CN112063540B

CN112063540B - Recombinant strain for producing beta-elemene or germacrene A

Info

Publication number: CN112063540B
Application number: CN202010995847.3A
Authority: CN
Inventors: 刘巍峰; 张伟欣; 孟祥锋
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2020-09-21
Filing date: 2020-09-21
Publication date: 2022-05-17
Anticipated expiration: 2040-09-21
Also published as: CN112063540A

Abstract

The invention provides a recombinant bacterium, wherein the starting strain of the recombinant bacterium is saccharomyces cerevisiae, a Germacrene A Synthase (GAS) is expressed in the recombinant bacterium, and the GAS is derived from Anabaena variabilis ATCC 29413; according to the invention, the efficiency of synthesizing the germacrene A by the yeast is effectively improved by optimizing the germacrene A synthesis module, the MVA pathway endogenous modification module, the synthase mutation modification module and the efflux channel module; the recombinant yeast can be efficiently used for producing beta-elemene and/or germacrene A.

Description

Recombinant strain for producing beta-elemene or germacrene A

Technical Field

The invention relates to the field of bioengineering, and in particular relates to a recombinant strain for producing beta-elemene or germacrene A.

Background

Beta-elemene is a sesquiterpene, which is naturally derived from Curcuma wenyujin Y.H. Chen et C.Ling of Zingiberaceae family and has the chemical name of 1-methyl-1-vinyl-2, 4-diisopropylcyclohexane. Beta-elemene is one of the main components of the national class II anticancer drug beta-elemene milk, has broad-spectrum antitumor effect, obvious curative effect and small side effect, and a plurality of clinical tests prove that the beta-elemene plays a certain role in treating superficial tumors such as lung cancer, liver cancer, ovarian cancer, gastric cancer and the like. In addition, the effect of beta-elemene under in vitro conditions depends on concentration and cancer cell types, and compared with therapeutic drugs such as adriamycin, vincristine taxol and the like, the beta-elemene has slight anaphylactic reaction, no adverse reaction such as neurotoxicity and the like is found, and the beta-elemene has low toxicity to normal cells. Therefore, the beta-elemene has good application value and market prospect as a high-efficiency and low-toxicity anticancer drug derived from plants.

The direct precursor of the beta-elemene is germacene A, which is a very important cyclic sesquiterpene and is usually used as an intermediate in the generation process of other sesquiterpenes, and the germacrene A is easy to undergo a copperrearrangement reaction under the acidic or heat treatment condition to cause a conformational change to form the beta-elemene.

The biosynthesis of germacrene A is based on the common precursors of terpenoid synthesis, isopentenyl diphosphate (IPP) and dimethylallyl Diphosphate (DMAPP), both of which originate from the methylerythrose-4-phosphate pathway (MEP pathway) and the MVA pathway.

The research of producing germacrene A by using microorganisms mainly focuses on using escherichia coli and saccharomyces cerevisiae as chassis cells, the genetic backgrounds of the two bacteria are clear, the gene operation method is mature, and the bacteria become a plant for heterogeneously synthesizing natural products. The germacrene A synthase is introduced by high permissive et al in 2012 and is expressed in saccharomyces cerevisiae and escherichia coli respectively, and the yield of germacrene A is extremely low and is 9.5 mu g/L and 128 mu g/L respectively. On the basis, the Escherichia coli is selected as an expression host for optimized synthesis of germacrene A, a heterologous MVA approach which is composed of 8 genes and can realize functional expression in the Escherichia coli is constructed, GAS from cyanobacteria is introduced, optimization of fermentation conditions is combined, and the yield reaches 6.33 mg/L. In 2017, Yating Hu et al used Saccharomyces cerevisiae as a host for producing germacrene A, and utilized metabolic engineering and synthetic biology techniques to improve metabolic flux, thereby realizing that the shake flask fermentation yield of germacrene A in Saccharomyces cerevisiae is 190.7 mg/L.

Although the prior art has already studied a lot on germacrene A, how to further improve the yield of germacrene A through condition optimization still remains a problem to be solved in the field.

Disclosure of Invention

In one aspect, the invention provides a recombinant bacterium, wherein a starting strain of the recombinant bacterium is Saccharomyces cerevisiae (Saccharomyces cerevisiae), the recombinant bacterium expresses Germacrene A Synthase (GAS), and the GAS is derived from Anabaena variabilis (Anabaena variabilis) ATCC 29413.

In one embodiment, the GAS has the amino acid sequence shown as seq id No. 1.

Further, the recombinant bacteria also express 3-hydroxy-3-methylglutaryl coenzyme A (HMGR1), preferably, the 3-hydroxy-3-methylglutaryl coenzyme A is truncated 3-hydroxy-3-methylglutaryl coenzyme A (tHMGR 1); more preferably, the amino acid sequence of tHMGR1 is shown in SEQ ID No. 2.

Furthermore, the recombinant bacterium also expresses farnesyl pyrophosphate synthase (ERG20), and preferably, the amino acid sequence of the ERG20 is shown as SEQ ID No. 3.

Further, the expression of squalene synthase (erg9) of the recombinant bacterium is down-regulated; the down-regulation of expression may be achieved by gene mutation or by promoter replacement; preferably, the expression of erg9 is down-regulated by replacing the promoter of itself with the HXT1(hexose transporter 1) promoter; more preferably, the amino acid sequence of erg9 is shown in SEQ ID No. 4; preferably, the HXT1 promoter has the sequence as shown in SEQ ID No. 11.

Further, the recombinant bacterium also comprises inactivation or inhibition of diacylglycerol pyrophosphate phosphatase 1(dpp1) and/or lipid phosphatase 1(lpp 1). Preferably, the amino acid sequence of dpp1 is shown in SEQ ID No.5, and the amino acid sequence of lpp1 is shown in SEQ ID No. 6.

Further, the recombinant bacterium also expresses an efflux channel protein pdr5 and/or SNQ2, preferably, the amino acid sequence of pdr5 is shown as SEQ ID No.8, and the amino acid sequence of SNQ2 is shown as SEQ ID No. 10.

In a preferred embodiment, the expression is overexpression.

Further, the amino acid sequence of GAS in the invention is a mutant sequence of SEQ ID No.1, specifically, on the basis of SEQ ID No.1, the 152 th H is mutated into Y, namely, the sequence shown in SEQ ID No. 9.

Furthermore, the starting strain is Saccharomyces cerevisiae (Saccharomyces cerevisiae) CEN. PK2-1D.

In another aspect, the present invention also provides a method for producing a recombinant bacterium, the method comprising the step of expressing a Germacrene A Synthase (GAS) derived from Anabaena variabilis ATCC 29413; preferably, the amino acid sequence of GAS is shown as SEQ ID No.1, or the amino acid sequence of GAS is a mutant sequence of SEQ ID No.1, specifically, on the basis of SEQ ID No.1, the 152 th H is mutated into Y, i.e., the sequence shown as SEQ ID No. 9.

Further, the method also comprises any one or any several of the following operations in the saccharomyces cerevisiae:

(1) expressing 3-hydroxy-3-methylglutaryl-coenzyme a (HMGR1), preferably said 3-hydroxy-3-methylglutaryl-coenzyme a is truncated 3-hydroxy-3-methylglutaryl-coenzyme a (tHMGR 1);

(2) expressing farnesyl pyrophosphate synthase (ERG 20);

(3) downregulating the expression of squalene synthase (erg 9);

(4) inactivating or inhibiting diacylglycerol pyrophosphate phosphatase 1(dpp 1);

(5) inactivating or inhibiting lipid phosphatase 1(lpp 1);

(6) expression of efflux channel proteins pdr5 and/or SNQ 2.

Further, the above expression is overexpression.

On the other hand, the invention also provides the application of the recombinant bacterium or the recombinant bacterium prepared by the method in the production of beta-elemene and/or germacrene A.

On the other hand, the invention also provides a method for preparing beta-elemene and/or germacrene A, which comprises the step of fermenting by using the recombinant bacterium or the recombinant bacterium prepared by the method.

On the other hand, the invention provides a Germacrene A Synthase (GAS) mutant, the amino acid sequence of the mutant is that on the basis of the sequence shown in SEQ ID No.1, the 152 th H is mutated into Y, namely the sequence shown in SEQ ID No. 9.

In another aspect, the present invention provides a vector comprising the above mutant, which includes a cloning vector or an expression vector.

In another aspect, the present invention also provides host cells comprising the above mutants, including but not limited to escherichia coli, saccharomyces cerevisiae.

In another aspect, the invention also provides the application of the mutant, the vector and the host cell in the production of beta-elemene and/or germacrene A.

In the present invention, the inactivation or inhibition may be achieved by mutating a gene in such a manner that the function of the gene is at least partially inactivated by gene deletion, gene insertion, or gene substitution; in a preferred embodiment, the at least partial inactivation is a complete inactivation of gene function.

In the invention, the expression comprises introducing a target gene into a host bacterium, or increasing the copy number of the target gene in the host bacterium, or replacing a promoter of the gene to improve the expression amount of the target gene. The target gene may be expressed in the host bacterium by an expression vector, or may be expressed by inserting the target gene into the genome of the host bacterium. In the invention, the host bacterium is preferably saccharomyces cerevisiae, and more preferably saccharomyces cerevisiae CEN.PK2-1D.

Preferably, the expression in the present invention is overexpression.

According to the invention, industrial strain Saccharomyces cerevisiae CEN. PK2-1D is used as a chassis cell, a germacrene A synthesis approach is divided into a germacrene A synthesis module, an MVA approach endogenous modification module, a synthase mutation modification module and an efflux channel module, and the efficiency of synthesizing germacrene A by yeast is effectively improved by regulating and optimizing each module, so that the yield reaches 656.65 mg/L.

The sequence information is as follows:

SEQIDNo.	description of the invention
		1	Anv germacrene A synthase
2	tHMGR1, truncated 3-hydroxy-3-methylglutaryl coenzyme A
		3	ERG20, farnesyl Pyrophosphate synthase
4	erg9, squalene synthase
		5	dpp1, diacylglycerol pyrophosphate phosphatase 1
6	lpp1, lipid phosphatase 1
		7	app1, actin 1
8	PDR5, efflux channel protein
		9	GAS mutant, H152Y
10	SNQ2, efflux channel protein
		11	HXT1(hexose transporter 1) promoter

Drawings

FIG. 1 shows the yields of germacrene A (Germacrene A) from different sources of germacrene A synthase-expressing strains.

FIG. 2 Effect of over-expression of tHMGR1 and ERG20 on germacene A content.

FIG. 3. Effect on germacrene A content by engineering the metabolic pathway of Saccharomyces cerevisiae.

FIG. 4 is a schematic diagram showing the effect of the 152 th site mutant of Anv on the production of germacrene A.

FIG. 5 is a graph showing the production of germacrene A in efflux channel overexpressing strains.

FIG. 6 is a graph showing germacrene A production and specific production in YEHP strain.

Detailed description of the preferred embodiments

The present invention will be further described with reference to the following examples, which are intended to be illustrative only and not to be limiting of the invention in any way, and any person skilled in the art can modify the present invention by applying the teachings disclosed above and applying them to equivalent embodiments with equivalent modifications. Any simple modification or equivalent changes made to the following embodiments according to the technical essence of the present invention, without departing from the technical spirit of the present invention, fall within the scope of the present invention.

The overexpression or knockout of the target gene in the invention is performed by the conventional operation in the field, such as:

the gene knockout utilizes the principle of homologous recombination, a primer with 70bp homologous sequences at the upstream and downstream of a target gene open reading frame at the 5' end is used for amplifying a knockout fragment, the knockout fragment is recovered, saccharomyces cerevisiae is transformed, a YPD plate containing 200 mug/mL of geneticin is coated, standing culture is carried out for 2-3 days at 30 ℃, a single colony is selected for culture, and then genome verification is extracted.

The yeast transformation method comprises the following steps: selecting a yeast single colony, culturing in a 3ml liquid YPD culture medium at 30 ℃ and 200rpm for 24h, collecting the thallus into a 2ml Ep tube, washing with sterile water for 2 times, adding 100 mu L of lithium acetate, mixing uniformly, placing on ice, sequentially adding a target fragment or plasmid (0.1-1 mu g), single-chain milt DNA (boiled for about 5min in advance), and 600 mu L of PEG4000, mixing uniformly, and standing at 30 ℃ for 30 min; 70 μ L of DMSO was added, heat shocked at 42 ℃ for 15min (with the period reversed every 5 min), and finally spread on corresponding auxotrophic plates.

Specific operations not mentioned in the embodiments of the present invention may be performed according to the techniques well known in the art.

Example 1 screening of Germacene A Synthase (GAS)

In order to construct a production platform of gemmaline A in Saccharomyces cerevisiae, GAS from different sources is screened in the example, a gemmaline A production platform is initially constructed, and Saccharomyces cerevisiae (Saccharomyces cerevisiae) CEN. PK2-1D is selected as a basal cell. GAS from different sources was transformed into Saccharomyces cerevisiae and expressed under the operation of the gal1 promoter.

By reference to the literature and alignment of NCBI sequences, there are currently over ten different Compositae GAS genes reported, including lettuce, yarrow, Artemisia annua, and the like. Firstly, four GASs with high enzyme specificity proved by in vitro experiments are selected, wherein the GASs are respectively AmGAS from Achillea millefolium, TmGAS from Taraxacum officinale, LTC2 from Lactuca sativa and NS1 from Nostoc sp.PCC7120, the industrial strain CEN.PK2-1D is used as a background strain, the GAS overexpression vectors are respectively transformed into yeast, and formed yeast strains are respectively yAmGAS, yTmGAS, yLTC2 and yNS 1; the content of germacrene A in the fermentation product was detected, as shown in FIG. 1, no germacrene A was detected by yAmGAS, yTmGAS and yLTC2, and germacrene A was clearly detected in strain yNS1 expressing NS 1.

Further, the inventors performed sequence alignment analysis of the amino acid sequence encoded by NS1 in a database, and selected six possible GAS with different similarity coefficients to NS1, expressed in saccharomyces cerevisiae from the sources of Anabaena variabilis ATCC29413, methylglyobolus mourosus KoM1, planomonosporasperica, nosoc sp.pcc7120, Calothrix sp.niess2100, Streptomyces platensis, abbreviated as: anv, Mem, Pls, Noc, Cal, Stp; the constructed recombinant expression strains are respectively yAnv, yMem, yPls, yNoc, yCal and yStp; as shown in FIG. 1, the yield of germacrene A in strain yAnv expressing Anv was the highest, 17.82mg/L, which was about 2 times that in strain NS 1; the amino acid sequence of the Anv is shown as SEQ ID No. 1.

Example 2 engineering of Strain yAnv to increase the yield of germacrene A

In this embodiment, truncated tHMGR1(truncated HMGR1, truncated 3-hydroxy-3-methylglutaryl coenzyme A, shown in SEQ ID No. 2) and FPP synthase (ERG20, Farnesyl pyrophosphate synthase, shown in SEQ ID No. 3) are overexpressed in strain yAnv (also referred to herein as YEMVA 1); strains YEMVA2 and YEMVA3 were obtained, respectively, where YEMVA2 was the overexpression of thhmgr 1 in yAnv and YEMVA3 was the overexpression of thhmgr 1 and ERG20 in yAnv.

As shown in fig. 2, the yield of germacrene a in YEMVA2 strain overexpressing tHMGR1 was about 4.43 times that in YEMVA1 strain; based on strain YEMVA2, ERG20 is further over-expressed to generate strain YEMVA3, and the yield of germacrene A is further improved to be about 1.4 times of the yield of strain YEMVA 2.

In this example, expression of strain erg9(squalene synthase 9, squalene synthase, SEQ ID No. 4) of YEMVA2 was further down-regulated, and its own promoter was replaced with HXT1(hexose transporter 1) (SEQ ID No. 11), to obtain strain YEMVA 4; on the basis of YEMVA4, knocking out dpp1(diacylglycerol pyrophosphate phosphatase 1; shown as SEQ ID No. 5) to obtain a strain YEMVA 5; on the basis of YEMVA5, knocking out lpp1(lipid phosphatase 1; shown as SEQ ID No. 6) to obtain a strain YEMVA 6; on the basis of YEMVA6, app1(actin patch protein 1; actin 1, shown as SEQ ID No. 7) is knocked out to obtain a strain YEMVA 7; further over-expression of ERG20(SEQ ID No. 3) was performed on the basis of YEMVA6 to obtain strain YEMVA8, and the gene manipulation performed by each strain is shown in FIG. 3A.

As shown in fig. 3, the erg9 expression was down-regulated by replacing the erg9 self promoter with HXT1p promoter on the basis of YEMVA2 strain, to obtain YEMVA4 strain with a yield of gimatene a about 2.4 times that of YEMVA2 strain. On the basis of the strain YEMVA4, the strain YEMVA5 is formed by knocking out dpp1, the yield of the strain YEMVA5 is similar to that of the strain YEMVA4, and the strain YEMVA6 is formed by knocking out lpp1, about 1.5 times of that of the strain YEMVA5, which shows that the double knocking out dpp1 and lpp1 can improve the yield of germacrene A. By continuously knocking out app1 on the basis of YEMVA6 strain, the yield of germacrene A in YEMVA7 strain is 180.53mg/L, which is reduced by about 10% compared with YEMVA6 strain. The yield of the germacrene A of the recombinant strain YEMVA8 is 226.82 +/-15.48 mg/L, and the result shows that the capacity of the strain for producing the germacrene A is greatly improved by referring to a figure 3B.

Example 3 Effect of site-directed mutagenesis Anv (Gimerane A synthase) on Gimerane A production

In this example, a germacrene A synthase Anv (SEQ ID No.1) was subjected to site-directed mutagenesis, and based on a constructed P.DELTA. -ERG20 strain (which is obtained by knocking out dpp1, lpp1, and replacing ERG9 promoter hxt1 in S.cerevisiae CEN. PK2-1D, and overexpressing tHMGR1, ERG20), a series of mutants were obtained by expressing Anv subjected to site-directed mutagenesis, and a Anv mutant that effectively improves synthesis ability was sought by detecting the change in the yield of germacrene A of the strain.

In order to increase the activity of germacrene A synthase Anv by site-directed mutagenesis, conserved amino acids A180, A181, A182, A183, L78, I75, L55, I215 and L54, which may be involved in substrate FPP binding, were subjected to corresponding point mutations; on the other hand, we also point-mutated the non-conserved amino acid H152 located on the surface of the protein distal to the catalytic center.

The results show that compared to wild type (strain P Δ -ERG20 expressing wild type Anv):

when the histidine at position 181 of Anv was mutated to glycine (G), the yield of germacrene a decreased by about 40%, while when the mutation was to cysteine (C), phenylalanine (F), leucine (L), tyrosine (Y), threonine (T), the yield of germacrene a was barely detectable;

when the 78 th leucine of Anv is mutated into alanine (A), threonine (T), isoleucine (I), glycine (G) and valine (V), the yield of germacrene A is reduced to different degrees; when the 75 th site isoleucine of Anv is mutated into alanine (A), phenylalanine (F) and leucine (L), the yield of germacrene A is reduced to different degrees;

when the leucine at the 55 th site of Anv is mutated into phenylalanine (F) and valine (V), the yield of the germacrene A is reduced to different degrees; when the mutation is tryptophan (W), alanine (A) and tyrosine (Y), the yield of the germacrene A is hardly detected;

when the 182 th leucine of Anv is mutated into threonine (T), glycine (G) and serine (S), the yield of germacrene A is reduced to different degrees;

when the 183 th leucine of Anv is mutated into phenylalanine (F), serine (S) and glycine (G), the yield of germacrene A is reduced to different degrees; whereas the production of germacrene A was barely detectable when mutated to threonine (T);

when the 215 th isoleucine of Anv is mutated into leucine (L), the yield of the germacrene A is reduced;

when alanine at position 180 is mutated to leucine (L) and leucine at position 54 is mutated to tyrosine (Y), germacrene a is hardly detectable;

histidine 152 distal to the active center of Anv protein was selected for mutagenesis: when histidine (H) at position Anv 152 is mutated into lysine (K), arginine (R), aspartic acid (D), alanine (A), glycine (G), leucine (L) and threonine (T), the yield of germacrene A is reduced, wherein the reduction of germacrene A yield is most obvious when the histidine (H) is mutated into lysine (K) and leucine (L), and is respectively reduced by 2.47 times and 8.865 times; however, when mutation was carried out to tyrosine (Y), the yield of germacrene a reached 323.81mg/L, and was increased by about 40% compared to the wild-type strain (strain P Δ Δ -ERG20 expressing wild-type Anv) (as shown in fig. 4), which was designated as H152Y, and the H152Y strain had only a sequence change of Anv compared to YEMVA8 (mutation of H to Y at position 152).

Example 4 Effect of efflux channel proteins on Geomalene A production

In order to further improve the yield of the germacrene A, an efflux channel endogenous to the saccharomyces cerevisiae is enhanced and expressed in the embodiment, the efflux channel of the germacrene A is definitely and effectively promoted, and finally the strain H152Y and the efflux channel are combined to obtain the strain with high yield of the germacrene A.

In this example, the effect on the production of s.cerevisiae germacrene A was first verified by overexpressing the efflux channel proteins pdr5, snq2, yor1, pdr1, pdr 3. An Anv overexpression vector is constructed by using an integrative plasmid pRS304, the formed plasmid is pIAAAV, then the pIAAAV vector is cut by Bsu36I enzyme to be completely linearized, a yeast P delta background strain (the strain is obtained by knocking out dpp1 and lpp1 in S.cerevisiae CEN.PK2-1D, replacing an erg9 promoter to hxt1 and overexpressing tHMGR1) is transformed by using a lithium acetate chemical transformation method, and the generated strain is YIAAV. YIAAV is taken as a chassis to respectively over-express pdr5, pdr3, pdr1 and pdr5^F185S、pdr3^Y276HSnq2, yor1, yielding YEEF1, YEEF2, YEEF3, YEEF4, YEEF5, YEEF6, YEEF7 strains, respectively; the results are shown in fig. 5, and compared with the control strain not expressing the efflux channel, the yield of germacrene a in the culture medium is improved by about 70% in the over-expressed strain YEEP1 of PDR5 (shown in seq id No. 8); in the SNQ2 (shown as SEQ ID No. 10) overexpression strain YEEP6, the yield of germacrene A is improved by about 27%; compared with the control strain, the other efflux channel expression strains have no obvious change in the yield of the germacrene A.

In order to obtain the optimal strain for producing germacrene A, yeast endogenous pathway modification, germacrene A synthase engineering and an efflux channel module are integrated, namely, an efflux channel protein PDR5 is further overexpressed on the basis of the strain H152Y to generate a recombinant strain YEHP, and the strain YEHP is subjected to fermentation culture and product detection.

As shown in FIG. 6, the yield of the germacrene A of the recombinant strain YEHP is gradually increased along with the prolonging of the fermentation culture time, and the yield is highest after the recombinant strain YEHP is cultured for 72 hours and reaches 656.65 +/-28.24 mg/L; the unit yield at 24h, 36h, 48h, 60h, 72h and 84h is respectively 80.79mg/L/OD, 92.78mg/L/OD, 92.56mg/L/OD, 89.37mg/L/OD, 108.61mg/L/OD and 92.33 mg/L/OD.

The above embodiments are merely illustrative of the technical ideas and features of the present invention, and the purpose thereof is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.

SEQUENCE LISTING

<110> Shandong university

<120> a recombinant strain for the production of beta-elemene or germacrene A

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610 615 620

Phe Met Arg Phe Arg Thr Thr Thr Gly Asp Ala Met Gly Met Asn Met

625 630 635 640

Ile Ser Lys Gly Val Glu Tyr Ser Leu Lys Gln Met Val Glu Glu Tyr

645 650 655

Gly Trp Glu Asp Met Glu Val Val Ser Val Ser Gly Asn Tyr Cys Thr

660 665 670

Asp Lys Lys Pro Ala Ala Ile Asn Trp Ile Glu Gly Arg Gly Lys Ser

675 680 685

Val Val Ala Glu Ala Thr Ile Pro Gly Asp Val Val Arg Lys Val Leu

690 695 700

Lys Ser Asp Val Ser Ala Leu Val Glu Leu Asn Ile Ala Lys Asn Leu

705 710 715 720

Val Gly Ser Ala Met Ala Gly Ser Val Gly Gly Phe Asn Ala His Ala

725 730 735

Ala Asn Leu Val Thr Ala Val Phe Leu Ala Leu Gly Gln Asp Pro Ala

740 745 750

Gln Asn Val Glu Ser Ser Asn Cys Ile Thr Leu Met Lys Glu Val Asp

755 760 765

Gly Asp Leu Arg Ile Ser Val Ser Met Pro Ser Ile Glu Val Gly Thr

770 775 780

Ile Gly Gly Gly Thr Val Leu Glu Pro Gln Gly Ala Met Leu Asp Leu

785 790 795 800

Leu Gly Val Arg Gly Pro His Ala Thr Ala Pro Gly Thr Asn Ala Arg

805 810 815

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

820 825 830

Leu Cys Ala Ala Leu Ala Ala Gly His Leu Val Gln Ser His Met Thr

835 840 845

His Asn Arg Lys Pro Ala Glu Pro Thr Lys Pro Asn Asn Leu Asp Ala

850 855 860

Thr Asp Ile Asn Arg Leu Lys Asp Gly Ser Val Thr Cys Ile Lys Ser

865 870 875 880

<210> 3

<211> 352

<212> PRT

<213> Saccharomyces cerevisiae

<400> 3

Met Ala Ser Glu Lys Glu Ile Arg Arg Glu Arg Phe Leu Asn Val Phe

1 5 10 15

Pro Lys Leu Val Glu Glu Leu Asn Ala Ser Leu Leu Ala Tyr Gly Met

20 25 30

Pro Lys Glu Ala Cys Asp Trp Tyr Ala His Ser Leu Asn Tyr Asn Thr

35 40 45

Pro Gly Gly Lys Leu Asn Arg Gly Leu Ser Val Val Asp Thr Tyr Ala

50 55 60

Ile Leu Ser Asn Lys Thr Val Glu Gln Leu Gly Gln Glu Glu Tyr Glu

65 70 75 80

Lys Val Ala Ile Leu Gly Trp Cys Ile Glu Leu Leu Gln Ala Tyr Phe

85 90 95

Leu Val Ala Asp Asp Met Met Asp Lys Ser Ile Thr Arg Arg Gly Gln

100 105 110

Pro Cys Trp Tyr Lys Val Pro Glu Val Gly Glu Ile Ala Ile Asn Asp

115 120 125

Ala Phe Met Leu Glu Ala Ala Ile Tyr Lys Leu Leu Lys Ser His Phe

130 135 140

Arg Asn Glu Lys Tyr Tyr Ile Asp Ile Thr Glu Leu Phe His Glu Val

145 150 155 160

Thr Phe Gln Thr Glu Leu Gly Gln Leu Met Asp Leu Ile Thr Ala Pro

165 170 175

Glu Asp Lys Val Asp Leu Ser Lys Phe Ser Leu Lys Lys His Ser Phe

180 185 190

Ile Val Thr Phe Lys Thr Ala Tyr Tyr Ser Phe Tyr Leu Pro Val Ala

195 200 205

Leu Ala Met Tyr Val Ala Gly Ile Thr Asp Glu Lys Asp Leu Lys Gln

210 215 220

Ala Arg Asp Val Leu Ile Pro Leu Gly Glu Tyr Phe Gln Ile Gln Asp

225 230 235 240

Asp Tyr Leu Asp Cys Phe Gly Thr Pro Glu Gln Ile Gly Lys Ile Gly

245 250 255

Thr Asp Ile Gln Asp Asn Lys Cys Ser Trp Val Ile Asn Lys Ala Leu

260 265 270

Glu Leu Ala Ser Ala Glu Gln Arg Lys Thr Leu Asp Glu Asn Tyr Gly

275 280 285

Lys Lys Asp Ser Val Ala Glu Ala Lys Cys Lys Lys Ile Phe Asn Asp

290 295 300

Leu Lys Ile Glu Gln Leu Tyr His Glu Tyr Glu Glu Ser Ile Ala Lys

305 310 315 320

Asp Leu Lys Ala Lys Ile Ser Gln Val Asp Glu Ser Arg Gly Phe Lys

325 330 335

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

340 345 350

<210> 4

<211> 444

<212> PRT

<213> Saccharomyces cerevisiae

<400> 4

Met Gly Lys Leu Leu Gln Leu Ala Leu His Pro Val Glu Met Lys Ala

1 5 10 15

Ala Leu Lys Leu Lys Phe Cys Arg Thr Pro Leu Phe Ser Ile Tyr Asp

20 25 30

Gln Ser Thr Ser Pro Tyr Leu Leu His Cys Phe Glu Leu Leu Asn Leu

35 40 45

Thr Ser Arg Ser Phe Ala Ala Val Ile Arg Glu Leu His Pro Glu Leu

50 55 60

Arg Asn Cys Val Thr Leu Phe Tyr Leu Ile Leu Arg Ala Leu Asp Thr

65 70 75 80

Ile Glu Asp Asp Met Ser Ile Glu His Asp Leu Lys Ile Asp Leu Leu

85 90 95

Arg His Phe His Glu Lys Leu Leu Leu Thr Lys Trp Ser Phe Asp Gly

100 105 110

Asn Ala Pro Asp Val Lys Asp Arg Ala Val Leu Thr Asp Phe Glu Ser

115 120 125

Ile Leu Ile Glu Phe His Lys Leu Lys Pro Glu Tyr Gln Glu Val Ile

130 135 140

Lys Glu Ile Thr Glu Lys Met Gly Asn Gly Met Ala Asp Tyr Ile Leu

145 150 155 160

Asp Glu Asn Tyr Asn Leu Asn Gly Leu Gln Thr Val His Asp Tyr Asp

165 170 175

Val Tyr Cys His Tyr Val Ala Gly Leu Val Gly Asp Gly Leu Thr Arg

180 185 190

Leu Ile Val Ile Ala Lys Phe Ala Asn Glu Ser Leu Tyr Ser Asn Glu

195 200 205

Gln Leu Tyr Glu Ser Met Gly Leu Phe Leu Gln Lys Thr Asn Ile Ile

210 215 220

Arg Asp Tyr Asn Glu Asp Leu Val Asp Gly Arg Ser Phe Trp Pro Lys

225 230 235 240

Glu Ile Trp Ser Gln Tyr Ala Pro Gln Leu Lys Asp Phe Met Lys Pro

245 250 255

Glu Asn Glu Gln Leu Gly Leu Asp Cys Ile Asn His Leu Val Leu Asn

260 265 270

Ala Leu Ser His Val Ile Asp Val Leu Thr Tyr Leu Ala Gly Ile His

275 280 285

Glu Gln Ser Thr Phe Gln Phe Cys Ala Ile Pro Gln Val Met Ala Ile

290 295 300

Ala Thr Leu Ala Leu Val Phe Asn Asn Arg Glu Val Leu His Gly Asn

305 310 315 320

Val Lys Ile Arg Lys Gly Thr Thr Cys Tyr Leu Ile Leu Lys Ser Arg

325 330 335

Thr Leu Arg Gly Cys Val Glu Ile Phe Asp Tyr Tyr Leu Arg Asp Ile

340 345 350

Lys Ser Lys Leu Ala Val Gln Asp Pro Asn Phe Leu Lys Leu Asn Ile

355 360 365

Gln Ile Ser Lys Ile Glu Gln Phe Met Glu Glu Met Tyr Gln Asp Lys

370 375 380

Leu Pro Pro Asn Val Lys Pro Asn Glu Thr Pro Ile Phe Leu Lys Val

385 390 395 400

Lys Glu Arg Ser Arg Tyr Asp Asp Glu Leu Val Pro Thr Gln Gln Glu

405 410 415

Glu Glu Tyr Lys Phe Asn Met Val Leu Ser Ile Ile Leu Ser Val Leu

420 425 430

Leu Gly Phe Tyr Tyr Ile Tyr Thr Leu His Arg Ala

435 440

<210> 5

<211> 289

<212> PRT

<213> Saccharomyces cerevisiae

<400> 5

Met Asn Arg Val Ser Phe Ile Lys Thr Pro Phe Asn Ile Gly Ala Lys

1 5 10 15

Trp Arg Leu Glu Asp Val Phe Leu Leu Ile Ile Met Ile Leu Leu Asn

20 25 30

Tyr Pro Val Tyr Tyr Gln Gln Pro Phe Glu Arg Gln Phe Tyr Ile Asn

35 40 45

Asp Leu Thr Ile Ser His Pro Tyr Ala Thr Thr Glu Arg Val Asn Asn

50 55 60

Asn Met Leu Phe Val Tyr Ser Phe Val Val Pro Ser Leu Thr Ile Leu

65 70 75 80

Ile Ile Gly Ser Ile Leu Ala Asp Arg Arg His Leu Ile Phe Ile Leu

85 90 95

Tyr Thr Ser Leu Leu Gly Leu Ser Leu Ala Trp Phe Ser Thr Ser Phe

100 105 110

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

115 120 125

Leu Asp Arg Cys Gln Pro Val Glu Gly Leu Pro Leu Asp Thr Leu Phe

130 135 140

Thr Ala Lys Asp Val Cys Thr Thr Lys Asn His Glu Arg Leu Leu Asp

145 150 155 160

Gly Phe Arg Thr Thr Pro Ser Gly His Ser Ser Glu Ser Phe Ala Gly

165 170 175

Leu Gly Tyr Leu Tyr Phe Trp Leu Cys Gly Gln Leu Leu Thr Glu Ser

180 185 190

Pro Leu Met Pro Leu Trp Arg Lys Met Val Ala Phe Leu Pro Leu Leu

195 200 205

Gly Ala Ala Leu Ile Ala Leu Ser Arg Thr Gln Asp Tyr Arg His His

210 215 220

Phe Val Asp Val Ile Leu Gly Ser Met Leu Gly Tyr Ile Met Ala His

225 230 235 240

Phe Phe Tyr Arg Arg Ile Phe Pro Pro Ile Asp Asp Pro Leu Pro Phe

245 250 255

Lys Pro Leu Met Asp Asp Ser Asp Val Thr Leu Glu Glu Ala Val Thr

260 265 270

His Gln Arg Ile Pro Asp Glu Glu Leu His Pro Leu Ser Asp Glu Gly

275 280 285

Met

<210> 6

<211> 274

<212> PRT

<213> Saccharomyces cerevisiae

<400> 6

Met Ile Ser Val Met Ala Asp Glu Lys His Lys Glu Tyr Phe Lys Leu

1 5 10 15

Tyr Tyr Phe Gln Tyr Met Ile Ile Gly Leu Cys Thr Ile Leu Phe Leu

20 25 30

Tyr Ser Glu Ile Ser Leu Val Pro Arg Gly Gln Asn Ile Glu Phe Ser

35 40 45

Leu Asp Asp Pro Ser Ile Ser Lys Arg Tyr Val Pro Asn Glu Leu Val

50 55 60

Gly Pro Leu Glu Cys Leu Ile Leu Ser Val Gly Leu Ser Asn Met Val

65 70 75 80

Val Phe Trp Thr Cys Met Phe Asp Lys Asp Leu Leu Lys Lys Asn Arg

85 90 95

Val Lys Arg Leu Arg Glu Arg Pro Asp Gly Ile Ser Asn Asp Phe His

100 105 110

Phe Met His Thr Ser Ile Leu Cys Leu Met Leu Ile Ile Ser Ile Asn

115 120 125

Ala Ala Leu Thr Gly Ala Leu Lys Leu Ile Ile Gly Asn Leu Arg Pro

130 135 140

Asp Phe Val Asp Arg Cys Ile Pro Asp Leu Gln Lys Met Ser Asp Ser

145 150 155 160

Asp Ser Leu Val Phe Gly Leu Asp Ile Cys Lys Gln Thr Asn Lys Trp

165 170 175

Ile Leu Tyr Glu Gly Leu Lys Ser Thr Pro Ser Gly His Ser Ser Phe

180 185 190

Ile Val Ser Thr Met Gly Phe Thr Tyr Leu Trp Gln Arg Val Phe Thr

195 200 205

Thr Arg Asn Thr Arg Ser Cys Ile Trp Cys Pro Leu Leu Ala Leu Val

210 215 220

Val Met Val Ser Arg Val Ile Asp His Arg His His Trp Tyr Asp Val

225 230 235 240

Val Ser Gly Ala Val Leu Ala Phe Leu Val Ile Tyr Cys Cys Trp Lys

245 250 255

Trp Thr Phe Thr Asn Leu Ala Lys Arg Asp Ile Leu Pro Ser Pro Val

260 265 270

Ser Val

<210> 7

<211> 587

<212> PRT

<213> Saccharomyces cerevisiae

<400> 7

Met Asn Ser Gln Gly Tyr Asp Glu Ser Ser Ser Ser Thr Ala Ala Thr

1 5 10 15

Ser Gly Pro Thr Ser Gly Asp Pro Arg Met Gly Lys Lys Gln Arg Phe

20 25 30

Met Asn Leu Ile Arg Thr Thr Lys Asp Val Tyr Ile Pro Asn Leu Thr

35 40 45

Ser Ser Ile Ser Gln Lys Thr Met Asp Gly Ile Arg Ser Thr Thr Asn

50 55 60

Ser Phe Glu Gly Tyr Asn Asp Leu Pro Met Glu Leu Pro His Asn Thr

65 70 75 80

Thr Ile Thr Tyr Phe Pro Thr Tyr Thr Thr Thr Asn Leu Val Asp Pro

85 90 95

Asp Gly Leu Ser Ala Pro Arg Lys Asp Phe Glu Thr Thr Val Arg Cys

100 105 110

Ala Val Ser Tyr Pro Gly Asn Pro Thr Ser Arg Arg Asn Arg Trp Leu

115 120 125

Leu Ser Leu Cys Lys Gln Tyr Leu Arg Thr Gly Thr Ala Glu Ala Asp

130 135 140

Val Ala Pro Val Val Pro Pro His Leu Glu Glu Asp Ser Gly Asp Leu

145 150 155 160

Asn Asp Ser Gln Ser Ser Ile Glu Ser Ser Leu Ser Ser Lys Ser Glu

165 170 175

Asn Arg Tyr Ser His Met Gly Ile Gln Glu Glu Asp Val Leu Asn Glu

180 185 190

Arg Ile Gln Gly Phe Leu Ser Lys Lys Val Pro Asn Thr Pro Val Val

195 200 205

Val Asp Leu Leu Pro Lys Asp Lys Leu Arg Gly Asp Thr Ala Ser Phe

210 215 220

Phe Gly Thr Thr Asp Ser Tyr Gly Asn Leu Leu Ile Lys Ala Glu Thr

225 230 235 240

Asp Phe Leu Pro Ser Lys Ile Asn Ile Thr Leu Asp Thr Pro Ile Glu

245 250 255

Gly His Ala Asp Pro Ile Ser Glu Thr Phe Pro Ala Asn Tyr Val Ser

260 265 270

Pro Tyr Gly Ile Gly Leu Ile Ser Asp Ile Asp Asp Thr Ile Lys His

275 280 285

Thr Gly Val Thr Gly Asp Arg Arg Ser Met Phe Arg Asn Val Phe Ile

290 295 300

His Asp Val Gln Ser Trp Val Ile Asp Gly Val Pro Leu Trp Tyr Lys

305 310 315 320

Thr Leu His Asp Val Ala Asp Val Asp Phe Phe Tyr Val Ser Asn Ser

325 330 335

Pro Ile Gln Thr Phe Thr Leu Leu Lys Gln Tyr Ile Cys Ala Asn Phe

340 345 350

Pro Pro Gly Pro Ile Phe Leu Lys Gln Tyr Ser Gly Asn Phe Phe Ser

355 360 365

Thr Ile Met Thr Ser Ser Ala Asn Arg Lys Ile Gln Pro Ile Ala Asn

370 375 380

Ile Leu Lys Asp Phe Pro Lys Lys Lys Phe Ile Leu Val Gly Asp Ser

385 390 395 400

Gly Glu His Asp Leu Glu Ala Tyr Thr Thr Thr Ala Leu Gln Phe Pro

405 410 415

Asn Gln Ile Leu Ala Ile Tyr Ile Arg Cys Cys Ser Asn Ser Met Ser

420 425 430

Asp Val Pro Ser His Asp Glu Glu Val Met Asn Glu Val Asn Asn Ile

435 440 445

Ile Glu Leu Gln Gln Arg Pro Met Gln Met Thr Lys Ser Thr Val Arg

450 455 460

Thr Arg Arg Arg Pro Pro Pro Pro Pro Ile Pro Ser Thr Gln Lys Pro

465 470 475 480

Ser Leu Thr Glu Glu Gln Thr Glu Ser Ile Arg Met Ser Arg Arg Asn

485 490 495

Lys Asp Glu Asn Asn Ala Lys Arg Val Ala Pro Pro Pro Leu Pro Asn

500 505 510

Arg Gln Leu Pro Asn Leu Asp Ala Asn Thr Tyr Tyr Val Pro Ser Ser

515 520 525

Gln Asn Asp Tyr Gly Met Tyr Gly Ala Phe Met Asp Lys Lys Ala Asp

530 535 540

Glu Trp Lys Arg Arg Val Met Asp Ser Ile Gln Lys Leu Ser Asn Gln

545 550 555 560

Asp Thr Thr Leu Met Phe Phe Ser Asp Pro Ala Leu Ser Leu Glu Asp

565 570 575

Ser Ile Arg Arg Ile Arg Glu Lys Tyr Ser Asn

580 585

<210> 8

<211> 1511

<212> PRT

<213> Saccharomyces cerevisiae

<400> 8

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

1 5 10 15

Ser Ser Ala Ser Ser Ser Thr Glu Asn Ala Ala Asp Leu His Asn Tyr

20 25 30

Asn Gly Phe Asp Glu His Thr Glu Ala Arg Ile Gln Lys Leu Ala Arg

35 40 45

Thr Leu Thr Ala Gln Ser Met Gln Asn Ser Thr Gln Ser Ala Pro Asn

50 55 60

Lys Ser Asp Ala Gln Ser Ile Phe Ser Ser Gly Val Glu Gly Val Asn

65 70 75 80

Pro Ile Phe Ser Asp Pro Glu Ala Pro Gly Tyr Asp Pro Lys Leu Asp

85 90 95

Pro Asn Ser Glu Asn Phe Ser Ser Ala Ala Trp Val Lys Asn Met Ala

100 105 110

His Leu Ser Ala Ala Asp Pro Asp Phe Tyr Lys Pro Tyr Ser Leu Gly

115 120 125

Cys Ala Trp Lys Asn Leu Ser Ala Ser Gly Ala Ser Ala Asp Val Ala

130 135 140

Tyr Gln Ser Thr Val Val Asn Ile Pro Tyr Lys Ile Leu Lys Ser Gly

145 150 155 160

Leu Arg Lys Phe Gln Arg Ser Lys Glu Thr Asn Thr Phe Gln Ile Leu

165 170 175

Lys Pro Met Asp Gly Cys Leu Asn Pro Gly Glu Leu Leu Val Val Leu

180 185 190

Gly Arg Pro Gly Ser Gly Cys Thr Thr Leu Leu Lys Ser Ile Ser Ser

195 200 205

Asn Thr His Gly Phe Asp Leu Gly Ala Asp Thr Lys Ile Ser Tyr Ser

210 215 220

Gly Tyr Ser Gly Asp Asp Ile Lys Lys His Phe Arg Gly Glu Val Val

225 230 235 240

Tyr Asn Ala Glu Ala Asp Val His Leu Pro His Leu Thr Val Phe Glu

245 250 255

Thr Leu Val Thr Val Ala Arg Leu Lys Thr Pro Gln Asn Arg Ile Lys

260 265 270

Gly Val Asp Arg Glu Ser Tyr Ala Asn His Leu Ala Glu Val Ala Met

275 280 285

Ala Thr Tyr Gly Leu Ser His Thr Arg Asn Thr Lys Val Gly Asn Asp

290 295 300

Ile Val Arg Gly Val Ser Gly Gly Glu Arg Lys Arg Val Ser Ile Ala

305 310 315 320

Glu Val Ser Ile Cys Gly Ser Lys Phe Gln Cys Trp Asp Asn Ala Thr

325 330 335

Arg Gly Leu Asp Ser Ala Thr Ala Leu Glu Phe Ile Arg Ala Leu Lys

340 345 350

Thr Gln Ala Asp Ile Ser Asn Thr Ser Ala Thr Val Ala Ile Tyr Gln

355 360 365

Cys Ser Gln Asp Ala Tyr Asp Leu Phe Asn Lys Val Cys Val Leu Asp

370 375 380

Asp Gly Tyr Gln Ile Tyr Tyr Gly Pro Ala Asp Lys Ala Lys Lys Tyr

385 390 395 400

Phe Glu Asp Met Gly Tyr Val Cys Pro Ser Arg Gln Thr Thr Ala Asp

405 410 415

Phe Leu Thr Ser Val Thr Ser Pro Ser Glu Arg Thr Leu Asn Lys Asp

420 425 430

Met Leu Lys Lys Gly Ile His Ile Pro Gln Thr Pro Lys Glu Met Asn

435 440 445

Asp Tyr Trp Val Lys Ser Pro Asn Tyr Lys Glu Leu Met Lys Glu Val

450 455 460

Asp Gln Arg Leu Leu Asn Asp Asp Glu Ala Ser Arg Glu Ala Ile Lys

465 470 475 480

Glu Ala His Ile Ala Lys Gln Ser Lys Arg Ala Arg Pro Ser Ser Pro

485 490 495

Tyr Thr Val Ser Tyr Met Met Gln Val Lys Tyr Leu Leu Ile Arg Asn

500 505 510

Met Trp Arg Leu Arg Asn Asn Ile Gly Phe Thr Leu Phe Met Ile Leu

515 520 525

Gly Asn Cys Ser Met Ala Leu Ile Leu Gly Ser Met Phe Phe Lys Ile

530 535 540

Met Lys Lys Gly Asp Thr Ser Thr Phe Tyr Phe Arg Gly Ser Ala Met

545 550 555 560

Phe Phe Ala Ile Leu Phe Asn Ala Phe Ser Ser Leu Leu Glu Ile Phe

565 570 575

Ser Leu Tyr Glu Ala Arg Pro Ile Thr Glu Lys His Arg Thr Tyr Ser

580 585 590

Leu Tyr His Pro Ser Ala Asp Ala Phe Ala Ser Val Leu Ser Glu Ile

595 600 605

Pro Ser Lys Leu Ile Ile Ala Val Cys Phe Asn Ile Ile Phe Tyr Phe

610 615 620

Leu Val Asp Phe Arg Arg Asn Gly Gly Val Phe Phe Phe Tyr Leu Leu

625 630 635 640

Ile Asn Ile Val Ala Val Phe Ser Met Ser His Leu Phe Arg Cys Val

645 650 655

Gly Ser Leu Thr Lys Thr Leu Ser Glu Ala Met Val Pro Ala Ser Met

660 665 670

Leu Leu Leu Ala Leu Ser Met Tyr Thr Gly Phe Ala Ile Pro Lys Lys

675 680 685

Lys Ile Leu Arg Trp Ser Lys Trp Ile Trp Tyr Ile Asn Pro Leu Ala

690 695 700

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

705 710 715 720

Pro Cys Ala Glu Tyr Val Pro Arg Gly Pro Ala Tyr Ala Asn Ile Ser

725 730 735

Ser Thr Glu Ser Val Cys Thr Val Val Gly Ala Val Pro Gly Gln Asp

740 745 750

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

755 760 765

Lys Asp Lys Trp Arg Gly Phe Gly Ile Gly Met Ala Tyr Val Val Phe

770 775 780

Phe Phe Phe Val Tyr Leu Phe Leu Cys Glu Tyr Asn Glu Gly Ala Lys

785 790 795 800

Gln Lys Gly Glu Ile Leu Val Phe Pro Arg Ser Ile Val Lys Arg Met

805 810 815

Lys Lys Arg Gly Val Leu Thr Glu Lys Asn Ala Asn Asp Pro Glu Asn

820 825 830

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

835 840 845

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

850 855 860

Ser Glu Ala Ile Phe His Trp Arg Asn Leu Cys Tyr Glu Val Gln Ile

865 870 875 880

Lys Ala Glu Thr Arg Arg Ile Leu Asn Asn Val Asp Gly Trp Val Lys

885 890 895

Pro Gly Thr Leu Thr Ala Leu Met Gly Ala Ser Gly Ala Gly Lys Thr

900 905 910

Thr Leu Leu Asp Cys Leu Ala Glu Arg Val Thr Met Gly Val Ile Thr

915 920 925

Gly Asp Ile Leu Val Asn Gly Ile Pro Arg Asp Lys Ser Phe Pro Arg

930 935 940

Ser Ile Gly Tyr Cys Gln Gln Gln Asp Leu His Leu Lys Thr Ala Thr

945 950 955 960

Val Arg Glu Ser Leu Arg Phe Ser Ala Tyr Leu Arg Gln Pro Ala Glu

965 970 975

Val Ser Ile Glu Glu Lys Asn Arg Tyr Val Glu Glu Val Ile Lys Ile

980 985 990

Leu Glu Met Glu Lys Tyr Ala Asp Ala Val Val Gly Val Ala Gly Glu

995 1000 1005

Gly Leu Asn Val Glu Gln Arg Lys Arg Leu Thr Ile Gly Val Glu

1010 1015 1020

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

1025 1030 1035

Ser Gly Leu Asp Ser Gln Thr Ala Trp Ser Ile Cys Gln Leu Met

1040 1045 1050

Lys Lys Leu Ala Asn His Gly Gln Ala Ile Leu Cys Thr Ile His

1055 1060 1065

Gln Pro Ser Ala Ile Leu Met Gln Glu Phe Asp Arg Leu Leu Phe

1070 1075 1080

Met Gln Arg Gly Gly Lys Thr Val Tyr Phe Gly Asp Leu Gly Glu

1085 1090 1095

Gly Cys Lys Thr Met Ile Asp Tyr Phe Glu Ser His Gly Ala His

1100 1105 1110

Lys Cys Pro Ala Asp Ala Asn Pro Ala Glu Trp Met Leu Glu Val

1115 1120 1125

Val Gly Ala Ala Pro Gly Ser His Ala Asn Gln Asp Tyr Tyr Glu

1130 1135 1140

Val Trp Arg Asn Ser Glu Glu Tyr Arg Ala Val Gln Ser Glu Leu

1145 1150 1155

Asp Trp Met Glu Arg Glu Leu Pro Lys Lys Gly Ser Ile Thr Ala

1160 1165 1170

Ala Glu Asp Lys His Glu Phe Ser Gln Ser Ile Ile Tyr Gln Thr

1175 1180 1185

Lys Leu Val Ser Ile Arg Leu Phe Gln Gln Tyr Trp Arg Ser Pro

1190 1195 1200

Asp Tyr Leu Trp Ser Lys Phe Ile Leu Thr Ile Phe Asn Gln Leu

1205 1210 1215

Phe Ile Gly Phe Thr Phe Phe Lys Ala Gly Thr Ser Leu Gln Gly

1220 1225 1230

Leu Gln Asn Gln Met Leu Ala Val Phe Met Phe Thr Val Ile Phe

1235 1240 1245

Asn Pro Ile Leu Gln Gln Tyr Leu Pro Ser Phe Val Gln Gln Arg

1250 1255 1260

Asp Leu Tyr Glu Ala Arg Glu Arg Pro Ser Arg Thr Phe Ser Trp

1265 1270 1275

Ile Ser Phe Ile Phe Ala Gln Ile Phe Val Glu Val Pro Trp Asn

1280 1285 1290

Ile Leu Ala Gly Thr Ile Ala Tyr Phe Ile Tyr Tyr Tyr Pro Ile

1295 1300 1305

Gly Phe Tyr Ser Asn Ala Ser Ala Ala Gly Gln Leu His Glu Arg

1310 1315 1320

Gly Ala Leu Phe Trp Leu Phe Ser Cys Ala Phe Tyr Val Tyr Val

1325 1330 1335

Gly Ser Met Gly Leu Leu Val Ile Ser Phe Asn Gln Val Ala Glu

1340 1345 1350

Ser Ala Ala Asn Leu Ala Ser Leu Leu Phe Thr Met Ser Leu Ser

1355 1360 1365

Phe Cys Gly Val Met Thr Thr Pro Ser Ala Met Pro Arg Phe Trp

1370 1375 1380

Ile Phe Met Tyr Arg Val Ser Pro Leu Thr Tyr Phe Ile Gln Ala

1385 1390 1395

Leu Leu Ala Val Gly Val Ala Asn Val Asp Val Lys Cys Ala Asp

1400 1405 1410

Tyr Glu Leu Leu Glu Phe Thr Pro Pro Ser Gly Met Thr Cys Gly

1415 1420 1425

Gln Tyr Met Glu Pro Tyr Leu Gln Leu Ala Lys Thr Gly Tyr Leu

1430 1435 1440

Thr Asp Glu Asn Ala Thr Asp Thr Cys Ser Phe Cys Gln Ile Ser

1445 1450 1455

Thr Thr Asn Asp Tyr Leu Ala Asn Val Asn Ser Phe Tyr Ser Glu

1460 1465 1470

Arg Trp Arg Asn Tyr Gly Ile Phe Ile Cys Tyr Ile Ala Phe Asn

1475 1480 1485

Tyr Ile Ala Gly Val Phe Phe Tyr Trp Leu Ala Arg Val Pro Lys

1490 1495 1500

Lys Asn Gly Lys Leu Ser Lys Lys

1505 1510

<210> 9

<211> 322

<212> PRT

<213> Artificial Sequence

<220>

<223> H152Y

<400> 9

Met Glu Lys Phe Thr Phe Pro Asn Leu Tyr Cys Pro Phe Pro Glu Arg

1 5 10 15

Lys Asn Pro Tyr Ser Glu Phe Leu Gln Asp Tyr Ala Leu Gln Trp Val

20 25 30

Ile Arg Phe Lys Leu Ile Asp Ser Glu Ser Leu Tyr Gln Arg Phe Ser

35 40 45

Lys Ala Lys Phe Tyr Leu Leu Thr Ala Gly Ala Tyr Pro His Cys Gln

50 55 60

Leu Glu Glu Leu Lys Ile Ala Asn Asp Val Ile Ser Trp Leu Phe Ile

65 70 75 80

Trp Asp Asp Gln Cys Asp Ile Ser Asp Leu Gly Lys Lys Pro Glu Leu

85 90 95

Leu Lys Thr Trp Cys Asn Arg Phe Leu Glu Ile Leu Asn Gly Ala Glu

100 105 110

Leu Thr Pro Asp Asp Leu Pro Leu Gly Phe Ala Leu Arg Asp Ile Arg

115 120 125

Asn Arg Ile Ile Asn Arg Gly Gly Ile Thr Phe Phe His His Phe Val

130 135 140

Arg Asn Phe Glu Asp Tyr Phe Tyr Gly Cys Ile Glu Glu Ala His Asn

145 150 155 160

Arg Val Asn Val Ser Val Pro Asp Val Glu Ala Tyr Ile Lys Ile Arg

165 170 175

Ser Ala Asn Ala Ala Ala Ala Leu Cys Leu Asn Leu Ile Glu Phe Cys

180 185 190

Asp Arg Val Met Ile Pro Tyr Ser Leu Arg Asn His Glu Thr Leu Lys

195 200 205

Lys Leu Thr Gln Met Thr Ile Asn Ile Leu Ala Trp Ser Asn Asp Ile

210 215 220

Phe Ser Ala Pro Arg Glu Ile Ala Asn Gly Glu Val His Asn Leu Val

225 230 235 240

Phe Val Ile His His His Gln Lys Ile Pro Leu Glu Lys Ala Met Leu

245 250 255

Ala Ala Ala Ala Met His Asn His Glu Val Gln Lys Leu Val Asn Leu

260 265 270

Glu Ser Lys Ile Ala Ser Phe Ser Ala Glu Thr Asp Ala Glu Ile Thr

275 280 285

Lys Tyr Ile Ser Gly Leu His Ala Trp Ile Arg Gly Asn Leu Asp Trp

290 295 300

Tyr Ala His Ser Gly Arg Tyr Gln Ile Thr Glu Lys Leu Glu Leu Leu

305 310 315 320

Ala Ser

<210> 10

<211> 1501

<212> PRT

<213> Saccharomyces cerevisiae

<400> 10

Met Ser Asn Ile Lys Ser Thr Gln Asp Ser Ser His Asn Ala Val Ala

1 5 10 15

Arg Ser Ser Ser Ala Ser Phe Ala Ala Ser Glu Glu Ser Phe Thr Gly

20 25 30

Ile Thr His Asp Lys Asp Glu Gln Ser Asp Thr Pro Ala Asp Lys Leu

35 40 45

Thr Lys Met Leu Thr Gly Pro Ala Arg Asp Thr Ala Ser Gln Ile Ser

50 55 60

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

65 70 75 80

Phe Ala Asp Ala Leu Ser Arg His Thr Thr Arg Ser Gly Ala Phe Asn

85 90 95

Met Asp Ser Asp Ser Asp Asp Gly Phe Asp Ala His Ala Ile Phe Glu

100 105 110

Ser Phe Val Arg Asp Ala Asp Glu Gln Gly Ile His Ile Arg Lys Ala

115 120 125

Gly Val Thr Ile Glu Asp Val Ser Ala Lys Gly Val Asp Ala Ser Ala

130 135 140

Leu Glu Gly Ala Thr Phe Gly Asn Ile Leu Cys Leu Pro Leu Thr Ile

145 150 155 160

Phe Lys Gly Ile Lys Ala Lys Arg His Gln Lys Met Arg Gln Ile Ile

165 170 175

Ser Asn Val Asn Ala Leu Ala Glu Ala Gly Glu Met Ile Leu Val Leu

180 185 190

Gly Arg Pro Gly Ala Gly Cys Ser Ser Phe Leu Lys Val Thr Ala Gly

195 200 205

Glu Ile Asp Gln Phe Ala Gly Gly Val Ser Gly Glu Val Ala Tyr Asp

210 215 220

Gly Ile Pro Gln Glu Glu Met Met Lys Arg Tyr Lys Ala Asp Val Ile

225 230 235 240

Tyr Asn Gly Glu Leu Asp Val His Phe Pro Tyr Leu Thr Val Lys Gln

245 250 255

Thr Leu Asp Phe Ala Ile Ala Cys Lys Thr Pro Ala Leu Arg Val Asn

260 265 270

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

275 280 285

Thr Ile Phe Gly Leu Arg His Thr Tyr Asn Thr Lys Val Gly Asn Asp

290 295 300

Phe Val Arg Gly Val Ser Gly Gly Glu Arg Lys Arg Val Ser Ile Ala

305 310 315 320

Glu Ala Leu Ala Ala Lys Gly Ser Ile Tyr Cys Trp Asp Asn Ala Thr

325 330 335

Arg Gly Leu Asp Ala Ser Thr Ala Leu Glu Tyr Ala Lys Ala Ile Arg

340 345 350

Ile Met Thr Asn Leu Leu Lys Ser Thr Ala Phe Val Thr Ile Tyr Gln

355 360 365

Ala Ser Glu Asn Ile Tyr Glu Thr Phe Asp Lys Val Thr Val Leu Tyr

370 375 380

Ser Gly Lys Gln Ile Tyr Phe Gly Leu Ile His Glu Ala Lys Pro Tyr

385 390 395 400

Phe Ala Lys Met Gly Tyr Leu Cys Pro Pro Arg Gln Ala Thr Ala Glu

405 410 415

Phe Leu Thr Ala Leu Thr Asp Pro Asn Gly Phe His Leu Ile Lys Pro

420 425 430

Gly Tyr Glu Asn Lys Val Pro Arg Thr Ala Glu Glu Phe Glu Thr Tyr

435 440 445

Trp Leu Asn Ser Pro Glu Phe Ala Gln Met Lys Lys Asp Ile Ala Ala

450 455 460

Tyr Lys Glu Lys Val Asn Thr Glu Lys Thr Lys Glu Val Tyr Asp Glu

465 470 475 480

Ser Met Ala Gln Glu Lys Ser Lys Tyr Thr Arg Lys Lys Ser Tyr Tyr

485 490 495

Thr Val Ser Tyr Trp Glu Gln Val Lys Leu Cys Thr Gln Arg Gly Phe

500 505 510

Gln Arg Ile Tyr Gly Asn Lys Ser Tyr Thr Val Ile Asn Val Cys Ser

515 520 525

Ala Ile Ile Gln Ser Phe Ile Thr Gly Ser Leu Phe Tyr Asn Thr Pro

530 535 540

Ser Ser Thr Ser Gly Ala Phe Ser Arg Gly Gly Val Leu Tyr Phe Ala

545 550 555 560

Leu Leu Tyr Tyr Ser Leu Met Gly Leu Ala Asn Ile Ser Phe Glu His

565 570 575

Arg Pro Ile Leu Gln Lys His Lys Gly Tyr Ser Leu Tyr His Pro Ser

580 585 590

Ala Glu Ala Ile Gly Ser Thr Leu Ala Ser Phe Pro Phe Arg Met Ile

595 600 605

Gly Leu Thr Cys Phe Phe Ile Ile Leu Phe Phe Leu Ser Gly Leu His

610 615 620

Arg Thr Ala Gly Ser Phe Phe Thr Ile Tyr Leu Phe Leu Thr Met Cys

625 630 635 640

Ser Glu Ala Ile Asn Gly Leu Phe Glu Met Val Ser Ser Val Cys Asp

645 650 655

Thr Leu Ser Gln Ala Asn Ser Ile Ser Gly Ile Leu Met Met Ser Ile

660 665 670

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

675 680 685

Phe Lys Trp Ile Ser Tyr Val Leu Pro Ile Arg Tyr Ala Phe Glu Ser

690 695 700

Met Leu Asn Ala Glu Phe His Gly Arg His Met Asp Cys Ala Asn Thr

705 710 715 720

Leu Val Pro Ser Gly Gly Asp Tyr Asp Asn Leu Ser Asp Asp Tyr Lys

725 730 735

Val Cys Ala Phe Val Gly Ser Lys Pro Gly Gln Ser Tyr Val Leu Gly

740 745 750

Asp Asp Tyr Leu Lys Asn Gln Phe Gln Tyr Val Tyr Lys His Thr Trp

755 760 765

Arg Asn Phe Gly Ile Leu Trp Cys Phe Leu Leu Gly Tyr Val Val Leu

770 775 780

Lys Val Ile Phe Thr Glu Tyr Lys Arg Pro Val Lys Gly Gly Gly Asp

785 790 795 800

Ala Leu Ile Phe Lys Lys Gly Ser Lys Arg Phe Ile Ala His Ala Asp

805 810 815

Glu Glu Ser Pro Asp Asn Val Asn Asp Ile Asp Ala Lys Glu Gln Phe

820 825 830

Ser Ser Glu Ser Ser Gly Ala Asn Asp Glu Val Phe Asp Asp Leu Glu

835 840 845

Ala Lys Gly Val Phe Ile Trp Lys Asp Val Cys Phe Thr Ile Pro Tyr

850 855 860

Glu Gly Gly Lys Arg Met Leu Leu Asp Asn Val Ser Gly Tyr Cys Ile

865 870 875 880

Pro Gly Thr Met Thr Ala Leu Met Gly Glu Ser Gly Ala Gly Lys Thr

885 890 895

Thr Leu Leu Asn Thr Leu Ala Gln Arg Asn Val Gly Ile Ile Thr Gly

900 905 910

Asp Met Leu Val Asn Gly Arg Pro Ile Asp Ala Ser Phe Glu Arg Arg

915 920 925

Thr Gly Tyr Val Gln Gln Gln Asp Ile His Ile Ala Glu Leu Thr Val

930 935 940

Arg Glu Ser Leu Gln Phe Ser Ala Arg Met Arg Arg Pro Gln His Leu

945 950 955 960

Pro Asp Ser Glu Lys Met Asp Tyr Val Glu Lys Ile Ile Arg Val Leu

965 970 975

Gly Met Glu Glu Tyr Ala Glu Ala Leu Val Gly Glu Val Gly Cys Gly

980 985 990

Leu Asn Val Glu Gln Arg Lys Lys Leu Ser Ile Gly Val Glu Leu Val

995 1000 1005

Ala Lys Pro Asp Leu Leu Leu Phe Leu Asp Glu Pro Thr Ser Gly

1010 1015 1020

Leu Asp Ser Gln Ser Ser Trp Ala Ile Ile Gln Leu Leu Arg Lys

1025 1030 1035

Leu Ser Lys Ala Gly Gln Ser Ile Leu Cys Thr Ile His Gln Pro

1040 1045 1050

Ser Ala Thr Leu Phe Glu Glu Phe Asp Arg Leu Leu Leu Leu Arg

1055 1060 1065

Lys Gly Gly Gln Thr Val Tyr Phe Gly Asp Ile Gly Lys Asn Ser

1070 1075 1080

Ala Thr Ile Leu Asn Tyr Phe Glu Arg Asn Gly Ala Arg Lys Cys

1085 1090 1095

Asp Ser Ser Glu Asn Pro Ala Glu Tyr Ile Leu Glu Ala Ile Gly

1100 1105 1110

Ala Gly Ala Thr Ala Ser Val Lys Glu Asp Trp His Glu Lys Trp

1115 1120 1125

Leu Asn Ser Val Glu Phe Glu Gln Thr Lys Glu Lys Val Gln Asp

1130 1135 1140

Leu Ile Asn Asp Leu Ser Lys Gln Glu Thr Lys Ser Glu Val Gly

1145 1150 1155

Asp Lys Pro Ser Lys Tyr Ala Thr Ser Tyr Ala Tyr Gln Phe Arg

1160 1165 1170

Tyr Val Leu Ile Arg Thr Ser Thr Ser Phe Trp Arg Ser Leu Asn

1175 1180 1185

Tyr Ile Met Ser Lys Met Met Leu Met Leu Val Gly Gly Leu Tyr

1190 1195 1200

Ile Gly Phe Thr Phe Phe Asn Val Gly Lys Ser Tyr Val Gly Leu

1205 1210 1215

Gln Asn Ala Met Phe Ala Ala Phe Ile Ser Ile Ile Leu Ser Ala

1220 1225 1230

Pro Ala Met Asn Gln Ile Gln Gly Arg Ala Ile Ala Ser Arg Glu

1235 1240 1245

Leu Phe Glu Val Arg Glu Ser Gln Ser Asn Met Phe His Trp Ser

1250 1255 1260

Leu Val Leu Ile Thr Gln Tyr Leu Ser Glu Leu Pro Tyr His Leu

1265 1270 1275

Phe Phe Ser Thr Ile Phe Phe Val Ser Ser Tyr Phe Pro Leu Arg

1280 1285 1290

Ile Phe Phe Glu Ala Ser Arg Ser Ala Val Tyr Phe Leu Asn Tyr

1295 1300 1305

Cys Ile Met Phe Gln Leu Tyr Tyr Val Gly Leu Gly Leu Met Ile

1310 1315 1320

Leu Tyr Met Ser Pro Asn Leu Pro Ser Ala Asn Val Ile Leu Gly

1325 1330 1335

Leu Cys Leu Ser Phe Met Leu Ser Phe Cys Gly Val Thr Gln Pro

1340 1345 1350

Val Ser Leu Met Pro Gly Phe Trp Thr Phe Met Trp Lys Ala Ser

1355 1360 1365

Pro Tyr Thr Tyr Phe Val Gln Asn Leu Val Gly Ile Met Leu His

1370 1375 1380

Lys Lys Pro Val Val Cys Lys Lys Lys Glu Leu Asn Tyr Phe Asn

1385 1390 1395

Pro Pro Asn Gly Ser Thr Cys Gly Glu Tyr Met Lys Pro Phe Leu

1400 1405 1410

Glu Lys Ala Thr Gly Tyr Ile Glu Asn Pro Asp Ala Thr Ser Asp

1415 1420 1425

Cys Ala Tyr Cys Ile Tyr Glu Val Gly Asp Asn Tyr Leu Thr His

1430 1435 1440

Ile Ser Ser Lys Tyr Ser Tyr Leu Trp Arg Asn Phe Gly Ile Phe

1445 1450 1455

Trp Ile Tyr Ile Phe Phe Asn Ile Ile Ala Met Val Cys Val Tyr

1460 1465 1470

Tyr Leu Phe His Val Arg Gln Ser Ser Phe Leu Ser Pro Val Ser

1475 1480 1485

Ile Leu Asn Lys Ile Lys Asn Ile Arg Lys Lys Lys Gln

1490 1495 1500

<210> 11

<211> 500

<212> DNA

<213> Artificial Sequence

<220>

<223> hxt1

<400> 11

gcattgagtc aaaagttttt ccgaagtgac ccagtgctct tttttttttt ccgtgaagga 60

ctgacaaata tgcgcacaag atccaatacg taatggaaat tcggaaaaac taggaagaaa 120

tgctgcaggg cattgccgtg ccgatctttt gtctttcaga tatatgagaa aaagaatatt 180

catcaagtgc tgatagaaga ataccactca tatgacgtgg gcagaagaca gcaaacgtaa 240

acatgagctg ctgcgacatt tgatggcttt tatccgacaa gccaggaaac tccaccatta 300

tctaatgtag caaaatattt cttaacaccc gaagttgcgt gtccccctca cgtttttaat 360

catttgaatt agtatattga aattatatat aaaggcaaca atgtccccat aatcaattcc 420

atctggggtc tcatgttctt tccccacctt aaaatctata aagatatcat aatcgtcaac 480

tagttgatat acgtaaaatc 500

Claims

1. A recombinant bacterium is a Saccharomyces cerevisiae (Saccharomyces cerevisiae) as a starting strain, a Germacrene A Synthase (GAS) is expressed in the recombinant bacterium, the GAS is derived from Anabaena variabilis (Anabaena variabilis) ATCC29413, and the amino acid sequence of the GAS is shown as SEQ ID No.1 or SEQ ID No. 9.

2. The recombinant strain as claimed in claim 1, wherein the recombinant strain further expresses any one or more of the following target genes: 3-hydroxy-3-methylglutaryl-coenzyme a (HMGR1), farnesyl pyrophosphate synthase (ERG 20); the amino acid sequence of HMGR1 is shown as SEQ ID No.2, and the amino acid sequence of ERG20 is shown as SEQ ID No. 3.

3. The recombinant strain according to any one of claims 1 to 2, wherein the recombinant strain further comprises any one or any several of the following operations:

(1) down-regulating the expression of squalene synthase (erg 9); (2) inactivating or inhibiting the expression of diacylglycerol pyrophosphate phosphatase 1(dpp 1); (3) inactivating or inhibiting the expression of lipid phosphatase 1(lpp 1); (4) expressed with efflux channel protein pdr5 and/or efflux channel protein SNQ 2;

the amino acid sequence of erg9 is shown as SEQ ID No.4, the amino acid sequence of dpp1 is shown as SEQ ID No.5, the amino acid sequence of lpp1 is shown as SEQ ID No.6, the amino acid sequence of pdr5 is shown as SEQ ID No.8, and the amino acid sequence of SNQ2 is shown as SEQ ID No. 10.

4.A method of producing a recombinant bacterium, the method comprising the step of expressing a Gemmaene A Synthase (GAS) in saccharomyces cerevisiae, the GAS being derived from Anabaena variabilis ATCC29413, the GAS having the amino acid sequence shown in SEQ ID No.1 or SEQ ID No. 9.

5. The method of claim 4, further comprising performing any one or any combination of the following in Saccharomyces cerevisiae:

(1) expressing 3-hydroxy-3-methylglutaryl coenzyme A (HMGR 1);

(2) expressing farnesyl pyrophosphate synthase (ERG 20);

(3) down-regulating the expression of squalene synthase (erg 9);

(5) inactivating or inhibiting lipid phosphatase 1(lpp 1);

(6) expressing the efflux channel protein pdr5 and/or the efflux channel protein SNQ 2;

the amino acid sequence of the HMGR1 is shown as SEQ ID No.2, the amino acid sequence of the ERG20 is shown as SEQ ID No.3, the amino acid sequence of the ERG9 is shown as SEQ ID No.4, the amino acid sequence of the dpp1 is shown as SEQ ID No.5, the amino acid sequence of the lpp1 is shown as SEQ ID No.6, the amino acid sequence of the pdr5 is shown as SEQ ID No.8, and the amino acid sequence of the SNQ2 is shown as SEQ ID No. 10.

6. Use of the recombinant bacterium of any one of claims 1-3 or the recombinant bacterium prepared by the method of any one of claims 4-5 in the production of β -elemene and/or germacrene A.

7. A method for producing β -elemene and/or germacrene a, said method comprising the step of fermenting a recombinant bacterium according to any one of claims 1 to 3 or a recombinant bacterium produced by a method according to any one of claims 4 to 5.

8. A mutant of Germacrene A Synthase (GAS), the amino acid sequence of which is shown in SEQ ID No. 9.

9. A vector or host cell comprising the mutant of claim 8.

10. Use of a mutant as claimed in claim 8, or a vector or host cell as claimed in claim 9, for the production of β -elemene and/or gemmaene a.