(-)-germacene a synthetase and preparation method therefor

Abstract

Provided are a (-)-germacene A synthetase and a preparation method therefor, which effectively achieve the synthesis of (+)-β-elemene.

Description

A (-)-germaene A synthase and preparation method thereof

[0001] Citation of Related Applications

[0002] This application claims priority to and the benefits of Chinese Patent Application No. 202310642542.8 filed with the State Intellectual Property Office of the People's Republic of China on June 1, 2023, and Chinese Patent Application No. 202310642522.0 filed with the State Intellectual Property Office of the People's Republic of China on June 1, 2023, the entire contents of which are hereby incorporated by reference into the text in their entirety. Technical Field

[0003] The invention belongs to the field of biotechnology and relates to a method for constructing (+)-beta-elemene and a related (-)-germaene A synthase. Background Art

[0004] β-elemene is a volatile sesquiterpenoid compound with a tulip-like flavor. Its oral emulsion and injection, as national Class II new drugs, have achieved significant clinical efficacy.

[0005] Currently, the biological pathway uses Germacrene A synthase (GAS) to catalyze the precursor substance Farnesyl pyrophosphate (FPP) to produce the sesquiterpenoid compound Germacrene A. Germacrene A can directly obtain β-elemene through a simple Cope rearrangement in vitro.

[0006] GAS is widely present in plants and microorganisms. Currently, many studies have used GAS expressed in recombinant bacteria to synthesize (+)-germanene A. The corresponding Cope rearrangement product is (-)-β-elemene. Recently, researchers have discovered the existence of (+)-β-elemene, whose structure is shown in Compound I: The name is (1R,2R,4S)-β-elemene. Its corresponding precursor compound is (-)-germazine A, as shown in Compound II: However, no biological method for synthesizing (-)-germazine A has been found so far.

[0007] Summary of the Invention

[0008] The invention provides a (-)-germarene A synthase for producing (+)-β-elemene through a biological pathway.

[0009] The present invention provides a protein for producing (-)-germane A, wherein the protein comprises the following segments: 1) a polypeptide having an amino acid sequence of X1Y1X2Y2W; 2) a polypeptide having an amino acid sequence of X3Y3Y4Y5Y6L; 3) a polypeptide having an amino acid sequence of LY7Y8Y9X4Y 10 R polypeptide; 4) amino acid sequence is Y 11 Y 12 Y 13 X5Y 14 of polypeptides.

[0010] Furthermore, the amino acid of X1 may be Y, H, E, Q, preferably Q or E or H, preferably Q or E, preferably E; the amino acid of Y1 may be S, A, L, T, preferably S, A, more preferably S; the amino acid of X2 may be L, A, T, preferably A, T, more preferably A; the amino acid of Y2 may be C, A, D, preferably A; the amino acid of X3 may be R, G, A, P, preferably A, G, more preferably A; the amino acid of Y3 may be L, F, M, preferably L, F, more preferably L The amino acid of Y4 may be L or I, preferably L; the amino acid of Y5 may be E, D or A, preferably D or E, more preferably D; the amino acid of Y6 may be I or V, preferably V; the amino acid of Y7 may be A, H, D, R or K, preferably A or D, more preferably A; the amino acid of Y8 may be D, E or N, preferably D; the amino acid of Y9 may be L, I or M, preferably L or I, more preferably L; the amino acid of X4 may be R, C, W or I, preferably C, W or I, preferably C or I, preferably C; Y 10 The amino acid can be R, K, Q, L, N, preferably R; Y 11 The amino acid can be R, W, Y, L, preferably R; Y 12 The amino acid may be N, A, V, K, Q, S, preferably A, S, V, preferably A, S, more preferably A; Y 13 The amino acid of X5 can be H, D, N, A, preferably D; the amino acid of X5 can be P, A, D, E, S, preferably P, E, D, S, preferably P, S, more preferably P; 14 The amino acid can be A, R, E, D, Q, L, preferably Q, A, preferably Q.

[0011] Furthermore, the present invention provides a protein for synthesizing (-)-germane A, which has the following segments: 1) a polypeptide with an amino acid sequence of X1SX2AW; 2) a polypeptide with an amino acid sequence of X3LLDVL; 3) a polypeptide with an amino acid sequence of LADX4RR; 4) a polypeptide with an amino acid sequence of RADX5Q; wherein the amino acid of X1 can be E, Q, H, preferably E, Q, preferably E; the amino acid of X2 can be A, D, preferably A; the amino acid of X3 can be A, G, preferably A; the amino acid of X4 can be C, I, preferably C; the amino acid of X5 can be P, S, preferably P.

[0012] Furthermore, the amino acid sites of the segment 1) correspond to positions 28-31 shown in SEQ ID NO.11; the amino acid sites of the segment 2) correspond to positions 101-106 shown in SEQ ID NO.11; the amino acid sites of the segment 3) correspond to positions 125-131 shown in SEQ ID NO.11; and the amino acid sites of the segment 4) correspond to positions 203-208 shown in SEQ ID NO.12.

[0013] Furthermore, the present invention provides a protein for synthesizing (-)-germane A, whose amino acid sequence is shown in SEQ ID NO.12, wherein positions 28-31 of the protein are replaced by X1SX2AW, positions 101-106 are replaced by X3LLDVL, positions 125-131 are replaced by LADX4RR, and positions 203-208 are replaced by RADX5Q, wherein the amino acid of X1 can be E, Q, or H, preferably E or Q, and preferably E; the amino acid of X2 can be A or D, preferably A; the amino acid of X3 can be A or G, preferably A; the amino acid of X4 can be C or I, preferably C; and the amino acid of X5 can be P or S, preferably P.

[0014] Furthermore, the protein is derived from Micromonospora, preferably from marine Micromonospora, preferably from marine Micromonospora AB-18-032, and the source refers to the different original source hosts of the gene.

[0015] The present invention provides a (-)-germaene A synthase for producing (-)-germaene A, which is used for producing (+)-β-elemene through a biological pathway.

[0016] The present invention provides a protein comprising a sequence selected from the group consisting of: 1) a sequence having at least 62% homology, preferably at least 63% homology, preferably at least 66% homology, and preferably 99% homology to the amino acid sequence shown in SEQ ID NO. 8; 2) or a sequence obtained by substituting and / or deleting, or adding / reducing one or more amino acids from the sequence shown in SEQ ID NO. 8; 3) or a fusion protein obtained by linking a tag to the N-terminus or C-terminus of the protein shown in SEQ ID NO. 8.

[0017] Furthermore, the protein is derived from Micromonospora, preferably from Micromonospora aurea, Micromonospora deserticola, Micromonospora CB01531, Micromonospora pieris, and more preferably from Micromonospora aurea, Micromonospora deserticola, Micromonospora CB01531. The source refers to the different original source hosts of the gene.

[0018] The present invention provides an engineered bacterium capable of producing (-)-germaene A, wherein the engineered bacterium comprises one or more enzymes capable of synthesizing (-)-germaene A, and the one or more enzymes are independently selected from:

[0019] (a) An enzyme capable of synthesizing (-)-germaene A having any one of SEQ ID NO: 12 to SEQ ID NO: 22, or having an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID NO: 12 to SEQ ID NO: 22.

[0020] In the art, the enzyme capable of synthesizing (-)-germaene A is also referred to as germacene A synthase (GAS) or terpene synthase (TPS).

[0021] The present invention provides an engineered bacterium capable of producing (-)-germaene A, wherein the engineered bacterium comprises one or more enzymes, and the one or more enzymes are independently selected from:

[0022] (a) a germacene A synthase (GAS) having any one of SEQ ID NO: 12 to SEQ ID NO: 18, or an amino acid sequence at least 80% identical to any one of SEQ ID NO: 12 to SEQ ID NO: 18, and / or

[0023] (b) a terpene synthase (TPS) having any one of SEQ ID NO: 19 to SEQ ID NO: 22, or an amino acid sequence that is at least 80% identical to any one of SEQ ID NO: 19 to SEQ ID NO: 22.

[0024] In certain embodiments, the engineered bacteria comprises a germacene A synthase (GAS) having any one of SEQ ID NO: 12 to SEQ ID NO: 18, or an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the amino acid sequence of any one of SEQ ID NO: 12 to SEQ ID NO: 18. In certain embodiments, the engineered bacteria comprises a germacene A synthase (GAS) having an amino acid sequence at least 95% identical to the amino acid sequence of any one of SEQ ID NO: 12 to SEQ ID NO: 18. In certain embodiments, the engineered bacteria comprises a germacene A synthase (GAS) having an amino acid sequence at least 99% identical to the amino acid sequence of any one of SEQ ID NO: 12 to SEQ ID NO: 18.

[0025] In certain embodiments, the engineered bacteria comprises a terpene synthase (TPS) having an amino acid sequence at least 80% (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to any one of SEQ ID NO: 19 to SEQ ID NO: 22. In certain embodiments, the engineered bacteria comprises a terpene synthase (TPS) having an amino acid sequence at least 95% identical to any one of SEQ ID NO: 19 to SEQ ID NO: 22. In certain embodiments, the engineered bacteria comprises a terpene synthase (TPS) having an amino acid sequence at least 99% identical to any one of SEQ ID NO: 19 to SEQ ID NO: 22.

[0026] In a further embodiment, the engineered bacteria comprises a germacene A synthase (GAS) having an amino acid sequence identical to any one of SEQ ID NO: 12 to SEQ ID NO: 18. In certain embodiments, the engineered bacteria comprises a germacene A synthase (GAS) having an amino acid sequence identical to any one of SEQ ID NO: 13 to SEQ ID NO: 15 and SEQ ID NO: 17 to SEQ ID NO: 18. In certain embodiments, the engineered bacteria comprises a germacene A synthase (GAS) having an amino acid sequence identical to any one of SEQ ID NO: 14 and SEQ ID NO: 17 to SEQ ID NO: 18.

[0027] In a further embodiment, the engineered bacteria comprises a terpene synthase (TPS) having an amino acid sequence identical to any one of SEQ ID NO: 19 to SEQ ID NO: 22. In some embodiments, the engineered bacteria comprises a terpene synthase (TPS) having an amino acid sequence identical to any one of SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 22. In certain embodiments, the engineered bacteria comprises a terpene synthase (TPS) having an amino acid sequence identical to any one of SEQ ID NO: 21 and SEQ ID NO: 22.

[0028] In further embodiments, the germacene A synthase (GAS) and / or terpene synthase (TPS) may have one or more amino acid mutations, including but not limited to substitutions, deletions, additions or changes.

[0029] In a further embodiment, the terpene synthase (TPS) has one or more amino acid mutations relative to the amino acid sequence of SEQ ID NO: 22. For example, the terpene synthase (TPS) may have a mutation at one or more of E28, A30, A101, C129, and P206 relative to the amino acid sequence of SEQ ID NO: 22. For example, the one or more amino acid mutations may be selected from one or more of E28Q, E28H, A30D, A101G, C129I, and P206S. In a further embodiment, the terpene synthase (TPS) has one or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 22, wherein the one or more amino acid substitutions are selected from E28Q, E28H, A30D, A101G, C129I, and P206S.

[0030] In certain embodiments, the terpene synthase (TPS) has an amino acid sequence identical to the amino acid sequence of SEQ ID NO: 29 to SEQ ID NO: 34.

[0031] In a further embodiment, the engineered bacteria contains an expression cassette of a nucleotide sequence encoding germarene A synthase (GAS) and / or terpene synthase (TPS).

[0032] In certain embodiments, the nucleic acid encoding a germacene A synthase (GAS) is independently selected from a nucleotide sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the nucleotide sequence of any one of SEQ ID NOs: 1-7. In certain embodiments, the nucleic acid encoding a germacene A synthase (GAS) has a nucleotide sequence that is at least 95% identical to the nucleotide sequence of any one of SEQ ID NOs: 1-7. In certain embodiments, the nucleic acid encoding a germacene A synthase (GAS) has a nucleotide sequence that is at least 99% identical to the nucleotide sequence of any one of SEQ ID NOs: 1-7.

[0033] In certain embodiments, the nucleic acid encoding a terpene synthase (TPS) is independently selected from a nucleotide sequence that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the nucleotide sequence of any one of SEQ ID NO: 8 to SEQ ID NO: 11 and SEQ ID NO: 23 to SEQ ID NO: 28. In certain embodiments, the nucleic acid encoding a terpene synthase (TPS) has a nucleotide sequence that is at least 95% identical to the nucleotide sequence of any one of SEQ ID NO: 8 to SEQ ID NO: 11 and SEQ ID NO: 23 to SEQ ID NO: 28. In certain embodiments, the nucleic acid encoding a terpene synthase (TPS) has a nucleotide sequence that is at least 99% identical to the nucleotide sequence of any one of SEQ ID NO: 8 to SEQ ID NO: 11 and SEQ ID NO: 23 to SEQ ID NO: 28.

[0034] In a specific embodiment, the present invention provides an engineered bacterium capable of producing (-)-germaene A, wherein the engineered bacterium comprises germacene A synthase (GAS) and / or terpene synthase (TPS), and / or comprises an expression cassette of a gene encoding the germacene A synthase (GAS) and / or an expression cassette of a gene encoding the terpene synthase (TPS).

[0035] In the present invention, the enzyme capable of synthesizing (-)-germaene A refers to an enzyme capable of converting farnesyl pyrophosphate into germacene.

[0036] In the present invention, germaene A synthase (GAS) refers to an enzyme that can convert farnesyl pyrophosphate into germaene and is primarily found in microorganisms of the genera Micromonospora, Nostoc, and Daldinia, as well as species of the genus Calothrix. For example, in the present invention, germaene A synthase (GAS) from Micromonospora marina, Micromonospora aurantiaca (strain ATCC 27029), Calothrix membranacea, Nostoc linckia FACHB-104, Nostoc carneum NIES-2107, Daldinia eschscholtzii, and Daldinia loculata can be used.

[0037] In the present invention, terpene synthase (TPS) refers to a microorganism that is capable of converting farnesyl pyrophosphate into germaceous ene and is primarily found in the genus Micromonospora. For example, terpene synthase (TPS) from Micromonospora deserti, Micromonospora sp. CB01531, Micromonospora sp. MH33, and Micromonospora maris AB-18-032 can be used in the present invention.

[0038] In a preferred embodiment of the present invention, codon-optimized genes are synthesized based on the cDNA sequence information of germarene A synthase (GAS) and terpene synthase (TPS) according to the codon preference of the engineered bacteria.

[0039] In a preferred embodiment, the engineered bacteria is yeast or Escherichia coli; preferably yeast; more preferably Saccharomyces cerevisiae.

[0040] In a more preferred embodiment, the engineered bacteria may be derived from wild-type yeast or E. coli, or from mutant yeast or E. coli obtained by mutagenesis or engineering. In an even more preferred embodiment, the engineered bacteria may be derived from wild-type yeast or E. coli, or from mutant yeast or E. coli obtained by mutagenesis or engineering that can produce farnesyl pyrophosphate (FPP).

[0041] In a preferred embodiment, in the case of yeast, the engineered bacteria of the present invention have increased farnesyl pyrophosphate synthase ERG20 activity.

[0042] In the present invention, increased farnesyl pyrophosphate synthase ERG20 activity can increase the sequential condensation of isopentenyl pyrophosphate (IPP) and its isomer dimethylallyl pyrophosphate (DMAPP) to improve the supply of the precursor farnesyl pyrophosphate (FPP).

[0043] Therefore, in a preferred embodiment of the present invention, the method for increasing the activity level of farnesyl pyrophosphate synthase ERG20 can be to introduce an expression cassette encoding a gene for farnesyl pyrophosphate synthase ERG20. For example, the farnesyl pyrophosphate synthase ERG20 encoded in the expression cassette encoding the gene for farnesyl pyrophosphate synthase ERG20 can be the farnesyl pyrophosphate synthase ERG20 contained in the engineered bacteria, or can be farnesyl pyrophosphate synthase ERG20 from the same species.

[0044] In a preferred embodiment, the amino acid sequence of the farnesyl pyrophosphate synthase ERG20 of the present invention comprises or is the amino acid sequence shown in SEQ ID NO: 97. Furthermore, the gene preferably comprises the polynucleotide shown in SEQ ID NO: 66.

[0045] In a further embodiment, the expression cassette further comprises a promoter and a terminator. Regarding the selection of the promoter and terminator, those skilled in the art can appropriately select a promoter and terminator suitable for the corresponding engineered bacteria according to the engineered bacteria.

[0046] In further embodiments, the expression cassette comprises a promoter and a terminator. In certain embodiments, the promoter is selected from TEF1, PGK1, or SSA1. In certain embodiments, the terminator is CYC1 or ADH1. In certain embodiments, the promoter is TEF1 and the terminator is CYC1. In certain embodiments, the promoter is PGK1 and the terminator is ADH1. In certain examples, the promoter is SSA1 and the terminator is ADH1.

[0047] For example, the expression cassette for GAS may be P PGK1 -GAS-T ADH1 For example, the expression cassette for TPS may be P PGK1 -TPS-T ADH1 For example, the expression cassette for ERG20 may be P TEF1 -ERG20-T CYC1 .

[0048] In a preferred embodiment, ERG20 may be in a different expression cassette from GAS and / or TPS.

[0049] For example, in a further embodiment, the expression cassette for ERG20 and GAS and / or TPS contains one or two of the following structures:

[0050] (a)P TEF1 -ERG20-T CYC1 -P PGK1 -GAS-T ADH1

[0051] (b)P TEF1 -ERG20-T CYC1 -P PGK1 -TPS-T ADH1 .

[0052] Furthermore, the TEF1 -ERG20-T CYC1 -P PGK1 -GAS-T ADH1 The expression cassette comprises a nucleotide sequence independently selected from any one of SEQ ID NO: 35 to SEQ ID NO: 36, SEQ ID NO: 40 to SEQ ID NO: 42, SEQ ID NO: 44 to SEQ ID NO: 45 that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the nucleotide sequence. In certain embodiments, the expression cassette comprising P TEF1 -ERG20-T CYC1 -P PGK1 -GAS-T ADH1 The expression cassette comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of any one of SEQ ID NO:35-SEQ ID NO:36, SEQ ID NO:40-SEQ ID NO:42, SEQ ID NO:44-SEQ ID NO:45. In certain embodiments, the expression cassette comprising P TEF1 -ERG20-T CYC1 -P PGK1 -GAS-T ADH1 The expression cassette comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of any one of SEQ ID NO:35-SEQ ID NO:36, SEQ ID NO:40-SEQ ID NO:42, SEQ ID NO:44-SEQ ID NO:45.

[0053] Furthermore, the TEF1 -ERG20-T CYC1 -P PGK1 -TPS-T ADH1The expression cassette comprises a nucleotide sequence independently selected from the group consisting of SEQ ID NO: 37 to SEQ ID NO: 39, SEQ ID NO: 43, and SEQ ID NO: 46 to SEQ ID NO: 51 that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the nucleotide sequence of any one of SEQ ID NO: 37 to SEQ ID NO: 39, SEQ ID NO: 43, and SEQ ID NO: 46 to SEQ ID NO: 51. In certain embodiments, the expression cassette comprising P TEF1 -ERG20-T CYC1 -P PGK1 -TPS-T ADH1 The expression cassette comprises a nucleotide sequence that is at least 95% identical to the nucleotide sequence of any one of SEQ ID NO:37-SEQ ID NO:39, SEQ ID NO:43, and SEQ ID NO:46-SEQ ID NO:51. In certain embodiments, the expression cassette comprising P TEF1 -ERG20-T CYC1 -P PGK1 -TPS-T ADH1 The expression cassette comprises a nucleotide sequence that is at least 99% identical to the nucleotide sequence of any one of SEQ ID NO:37 to SEQ ID NO:39, SEQ ID NO:43, and SEQ ID NO:46 to SEQ ID NO:51.

[0054] In some embodiments, ERG20 may be in the same expression cassette as GAS and / or TPS, for example, the ERG20 and GAS and / or TPS are expressed in the form of a fusion protein. That is, the engineered bacteria contain ERG20-GAS fusion protein and / or ERG20-TPS fusion protein. In the present invention, the "-" used when referring to gene expression cassettes and fusion proteins does not indicate the order in which the reagents of the genes or proteins referred to are connected, unless otherwise expressly specified. For example, the term "ERG20-GAS fusion protein" includes both a fusion protein in which ERG20 is connected to the N-terminus of GAS and a fusion protein in which ERG20 is connected to the C-terminus of GAS.

[0055] Furthermore, the encoding nucleic acid for expressing the fusion protein of gemmaene A synthase and / or the fusion protein of terpene synthase (TPS) in vivo is introduced into the yeast or Escherichia coli.

[0056] In the present invention, ERG20 can be directly connected to GAS and / or TPS, or there may be a spacer sequence between ERG20 and GAS and / or TPS, for example, 2-40 amino acids, preferably 5-20 amino acids. In a preferred embodiment of the present invention, ERG20 can be fused to the N-terminus or C-terminus of GAS and / or TPS. Furthermore, the germarene A synthase and / or terpene synthase (TPS) is connected to the ERG20 via a connecting peptide. In a preferred embodiment, the connecting peptide is selected from one or more of GGGGS, YGQ, PGGH, YRSQI, VIPFIS, FLYLKF, WRFSPKLQ or HHVQESQCISTV, or a repeated connecting peptide consisting of multiple repeats of the connecting peptide. In certain embodiments, the connecting peptide is preferably GGGGS.

[0057] The ERG20-GAS fusion protein may have the sequences shown in SEQ ID NO: 67 to SEQ ID NO: 78, and the polynucleotide sequences thereof may be the sequences shown in SEQ ID NO: 79 to SEQ ID NO: 90. The ERG20-TPS fusion protein may have the sequences shown in SEQ ID NO: 68 to SEQ ID NO: 71, SEQ ID NO: 74, and SEQ ID NO: 76, and the polynucleotide sequences thereof may be the sequences shown in SEQ ID NO: 80 to SEQ ID NO: 83, SEQ ID NO: 86, and SEQ ID NO: 88. The ERG20-GAS fusion protein may have the sequences shown in SEQ ID NO: 67, SEQ ID NO: 72, SEQ ID NO: 73, SEQ ID NO: 75, SEQ ID NO: 77, and SEQ ID NO: 78, and the polynucleotide sequences thereof may be the sequences shown in SEQ ID NO: 79, SEQ ID NO: 84 to SEQ ID NO: 87, SEQ ID NO: 89, and SEQ ID NO: 90.

[0058] Furthermore, the expression cassette of the fusion protein contains one or more of the following structures:

[0059] (a)P PGK1 -ERG20-GGGGS-GAS-T ADH1

[0060] (b)P PGK1 -ERG20-GGGGS-TPS-T ADH1

[0061] (c)P SSA1 -GAS-GGGGS-ERG20-T ADH1

[0062] (d)P SSA1 -TPS-GGGGS-ERG20-T ADH1 .

[0063] In certain embodiments, the PGK1 -ERG20-GGGGS-GAS-T ADH1 or P SSA1 -GAS-GGGGS-ERG20-T ADH1 The expression cassette comprises a nucleotide sequence independently selected from the group consisting of SEQ ID NO: 52, SEQ ID NO: 57-SEQ ID NO: 58, SEQ ID NO: 60, and SEQ ID NO: 62-SEQ ID NO: 63 that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the nucleotide sequence of any one of SEQ ID NO: 52, SEQ ID NO: 57-SEQ ID NO: 58, SEQ ID NO: 60, and SEQ ID NO: 62-SEQ ID NO: 63. In certain embodiments, the expression cassette comprising P PGK1 -ERG20-GGGGS-GAS-T ADH1 or P SSA1 -GAS-GGGGS-ERG20-T ADH1 The expression cassette has a nucleotide sequence that is at least 95% identical to the nucleotide sequence of any one of SEQ ID NO:52, SEQ ID NO:57-SEQ ID NO:58, SEQ ID NO:60, and SEQ ID NO:62-SEQ ID NO:63. In certain embodiments, the expression cassette comprising P PGK1 -ERG20-GGGGS-GAS-T ADH1 or P SSA1 -GAS-GGGGS-ERG20-T ADH1 The expression cassette has a nucleotide sequence that is at least 99% identical to the nucleotide sequence of any one of SEQ ID NO:52, SEQ ID NO:57-SEQ ID NO:58, SEQ ID NO:60, and SEQ ID NO:62-SEQ ID NO:63.

[0064] Furthermore, the PGK1 -ERG20-GGGGS-TPS-T ADH1 or P SSA1 -TPS-GGGGS-ERG20-T ADH1The expression cassette comprises a nucleotide sequence independently selected from any one of SEQ ID NO: 53 to SEQ ID NO: 56, SEQ ID NO: 59, and SEQ ID NO: 61 that is at least 90% (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) identical to the nucleotide sequence. In certain embodiments, the expression cassette comprising P PGK1 -ERG20-GGGGS-TPS-T ADH1 or P SSA1 -TPS-GGGGS-ERG20-T ADH1 The expression cassette has a nucleotide sequence that is at least 95% identical to the nucleotide sequence of any one of SEQ ID NOs: 53-56, SEQ ID NOs: 59, and SEQ ID NOs: 61. In certain embodiments, the expression cassette comprising P PGK1 -ERG20-GGGGS-TPS-T ADH1 or P SSA1 -TPS-GGGGS-ERG20-T ADH1 The expression cassette has a nucleotide sequence that is at least 99% identical to the nucleotide sequence of any one of SEQ ID NO: 53 to SEQ ID NO: 56, SEQ ID NO: 59, and SEQ ID NO: 61.

[0065] Furthermore, the engineered bacteria is yeast or Escherichia coli; preferably yeast; more preferably Saccharomyces cerevisiae.

[0066] In some embodiments, in the yeast engineered bacteria, the engineered bacteria further include one or more of the following characteristics:

[0067] reducing the activity of squalene synthase ERG9, such as reducing its expression level;

[0068] Reduced activity, e.g., reduced expression level, of the genes DPP1 and / or LPP1 responsible for farnesol synthesis;

[0069] increasing the activity of acetyl-CoA C-acetyltransferase / 3-hydroxy-3-methylglutaryl-CoA reductase EfmvaE, such as increasing its expression level;

[0070] Increased activity of 3-hydroxy-3-methylglutaryl-CoA reductase HMGr, such as increased expression levels;

[0071] Increased activity of mevalonate kinase ERG12, such as increased expression levels;

[0072] Increased activity of phosphomevalonate kinase ERG8, such as increased expression levels;

[0073] Increased activity of mevalonate pyrophosphate decarboxylase ERG19, such as increased expression levels;

[0074] Increased activity of acetyl-CoA-acetyltransferase ERG10, such as increased expression levels;

[0075] Increased activity of 3-hydroxy-3-methylglutaryl-CoA synthetase ERG13, such as increased expression levels;

[0076] Reduced activity of GAL80, which is responsible for regulating expression of the GAL promoter, such as reduced expression levels;

[0077] Increased isopentenyl pyrophosphate isomerase IDI1 activity, such as increased levels; or

[0078] Increased activity of 3-hydroxy-3-methylglutaryl-CoA reductase 1 tHMG1, such as increased expression level.

[0079] In some embodiments, in the yeast engineered bacteria, the engineered bacteria further include one or more of the following characteristics:

[0080] reducing the activity, such as expression level, of squalene synthase ERG9;

[0081] Reduced expression levels of DPP1 and / or LPP1, genes responsible for farnesol synthesis;

[0082] Increased activity of acetyl-CoA-acetyltransferase / 3-hydroxy-3-methylglutaryl-CoA reductase EfmvaE;

[0083] Increased activity of 3-hydroxy-3-methylglutaryl-CoA reductase HMGr;

[0084] Increased activity of mevalonate kinase ERG12;

[0085] Increased activity of phosphomevalonate kinase ERG8;

[0086] Increased activity of mevalonate pyrophosphate decarboxylase ERG19;

[0087] Increased activity of acetyl-CoA-acetyltransferase ERG10;

[0088] Increased activity of 3-hydroxy-3-methylglutaryl-CoA synthetase ERG13; or

[0089] Increased activity of 3-hydroxy-3-methylglutaryl-CoA reductase 1 tHMG1.

[0090] In some embodiments, in the yeast engineered bacteria, the engineered bacteria further include one or more of the following characteristics:

[0091] reduced expression levels of LPP1, the gene responsible for farnesol synthesis;

[0092] Increased activity of acetyl-CoA-acetyltransferase / 3-hydroxy-3-methylglutaryl-CoA reductase EfmvaE;

[0093] Reduced activity of GAL80, which is responsible for regulating expression of the GAL promoter;

[0094] Increased activity of isopentenyl pyrophosphate isomerase IDI1;

[0095] Increased activity of mevalonate kinase ERG12;

[0096] Increased activity of phosphomevalonate kinase ERG8;

[0097] increased activity of mevalonate pyrophosphate decarboxylase ERG19; or

[0098] Increased activity of 3-hydroxy-3-methylglutaryl-CoA synthetase ERG13.

[0099] The increase in its activity can be achieved by changing its promoter, enzyme modification, or increasing its expression level.

[0100] In a specific embodiment, the ERG9 promoter P ERG9 Change to P HXT1 , to reduce the expression level of squalene synthase ERG9 in yeast;

[0101] knocking out the genes DPP1 and / or LPP1 responsible for farnesol synthesis in yeast to reduce the levels of the genes DPP1 and / or LPP1 in yeast;

[0102] The activity of acetyl-CoA-acetyltransferase / 3-hydroxy-3-methylglutaryl-CoA reductase EfmvaE in yeast is increased by introducing an expression cassette encoding an acetyl-CoA-acetyltransferase / 3-hydroxy-3-methylglutaryl-CoA reductase EfmvaE gene into yeast;

[0103] The activity of 3-hydroxy-3-methylglutaryl-CoA reductase HMGr in yeast is increased by introducing a gene expression cassette encoding 3-hydroxy-3-methylglutaryl-CoA reductase HMGr into yeast;

[0104] The activity of mevalonate kinase ERG12 in yeast is increased by introducing a gene expression cassette encoding mevalonate kinase ERG12 into yeast;

[0105] The activity of phosphomevalonate kinase ERG8 in yeast is increased by introducing a gene expression cassette encoding phosphomevalonate kinase ERG8 into yeast;

[0106] The activity of mevalonate pyrophosphate decarboxylase ERG19 in yeast is increased by introducing a gene expression cassette encoding mevalonate pyrophosphate decarboxylase ERG19 into yeast;

[0107] The activity of acetyl-CoA-acetyltransferase ERG10 in yeast is increased by introducing an expression cassette encoding an acetyl-CoA-acetyltransferase ERG10 gene into yeast;

[0108] The activity of 3-hydroxy-3-methylglutaryl-CoA synthetase ERG13 in yeast is increased by introducing a gene expression cassette encoding 3-hydroxy-3-methylglutaryl-CoA synthetase ERG13 into yeast;

[0109] By knocking out GAL80 in yeast, the level of GAL80 in yeast was reduced;

[0110] Increasing the activity of isopentenyl pyrophosphate isomerase IDI1 in yeast by introducing an expression cassette encoding an isopentenyl pyrophosphate isomerase IDI1 gene into yeast; or

[0111] The activity of 3-hydroxy-3-methylglutaryl-CoA reductase 1tHMG1 in yeast is increased by introducing a gene expression cassette encoding 3-hydroxy-3-methylglutaryl-CoA reductase 1tHMG1 into yeast.

[0112] In one embodiment, the squalene synthase ERG9 encoding gene expression cassette may include a promoter P HXT1 Alternatively, the squalene synthase ERG9 encoding gene expression cassette may include a promoter P ERG9 , the gene encoding squalene synthase ERG9.

[0113] In one embodiment, the promoter P HXT1The nucleotide sequence is the sequence shown by SEQ ID NO:91. In an embodiment of the present invention, the above-mentioned expression cassettes can be integrated into the genome of the yeast cell respectively. The above-mentioned expression cassettes can also be connected to each other and integrated into the genome of the Saccharomyces cerevisiae cell. For example, all expression cassettes can be connected in series and integrated into the genome of the Saccharomyces cerevisiae cell. Alternatively, the above-mentioned expression cassettes can be constructed into multiple expression modules and then integrated into the genome of the Saccharomyces cerevisiae cell. In the present invention, an expression module refers to two or more expression cassettes that are operably connected. In the present invention, the expression cassette and / or expression module can be integrated into one or more sites. In the present invention, CRISPR / Cas9 is preferably used for the integration of the expression cassette and / or expression module.

[0114] The yeast of the present invention may be any Saccharomyces cerevisiae available in the art, for example, commercially available Saccharomyces cerevisiae CEN.PK2-1D, Saccharomyces cerevisiae INVSc1, Saccharomyces cerevisiae BY4742, Saccharomyces cerevisiae YPH499 or Saccharomyces cerevisiae W303-1B.

[0115] The encoding gene expression cassettes of the present invention are integrated into the Saccharomyces cerevisiae genome; or, these encoding gene expression cassettes exist in the Saccharomyces cerevisiae cells in the form of plasmids.

[0116] In some embodiments, the plasmid vector is selected from pESC-HIS, pESC-URA, pESC-TRP and pESC-TRP (Invitrogen, USA).

[0117] The enzyme of the present invention can be:

[0118] (a) naturally occurring wild-type enzyme;

[0119] (b) a polypeptide of a wild-type enzyme formed by substitution, deletion or addition of one or more amino acid residues, or by addition of a signal peptide sequence, and having the corresponding activity;

[0120] (c) a polypeptide having a sequence comprising the polypeptide sequence described in (a) or (b);

[0121] (d) A polypeptide having an amino acid sequence identity of ≥85% or ≥90% (preferably ≥95%) to that of the wild-type enzyme and having the activity of the wild-type enzyme.

[0122] In the present invention, the coding gene used may be a natural polynucleotide sequence (e.g., a cDNA sequence, a genomic sequence, or RNA, etc.) encoding any one of (a)-(d) above, or a degenerate variant thereof. As used herein, "degenerate variant" in the present invention refers to a polynucleotide sequence that can encode any one of the proteins in (a)-(d) above, but differs from the natural polynucleotide sequence. Codon-optimized DNA sequences are preferably used. Polynucleotides can generally be obtained by PCR amplification, recombinant methods, or artificial synthesis.

[0123] In Saccharomyces cerevisiae, the exogenous gene expression cassette can be integrated into the genome for expression, or it can be expressed in the form of a plasmid free from the genome.

[0124] The Saccharomyces cerevisiae episomal vector may be a commercially available vector or any vector having the same function. For example, the episomal vector may be a pESC series vector, including pESC-HIS, pESC-URA, pESC-TRP, and pESC-TRP; pYES2; or pAUR123 (Invitrogen, USA).

[0125] Homologous recombination is the primary method for integrating foreign genes into the yeast genome. Homologous recombination involves amplifying upstream and downstream sequences of the integration site as upstream or downstream homology arms, constructing a target gene expression cassette (comprising a promoter, target gene, and terminator), typically with a selection marker gene in the upstream homology arm. These components are then ligated in the order of upstream homology arm, gene expression cassette, and downstream homology arm to form a fragment suitable for homologous recombination. The resulting fragment suitable for homologous recombination is then introduced into Saccharomyces cerevisiae, and positive transformants are screened for the selection marker, resulting in engineered, integrated Saccharomyces cerevisiae strains.

[0126] The integration site in the Saccharomyces cerevisiae genome can be selected from the following sites: the δ site, 1-10 random positions among multiple δ genes on the Saccharomyces cerevisiae chromosome; the rDNA site, 1-10 random positions among multiple ribosomal genes on the Saccharomyces cerevisiae chromosome; the HIS3 site, the position of the HIS3 gene in the histidine biosynthesis pathway on the Saccharomyces cerevisiae chromosome; or the Trp1 site, the position of the Trp1 gene in the tryptophan biosynthesis pathway on the Saccharomyces cerevisiae chromosome.

[0127] Available Saccharomyces cerevisiae gene integration screening markers can be any screening marker known to those skilled in the art, as long as the screening markers used when integrating different fragments into the same Saccharomyces cerevisiae strain are different from each other. Common screening markers include auxotrophic screening markers and resistance screening markers. Among them, auxotrophic screening markers can be selected from LEU, HIS, URA or TRP. Resistance screening markers can be G418 or HYG.

[0128] The homologous recombination fragment or recombinant plasmid is introduced into Saccharomyces cerevisiae using methods known in the art. Various transformation methods known to those skilled in the art, such as electroporation and lithium acetate chemical transformation, can be used to transform Saccharomyces cerevisiae.

[0129] The activity level of an enzyme or gene in Saccharomyces cerevisiae can be reduced by reducing the gene corresponding to the enzyme or the expression level of the gene or reducing the activity of the gene corresponding to the enzyme or the gene.

[0130] In constructing the working strains of the present invention, any method known to those skilled in the art can be used to reduce the expression level of the target gene or reduce the enzyme activity (including inactivating the target gene), such methods include but are not limited to: replacement of promoter elements, gene knockout, site-directed mutagenesis, or RNA interference (RNAi). In embodiments of the present invention involving RNAi, there is no particular limitation on the method for achieving RNAi, and various RNAi techniques well known to those skilled in the art can be used. For example, small interfering RNA (siRNA), antisense nucleic acids, microRNA, etc. can be used to hinder the transcription or translation of the target gene, thereby causing a decrease in the expression level of the target gene.

[0131] When E. coli is used as the engineered bacterium, the GAS or TPS expression cassette can use a promoter derived from E. coli itself, such as glnAP2.

[0132] In the case of Escherichia coli, the engineered bacteria may contain genes related to the MVA pathway.

[0133] In a preferred embodiment, the engineered E. coli bacteria may have reduced activity of the glnL gene. For example, the glnL gene in the engineered E. coli bacteria may be knocked out.

[0134] The present invention further provides a host cell capable of producing (-)-germaene A, wherein the host cell comprises one or more nucleic acids, each of which encodes:

[0135] (a) a germacene A synthase (GAS) having an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID NO: 12 to SEQ ID NO: 18, and / or

[0136] (b) a terpene synthase (TPS) having an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID NO: 19 to SEQ ID NO: 22 and SEQ ID NO: 29 to SEQ ID NO: 34.

[0137] Furthermore, the nucleic acids are independently selected from:

[0138] (a) a nucleotide sequence that is at least 90% identical to the nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 7; preferably a nucleotide sequence that is at least 95% identical; more preferably a nucleotide sequence that is at least 99% identical;

[0139] (b) a nucleotide sequence that is at least 90% identical to the nucleotide sequence of any one of SEQ ID NO: 8 to SEQ ID NO: 11 and SEQ ID NO: 23 to SEQ ID NO: 28; preferably a nucleotide sequence that is at least 95% identical; more preferably a nucleotide sequence that is at least 99% identical.

[0140] In certain embodiments, the nucleic acid is independently selected from a nucleotide sequence that is at least 90% identical to the nucleotide sequence of any one of SEQ ID NO:35-SEQ ID NO:51; preferably, a nucleotide sequence that is at least 95% identical; more preferably, a nucleotide sequence that is at least 99% identical.

[0141] In certain embodiments, the host cell is an Escherichia coli cell or an Escherichia coli strain, a yeast cell or a yeast strain; preferably a yeast cell or a yeast strain; more preferably a Saccharomyces cerevisiae cell or a Saccharomyces cerevisiae strain.

[0142] In a further embodiment, the Saccharomyces cerevisiae is obtained by replacing the ERG9 promoter in CEN.PK2-1D Saccharomyces cerevisiae with P HXT1 ; After knocking out LPP1 and DPP1 and inserting P TEF1 -EfmvaE-T ADH2 and P TEF1 -HMGr(opt)-T PGI1 ; Insert HIS3-P at chromosome 1622b TEF1 -ERG10-P FBA1 -ERG13-P TEF2 -tHMG1; URA3-P inserted at the YPRC15C locus on the chromosome TEF1 -ERG12-P FBA1 -ERG8-P TEF2 In certain embodiments, the Saccharomyces cerevisiae is obtained by knocking out LPP1 and GAL80 in CEN.PK2-1D Saccharomyces cerevisiae and inserting P TEF1 -EfmvaE-T ADH2 and P TEF2 -IDI1; URA3-P inserted at the YPRC15C locus on the chromosome TEF1 -ERG12-P FBA1 -ERG8-P TEF2-ERG19; HIS3-ERG13-P inserted at chromosome 1622b GAL1 -P GAL10 -EfmvaE-T ADH2 ; Insert TRP at the trp1-289 site on chromosome.

[0143] In the present invention, the term "HMGr (opt)" refers to a codon-optimized sequence of the HMGr gene used, for example, a sequence codon-optimized for yeast cells.

[0144] The present invention further provides a method for producing (-)-germarene A, comprising the following steps: fermenting the above-mentioned engineered bacteria and host cells to obtain (-)-germarene A.

[0145] The present invention further provides the use of the above-mentioned engineered bacteria and host cells in producing (+)-β-elemene.

[0146] Furthermore, the present invention provides a method for producing (-)-germane A, characterized in that the method comprises:

[0147] 1) Provide farnesyl pyrophosphate (FPP) precursor,

[0148] 2) contacting the FPP precursor with an enzyme for catalyzing the production of (-)-germarene A from the FPP precursor. Preferably, the enzyme is selected from the above-mentioned proteins.

[0149] Furthermore, the enzyme is an enzyme in a microorganism that provides a precursor heterologously.

[0150] Furthermore, the microorganism is yeast or Escherichia coli.

[0151] The present invention further provides a genetically modified host cell capable of producing (-)-germarene A, comprising 1) one or more heterologous nucleic acids encoding one or more enzymes in the mevalonate pathway for producing farnesyl pyrophosphate; and 2) a heterologous nucleic acid encoding an enzyme for producing (-)-germarene A, wherein the enzyme is selected from the above-mentioned protein.

[0152] The present invention provides a method for producing (+)-β-elemene, comprising the following steps:

[0153] 1) fermenting the engineered bacteria or host cells to obtain a fermentation liquid;

[0154] 2) extracting the fermentation broth with an organic solvent and collecting the organic phase;

[0155] 3) heating the organic phase to obtain (+)-β-elemene.

[0156] In a further embodiment, the fermentation comprises yeast fermentation or E. coli fermentation. Yeast fermentation or E. coli fermentation can be performed using methods known in the art.

[0157] In some embodiments, the fermentation comprises a two-phase fermentation or a single-phase fermentation.

[0158] In some embodiments, the fermentation temperature is 20-40°C. In some embodiments, the fermentation temperature is 20-37°C. In some embodiments, the fermentation temperature is 37°C. In some embodiments, the fermentation temperature may be 20-30°C. In some embodiments, the fermentation temperature may be 25-30°C. In some embodiments, the fermentation temperature is 25°C.

[0159] In some embodiments, the fermentation speed is 100-500 rpm. In some embodiments, the fermentation speed is 200-300 rpm.

[0160] In some embodiments, the fermentation time is 1-240 hours. In some embodiments, the fermentation time is 40-120 hours.

[0161] In some embodiments, the fermentation is a single-phase yeast fermentation. For example, the single-phase yeast fermentation comprises inoculating a single clone of the engineered bacteria into a fermentation medium, fermenting at a temperature of 30°C and a rotational speed of 250 rpm for 40 hours. Prior to inoculation into the fermentation medium, the bacteria may undergo expansion culture, including one or more single-stage expansion cultures.

[0162] In some embodiments, the fermentation is a yeast biphasic fermentation. For example, the yeast biphasic fermentation includes inoculating a single clone of the engineered bacteria into an expansion medium, culturing at a fermentation temperature of 30°C and a fermentation speed of 250 rpm with shaking for 24 hours; transferring 5 vol%-10 vol% of the bacterial solution from the 24-hour culture to a flask containing fermentation medium and n-dodecane, and culturing at a fermentation temperature of 30°C and a fermentation speed of 250 rpm for 120 hours. In some embodiments, the fermentation is an Escherichia coli biphasic fermentation, including inoculating a single clone of the engineered bacteria into an expansion medium, culturing at a fermentation temperature of 37°C and a fermentation speed of 250 rpm overnight; transferring 5 vol%-10 vol% of the bacterial solution from the overnight culture to a flask containing fermentation medium and n-dodecane, and culturing at a fermentation temperature of 37°C and a fermentation speed of 250 rpm for 24 hours. In biphasic fermentation, one or more stages of amplification culture may be used.

[0163] In some embodiments, the germacene A is extracted by extraction or chromatography. In some embodiments, the organic solvent used for extraction includes but is not limited to n-dodecane, isopropyl myristate, n-hexane, n-heptane, etc. In some embodiments, the organic solvent used for extraction is n-dodecane.

[0164] In some embodiments, the (-)-germanene A can be converted into (+)-β-elemene by heating.

[0165] Technical Effects

[0166] The method of the invention can realize the efficient synthesis of (-)-germaene A, has simple and effective construction and fermentation methods, and is easy to realize industrialization.

[0167] definition

[0168] Unless otherwise indicated, the following terms used in this disclosure have the following meanings. A particular term should not be considered as undefined or unclear unless otherwise defined, but should be understood according to its ordinary meaning in the art. When a trade name appears in this article, it is intended to refer to the corresponding commercial product or its active ingredient. The term "wild type" refers to a naturally occurring polynucleotide sequence encoding a protein or a portion thereof, or a protein sequence or a portion thereof, as normally found in the body.

[0169] The term "nucleic acid" is well known in the art. "Nucleic acid" as used herein generally refers to a molecule (i.e., chain) of DNA, RNA, or their derivatives or analogs containing a nucleobase. Nucleobases include, for example, naturally occurring purines or pyrimidine bases (e.g., adenine "A", guanine "G", thymine "T" or cytosine "C") found in DNA or RNA. The term "nucleic acid" encompasses the terms "oligonucleotide" and "polynucleotide."

[0170] A "promoter" is a region of a nucleic acid sequence that controls the initiation and rate of transcription. It may contain elements to which regulatory proteins and molecules (such as RNA polymerase and other transcription factors) can bind to initiate specific transcription of a nucleic acid sequence.

[0171] A "terminator" is a sequence-based element that defines the end of a transcription unit (e.g., a gene) and initiates the release of newly synthesized RNA from the transcription machinery. The terminator is located downstream of the gene to be transcribed, typically directly following any 3' regulatory elements, such as a polyadenylation or poly(A) signal.

[0172] Unless the context clearly indicates otherwise, the singular terms "a / an / the" include plural referents. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise.

[0173] The word "comprise" or "comprises" and its English variations such as comprises or comprising should be understood as having an open and non-exclusive meaning, ie, "including but not limited to".

[0174] All patents, patent applications and other identified publications are expressly incorporated herein by reference for the purposes of description and disclosure. Any citation of these publications herein does not constitute an admission that the publications become part of the common general knowledge in the art. BRIEF DESCRIPTION OF THE DRAWINGS

[0175] FIG1 shows a schematic diagram of the metabolic pathway for the biosynthesis of (-)-germarene A in an engineered bacterium using yeast as a starting strain. DETAILED DESCRIPTION

[0176] The technical solution of the present invention is further described in detail below in conjunction with the description and specific implementation examples, but the implementation of the present invention is not limited thereto. Equivalent replacements, combinations, improvements or modifications made by those skilled in the art to the technical solution of the present invention based on the description of the present invention should all be included in the scope of protection of the present invention.

[0177] Unless otherwise indicated, the experimental methods used below are routine methods well known to those skilled in the art. For example, they can be performed using the standard procedures described in the following works: Sambrook et al., Molecular Cloning: A Laboratory Manual (3rd ed.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2001; Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA, 1995; and Juan S. Bonifacino et al., Current Protocols in Cell Biology, John Wiley and Sons, Inc.

[0178] For information on plasmid pJBEI-6409 and plasmid pBbA5c-MM, refer to Pamela et al., Nature Communication, 2011; Jorge et al., Metabolic Engineering, 19 (2013) 33-41.

[0179] Identification of (-)-germarene A: Based on the disclosure in Alfred J. Weinheimer et al., Tetrahedron Letters No. 7, pp. 497-500, 1970.

[0180] Example 1

[0181] 1. Plasmid Preparation

[0182] The promoter TEF1 (SEQ ID NO: 92), PGK1 (SEQ ID NO: 93) and ERG20 gene fragment (SEQ ID NO: 66) were obtained from Saccharomyces cerevisiae by conventional methods, and the linear vector fragment pESC-LEU-T was obtained by PCR amplification from the plasmid pESC-LEU (Miaoling Biotechnology, China). CYC1 -T ADH1 . Using the full-strength gold seamless cloning kit The linear vector fragment was ligated with the TEF1, PGK1, and ERG20 fragments using the Seamless Cloning and Assembly Kit (Quanshijin, China). The ligation product was then transformed into Trans5α competent cells (Quanshijin, China), plated on LB plates containing ampicillin, and cultured at 37°C for 16 h. A single clone was picked and cultured overnight at 37°C. The plasmid was extracted and sequenced to obtain the yeast expression vector pESC-LEU-P TEF1 -ERG20-T CYC1 -P PGK1 -T ADH1 .

[0183] The nucleotide sequences encoding MmGAS, MaGAS, MdeTPS, MmsTPS, MmoTPS, CmGAS, NiGAS, NcGAS, VmaTPS, DeGAS, and DlGAS were synthesized by conventional means according to the nucleotide sequences in Table 1 using the full-length Gold Seamless Cloning Kit. The synthesized gene fragments were cloned into the yeast expression vector pESC-LEU-P using the Seamless Cloning and Assembly Kit (Quanshijin, China). TEF1 -ERG20-T CYC1 -P PGK1 -T ADH1The ligation product was transformed into Trans 5α competent cells (Quanshijin, China), spread on LB plates containing ampicillin, and cultured at 37°C for 16 h. After picking a single clone and culturing it at 37°C overnight, the plasmid was extracted and sequenced to obtain the plasmids shown in Table 1.

[0184] Table 1 Enzyme related information and constructed recombinant plasmids

[0185] 2. Construction of Saccharomyces cerevisiae strain CEN11

[0186] CEN.PK2-1D (Euroscarf, Germany) was selected for transformation. Donor DNA was constructed by conventional methods and gene editing was performed as shown in Table 2 below (Amanda Reider Apel. et al., 2016). The ERG9 promoter was replaced with HXT1 to downregulate the expression level of ERG9, and the LPP1 and DPP1 genes were knocked out while the EfmvaE and HMGr (opt) genes were inserted. The HIS3, ERG10, ERG13, and tHMG1 genes were inserted at the neutral site 1622b, and the URA3, ERG12, ERG8, and ERG19 genes were inserted at the neutral site YPRC15C. Finally, the recombinant Saccharomyces cerevisiae strain CEN11 was obtained.

[0187] Table 2

[0188] The CEN11 monoclonal cells were picked and transferred to 3 mL SD-His Ura (Panjinuo, China) liquid medium and cultured at 30°C for 24 h. The cells were then transferred to 50 mL SD-His Ura (Panjinuo, China) liquid medium at a 5% transfer volume and cultured until the OD 600 =0.6-0.8, the cells were collected and CEN11 competent cells were prepared using ZYMO Frozen-EZ Yeast Transformation II Kit (Zymo Research, USA) according to the kit's operating procedures.

[0189] 3. Construction of engineered bacteria

[0190] According to the ZYMO Frozen-EZ Yeast Transformation II Kit (Zymo Research, USA) procedure, different plasmids (see Table 3) were transformed into the above-mentioned CEN11 competent cells, coated on SD-Leu His Ura plates, and cultured at 30°C for about 65 hours to obtain the corresponding engineered bacteria (see Table 3 below).

[0191] Table 3

[0192] 4. Preparation and yield of (-)-germaene

[0193] 4.1 Culture medium preparation

[0194] Calcium chloride mother liquor: prepared according to Example 3 of the prior art CN108060092B.

[0195] Trace metal salt mother liquor: prepared according to Example 3 of the prior art CN108060092B.

[0196] Vitamin mother solution: 0.2g / L biotin, 0.8g / L sodium p-aminobenzoate, 4g / L niacin, 4g / L calcium pantothenate, 4g / L pyridoxine hydrochloride, 4g / L thiamine hydrochloride, 100g / L inositol.

[0197] F1 fermentation medium: 25 g / L glucose, 15 g / L ammonium sulfate, 6.15 g / L magnesium sulfate heptahydrate, 0.72 g / L zinc sulfate heptahydrate, 8 g / L potassium dihydrogen phosphate, 2 ml / L calcium chloride mother liquor, 10 ml / L trace metal salt mother liquor, 3 ml / L vitamin mother liquor, 20 mg / L tryptophan.

[0198] 4.2 Fermentation of engineered Saccharomyces cerevisiae

[0199] Single clones of the engineered bacteria obtained in step 3 were transferred to 3 mL of SD-Leu (Panjinuo, China) liquid medium and cultured with shaking at 30°C and 250 rpm for 24 h. A 5% transfer volume was then added to a 100 mL Erlenmeyer flask containing 20 mL of F1 fermentation medium and 2 mL of n-dodecane, and cultured at 30°C and 250 rpm for 120 h. The n-dodecane layer above the fermentation broth was aspirated and diluted 10-fold with ethanol for yield analysis. The results are shown in Table 4.

[0200] Table 4 *Indicates: trace amount, large detection error

[0201] Example 2

[0202] 1. Construction of yeast expression plasmid for VmaTPS point mutant

[0203] Plasmid pESC-LEU-P TEF1 -ERG20-T CYC1 -P PGK1 -VmaTPS-T ADH1Using the template as a template, point mutation primers were designed for full plasmid amplification (primers are shown in Table 5). The product was digested with DpnI (New England Biolabs, USA) at 37°C for 3 h. The target fragment was purified using the TaKaRa MiniBEST DNA Fragment Purification Kit Ver. 4.0 (TaKaRa, Japan) and transformed into fully gold Trans 5α competent cells. The cells were plated on LB plates containing ampicillin and grown overnight at 37°C. Single colonies were selected for colony PCR verification. After inoculation and culture of positive clones, plasmids were extracted and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing verification of the correct mutation. The sequence of the mutant enzyme and related information are shown in Table 6.

[0204] Table 5 Primer sequences used to introduce point mutations

[0205] Table 6 Relevant information of mutant enzymes and constructed recombinant plasmids

[0206] 2. Construction of engineered bacteria

[0207] According to step 2 (construction of Saccharomyces cerevisiae strain CEN11) and step 3 (construction of engineered bacteria) in Example 1, the engineered bacteria in Table 7 were obtained.

[0208] Table 7 Engineering bacteria and corresponding plasmids

[0209] 3. Preparation and yield of (-)-germaene

[0210] Fermentation culture and yield detection were carried out according to step 4 (preparation of (-)-germaene) in Example 1. The results are shown in Table 8 below:

[0211] Table 8 Yield of (-)-germaene *Indicates: trace amount, large detection error

[0212] Example 3

[0213] 1. Plasmid Construction

[0214] In order to construct the expression vector of the fusion protein, GGGGS was selected as the connecting peptide (Qi Liu et al, 2023), and the ERG20 gene fragment (SEQ ID NO: 66) was obtained from Saccharomyces cerevisiae by conventional methods. Seamless Cloning and Assembly Kit (Quanshijin, China) was used to mix the synthesized MaGAS, MdeTPS, MmsTPS, MmoTPS, VmaTPS, DeGAS, and DlGAS gene fragments (sequences of which are shown in Table 1) at a molar mass ratio of 1:1 according to the kit instructions, and then cloned into the yeast expression vector pESC-LEU-P PGK1 -T ADH1 The ligation product was transformed into Trans 5α competent cells (Quanshijin, China), spread on LB-Amp resistant plates, and cultured at 37°C for 16 h. After picking a single clone and culturing it at 37°C overnight, the plasmid was extracted and sequenced to obtain the plasmids shown in Table 10.

[0215] 2. Construction of Engineered Bacteria

[0216] According to step 2 (construction of Saccharomyces cerevisiae strain CEN11) and step 3 (construction of engineered bacteria) in Example 1, the engineered bacteria shown in Table 9 were obtained.

[0217] Table 9 Engineering bacteria and corresponding plasmids

[0218] 3. Preparation and yield of (-)-germaene

[0219] Fermentation culture and yield detection were carried out according to step 4 (preparation of (-)-germaene) in Example 1. The results are shown in Table 10:

[0220] Table 10 Yield of (-)-germaene

[0221] Example 4

[0222] 1. Construction of Saccharomyces cerevisiae strain CEN124

[0223] To achieve the synthesis of germaene A in Saccharomyces cerevisiae, CEN.PK2-1D (Euroscarf, Germany) was selected for transformation. Donor DNA was constructed by conventional means and gene editing was performed as shown in Table 11 below (Amanda Reider Apel et al., 2016). The LPP1 and GAL80 genes were knocked out and the EfmvaE and IDI1 genes were inserted simultaneously. The URA3, ERG12, ERG8, and ERG19 genes were inserted at the neutral site YPRC15C, and the HIS3, ERG13, and EfmvaE genes were inserted at the neutral site 1622b. The TRP gene was backfilled in situ, and the ERG9 promoter was replaced with HXT1 and then with the original ERG9 promoter. Finally, the recombinant Saccharomyces cerevisiae strain CEN124 was obtained.

[0224] Table 11

[0225] 2. Preparation of competent CEN124 yeast

[0226] The CEN124 monoclone was picked and transferred to 3 mL SD-Trp His Ura (Panjinuo, China) liquid medium and cultured at 30°C for 24 h. Then, the clone was transferred to 50 mL YPD liquid medium at a 5% transfer volume and cultured until the OD 600 =0.6-0.8, the bacteria were collected and CEN124 competent cells were prepared using ZYMO Frozen-EZ Yeast Transformation II Kit (Zymo Research, USA) according to the kit operation procedure.

[0227] 3. Construction of engineered bacteria

[0228] According to the ZYMO Frozen-EZ Yeast Transformation II Kit (Zymo Research, USA) kit operating procedures, different plasmids (see Table 12) were transformed into the above-mentioned CEN124 competent cells, coated on SD-Leu plates, and cultured at 30°C for 48 hours to obtain the corresponding engineered bacteria (see Table 12).

[0229] Table 12 Engineering bacteria and corresponding plasmids

[0230] 4. Preparation and yield of (-)-germaene

[0231] 4.1 Culture medium preparation

[0232] Calcium chloride mother liquor: prepared according to Example 3 of the prior art CN108060092B.

[0233] Trace metal salt mother liquor: prepared according to Example 3 of the prior art CN108060092B.

[0234] Vitamin mother solution: 0.2g / L biotin, 0.8g / L sodium p-aminobenzoate, 4g / L niacin, 4g / L calcium pantothenate, 4g / L pyridoxine hydrochloride, 4g / L thiamine hydrochloride, 100g / L inositol.

[0235] Succinic acid mother liquor: 60 g / L succinic acid, adjust pH to 5.05 with NaOH.

[0236] F2 fermentation medium: 25 g / L glucose, 15 g / L ammonium sulfate, 6.15 g / L magnesium sulfate heptahydrate, 0.72 g / L zinc sulfate heptahydrate, 8 g / L potassium dihydrogen phosphate, 2 ml / L calcium chloride mother liquor, 10 ml / L trace metal salt mother liquor, 3 ml / L vitamin mother liquor, 100 mL / L succinic acid mother liquor.

[0237] 4.2 Fermentation of engineered Saccharomyces cerevisiae

[0238] Single clones of the engineered bacteria obtained in step 3 were transferred to 3 mL of SD-Leu (Panjinuo, China) liquid medium and cultured at 30°C, 250 rpm, and shaken for 24 hours. A 5% transfer volume was used to transfer the 24-hour culture broth to a 100 mL Erlenmeyer flask containing 20 mL of F2 fermentation medium and cultured at 30°C, 250 rpm, for 40 hours. 750 μL of the 40-hour fermentation broth was transferred to a 2 mL centrifuge tube, 300 μL of n-dodecane was added, and the cells were ground with glass beads for 30 minutes. The n-dodecane layer was centrifuged and diluted 10-fold with ethanol for yield analysis. The results are shown in Table 13:

[0239] Table 13 Yield of (-)-germaene

[0240] Example 5 Escherichia coli system

[0241] 1. Construction of pCDF-glnAP2-MaGAS recombinant plasmid

[0242] The promoter fragment glnAP2 (SEQ ID NO: 94) was obtained from the Escherichia coli W3110 genome using conventional methods. The T7 promoter fragment in the plasmid pCDFDuet-1 (Miaoling Biotechnology, China) was replaced with glnAP2 to generate the plasmid pCDF-glnAP2. The constructed plasmid pCDF-glnAP2 was used as a template for reverse amplification to generate a linear vector. Simultaneously, the target fragment containing homology arms was amplified using the synthesized plasmid pET-28a-MaGAS (BGI, China). Details of the primers used are shown in Table 14.

[0243] Fragment amplification was performed using the high-success-rate PCR enzyme TOYOBO KOD FX (TOYOBO, Japan). The amplification system consisted of 12.5 μL of 2× PCR Buffer, 5 μL of 2 mM dNTPs, 0.75 μL of each primer, 10 ng of template, 0.5 μL of KOD FX, and ddH₂O to a total volume of 25 μL. The pCDF-glnAP2 fragment amplification conditions were: 94°C denaturation for 5 minutes (1 cycle); 30 cycles of denaturation at 98°C for 10 seconds, annealing at 53°C for 30 seconds, and extension at 68°C for 2.5 minutes; and 1 cycle of extension at 68°C for 2 minutes. The amplification conditions of MaGAS fragment were as follows: pre-denaturation at 94°C for 5 min (1 cycle); denaturation at 98°C for 10 s, annealing at 60°C for 30 s, and extension at 68°C for 1 min, with the annealing temperature decreasing by 1°C in each subsequent cycle until it reached 50°C, for a total of 10 cycles; denaturation at 98°C for 10 s, annealing at 50°C for 30 s, and extension at 68°C for 1 min (20 cycles); and extension at 68°C for 2 min (1 cycle).

[0244] Table 14 Primer sequences

[0245] After the reaction, the fragments were digested with DpnI at 37℃ for 2h and then electrophoresed on 1% agarose gel. The target fragments were recovered by cutting the gel. The linearized vector pCDF-glnAP2 was homologously recombined with the target MaGAS fragment (SEQ ID NO. 2) using a Seamless Cloning and Assembly Kit (Quanshijin, China). The cells were then transformed into Quanshijin Trans 5α competent cells, plated on LB plates containing spectinomycin, and cultured overnight at 37°C. Single colonies were identified and verified by colony PCR using primers glnAP2-check-F and pCDF-check-R (see Table 16). Positive colonies were inoculated and cultured, and plasmids were extracted and sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing. The successfully constructed plasmid was named pCDF-glnAP2-MaGAS (see SEQ ID NO. 64).

[0246] 2. Construction of pLacUV5-MVA recombinant plasmid

[0247] The fragments containing the upstream and downstream genes of the MVA pathway, the replication center, and the resistance gene fragment (SEQ ID NO: 95-SEQ ID NO: 96) were obtained from plasmid pJBEI-6409 (Miaoling Biotechnology, China) and plasmid pBbA5c-MM (Miaoling Biotechnology, China) by conventional methods. Each fragment was homologously recombined using the Seamless Cloning and Assembly Kit (Quanshijin, China), and then transformed into Quanshijin Trans 5α competent cells. The cells were plated on LB plates containing chloramphenicol and cultured at 37°C overnight. Single clones were picked for culture, and the plasmids were extracted and sequenced to obtain the plasmid pLacUV5-MVA.

[0248] 3. Construction of Escherichia coli W3110L strain

[0249] In order to achieve the synthesis of germaene A in Escherichia coli, Escherichia coli W3110 (ToloBio, China) was selected for transformation. Donor DNA was constructed by conventional methods and gene editing was performed as shown in Table 15 (Yu Jiang. et al, 2015). The Escherichia coli W3110L strain was obtained by knocking out the glnL gene.

[0250] Table 15

[0251] 3. Preparation of competent E. coli W3110L

[0252] Take the W3110L monoclonal clone obtained in step 2 and culture it in LB liquid medium at 37°C and 250 rpm overnight. Then transfer it to 50 mL of fresh LB liquid medium at a transfer volume of 1% and culture it until the OD 600 =0.4-0.6, collect the bacteria, and prepare electroporation competent cells according to the Cold Spring Harbor Laboratory Manual.

[0253] 4. Construction of engineered bacteria

[0254] Take the W3110L competent cells from step 3, add the constructed plasmid pLacUV5-MVA (see SEQ ID NO. 65), then add the constructed plasmid pCDF-glnAP2-MaGAS, mix well, transfer to a cuvette, and electroporate under the following conditions: 1.8 kV, 25 uF, 200 Ω. After electroporation, add 1 mL of 37°C preheated SOC to resuspend, transfer to a centrifuge tube, and resuspend at 37°C for 1 hour. Then, spread on LB plates containing spectinomycin and chloramphenicol and culture at 37°C overnight. The resulting engineered strain was named W3110L-MVA-MaGAS.

[0255] 5. Preparation of (-)-germaene and its content detection

[0256] 5.1 Medium Preparation: M9 medium was prepared according to Cold Spring Harbor Protocols (https: / / cshprotocols.cshlp.org / ).

[0257] 5.2 Fermentation of Engineered Escherichia coli: A single clone of the engineered bacteria obtained in step 4 was transferred to 3 mL of LB-Spec+Chlor liquid medium and cultured overnight at 37°C, 250 rpm. A 5% transfer volume of the overnight culture solution was transferred to a 250 mL Erlenmeyer flask containing 20 mL of M9 medium and 2 mL of n-dodecane. The culture was cultured at 37°C, 250 rpm for 24 h. The yield was determined. The results are shown in Table 16:

[0258] Table 16

[0259] Example 6 Preparation of (+)-β-elemene

[0260] To the (-)-germane A fermentation broth obtained in Example 1-5, n-dodecane was added at a ratio of 1:2 (V n-dodecane:V fermentation broth), and the mixture was stirred and extracted for 16 hours. The organic phase was heated at 150°C for 2 hours to obtain a conversion solution. The conversion solution was then subjected to silver nitrate-silica gel and silica gel column chromatography, respectively, to obtain (+)-β-elemene. 1 H NMR(CDCl3,400MH z):1.03(3H,s,H-15), 1.48(3H,m,H-2,3a), 1.64(1H,m,H-5a), 1.56(2H,m,H-3b,5b) ,1.74(3H,s,H-11), 1.77(3H,s,H-14), 1.96(1H,m,H-6), 2.04(1H,m,H-4), 4.61(1H,s ,H-10a), 4.73(1H,s,H-10b), 4.74(1H,brs,H-13a), 4.85(1H,brs,H-13b), 4.92(1H, d, J = 11.2 Hz, H-8a), 4.93 (1H, d, J = 17.6 Hz, H-8b), 5.85 (1H, dd, J = 17.6, 11.2 Hz, H-7).

[0261] 13 C NMR (CDCl3, 100MHz): 16.6 (C-15), 21.1 (C-14), 24.8 (C-11), 26.8 (C-5), 32.9 (C-3), 39.8 (C-1), 39.9 (C-2) , 45.7(C-6), 52.7(C-4), 108.2(C-13), 109.8(C-8), 112.1(C-10), 147.7(C-9), 150.3(C-12), 150.4(C-7).

Claims

1. An engineered bacterium capable of producing (-)-germaneene A, wherein the engineered bacterium comprises one or more enzymes independently selected from: (a) a germacene A synthase (GAS) having any one of SEQ ID NO: 12 to SEQ ID NO: 18, or having an amino acid sequence that is at least 80% identical to any one of SEQ ID NO: 12 to SEQ ID NO: 18, and / or (b) a terpene synthase (TPS) having any one of SEQ ID NO: 19 to SEQ ID NO: 22, or an amino acid sequence that is at least 80% identical to any one of SEQ ID NO: 19 to SEQ ID NO:

22.

2. The engineered bacteria according to claim 1, in, The engineered bacteria comprises a germacene A synthase (GAS) having an amino acid sequence that is at least 81%, preferably at least 85%, more preferably at least 90%, further preferably at least 95%, and most preferably at least 99% identical to the amino acid sequence of any one of SEQ ID NO: 12 to SEQ ID NO:

18.

3. The engineered bacteria according to claim 1, in, The engineered bacteria comprises a terpene synthase (TPS) having an amino acid sequence that is at least 81%, preferably at least 85%, more preferably at least 90%, further preferably at least 95%, and most preferably at least 99% identical to the amino acid sequence of any one of SEQ ID NO: 19 to SEQ ID NO:

22.

4. The engineered bacteria according to claim 1 or 2, in, The engineered bacteria comprises a gimaecene A synthase (GAS) having an amino acid sequence identical to any one of SEQ ID NO: 12-SEQ ID NO: 18; preferably a gimaecene A synthase (GAS) having an amino acid sequence identical to any one of SEQ ID NO: 13-SEQ ID NO: 15 and SEQ ID NO: 17-SEQ ID NO: 18; more preferably a gimaecene A synthase (GAS) having an amino acid sequence identical to SEQ ID NO: 14 and SEQ ID NO: 17-SEQ ID NO:

18.

5. The engineered bacteria according to claim 1 or 3, in, The engineered bacteria comprises a terpene synthase (TPS) having an amino acid sequence of any one of SEQ ID NO: 19, SEQ ID NO: 21, and SEQ ID NO: 22; preferably comprises a terpene synthase (TPS) having an amino acid sequence of any one of SEQ ID NO: 21 and SEQ ID NO:

22.

6. According to the engineered bacteria described in claim 1, 3 or 5, in, The terpene synthase (TPS) has one or more amino acid substitutions relative to the amino acid sequence of SEQ ID NO: 22; preferably, the terpene synthase (TPS) has an amino acid substitution at one or more of E28, A30, A101, C129 and P206 relative to the amino acid sequence of SEQ ID NO: 22; more preferably, the one or more amino acid substitutions are selected from one or more of E28Q, E28H, A30D, A101G, C129I and P206S.

7. The engineered bacteria according to any one of claims 1 to 6, in, The engineered bacteria contain an expression box of a nucleotide sequence encoding germarene A synthase (GAS) and / or terpene synthase (TPS).

8. The engineered bacteria according to claim 7, in, The nucleic acid encoding germarene A synthase (GAS) comprises a nucleotide sequence independently selected from the group consisting of at least 90%, preferably at least 95%, more preferably at least 99% identical to the nucleotide sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 7; or The nucleic acid encoding a terpene synthase (TPS) comprises a nucleotide sequence independently selected from the group consisting of at least 90%, preferably at least 95%, more preferably at least 99% identical to the nucleotide sequence of any one of SEQ ID NO:8-SEQ ID NO:11 and SEQ ID NO:23-SEQ ID NO:

28.

9. The engineered bacteria according to any one of claims 1 to 8, in, The engineered bacteria has increased farnesyl pyrophosphate synthase ERG20 activity; preferably, the engineered bacteria further comprises an expression cassette encoding farnesyl pyrophosphate synthase ERG20.

10. The engineered bacteria according to claim 9, in, The farnesyl pyrophosphate synthase ERG20 and the jimarenene A synthase and / or the terpene synthase (TPS) are in the same expression cassette; preferably, the farnesyl pyrophosphate synthase ERG20 and the jimarenene A synthase and / or the terpene synthase (TPS) are in the form of a fusion protein; more preferably, in the fusion protein, the farnesyl pyrophosphate synthase ERG20 and the jimarenene A synthase and / or the terpene synthase (TPS) are connected by a connecting peptide, and the connecting peptide is selected from GGGGS, YGQ, PGGH, YRSQI, VIPFIS, FLYLKF, WRFSPKLQ or HHVQESQCISTV; preferably GGGGS.

11. The engineered bacteria according to any one of claims 1 to 10, in, The engineered bacteria is yeast or Escherichia coli; preferably yeast; more preferably Escherichia coli or Saccharomyces cerevisiae.

12. The engineered bacteria according to any one of claims 1 to 11, in, In the yeast engineered bacteria, the engineered bacteria further include one or more of the following characteristics: Reduce the activity of squalene synthase ERG9; Reduced activity of the genes DPP1 and / or LPP1 responsible for farnesol synthesis; Increase the activity of acetyl-CoA C-acetyltransferase / 3-hydroxy-3-methylglutaryl-CoA reductase EfmvaE; Increased activity of 3-hydroxy-3-methylglutaryl-CoA reductase HMGr; Increased activity of mevalonate kinase ERG12; Increased activity of phosphomevalonate kinase ERG8; Increased activity of mevalonate pyrophosphate decarboxylase ERG19; Increased activity of acetyl-CoA-acetyltransferase ERG10; Increased activity of 3-hydroxy-3-methylglutaryl-CoA synthetase ERG13; Reduced activity of GAL80, which is responsible for regulating the expression of the GAL promoter; Increased activity of isopentenyl pyrophosphate isomerase IDI1; or Increased activity of 3-hydroxy-3-methylglutaryl-CoA reductase 1 tHMG1.

13. The engineered bacteria according to any one of claims 1 to 11, in, In the E. coli engineered bacteria, the GAS or TPS expression cassette in the engineered bacteria can use a promoter from E. coli itself, preferably glnAP2; Alternatively, the engineered Escherichia coli has reduced activity of the glnL gene.

14. The engineered bacteria according to claim 13, in, In the engineered Escherichia coli, the engineered bacteria contain an introduced gene of the MVA pathway.

15. A host cell capable of producing (-)-germaene A, in, The host cell comprises one or more nucleic acids, each of which encodes: (a) a germacene A synthase having an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID NO: 12 to SEQ ID NO: 18 independently (GAS), and / or (b) a terpene synthase (TPS) having an amino acid sequence that is at least 80% identical to the amino acid sequence of any one of SEQ ID NO: 19 to SEQ ID NO: 22 and SEQ ID NO: 29 to SEQ ID NO:

34.

16. The host cell according to claim 15, in, The host cell is an Escherichia coli cell or an Escherichia coli strain, a yeast cell or a yeast strain; preferably a yeast cell or a yeast strain; more preferably a Saccharomyces cerevisiae cell or a Saccharomyces cerevisiae strain.

17. A method for producing (-)-germaene A, comprising the following steps: fermenting the engineered bacteria according to any one of claims 1 to 14 and the host cell according to claim 15 or 16 to obtain (-)-germaene A.

18. Use of the engineered bacteria according to any one of claims 1 to 14, or the host cell according to claim 15 or 16 in producing (+)-β-elemene.

19. A method for producing (+)-β-elemene, include: 1) fermenting the engineered bacteria according to any one of claims 1 to 14, or the host cell according to claim 15 or 16 to obtain a fermentation liquid; 2) extracting the fermentation broth with an organic solvent and collecting an organic phase; 3) heating the organic phase to obtain (+)-β-elemene.

20. The method according to claim 19, in, The organic solvent is selected from one or more of n-dodecane, isopropyl myristate, n-hexane and n-heptane.

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