassium dihydrogen phosphate, 2 mL of calcium chloride mother liquid, 10 mL of trace metal salt mother liquid; 12 mL of vitamin mother liquid, and lg of tryptophan, wherein the calcium chloride mother liquid is 19.2 g/L aqueous solution of calcium chloride dehydrate, wherein the trace metal salt mother liquid contains per L volume: 19.1 g of disodium ethylenediamine tetraacetate; 10.2 g of zinc sulfate heptahydrate; 0.5 g of manganese chloride tetrahydrate; 0.86 g of cobalt chloride hexahydrate; 0.78 g of copper sulfate pentahydrate; 0.56 g of sodium molybdate dihydrate; and 5.12 g of iron sulphite heptahydrate, wherein the vitamin mother liquid contains per L volume: 0.05 g of biotin; 0.2 g of sodium p-aminobenzoate; 1 g of niacin; 1 g of calcium pantothenate; 1 g pyridoxine hydrochloride; 1 g of thiamine hydrochloride; and 25 g of inositol.

45. The method of claim 42, wherein the fed-batch medium contains per L volume: 800 g of glucose, 5.125 g of magnesium sulfate heptahydrate, 3.5 g of potassium sulfate, 0.28 g of sodium sulfate, 9 g of potassium dihydrogen phosphate and lg of tryptophan.

46. The method of claim 40, wherein: the organic solvent is n-dodecane; and the heating is at 100-380 C. for 1 hour.

Description

BRIEF DESCRIPTION OF THE DRAWING

[0106] FIG. 1 shows the germacrene A biosynthetic pathway.

[0107] FIG. 2 is a GC-MS test chromatomap.

DETAILED DESCRIPTION OF THE INVENTION

[0108] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0109] Unless otherwise specified, the materials, reagents and the like used in the following examples are commercially available.

[0110] FIG. 1 shows the germacrene A biosynthetic pathway.

EXAMPLE 1

Preparation of Target Genes and Plasmids Used

[0111] 1. Preparation of Target Genes

[0112] (1) Acquisition of ADH2, ALD6, ASC1, MF1, TEF1 and CYC1

[0113] 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 CYC1.

[0114] PCR amplification kit TAKARA PrimeSTARHS DNApolymerase was used to formulate an amplification system (TAKARA). The amplification system included: 5PS Buffer 10 L, dNTPMix 4 L, primers 1 L for each, genomic DNA template 1 L, PrimeSTARHS polymerase (2.5 U/L) 0.5 L, distilled water supplemented to a total volume of 50 L. The amplification conditions were: pre-denaturation at 98 C. for 3 minutes (1 cycle); denaturation at 98 C. for 10 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-00001 TABLE1 Primersequences Genefragment Primername Primersequence(5.fwdarw.3) ADH2 SexA1-ADH2 GCGACCWGGTATGTCTATTCCAGAAACTCAAAAAGC ADH2-Asc1 GCGGCGCGCCTTATTTAGAAGTGTCAACAACGTATC ALD6 SexA1-ALD6 TCGCGACCWGGTAAAACAATGACTAAGCTACACTTTGAC ALD6-Asc1 TCGCGGCGCGCCTTACAACTTAATTCTGACAGCT ACS1 SexA1-Asc1 TCGCGACCWGGTAAAACAATGTCGCCCTCTGCCGTACAATC ACS1-Asc1 TCGCGGCGCGCCTTACAACTTGACCGAATCAATTAG TEF1 Sac11-TEF1 GCGCCGCGGAGTGATCCCCCACACACCATAGCTT TEF1-SexA1 TGGCGACCWGGTTTTGTAATTAAAACTTAGATTAGA MF1 BamH1-pMF1 GCGGGATCCGGGAAGACATGCTTAACAAGAAGAT pMF1-SexA1 GCGACCTGGTTCTTTTAATCGTTTATATTGTGTAT CYC1 Asc1-CYC1 GCGGCGCGCCCCGCTGATCCTAGAGGGCCGCATCA CYC1-Sac11 GCGCCGCGGGCGCGTTGGCCGATTCATTAATGCA

[0115] (2) Acquisition of Farnesyl Pyrophosphate Synthase Gene SynSmFPS from Salvia miltiorrhiza and Germacrene A Synthetase Gene STpGMA from Tanacetum parthenium

[0116] 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 SynSmFPSgene and STpGMASgene were constructed, respectively.

[0117] (3) Acquisition of Farnesyl Pyrophosphate Synthase Gene ERG20-GGGS from Yeast and Germacrene A Synthetase Gene GGGS-LsLTC2 from Lettuce

[0118] 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 L 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 L 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 L of milliQ DEPC-treated water was added to fully dissolve the precipitate, and stored at 70 C.

[0119] 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 L, dNTP 1 L, total RNA 1 L (200 ng), H.sub.2O 6 L, Total 10 L; 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 L, RNAs Inhibiter 0.5 L, R-Transcription 1 L, H.sub.2O 4.5 L; 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.

[0120] 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 (the one of positions 13-1686 in SEQ ID NO.11 was ERG20) and 1688 bp of GGGS-LsLTC2 (the one of positions 1-1056 in SEQ.ID NO.12 was LsLTC2).

[0121] 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: 5Phusion HF Buffer 10 L, dNTP (10 mM each dNTP) 1 L, DNA template 20 ng, primers (10 M) 1.5 L for each, Phusion High-Fidelity DNA Polymerase (2.5 U/L) 0.5 L, and distilled water supplemented to a total volume of 50 L. 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 1 min (30 cycles); and extension at 72 C. for 10 min (1 cycle).

TABLE-US-00002 TABLE2 Primersequences Genefragment Primername Primersequence(5.fwdarw.3) ERG20-GGGS SEXA1-ERG20 GCGACCWGGTAAAACAATGGCTTCAGAAAAAGAAATTAGGAG ERG20-GGGS CTTTCCCATAGAACCACCACCCTATTTGCTTCTCTTGTAAACTTTG GGGS-LSLTC2 GGGS-LSLTC2 GGTGGTGGTTCTATGGCAGCAGTTGACACTAA LSLTC2-ASC1 GCGGGCGCGCCTTACATGGTACAGAACCAACAAAT

[0122] 2. Construction of Recombinant Plasmids

[0123] (1) Plasmid pM2-ADH2

[0124] 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.

[0125] (2) Plasmid pM4-ACS1

[0126] 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.

[0127] (3) Plasmid pM3-ALD6

[0128] 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.

[0129] (4) Construction of Plasmids pRS313-LEU2-P.sub.TEF1-STpGMAS-T.sub.CYC1 and pRS425-LEU2-P.sub.TEF1-STpGMAS-T.sub.CYC1

[0130] 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;

[0131] 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;

[0132] pUC57-STpGMAS was enzyme digested by using SexA1 and Asc1, and 1694 bp of STpGMAS was recovered.

[0133] 50 ng of each of the enzyme-digested products TEF1, CYC1 and STpGMAS was added into a ligation system including: 2 L of 10T4 DNA Ligase Reaction Buffer (NEB), 1 L of T4 DNA Ligase (NEB, 400,000 cohesive end units/ml), distilled water supplemented to 20 L; they reacted at room temperature for 2 hours to obtain a ligation product.

[0134] 1 L of the ligation product was added into a PCR system (Phusion High-Fidelity PCR Master Mix with HF Buffer kit, NEB) including: 5Phusion HF Buffer 10 L, dNTP (10 mM each dNTP) 1 L, DNA template 20 ng, and primers Sac11-TEF1 and CYC1-Sac11 (10 M) in Table 3, 1.5 L for each, Phusion High-Fidelity DNA Polymerase (2.5 U/L) 0.5 L, and distilled water supplemented to a total volume of 50 L. 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.

[0135] The amplification product was purified, and then enzyme digested by using SacII. The target fragment SacII-TEF1-STpGMAS-CYC1-SacII was recovered from gel, and prepared to use.

[0136] 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 L of NEB buffer and 1 L of CIP dephosphorylation enzyme (NEB) were then added, and distilled water was supplemented to 40 L; it was treated at 37 C. for 1 h, and EDTA with the final concentration of 10 mol 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.

[0137] 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 L 10T4 DNA Ligase Reaction Buffer (NEB)), 1 L T4 DNA Ligase (NEB, 400,000 cohesive end units/ml), distilled water supplemented to 20 L; 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.

[0138] 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. Using pRS425 as a template, LEU2 (1808 bp) was amplified by using the primers in Table 3.

[0139] The amplification system included: 5Phusion HF Buffer 10 L, dNTP (10 mM each dNTP) 1 L, DNA template 20 ng, primers (10 M) 1.5 L for each, Phusion High-Fidelity DNA Polymerase (2.5 U/L) 0.5 L, and distilled water supplemented to a total volume of 50 L. 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).

[0140] The target fragment was purified from gel. 2 L of 10T4 DNA Ligase Reaction Buffer (NEB) and 1 L of T4 Polynucleotide kinase (NEB) were added into the product of LEU2 fragment, and distilled water was supplemented to a total volume of 20 L. 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-00003 TABLE3 Primersequences Genefragment Primername Primersequence(5.fwdarw.3) TEF1-STpGMAS- Sac11-TEF1 GCGCCGCGGAGTGATCCCCCCACACACCATAGCTT CYC1 CYC1-SAC11 GCGCCGCGGGCGCGTTGGCCGATTCATTAATGCA pRS313-TEF1- V313-to-R CTTTGCCTTCGTTTATCTTGC STpGMAS-CYC1 V313-to-F TATATGTATACCTATGAATGTCAG LEU2 Bsp-Leu-F TGGcgTCCGGATTAAGCAAGGATTTTCTTAACTTCTTC Bsp-Leu-F TGGcgTCCGGAGATGCGGTATTTTCTCCTTACGCA

[0141] (5) Construction of Plasmid pRS425-LEU2-P.sub.TEF1-SynSmFPS-GGGS-STpGMAS-T.sub.CYC1

[0142] Using pUC57-SynSmFPS and pUC57-STpGMAS as templates, 1080 bp of SynSmFPS-GGGS and 1704 bp of GGGS-STpGMAS were obtained by amplification using the primers in Table 4.

[0143] The amplification system included: 5Phusion HF Buffer 10 L, dNTP (10 mM each dNTP) 1 L, DNA template 20 ng, primers (10 M) 1.5 L for each, Phusion High-Fidelity DNA Polymerase (2.5 U/L) 0.5 L, and distilled water supplemented to a total volume of 50 L. 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).

[0144] SynSmFPS-GGGS and GGGS-STpGMAS were used together as templates, and 2767 bp of SynSmFPS-GGGS-STpGMAS fragment was obtained by amplification using the primers in Table 4 (SexA1-SynSmFPS and STpGMAS-Asc1).

[0145] The amplification system included: 5Phusion HF Buffer 10 L, dNTP (10 mM each dNTP) 1 L, DNA templates SynSmFPS-GGGS and GGGS-STpGMAS 20 ng for each, primers (10 M) 1.5 L for each, Phusion High-Fidelity DNA Polymerase (2.5 U/L) 0.5 L, and distilled water supplemented to a total volume of 50 L. 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).

[0146] The amplification product was purified, and then enzyme digested with SexA1 and Asc1, and the target fragment SexA1-SynSmFPS-GGGS-STpGMAS-Asc1 (2760 bp) was recovered from gel, and prepared to use.

[0147] 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-STpGMAS-Asc1 was added into the ligation system including: 2 L 10T4 DNA Ligase Reaction Buffer (NEB), 1 L T4 DNA Ligase (NEB, 400,000 cohesive end units/ml), and distilled water supplemented to 20 L; 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-STpGMAS-T.sub.CYC1 was obtained.

TABLE-US-00004 TABLE4 Primersequences Genefragment Primername Primersequence(5.fwdarw.3) SynSm-FPS- SEXA1-SynSmFPS ACCTGGTAAAACAATGGCTAATTTGAATG GGGS GTGAATC SynSmFPS-GGGS TGCTGCCATAGAACCACCACCTTTTTGTC TTTTATAGATTTTACC GGGS-STpGMAS GGGS-STpGMAS GGTGGTGGTTCTATGGCAGCAGTACAAG CAACCAC STpGMAS-Asc1 GGCGCGCCTCAGACTGGCAAGGAATCTA SynSmFPS- SexA1-SynSmFPS ACCTGGTAAAACAATGGCTAATTTGAATG GGGS-STpGMAS GTGAATC STpGMAS-Asc1 GGCGCGCCTCAGACTGGCAAGGAATCTA

[0148] (6) Construction of Plasmid pRS425-LEU2-P.sub.MF1-SynSmFPS-GGGS-STpGMAS-T.sub.CYC1

[0149] MF1 obtained in the above 1. Preparation of target genes and plasmid pRS425-LEU2-P.sub.TEF1-SynSmFPS-GGGS-STpGMAS-T.sub.CYC1 constructed in the above item (5) were double enzyme digested by using BamH1 (purchased from TaKaRa) and SexA1, respectively. 814 bp target promoter gene MF1 and 9898 bp vector fragment pRS425-LEU2- . . . -SynSmFPS-GGGS-STpGMAS-T.sub.CYC1 were purified from gel and the two (50 ng for each) were added into a ligation system including: 2 L 10T4 DNA Ligase Reaction Buffer (NEB), 1 L T4 DNA Ligase (NEB, 400,000 cohesive end units/ml), and distilled water supplemented to 20 L; 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-STpGMAS-T.sub.CYC1.

[0150] (7) Construction of Plasmid pM2-ERG20-GGGS-LsLTC2

[0151] Using ERG20-GGGS and GGGS-LsLTC2 together as templates, an ERG20-GGGS-LsLTC2 fragment of about 2744 bp was obtained by amplification using the primers (SexA1-ERG20 and LsLTC2-Asc1) in Table 5.

[0152] The amplification system included: 5Phusion HF Buffer 10 L, dNTP (10 mM each dNTP) 1 L, DNA templates ERG20-GGGS and GGGS-LsLTC2 20 ng for each, primers (10 M) 1.5 L for each, Phusion High-Fidelity DNA Polymerase (2.5 U/L) 0.5 L, and distilled water supplemented to a total volume of 50 L. 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).

[0153] The amplification product was purified, and then enzyme digested with SexA1 and Asc1, and the target fragment SexA1-ERG20-GGGS-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-LsLTC2.

TABLE-US-00005 TABLE5 Primersequences Genefragment Primername Primersequence(5.fwdarw.3) ERG20-GGGS SEXA1-ERG20 GCGACCWGGTAAAACAATGGCTTCAGAAAAAGAAATTAGGAG ERG20-GGGS CTTTCCCATAGAACCACCACCCTATTTGCTTCTCTTGTAAACTTTG GGGS-LSLTC2 GGGS-LSLTC2 GGTGGTGGTTCTATGGCAGCAGTTGACACTAA LSLTC2-ASC1 GCGGGCGCGCCTTACATGGTACAGAACCAACAAAT ERG20-GGGS- SEXA1-ERG20 GCGACCWGGTAAAACAATGGCTTCAGAAAAAGAAATTAGGAG STPGMAS LSLTC2-ASC1 GCGGGCGCGCCTTACATGGATACAGAACCAACAAAT

[0154] (8) Construction of Plasmid pEASY-NDT80-HIS3

[0155] 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.

[0156] The amplification system included: 5Phusion HF Buffer 10 L, dNTP (10 mM each dNTP) 1 L, DNA template 20 ng, primers (10 M) 1.5 L for each, Phusion High-Fidelity DNA Polymerase (2.5 U/L) 0.5 L, and distilled water supplemented to a total volume of 50 L. 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).

[0157] 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-00006 TABLE6 Primers Genefragment Primername Template Primersequence(5.fwdarw.3) NDT NDT80-up- GenomicDNA GCGGTTTAAACGTTCGACCATATTGATGAAGAGT PmeI ofNK2-SQ GGGTAGG NDT8-down CTGTTCCATTGATTTCTTCTCTATTGTTATATC HIS3 Bsp-HIS-F pRS313 TGGCCGTCCGGATCGCGCGTTTCGGTGATGACGG Pme1-HIS-R GCGGTTTAAACGTGTCACTACATAAGAACACCT

[0158] 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 L NEB buffer (reaction buffer, purchased from NEB (Beijing) Co., Ltd.) and 1 L CIP dephosphorylation enzyme (NEB) were added, and distilled water was supplemented to 40 L; it was treated at 37 C. for 1 h, to which EDTA at a final concentration of 10 mol 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.

[0159] HIS3 (30 ng) was purified from gel, 4 L of 10T4 DNA Ligase Reaction Buffer (NEB) and 1 L of T4 Polynucleotide kinase (NEB) were added, and distilled water was supplemented to 40 L, 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.

[0160] The information of plasmids constructed above was shown in Table 7 below:

TABLE-US-00007 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- Containing P.sub.TEF1-SynSmFPS-T.sub.CYC1 STpGMAS-T.sub.CYC1 cassette, LEU2, low-copy plasmid pRS425-LEU2-P.sub.TEF1- Containing P.sub.TEF1-SynSmFPS-T.sub.CYC1 STpGMAS-T.sub.CYC1 cassette, LEU2, high-copy plasmid pRS425-LEU2-P.sub.TEF1- Containing P.sub.TEF1-SynSmFPS-GGGS- SynSmFPS-GGGS- STpGMAS-T.sub.CYC1 cassette, STpGMAS-T.sub.CYC1 LEU2, high-copy plasmid pRS425-LEU2-P.sub.MF1- Containing P.sub.MF1-SynSmFPS-GGGS- SynSmFPS-GGGS- STpGMAS-T.sub.CYC1 cassette, STpGMAS-T.sub.CYC1 LEU2, high-copy plasmid pEASY-NDT80-HIS3 NDT80, HIS3

[0161] (9) Construction of Plasmid pEASY-rDNA-TRP1

[0162] 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.

[0163] The amplification system included: 5Phusion HF Buffer 10 L, dNTP (10 mM each dNTP) 1 L, DNA template 20 ng, primers (10 M) 1.5 L for each, Phusion High-Fidelity DNA Polymerase (2.5 U/L) 0.5 L, and distilled water supplemented to a total volume of 50 L. 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) .

[0164] 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-00008 TABLE8 Primers Genefragment Primername Template Primersequence(5.fwdarw.3) rDNA rDNA-up-F GenomicDNA ATGAGAGTAGCAAACGTAAGTCT rDNA-R-PmeI ofNK2-SQ GCGGTTTAAACTTTCCTCTAATCAGGTTC CACCA TRP1 SSP-TRP1-F pRS314 TGGCGTCCGGATACAATCTTGATCCGGA GCT BSP-TRP1-F TGGCGTCCGGACACAAACAATACTTAAA TAAATAC

[0165] pEASY-rDNA was enzyme digested by using PmeI, and 5122 bp target fragment (30 ng) was purified from gel, 4 L NEB buffer and 1 L CIP dephosphorylation enzyme (NEB) was added, and distilled water supplemented to a total volume of 40 L; it was treated at 37 C. for 1 h, to which EDTA at a final concentration of 10 mol 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.

[0166] TRP1 (30 ng) was purified from gel, 4 L of 10T4 DNA Ligase Reaction Buffer (NEB) and 1 L of T4 Polynucleotide kinase (NEB) were added, and distilled water was supplemented to a total volume of 40 L, 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

[0167] 1. Preparation of Yeast Competent Cells

[0168] 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 1 min 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, pH 7.5), 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 L.

[0169] 2. Construction of Strain FPP-001

[0170] 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

[0171] 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:

[0172] 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).

[0173] The amplification system included: 5Phusion HF Buffer 10 L, dNTP (10 mM each dNTP) 1 L, DNA template 20 ng, primers (10 M) 1.5 L for each, Phusion High-Fidelity DNA Polymerase (2.5 U/L) 0.5 L, and distilled water supplemented to a total volume of 50 L. 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-00009 TABLE9 Primers Amplification module PCRtemplate fragmentname Primername Primersequence(5.fwdarw.3) M1 pEASY-NDT80- NDT80-HIS3-up X1-M-pEASY-r-t-F CTTGCAAATGCCTATTGT HIS3 GCAGATGTTATAATATCCT GTGCGTTTAATTAAGGCCT CGTATGTTGTGTGGAATT GT NDT80-interg-2 CTGGCTTTAAAAAATGGA TAAAAAGGGATG M2 pM2-ADH2 P -ADH2-T -M-pEASY-PGK1-F CTGTTTCCTGTGTGAAAT TGTTATCCGCTCACAATT CCACACAACATACGAGCC TTAATTAAACGCACAGAT ATTATAAC 3G-1-M-ADH -TDH3- CCTCCGCGTCATTAAACT R TCTTGTTGTTGACGCTAA CATTCAACGCTAGTATTC GGCATGCCGGTAGAGGTG TGG M3 pM4-ACS1 P -ACS1-T 3G-3-M-ADH -TDH3- CAGGTATAGCATGAGGTC F GCTCTTATTGACCACACC TCTACCGGCATGCCGAAT ACTAGCGTTGAATGTTAG CGTC 3G-3-M- -TEF1-R AGGAGTAGAAACATTTTG AAGCTATGGTGTGTGGGG GATCACTTTAATTAATCT ATATAACAGTTGAAATTT GGA M4 pM3-ALD6 P -ALD6-T 3G-2-M- -TEF1-F GTCATTTTCGCGTTGAGA AGATGTTCTTATCCAAAT TTCAACTGTTATATAGAT TAATTAAAGTGATCCCCC ACAC M-CYC1-pEASY-R CGTATTACAATTCACTGG CCGTCGTTTTACAACGTC GTGACTGGGAAAACCCTG GCGCGTTGGCCGATTCAT TAATGC M5 pEASY-NDT80- NDT80-HIS3- NDT80-interg-1 CATCATAAGGAATTCCGG HIS3 down GATTCTCCCCAT X2-M-pEASY-r-t-R CGAAGGCTTTAATTTGCA AGCTGCGGCCCTGCATTA ATGAATCGGCCAACGCGC CAGGGTTTTCCCAGTCAC GACGTTG indicates data missing or illegible when filed

[0174] 2) Construction of Strain FPP-001

[0175] 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 g (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.

[0176] 3 Construction of Strains ELE-001 and ELE-002

[0177] 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.

[0178] 4 Construction of Strain ELE-011

[0179] FPP-001 competent cells were prepared according to the steps in the above item 3. Then, plasmid pRS425-LEU2-P.sub.TEF1-SynSmFPS-GGGS-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.

[0180] 5 Construction of Strains ELE-012 to ELE-019

[0181] Using plasmid pRS425-LEU2-P.sub.TEF1-SynSmFPS-GGGS-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 of the fusion protein SynSmFPS-GGGS-STpGMAS in the vector were replaced with 3A001, 4A001, 5A002, 6A005, 6B004, 8A005, 12A003, respectively (as shown in Table 10).

[0182] Using plasmid pRS425-LEU2-P.sub.MF1-SynSmFPS-GGGS-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 of the fusion protein SynSmFPS-GGGS-STpGMAS in the vector was replaced with 8A005.

[0183] Table 10 Showing the nucleotide sequences and amino acid sequences of linker peptides

TABLE-US-00010 Linker Aminoacidsequence peptidename Nucleotidesequence(5.fwdarw.3) oflinkerpeptide 3A001 TACGGTCAG YGQ 4A001 CCGGGGGGACAC PGGH 5A002 TATAGAAGTCAAATC YRSQI 6A005 GTGATACCTTTTATTTCA VIPFIS 6B004 TTTTTGTATCTTAAGTTT FLYLKF 8A005 TGGCGGTTCTCGCCGAAGCTTCAG WRFSPKLQ 12A003 CACCACGTGCAGGAGTCACAATGTATTTCCACAGTG HHVQESQCISTV

[0184] The specific reaction conditions were as follows:

[0185] The above amplification system included: 5Phusion HF Buffer 10 L, dNTP (10 mM each dNTP) 1 L, DNA template 20 ng, primers (as shown in Table 11) (10 M) 1.5 L for each, Phusion High-Fidelity DNA Polymerase (2.5 U/L) 0.5 L, and distilled water supplemented to a total volume of 50 L. 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).

[0186] The amplification product was digested by using DpnI enzyme from Fermentas Company after being purified. The system thereof included: 5Fast Digest Green Buffer 4 L, purified product 34 L, DpnI 2 L. The enzyme digestion temperature and reaction time were 37 C. and 1 h, respectively. Finally, it was recovered from gel and stored.

TABLE-US-00011 TABLE11 LinkerPeptide Primername Primersequence(5.fwdarw.3) 3A001 50bp3A001STpGmA CAAGCAGTTTTGAAATCATTTTTGGGTAAAATCTATAAAAGACA AAAATACGGTCAGATGGCAGCAGTACAAGCAACCAC SynSmFPSLinkerR TTTTTGTCTTTTATAGATTTTACC 4A001 50bp4A001STpGmA CAAGCAGTTTTGAAATCATTTTTGGGTAAAATCTATAAAAGACA AAAACCGGGGGGACACATGGCAGCAGTACAAGCAACCAC SynSmFPSLinkerR TTTTTGTCTTTTATAGATTTTACC 5A002 50bp5A001STpGmA CAAGCAGTTTTGAAATCATTTTTGGGTAAAATCTATAAAAGACA AAAATATAGAAGTCAAATCATGGCAGCAGTACAAGCAACCAC SynSmFPSLinkerR TTTTTGTCTTTTATAGATTTTACC 6A005 50bp6A005STpGmA CAAGCAGTTTTGAAATCATTTTTGGGTAAAATCTATAAAAGACAA AAAGTGATACCTTTTATTTCAATGGCAGCAGTACAAGCAACCAC SynSmFPSLinkerR TTTTTGTCTTTTATAGATTTTACC 6B004 50bp6B004STpGmA CAAGCAGTTTTGAAATCATTTTTGGGTAAAATCTATAAAAGACAA AAATTTTTGTATCTTAAGTTTATGGCAGCAGTACAAGCAACCAC SynSmFPSLinkerR TTTTTGTCTTTTATAGATTTTACC 8A005 50bp8A005STpGmA CAAGCAGTTTTGAAATCATTTTTGGGTAAAATCTATAAAAGACA AAAATGGCGGTTCTCGCCGAAGCTTCAGATGGCAGCAGTACA AGCAACCAC SynSmFPSLinkerR TTTTTGTCTTTTATAGATTTTACC 12A003 50bp12A003STpGmA CAAGCAGTTTTGAAATCATTTTTGGGTAAAATCTATAAAAGACA AAAACACCACGTGCAGGAGTCACAATGTATTTCCACAGTGATG GCAGCAGTACAAGCAACCAC SynSmFPSLinkerR TTTTTGTCTTTTATAGATTTTAGG

[0187] 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.

[0188] 6 Construction of Recombinant Strain ELE-020

[0189] 1) Preparation of P.sub.PGK1-ERG20-GGGS-LsLTC2-T.sub.ADH1, P.sub.TEF1-SynSmFPS-GGGS-STpGMAS-T.sub.CYC1, rDNA-TRP1-Up, and rDNA-TRP1-Down

[0190] P.sub.PGK1-ERG20-GGGS-LsLTC2-T.sub.ADH1 and P.sub.TEF1-SynSmFPS-GGGS-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:

[0191] The functional modules were obtained by PCR using templates and primers described in Table 12, respectively:

[0192] M1 (rDNA-TRP1-up),

[0193] M2 (P.sub.PGK1-ERG20-GGGS-LsLTC2-T.sub.ADH1),

[0194] M3 (P.sub.TEF1-SynSmFPS-GGGS-STpGMAS-T.sub.CYC1),

[0195] M4 (rDNA-TRP1-down).

[0196] The amplification system included: 5Phusion HF Buffer 10 L, dNTP (10 mM each dNTP) 1 L, DNA template 20 ng, primers (10 M) 1.5 L for each, Phusion High-Fidelity DNA Polymerase (2.5 U/L) 0.5 L, and distilled water supplemented to a total volume of 50 L. 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-00012 TABLE12 Primers Amplification Module PCRtemplate fragmentname Primername Primersequence(5-3) M1 pEASY-rDNA- rDNA-TRP1-up X1-M-pEASY-r-t-F CTTGCAAATGCCTATTGTGCA TRP1 GATGTTATAATATCTGTGCGTT TAATTAAGGCTCGTATGTTGT GTGGAATTGT X1-r-t-R-rDNA CTCACTATTTTTTACTGCGGA AGCGG M2 pM2-ERG20- P.sub.PGK1-ERG20- 1-M-pEASY-PGK1-F CTGTTTCCTGTGTGAAATTGTT GGGS-LsLTC2 GGGS-LTC2- ATCCGCTCACAATTCCACACA T.sub.ADH1 ACATACGAGCCTTAATTAAAC GCACAGATATTATAAC 1-M-ADHt-TEF1-R GGAGTAGAAACATTTTGAAG CTATGGTGTGTGGGGGATCAC TTTAATTAATCGGCATGCCGG TAGAGGTG M3 pRS425-LEU2- P.sub.TEF1-SynSmFPS- 2-M-ADHt-TEF1-F GGTATAGCATGAGGTCGCTCT P.sub.TEF1- GGGS- TATTGACCACACCTCTACCGG SynSmFPS- STpGMAS-T.sub.CYC1 CATGCCGATTAATTAAAGTGA GGGS- TCCCCCA STpGMAS- M-CYC1-pEASY-R CGTATTACAATTCACTGGCCG T.sub.CYC1 TCGTTTTACAACGTCGTGACT GGGAAAACCCTGGCGCGTTG GCCGATTCATTAATGC M4 pEASY-rDNA- rDNA-TRP1- X2-r-t-F-rDNA GAACTGGGTTACCCGGGGCA TRP1 down CCTGTC X2-M-pEASY-r-t-R CGAAGGCTTTAATTTGCAAG CTGCGGCCCTGCATTAATGAA TCGGCCAACGCGCCAGGGTT TTCCCAGTCACGACGTTG

[0197] 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 g (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.

[0198] 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.

[0199] The information of all the above engineering strains was shown in Table 13.

TABLE-US-00013 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, SD-Ura P.sub.ENO2-IDI1-T.sub.PDC1, 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 (MATura3-52; trp1-289; leu2-3, 112; his31; MAL2-8C, SUC2) FPP-001 P.sub.PGK1-ADH2-T.sub.ADH1, P.sub.TEF1-ALD6-T.sub.CYC1, SD-Ura-His P.sub.TDH3-ACS1-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 SD-Ura-His-Leu pRS313-LEU2-P.sub.TEF1-STpGMAS-T.sub.CYC1 ELE-002 FPP-001 transferred with pRS425-LEU2-P.sub.TEF1- SD-Ura-His-Leu STpGMAS-T.sub.CYC1 ELE-011 FPP-001 transferred with pRS425-LEU2-P.sub.TEF1- SD-Ura-His-Leu SynSmFPS-GGGS-STpGMAS-T.sub.CYC1 ELE-012 FPP-001 transferred with pRS425-LEU2-P.sub.TEF1- SD-Ura-His-Leu SynSmFPS-3A001-STpGMAS-T.sub.CYC1 ELE-013 FPP-001 transferred with pRS425-LEU2-P.sub.TEF1- SD-Ura-His-Leu SynSmFPS-4A001-STpGMAS-T.sub.CYC1 ELE-014 FPP-001 transferred with pRS425-LEU2-P.sub.TEF1- SD-Ura-His-Leu SynSmFPS-5A002-STpGMAS-T.sub.CYC1 ELE-015 FPP-001 transferred with pRS425-LEU2-P.sub.TEF1- SD-Ura-His-Leu SynSmFPS-6A005-STpGMAS-T.sub.CYC1 ELE-016 FPP-001 transferred with pRS425-LEU2-P.sub.TEF1- SD-Ura-His-Leu SynSmFPS-6B004-STpGMAS-T.sub.CYC1 ELE-017 FPP-001 transferred with pRS425-LEU2-P.sub.TEF1- SD-Ura-His-Leu SynSmFPS-8A005-STpGMAS-T.sub.CYC1 ELE-018 FPP-001 transferred with pRS425-LEU2-P.sub.TEF1- SD-Ura-His-Leu SynSmFPS-12A003-STpGMAS-T.sub.CYC1 ELE-019 FPP-001 transferred with pRS425-LEU2-P.sub.TEF1- SD-Ura-His-Leu SynSmFPS-8A005-STpGMAS-T.sub.CYC1 ELE-020 P.sub.PGK1-ERG20-GGGS-LsLTC2-T.sub.ADH1, SD-Ura-His-Leu-Trp P.sub.TEF1-SynSmFPS-GGGS-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 -Elemene

[0200] 1. Engineering Strain Culture and Product Extraction

[0201] 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 1 d. 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.

[0202] 2. -Elemene Conversion and its Qualitative and Quantitative Analyses

[0203] 1) -Elemene Conversion

[0204] 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.

[0205] 2) Detection

[0206] The converted material was diluted 10 times with n-hexane, filtered through an organic nylon membrane (0.22 m), and detected by using GC-MS. Testing equipment: Agilent GCMSD Agilent 7890A/5975C; GC-MS measurement conditions: inlet temperature 250 C., injection volume 1 L, 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 -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 -elemene produced by all engineering yeast strains prepared in Example 2.

[0207] As a result, the yield of each engineering strain after fermentation for 6 days was as follows:

[0208] 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 -elemene of ELE-001 reached 9.3 mg/L, and the yield of -elemene of ELE-002 reached 22.1 mg/L; Engineering strain ELE-011 was obtained by introducing high copy number of fusion protein gene SynSmFPS-GGGS-STpGMAS based on FPP-001, and the yield of -elemene reached 101.1 mg/L.

[0209] 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.

[0210] Engineering strain ELE-020 was obtained by the recombination and introduction of fusion protein genes P.sub.PGK1-ERG20-GGGS-LsLTC2-T.sub.ADH1 and P.sub.TEF1-SynSmFPS-GGGS-STpGMAS-T.sub.CYC1 based on ELE-019.

[0211] The yields of -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.

[0212] 3. Bioreactor Fermentation Culture

[0213] 1) Medium Formulation

[0214] The calcium chloride mother liquid: 19.2 g/L aqueous solution of calcium chloride dihydrate.

[0215] 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.

[0216] 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.

[0217] 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.

[0218] 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.

[0219] 2) Fermentation of Engineering Strain ELE-019

[0220] 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 L 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.

[0221] 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.: BioFlo320).

[0222] 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.

[0223] 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.

[0224] 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 -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

[0225] 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 -elemene and/or germacrene A, and provides a potent strain and research basis for the biosynthesis of anti-cancer raw material -elemene.