CN108913732B - Method for heterologous production of monacolin J and application - Google Patents

Abstract

The invention relates to transformation and application of pichia pastoris engineering bacteria for heterologous production of monacolin J. In the invention, the yeast engineering strain is optimized and modified by a gene recombination technology to obtain the yeast engineering strain capable of producing monacolin J or a dihydro monacolin L intermediate product thereof. The yield of the target product of the yeast engineering bacteria is obviously increased, and a new way for industrially producing monacolin J and downstream products thereof is provided.

Description

Method for heterologous production of monacolin J and application

Technical Field

The invention belongs to the field of bioengineering; more specifically, the invention relates to the transformation and application of pichia pastoris engineering bacteria for heterogeneously producing monacolin J.

Background

Simvastatin is an artificial semisynthetic drug and is widely used as a hypolipidemic drug because it has an obvious inhibitory effect on cholesterol biosynthesis in the human body. Currently, the industrial production of simvastatin is mainly derived from the biological or chemical transformation of the precursor monacolin J, which is obtained from the hydrolysis of the fermentation product lovastatin of aspergillus terreus. In the production process of simvastatin, monacolin J can be converted into simvastatin in one step through enzyme catalysis, and the process of hydrolyzing lovastatin to obtain monacolin J is hydrolysis by a full chemical method, so that the steps are complex, byproducts are more, and a catalyst needs to be introduced. The monacolin J is heterogeneously synthesized by using the microbial chassis cells in a one-step method, can replace a chemical hydrolysis method, avoids the problems, and provides a new method for the industrial production of monacolin J and simvastatin.

Pichia pastoris (Pichia pastoris) is an excellent microbial underpan cell, has received much attention because it can express a wide range of recombinant proteins, and has been used as a generally recognized safe microorganism to successfully express more than 1000 recombinant proteins. Pichia pastoris can grow rapidly to high density in defined media, with the ability to perform complex post-translational protein modifications to direct correct protein folding. In addition, pichia pastoris is a natural methylotrophic microorganism that can grow to high density with strong methanol metabolization capacity under conditions where methanol is the sole carbon source. Methanol is a byproduct in the coal chemical industry, can be synthesized from natural gas and also can be obtained by reducing carbon dioxide in the air, and compared with common fermentation type sugars, the methanol has stronger reducing power and can provide more driving force for the synthesis of heterologous products. The large amount, low price and renewable characteristics of methanol promote the gradual development of methanol into a good raw material of biochemical derivative products. Meanwhile, pichia pastoris has a mature and matched heterologous expression vector, researches on transformation, screening and scale amplification of pichia pastoris have been carried out for many years, and pichia pastoris can be used as a good chassis cell to synthesize and produce high-value-added biological products.

Disclosure of Invention

The invention aims to provide a method for heterogeneously synthesizing monacolin J by mixed co-culture of recombinant yeast engineering bacteria by taking ethanol as a substrate.

In a first aspect of the invention, there is provided a method for the heterologous production of dihydromonacolin L comprising: (1) providing engineering yeast bacteria, and allowing the engineering yeast bacteria to express exogenous expression cassettes of the following genes: lovB, lovC, lovG, npgA, and driving expression of the transcriptional activator uta with an ethanol inducible promoter; (2) and (2) culturing the yeast engineering bacteria in the step (1) by taking ethanol as a carbon source, a precursor and/or an inducer, thereby generating a product, namely the dihydromonacolin L.

In another aspect of the invention, there is provided a method for heterologous production of monacolin J comprising: (a) providing engineering yeast bacteria, and allowing the engineering yeast bacteria to express exogenous expression cassettes of the following genes: lovB, lovC, lovG, npgA, and driving expression of the transcriptional activator uta with an ethanol inducible promoter; (b) providing engineering yeast bacteria, and allowing the engineering yeast bacteria to express exogenous expression cassettes of the following genes: lovA, cpr, and ethanol inducible promoter to drive expression of transcriptional activator uta; (c) and (b) culturing the mixed bacteria of the engineering yeast bacteria in the step (a) and the step (b) by using ethanol as a carbon source, a precursor and/or an inducer, thereby generating the product monacolin J.

In a preferred embodiment, the engineered yeast strain in (1) or (a) further expresses exogenous alcohol dehydrogenase ADH, acetyl-coa synthetase ACS and acetyl-coa decarboxylase ACC.

In another preferred embodiment, P is used in (1) or (a)_AOX1RAs promoters driving the expression of lovB, lovC, lovG, npgA; or

(b) In (1) with P_AOX1RAs a promoter to drive expression of lovA, cpr; or

(1) Or in (a) or (b) with P_ICL1Is an ethanol inducible promoter.

In another preferable example, (c) in the mixed bacteria of the engineering yeast bacteria (a) and (b), the ratio of the bacteria (a) to the bacteria (b) is 1: 0.1-1; preferably 1: 0.2-0.5 (the ratio of the number of two yeast cells in the flora); or, in the step (2) or (c), a yeast basic nitrogen source culture medium added with ethanol (0.2-1%, preferably 0.3-0.8%, more preferably 0.4-0.6%) is used for culturing, and glucose is used as a carbon source for feeding in the early stage of fermentation; after the wet weight of the cells reaches 200-400 g/L (preferably 250-350 g/L; more preferably 280-320 g/L), the carbon source is switched to ethanol for feeding.

In another preferred embodiment, said genes lovB, lovC, lovG, npgA, lovA, cpr are derived from Aspergillus terreus.

In another preferred embodiment, the lovB gene has the nucleotide sequence shown in SEQ ID NO. 1, or a degenerate sequence thereof, or a nucleotide sequence encoding an isofunctional protein which is 70% or more (preferably 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, e.g., 98% to 99%) identical to the sequence shown in SEQ ID NO. 1.

In another preferred embodiment, the lovC gene has the nucleotide sequence shown in SEQ ID NO. 2, or a degenerate sequence thereof, or a nucleotide sequence encoding an isofunctional protein which is 70% or more (preferably 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, e.g., 98% to 99%) identical to the sequence shown in SEQ ID NO. 2.

In another preferred embodiment, the lovG gene has the nucleotide sequence shown in SEQ ID NO. 3, or a degenerate sequence thereof, or a nucleotide sequence encoding an isofunctional protein having an identity of more than 70% (preferably more than 80%, more preferably more than 90%, more preferably more than 93%, more preferably more than 95%, more preferably more than 97%, e.g., 98% to 99%) to the sequence shown in SEQ ID NO. 3.

In another preferred embodiment, the npgA gene has the nucleotide sequence shown in SEQ ID NO. 4, or a degenerate sequence thereof, or a nucleotide sequence encoding an isofunctional protein which is 70% or more (preferably 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, e.g., 98% to 99%) identical to the sequence shown in SEQ ID NO. 4.

In another preferred embodiment, the lovA gene has the nucleotide sequence shown in SEQ ID NO. 5, or a degenerate sequence thereof, or a nucleotide sequence encoding an isofunctional protein which is 70% or more (preferably 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, e.g., 98% to 99%) identical to the sequence shown in SEQ ID NO. 5.

In another preferred embodiment, the cpr gene has the nucleotide sequence shown in SEQ ID NO. 6, or a degenerate sequence thereof, or a nucleotide sequence encoding an isofunctional protein which is 70% or more (preferably 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, e.g., 98% to 99%) identical to the sequence shown in SEQ ID NO. 6.

In another preferred embodiment, the uta gene has the nucleotide sequence shown in SEQ ID NO. 7, or a degenerate sequence thereof, or a nucleotide sequence encoding an isofunctional protein which is 70% or more (preferably 80% or more, more preferably 90% or more, still more preferably 93% or more, still more preferably 95% or more, still more preferably 97% or more, e.g., 98% to 99%) identical to the sequence shown in SEQ ID NO. 6.

In another preferred embodiment, the gene encoding alcohol dehydrogenase ADH has the nucleotide sequence shown in SEQ ID NO:20, or a degenerate sequence thereof, or a nucleotide sequence encoding a functional protein which is identical to the nucleotide sequence shown in SEQ ID NO:20 by more than 70% (preferably more than 80%, more preferably more than 90%, more preferably more than 93%, more preferably more than 95%, more preferably more than 97%, for example, 98% to 99%).

In another preferred embodiment, the ACS-encoding gene has the nucleotide sequence shown in SEQ ID NO:21, or a degenerate sequence thereof, or a nucleotide sequence encoding a functional protein which is 70% or more (preferably 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, e.g., 98% to 99%) identical to the sequence shown in SEQ ID NO: 21.

In another preferred embodiment, the coding gene of acetyl-CoA decarboxylase ACC has the nucleotide sequence shown in SEQ ID NO. 22, or a degenerate sequence thereof, or a nucleotide sequence encoding a functional protein which is 70% or more (preferably 80% or more, more preferably 90% or more, more preferably 93% or more, more preferably 95% or more, more preferably 97% or more, e.g., 98% to 99%) identical to the sequence shown in SEQ ID NO. 22.

In another preferred embodiment, the promoter P_AOX1RThe gene sequence of (A) has a nucleotide sequence shown as SEQ ID NO. 8.

In another preferred embodimentPromoter P_ICL1The gene sequence of (A) has a nucleotide sequence shown in SEQ ID NO. 9.

In another aspect of the present invention, there is provided a yeast engineering bacterium for producing dihydromonacolin L, which comprises an exogenous expression cassette of the following genes: lovB, lovC, lovG, npgA, and transcriptional activator uta; preferably, it further comprises the genes encoding exogenous alcohol dehydrogenase ADH, acetyl-coa synthetase ACS and acetyl-coa decarboxylase ACC.

In another aspect of the present invention, there is provided an engineered yeast strain for producing monacolin J, comprising an exogenous expression cassette for genes of the following group: lovA, cpr, and transcriptional activator uta.

In a preferred embodiment, the yeast engineering bacteria are methanol nutritional yeast; preferably, the methylotrophic yeast comprises pichia pastoris.

In another preferred embodiment, the pichia is pichia GS 115.

In another preferred embodiment, the yeast engineering bacteria are used for producing dihydromonacolin L or monacolin J by using ethanol as a carbon source, a precursor and/or an inducer.

In another aspect of the present invention, a kit for producing monacolin J or an intermediate thereof is provided, wherein the kit comprises the engineered yeast.

In another aspect of the present invention, there is provided a kit for producing monacolin J or an intermediate thereof, said kit comprising a construct comprising expression cassettes for the following genes: lovB, lovC, lovG, npgA, transcriptional activator uta, and/or another construct comprising an expression cassette for the following group of genes: lovA, cpr, transcriptional activator uta.

In another preferred embodiment, the kit further comprises a culture medium containing ethanol as a carbon source, precursor and/or inducer.

Other aspects of the invention will be apparent to those skilled in the art in view of the disclosure herein.

Drawings

FIG. 1, Artificial transcription System P_ICL1-UTA_P_AOX1RAnd control GFP expression intensity in ethanol or methanol conditions.

FIG. 2 is a schematic diagram of the fermentation production of dihydromonacolin L by a Pichia pastoris engineering strain; wherein, DML, dihydromonacolin L; WT, Pichia pastoris wild type strain.

FIG. 3 is a schematic diagram of the production of monacolin J by mixed culture of two strains; wherein MJ, monacolin J; WT, pichia pastoris wild type.

FIG. 4 is a graph showing fermentation yields of monacolin J produced by mixed culture of two strains at different initial inoculation ratios; MJ, monacolin J, among others.

FIG. 5 is a graph showing the fermentation profile of a 5L reactor for producing monacolin J by mixed culture with optimal initial inoculation ratios of two strains; MJ, monacolin J, among others.

Detailed Description

Through intensive research, the inventor optimizes and reforms the yeast engineering strain by a gene recombination technology to obtain the yeast engineering strain capable of producing monacolin J or an intermediate product dihydro monacolin L thereof. The yield of the target product of the yeast engineering bacteria is obviously increased, and a new way for industrially producing monacolin J and downstream products thereof is provided.

Term(s) for

As used herein, the term "expression cassette" or "gene expression cassette" refers to a gene expression system that contains all the necessary elements required for expression of a polypeptide of interest, typically including the following elements: a promoter, a gene sequence encoding a polypeptide, a terminator; in addition, the protein also can selectively comprise a signal peptide coding sequence and the like; these elements are operatively connected.

As used herein, the term "expression construct" refers to a recombinant DNA molecule comprising a desired nucleic acid coding sequence, which may comprise one or more gene expression cassettes. The "construct" is typically contained in an expression vector.

As used herein, the term "exogenous" or "heterologous" refers to the relationship between two or more nucleic acids or protein sequences from different sources, or the relationship between a protein (or nucleic acid) from a different source and a host cell. For example, a nucleic acid is exogenous to a host cell if the combination of the nucleic acid and the host cell is not normally naturally occurring. A particular sequence is "foreign" to the cell or organism into which it is inserted.

Gene and expression system thereof

In the invention, the high-efficiency production of the monacolin J or the intermediate dihydro monacolin L thereof in the yeast engineering bacteria is realized by transforming polygene combination in the yeast engineering bacteria. Genes used for the production of dihydromonacolin L include: lovB, lovC, lovG, npgA; genes used for production of monacolin J include: lovB, lovC, lovG, npgA, lovA, cpr. In a preferred embodiment of the invention, the genes lovB, lovC, lovG, npgA, lovA, cpr from A.terreus have the nucleotide sequences shown in SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, respectively.

In order to optimize the yield of the monacolin J or the intermediate dihydromonacolin L thereof, the inventor carries out further optimization on engineering bacteria and a fermentation medium, and realizes high yield of the dihydromonacolin L or the monacolin J by taking ethanol as a substrate. In a preferred form of the invention, an ethanol inducible promoter (preferably P) is used_ICL1) To drive the expression of the transcription activator UTA and simultaneously drive the expression of the monacolin J synthesis related gene. Since UTA has the activity of recruiting RNA polymerase, it is also capable of synthesizing the promoter (e.g., P) of the gene involved in monacolin J_AOX1R) Binding, and thus ethanol inducible promoter (preferably P)_ICL1) The ethanol induction signal is amplified by the artificial transcription device, so that the purpose of enhancing the expression of the monacolin J synthesis related gene is achieved. In a preferred embodiment of the invention, the promoter P_ICL1The nucleotide sequence of (A) is shown in SEQ ID NO. 9, the nucleotide sequence of a transcription activator uta is shown in SEQ ID NO. 7, and a promoter P_AOX1RThe nucleotide sequence of (A) is shown in SEQ ID NO. 8.

In a preferred embodiment of the present invention, in order to further increase the metabolic flux of ethanol conversion to acetyl-coa and malonyl-coa, the present inventors overexpressed ethanol dehydrogenase ADH, acetyl-coa synthetase ACS and acetyl-coa decarboxylase ACC, further increasing the yield of dihydromonacolin L.

The above-mentioned gene may be naturally occurring, for example, it may be isolated or purified from a plant or microorganism. In addition, the gene can also be artificially prepared, for example, the gene can be obtained according to the conventional genetic engineering recombination technology, or the gene can be obtained by an artificial synthesis method. In a preferred embodiment of the invention adh has the nucleotide sequence shown in SEQ ID NO. 20, acs has the nucleotide sequence shown in SEQ ID NO. 21, and acc has the nucleotide sequence shown in SEQ ID NO. 22.

The nucleotide sequence of the above-mentioned gene may be the same as the sequences shown in SEQ ID NOS: 1 to 9 and 20 to 22, or may be a degenerate variant thereof. As used herein, "degenerate variant" means in the present invention a nucleic acid sequence that encodes a protein having the same function but differs from a sequence selected from the group consisting of SEQ ID NOs 1-9, 20-22.

The genes may include: a coding sequence encoding only the mature polypeptide; the coding sequence for the mature polypeptide and various additional coding sequences; the coding sequence (and optionally additional coding sequences) as well as non-coding sequences for the mature polypeptide.

The invention also relates to variants of said genes, which encode polypeptides which differ in amino acid sequence from their corresponding wild-type polypeptide, being fragments, analogues or derivatives of the wild-type polypeptide. The variant of the polynucleotide may be a naturally occurring allelic variant or a non-naturally occurring variant. These nucleotide variants include substitution variants, deletion variants and insertion variants. As is known in the art, an allelic variant is a substitution of a polynucleotide, which may be a substitution, deletion, or insertion of one or more nucleotides, without substantially altering the function of the polypeptide encoded thereby.

The present invention also relates to polynucleotides which hybridize to the above-described sequences and which have at least 70%, preferably at least 80%, identity between the two sequences, more preferably at least 80%. The present invention particularly relates to polynucleotides which hybridize under stringent conditions to the polynucleotides of the present invention. In the present invention, "stringent conditions" mean: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 XSSC, 0.1% SDS, 60 ℃; or (2) adding denaturant during hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1% Ficoll, 42 deg.C, etc.; or (3) hybridization occurs only when the identity between two sequences is at least 90% or more, preferably 95% or more. Also, the polynucleotides that hybridize to the polypeptide encoded by the polynucleotide have the same biological functions and activities as the corresponding wild-type polypeptide.

It is to be understood that while multiple genes of the present invention are preferably obtained from a particular species, their homologous genes (e.g., having greater than 80%, such as 90%, 95%, even 98%, 99% sequence identity) obtained from other species are also within the contemplation of the present invention. Methods and means for aligning sequence identity are also well known in the art, for example BLAST.

The full-length sequence of each gene of the present invention or a fragment thereof can be obtained by a PCR amplification method, a recombination method, or an artificial synthesis method. For PCR amplification, primers can be designed based on the nucleotide sequences disclosed herein, particularly the open reading frame sequences, and the sequences can be amplified. When the sequence is longer, two or more PCR amplifications can be carried out, and then the amplified fragments are spliced together according to the correct sequence.

The invention also relates to a vector comprising said polynucleotide, and a host cell genetically engineered with said vector.

In the present invention, the sequence of each gene may be inserted into a recombinant expression vector. The term "recombinant expression vector" refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus, or other vector well known in the art. In general, any plasmid or vector can be used as long as it can replicate and is stable in the host. An important feature of expression vectors is that they generally contain an origin of replication, a promoter, a marker gene and translation control elements.

The sequences of the genes can be respectively inserted into recombinant expression vectors, and a plurality of the recombinant expression vectors are co-transferred into host cells; the expression cassettes of multiple genes can also be inserted into the same recombinant expression vector in tandem and transferred into host cells. The recombinant expression vector may further comprise an expression control sequence operably linked to the sequence of the gene to facilitate expression of the protein. It is understood that recombinant expression vectors can be conveniently constructed by those skilled in the art having the benefit of the teachings of the present invention. The obtained recombinant expression vector is also included in the present invention.

In the expression regulation sequence or the expression cassette, an inducible or constitutive promoter can be applied according to different requirements, and the inducible promoter can realize more controllable protein expression and compound production, thereby being beneficial to industrial application.

Vectors containing the appropriate gene sequences and appropriate promoter or control sequences described above may be used to transform appropriate host cells to enable expression of the protein. In the present invention, the host cell is preferably a yeast engineering bacterium, more preferably a methylotrophic yeast cell such as Pichia pastoris.

Synthesis method

The invention provides a Monacolin J heterologous production method taking ethanol as a carbon source, a precursor and an inducer, which verifies that the Pichia pastoris can supply sufficient precursor acetyl coenzyme A by taking ethanol as a substrate by detecting the growth of the Pichia pastoris in the ethanol and determining the flow direction of an ethanol metabolism passage. The transcription regulation genetic circuit in the methanol nutritional yeast cell is artificially modified, so that the transcription strength of an ethanol induction promoter is improved, and the expression of heterologous pathway enzymes is enhanced. The yeast expression system and the fermentation method of the invention enable the heterologous synthesis amount of monacolin J in pichia pastoris to reach 2.2 g/L.

In order to further improve the production of the monacolin J in the pichia pastoris, the invention improves the metabolism flux of the cytoplasmic acetyl coenzyme A in the pichia pastoris by taking ethanol as a substrate, and simultaneously improves the transcription expression of heterologous enzyme genes by constructing an ethanol-induced transcription device through artificial design. On the premise of ensuring sufficient precursors and high-efficiency expression of enzyme, the synthesis of the product monacolin J is promoted.

The dihydromonacolin L is catalyzed and oxidized by an enzyme LovA to synthesize monacolin J, and in order to avoid metabolic pressure caused by introducing excessive exogenous genes into a single cell, the inventor provides a strategy for constructing double-strain co-culture. The inventors also found that the initial mixing ratio of the two strains in the co-culture system affects the growth of the strains and the synthesis of the products to some extent, so the inventors optimized the initial inoculation ratio to obtain a better initial inoculation ratio.

Acetyl coenzyme A is a core intermediate metabolite of cell basic metabolism, is formed by connecting an acetic acid molecule and coenzyme A through a thioester bond, can provide acyl groups for various biochemical reactions in cells, is a substrate for protein acylation, and participates in enzyme transcription and function regulation. Meanwhile, acetyl coenzyme A is also a precursor molecule of a series of bioactive substances, including fatty acid, terpenoid, polyketide and the like. With the intensive research on the synthesis of target compounds by using the concept of microbial factories, people pay more attention to the problem of precursor supply in microbial hosts, and the energy flow of substances generated by carbon source metabolism is promoted to flow to acetyl coenzyme A more by reasonably modifying an acetyl coenzyme A metabolic network, so that the energy flow is used as a substrate of the target compounds to promote the synthesis of final products. Ethanol is a precursor that can be used to increase the supply of acetyl-coa, and the synthesis of acetyl-coa derivatives can be enhanced by the addition of ethanol.

Heterologous pathway construction and metabolic engineering in a single host cell often burdens the growth and metabolism of the host cell, causing a decrease in the amount of synthesized end product. In the invention, by rationally designing a multi-cell co-culture flora system, distributing a complex biosynthesis pathway to independent microbial cells and adjusting the inoculation proportion of each cell, a heterologous pathway can be optimized, the metabolic pressure can be relieved and the product synthesis can be promoted in the flora co-culture system. The synthetic biological element is combined with the traditional molecular operation technology for use, so that a promoter transcription regulation network can be artificially designed and constructed, the transcription strength of the promoter is enhanced, and the expression of heterologous proteins is further improved.

In the present inventionIn the specific example, firstly, a recombinant pichia pastoris strain for synthesizing the dihydromonacolin L by using ethanol as a substrate is constructed. Use of an artificially designed promoter P_AOX1RTo drive the expression of the genes lovB, lovC, lovG, npgA, using the promoter P_ICL1Drives the expression of the transcription activator UTA, and can produce the dihydromonacolin L by using ethanol as a substrate through culture and fermentation. In order to further improve the metabolic flux of converting ethanol into acetyl coenzyme A and malonyl coenzyme A, the invention overexpresses alcohol dehydrogenase ADH, acetyl coenzyme A synthetase ACS and acetyl coenzyme A decarboxylase ACC, and further improves the synthesis of dihydromonacolin L to obtain the high-yield strain P.p/DML of the dihydromonacolin L. The recombinant bacterium P.p/DML can produce dihydromonacolin L through fermentation, and the dihydromonacolin L enters the recombinant bacterium P.p/sAR to be catalyzed and oxidized to synthesize monacolin J. Wherein the recombinant strain comprises a constitutive promoter P_GAPExpression cassette for driving expression of transcription activator UTA, and artificially synthesized promoter P_AOX1RExpression cassettes driving expression of lovA and cpr.

In a specific embodiment of the invention, the superior fermentation strategy obtained by shake flask horizontal culture was scaled up to a 5L reactor for process scaling up pilot scale to evaluate the large-scale productivity of monacolin J. The co-cultured strains P.p/DML and P.p/sAR were separately cultured to logarithmic growth phase, and mixed inoculation was performed according to the optimum initial inoculation ratio of the shake flask. In the early stage of fermentation, glucose is used as a carbon source to maintain the rapid growth of the strain, and the promoter P is inhibited_ICL1Initiate, repress heterologous pathway expression. When the wet weight of the thalli grows to 300g/L, switching a carbon source to ethanol, starting heterologous pathway expression, sampling at intervals of 8h to measure the wet weight of the thalli and the yield of the monacolin J, and finally obtaining the yield of the monacolin J of 2.2g/L in 96 h.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Material

The LLB culture medium is as follows: 1% (w/v) peptone, 0.5% (w/v) yeast powder, 0.5% (w/v) sodium chloride.

YPD medium was: 2% (w/v) peptone, 1% (w/v) yeast powder, 2% (w/v) glucose.

The MGY culture medium is: 0.67% (w/v) Yeast nitrate base (YNB, Yeast basic nitrogen source medium), 1% (w/v) glycerol.

Ethanol fermentation of pichia pastoris production strains, using YNE medium: 1.34% (w/v) yeast basic nitrogen source medium (YNB), 0.5% (v/v) ethanol, ethanol was added every 24 h.

Sterilizing solid and liquid culture medium at 121 deg.C under high pressure for 20min, preparing 50% (w/v) glucose solution, filtering with 0.22 μm sterile filter membrane, and adding corresponding volume of glucose mother liquor in ultra-clean bench according to final concentration.

The use amount of antibiotics is as follows: ampicillin, 50. mu.g/ml; bleomycin, 50 μ g/ml for Escherichia coli culture; 100 mu g/ml is used for culturing pichia pastoris; geneticin, 250. mu.g/ml.

Peptone, yeast powder and malt extract were purchased from beijing bayer died biotechnology limited.

Basic nitrogen source medium YNB was purchased from Sigma.

Glucose, glycerol, methanol, ethanol, ethyl acetate, ammonia water, potassium dihydrogen phosphate, phosphoric acid, sodium chloride, and the like are available from national drug group chemical agents, ltd.

The DNA purification and recovery kit, the plasmid miniextraction kit and the yeast genome extraction kit are purchased from Tiangen Biotechnology (Beijing) Co., Ltd.

PCR primers, gene synthesis, plasmid sequencing and the like are completed by Suzhou Jinzhi Biotechnology GmbH, and PCR high-fidelity enzyme and restriction enzyme are purchased from TaKaRa.

Both the single-fragment seamless cloning kit and the multi-fragment seamless assembly kit were purchased from Nanjing Novozam.

Example 1 construction of a plasmid for heterologous expression of the Monacolin J biosynthesis Gene

1.pZ_UTA_P_AOX1RConstruction of the BCGN plasmid

Starting with the Pichia commercial plasmid pPIC ZB (Invitrogen), according to the promoter P_AOX1RThe promoter fragment was synthesized, the plasmid pPIC ZB was digested with BglII and XhoI, the original AOX1 promoter fragment was removed by agarose electrophoresis, and the promoter P was isolated_AOX1RCarrying out seamless cloning on the fragment and the plasmid fragment after enzyme digestion, and obtaining the plasmid pZ _ P through monoclonal screening and sequencing verification_AOX1R。

UTA fragments are synthesized according to UTA nucleotide sequence, and simultaneously, a primer is designed and a pichia pastoris genome is used as a template for amplification to obtain a promoter P_ICL1Fragment, mixing uta fragment with P by OverlapPCR technique_ICL1The fragments are ligated in vitro, the ligated P_ICL1Seamless cloning of the UTA fragment and the cleaved pPIC ZB fragment to obtain the plasmid pZ _ P_ICL1-UTA。

The gene fragments of lovB (SEQ ID NO:1), lovC (SEQ ID NO:2), lovG (SEQ ID NO:3) and npgA (SEQ ID NO:4) were PCR-amplified and each gene fragment was separately ligated with linearized plasmid pZ _ P_AOX1RSeamless cloning is carried out to obtain plasmids pZ _ P respectively_AOX1R-LovB、pZ_P_AOX1R-LovC、pZ_P_AOX1RLovG and pZ _ P_AOX1R-NpgA。

Plasmid pZ _ P was digested with the endonuclease BamHI alone_AOX1RLovB, design primer pair TT-lacOcACOX-F (SEQ ID NO:10) and BamH-TT-R (SEQ ID NO:11) as plasmid pZ _ P_AOX1RUsing LovC as a template, obtaining an expression cassette fragment by PCR amplification, and mixing the fragment with a linearized plasmid pZ _ P_AOX1RSeamless cloning of LovB, and obtaining of plasmid pZ _ P by colony PCR verification_AOX1R-BC. By continuing the same procedure, the expression cassette fragments of lovG and npgA can be inserted further on the basis of this plasmid, resulting in plasmid pZ _ P_AOX1R-BCGN。

In plasmid pZ _ P_ICL1On the basis of UTA plasmid, primer pairs TT-pICL 1-F (SEQ ID NO:12) and BamH-TT-R (SEQ ID NO:11) are used for obtaining P through PCR amplification_ICL1Driving expression of expression UTAA cassette fragment. The linearized plasmid pZ _ P_AOX1RCarrying out seamless cloning transformation on the BCGN and the expression cassette fragment to obtain an expression plasmid pZ _ UTA _ P_AOX1R-BCGN。

2.pK_UTA_P_AOX1RConstruction of the-sAR plasmid

Plasmid pZ _ P was double digested with XhoI and SalI_AOX1R(ii) a Obtaining gene fragments of lovA (SEQ ID NO:5) and cpr (SEQ ID NO:6) by primer PCR amplification, and respectively mixing each gene fragment with linearized plasmid pZ _ P_AOX1RCarrying out seamless cloning to obtain a plasmid pZ _ P_AOX1R-LovA、pZ_P_AOX1R-CPR。

Primer pairs TT-KanaHis-F (SEQ ID NO:13), Amp-KanaHis-R (SEQ ID NO:14), KanaHis-Amp-F (SEQ ID NO:15) and lacOcaOX-Amp-R (SEQ ID NO:16) are designed, and plasmid pPIC3.5K is used as a template for PCR amplification. Simultaneously designing primer pairs Amp-lacOcAOX-F (SEQ ID NO:17) and KanaHis-TTSpe-R (SEQ ID NO:18) to obtain plasmid pZ _ P_AOX1RCarrying out PCR amplification by taking LovA as a template to obtain an expression cassette fragment, carrying out multi-fragment seamless cloning on the expression cassette fragment and a linearized plasmid to obtain a plasmid pK _ P_AOX1R-LovA。

Plasmid pK _ P linearized with SpeI_AOX1RLovA, plasmid pZ _ P using primer pair TT-lacOcAOX-F (SEQ ID NO:10) and KanaHis-TTSpe-R (SEQ ID NO:18)_AOX1RThe plasmid pK _ P can be obtained by taking CPR as a template, obtaining a fragment through PCR amplification and carrying out seamless cloning on the fragment and the linearized plasmid_AOX1R-sAR. The linearized plasmid pK _ P was digested further with the endonuclease SpeI_AOX1RsA, plasmid pZ-P using primers TT-pGAP-F (SEQ ID NO:19) and BamH-TT-R (SEQ ID NO:11)_GAP-UTA is used as a template, an expression cassette fragment can be obtained through PCR amplification, the linearized plasmid and the expression cassette fragment are respectively subjected to single-fragment seamless cloning, and a plasmid pK _ UTA _ P can be obtained_AOX1R-sAR。

Example 2, P_ICL1Promotion of ethanol expression by driving expression of UTA

Plasmid pZ _ P prepared as described above_AOX1RXho and Sal double digestion linearized plasmid, primer design and amplification of eGFP reporter gene fragment, and obtaining P_ICL1UTA by seamless Assembly of kitsCloning and screening to obtain plasmid pZ _ P_ICL1-UTA_P_AOX1R-eGFP. At the same time, with P_ICL1Drive expression of eGFP, P_AOX1Driving expression of eGFP as a control. Linearizing the plasmid, electrically transforming a pichia pastoris wild strain, and screening a transformant. And carrying out genotype identification on the obtained monoclonal transformant, and fermenting by using ethanol or methanol as a carbon source.

As shown in FIG. 1, an artificial transcription system P was used_ICL1-UTA_P_AOX1RGFP expression intensity in ethanol compared to the original P_ICL1The strength of the promoter is greatly improved and is far higher than that of the original P_AOX1Intensity of GFP expression under methanol and ethanol conditions. The artificial transcription device provided by the invention is proved to be capable of remarkably improving the expression level of heterologous proteins.

Example 3 obtaining of Co-culture Strain producing Monacolin J

1. Construction of Dihydromonacolin L producing Strain

Plasmid pZ _ UTA _ P prepared as described above_AOX1RBCGN, using SpeI single enzyme digestion, after purification and recovery, transforming Pichia wild strain electrically, using bleomycin resistant YPD solid plate to screen transformant. And carrying out genotype identification on the obtained monoclonal transformant, extracting a metabolite in a fermentation liquor after ethanol induction fermentation, and carrying out high performance liquid chromatography analysis to synthesize a recombinant bacterium of the dihydromonacolin L, namely the pichia pastoris strain for producing the dihydromonacolin L, which is named as P.p/DML.

2. Metabolic engineering optimization for improving production of dihydromonacolin L

To further enhance the supply of acetyl-CoA and the synthesis of dihydromonacolin L, alcohol dehydrogenase ADH (SEQ ID NO:20), acetyl-CoA synthetase ACS (SEQ ID NO:21) and acetyl-CoA decarboxylase ACC (SEQ ID NO:22) were further overexpressed on the basis of the dihydromonacolin L-producing strain.

The genome of the saccharomyces cerevisiae is used as a template, and a primer is designed to obtain an ethanol dehydrogenase gene adh and an acetyl coenzyme A synthetase gene acs through PCR amplification. The acetyl coenzyme A decarboxylase acc can be obtained by taking a pichia pastoris genome as a template and designing a primer for PCR amplification. Using the endonucleases BspT104I and KpnIDouble restriction enzyme plasmid pGAPZ alpha, removing signal peptide fragments by DNA gel electrophoresis to obtain linearized plasmids, respectively carrying out seamless cloning on the linearized plasmids and three gene fragments, respectively transforming escherichia coli, and obtaining P by colony PCR verification and sequencing result comparison_GAPPlasmid pZ-P for expression of respective enzyme genes_GAP-ADH、pZ-P_GAPACS and pZ-P_GAP-ACC. Designing a primer pair, and removing the HIS4 gene by PCR amplification by using a pichia pastoris commercial vector pPIC3.5K as a template to obtain a plasmid pKdH from which the HIS4 screening marker gene is removed. Simultaneously with plasmid pZ-P_GAPADH and pZ-P_GAPACS is taken as a template, and PCR amplification is carried out to obtain the gene expression cassette. The gene expression cassette and the linearized plasmid pKdH are respectively subjected to multi-fragment seamless cloning to obtain the plasmid pKdH-P_GAPADH and pKdH-P_GAP-ACS. Plasmid pKdH-P was digested with SpeI alone_GAPThe ADH is linearized, the linearized plasmid and the expression cassette fragment are seamlessly cloned and transformed, and the plasmid pKdH-P can be obtained through screening_GAP-ADH+P_GAP-ACS. The plasmid is linearized by BlnI, competent cells of pichia pastoris P.p/DML are electrically transformed, a YPD culture medium added with geneticin is used for screening transformants and carrying out genotype verification, and pichia pastoris strains with over-expressed ADH and ACS can be obtained.

Using BamHI and BlnI double digestion plasmid pPIC3.5K, primers were designed and expressed as pZ-P_GAPusing-ACC as template, obtaining P by PCR amplification_GAPThe ACC-TT fragment and the ACC-TT fragment are subjected to seamless cloning, and the plasmid pK-P can be obtained through colony PCR screening and verification_GAP-ACC. Linearizing the plasmid, electrically transforming pichia pastoris competent cells which over-express ADH and ACS, screening transformants and genotype verification by using an MGY plate, simultaneously screening single copy integration of each expression plasmid, obtaining an ADH, ACS and ACC over-expressed dihydromonacolin L production strain P.p/DML _ OE, extracting a fermentation product after ethanol induction fermentation of the obtained pichia pastoris transformant, and carrying out quantitative analysis by high performance liquid chromatography, wherein as shown in figure 2, compared with the yield of the dihydromonacolin L of the strain before optimization, the yield of the dihydromonacolin L is increased by 195%, and the optimized strain is named as P.p/DML _ OE (P.p/DML with over expression).

3. Construction of Strain catalyzing Synthesis of Monacolin J from DihydroMonacolin L

Plasmid pK _ UTA _ P was digested with BlnI_AOX1RsAR, electrically transforming Pichia pastoris wild strain after purification and recovery, and screening transformants by using geneticin resistant YPD solid plates. The obtained monoclonal transformant was subjected to genotype identification.

The obtained product was transformed into pK _ UTA _ P_AOX1R-sAR, co-cultured with strain P.p/DML _ OE for fermentation. Then, the metabolite in the fermentation broth was extracted and analyzed by high performance liquid chromatography, as shown in fig. 3, to determine that the fermentation product was monacolin J.

The above recombinant Pichia pastoris strain for catalyzing the conversion of dihydromonacolin L to monacolin J was named P.p/sAR.

Example 4 Co-cultivation of recombinant strains to produce Monacolin J

1. Determination of optimal initial inoculation ratio of Co-cultured Strain producing Monacolin J

After repeated research, the inventor finds that in the P.p/DML and P.p/sAR double-strain co-culture system, different initial inoculation ratios enable the co-culture system to be in different stable states. Therefore, the present inventors considered that it was necessary to optimize the initial inoculation ratio of the two strains in the co-culture system.

The inventors set the initial inoculation ratios of strain P.p/DML to strain P.p/sAR to 1:0.1, 1:0.2, 1:0.5, 1 in this order; 1, 1:1.5, 1:2, the effect of different inoculation ratios on throughput was observed.

The production amounts of monacolin J in each group are shown in fig. 4, and it can be seen that the production amounts of monacolin J are significantly desirable when the initial inoculation ratios of strain P.p/DML and strain P.p/sAR are 1: 0.2-0.5; whereas at an initial inoculation ratio of 1:0.2, the production of monacolin J reached a maximum of 156 mg/L.

2. High-density fermentation process of recombinant engineering bacteria 5L reactor

The present inventors scaled up a co-culture system with an initial inoculation ratio of 1:0.2 for strain P.p/DML and strain P.p/sAR into a 5L reactor for fermentation pilot scale. Respectively culturing strains P.p/DML and P.p/sAR in a shake flask to logarithmic growth phase, measuring the bacterial concentration, mixing at a ratio of 1:0.2, inoculating into a 5L reactor, controlling the pH to be 4.5, and controlling the dissolved oxygen to be 30-70%. And in the early stage of fermentation, glucose is used as a carbon source for feeding and feeding, the feeding rate of the glucose is controlled from low to high and is gradually increased from 24g/h to 40g/h, and the residual concentration of the glucose in the culture medium is detected, so that the large accumulation of the glucose caused by too fast feeding of the glucose is avoided. And after the cell grows at a high speed until the wet weight of the thalli reaches 300g/L, switching a carbon source to ethanol feeding, wherein the ethanol feeding rate is increased from slow to fast and gradually from 0g/h to 24g/h, monitoring the residual concentration of ethanol in the fermentation liquid during the period to avoid over-high ethanol accumulation, maintaining the feeding rate at 24g/h until the thalli is fermented to 110h after the thalli adapts to the ethanol, and maintaining the pH at 4.5 and the dissolved oxygen at 30-70 percent during the period. The yield during fermentation was determined. As shown in FIG. 5, at 96h of fermentation, the co-cultivation system could produce 2.2g/L of monacolin J.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Sequence listing

<110> university of east China's college of science

<120> method for heterologous production of monacolin J and application thereof

<130> 184446

<160> 22

<170> SIPOSequenceListing 1.0

<210> 1

<211> 9117

<212> DNA

<213> Aspergillus terreus (Aspergillus terreus)

<400> 1

atggctcaat ctatgtatcc taatgagcct attgtcgtgg tcggcagtgg ttgtcgcttc 60

cctggtgacg ccaacacacc ctccaagctc tgggagctac tccagcatcc tcgcgatgtg 120

cagagtcgaa tccccaaaga acgatttgac gtcgacacat tttatcaccc ggacgggaag 180

caccacgggc gaacaaatgc accctacgcc tatgttctcc aagacgatct gggcgccttc 240

gatgcggcct tcttcaatat ccaggctgga gaggccgaga gtatggaccc ccagcaccgg 300

ctgttgctgg agacggtgta cgaggccgta acgaatgctg gaatgcgtat ccaggatctg 360

cagggaactt cgactgctgt ttacgtcggg gtgatgacgc acgactatga gactgtctca 420

acccgcgacc tggagagcat ccccacctac tcggcgacgg gtgtcgcggt cagtgttgcg 480

tccaaccgca tctcgtattt ttttgactgg catggaccaa gtatgacgat cgatacggca 540

tgcagctcgt cgttggttgc cgttcatctg gcggtgcaac agctacggac gggtcaaagc 600

tccatggcaa ttgctgcggg tgcgaatctg attctggggc ccatgacatt cgtccttgaa 660

agcaaattga gcatgctatc cccctcgggt cgatcccgca tgtgggacgc cggagctgac 720

ggctatgcca gaggcgaagc tgtttgctct gtagtgttga agacattgag tcaagccttg 780

cgcgatgggg acacgattga atgtgtcatc cgagaaactg gggtgaatca agatggccga 840

acgaccggaa ttacgatgcc gaaccatagt gctcaggagg cactcatcaa ggctacctac 900

gcccaggctg gccttgacat caccaaggcc gaggacaggt gccaattctt cgaggctcat 960

gggactggta ctccggccgg agatccccag gaggcggagg ccattgcaac agccttcttc 1020

ggccacgagc aggtagcacg cagcgacgga aacgagaggg cccctctgtt cgtgggcagt 1080

gcgaaaactg ttgtcgggca caccgagggc acggccggtc tggctggtct catgaaggcg 1140

tcgttcgctg tccgccatgg ggtaatcccc cccaacctgc tgttcgacaa aatcagcccg 1200

cgagtcgccc cattctataa aaacctgagg attccgacag aagctaccca atggccagct 1260

ctcccacccg gacaaccgcg ccgcgccagt gtcaactcct ttggattcgg cggcacgaat 1320

gcgcatgcca ttattgagga atacatggag ccagagcaaa accagctgcg agtctcgaat 1380

aatgaggact gcccacccat gaccggtgtc ctgagtttac ccttagtcct ctcggcgaag 1440

tcccagcgct ccttaaagat aatgatggag gagatgctgc aattccttga gtctcacccc 1500

gagatacact tgcacgacct cacctggtcc ttactgcgca agcggtcagt tctacccttc 1560

cgccgggcta ttgtcggcca tagtcatgaa accatccgcc gggctttgga ggatgccatc 1620

gaggatggta ttgtgtcgag cgacttcact acggaggtca gaggccagcc atcggtgttg 1680

ggaatcttca ccgggcaggg ggcgcagtgg ccggggatgt taaagaatct gatagaggca 1740

tcgccatatg tgcggaacat agtgagggag ctggacgact ccctgcagag cttgccggaa 1800

aaataccggc cctcgtggac gctactggac cagttcatgc tagaaggaga ggcctccaac 1860

gtccaatatg ctactttctc ccagccatta tgctgcgcgg tgcaaattgt cctggtccgt 1920

ctccttgaag ccgcgagaat acgattcacg gctgttgttg gacatagctc cggcgaaatt 1980

gcttgcgcct ttgctgccgg gctcatcagt gcctcgttgg cgattcggat tgcttactta 2040

cgtggagtcg tctcggcagg gggcgccaga ggcacaccgg gagccatgtt ggccgccggg 2100

atgtcctttg aggaagcaca agagatctgc gagttggatg cctttgaggg ccgcatctgc 2160

gtggctgcca gcaattcccc agacagtgta actttctctg gcgacgcgaa cgcaattgat 2220

cacctgaagg gcatgttgga ggatgagtcc acttttgcga gactgctcaa ggtcgataca 2280

gcgtaccact cgcatcatat gcttccatgt gcagacccat atatgcaagc cctagaagag 2340

tgtggttgtg ctgttgccga tgcaggttcc ccagccggaa gtgtaccctg gtattcgtcc 2400

gtggacgccg agaacaggca aatggcagca agagacgtga ccgccaagta ctggaaagat 2460

aacttagtat ctccggtgct attctcccac gcagtgcagc gggcagtcgt cacgcacaag 2520

gcgctggata tcgggattga agtgggctgt cacccagctc tcaagagccc atgcgtcgcc 2580

accatcaagg atgtcctatc tggggttgac ctggcgtata caggttgctt ggagcgagga 2640

aagaatgatc tcgattcatt ctctcgagca ctggcatatc tctgggaaag gtttggtgcc 2700

tccagtttcg atgcggacga gttcatgcgt gcagtcgcgc ctgatcggcc ctgtatgagt 2760

gtgtcgaagc tcctaccggc ctatccatgg gaccgctctc gtcgctactg ggtggaatcc 2820

cgagcaactc gccaccatct tcgagggccc aagccccatc ttctattagg aaagctctcc 2880

gaatacagca ctccgctaag cttccagtgg ctgaattttg tgcgcccacg agacattgaa 2940

tggcttgatg gacatgcatt gcaaggccag actgtcttcc ctgcggccgg ctatatcgtc 3000

atggcaatgg aagcagcctt aatgattgct ggcacccacg caaagcaggt caagttactg 3060

gagatcttgg atatgagcat tgacaaggcg gtgatatttg acgacgaaga cagcttggtt 3120

gagctcaacc tgacagctga cgtgtctcgc aacgccggcg aagcaggttc aatgaccata 3180

agcttcaaga tcgattcctg tctatcgaag gagggtaacc tatccctatc agccaagggc 3240

caactggccc taacgataga agatgtcaat cccaggacga cttccgctag cgaccagcac 3300

catcttcccc cgccagaaga ggaacatcct catatgaacc gtgtcaacat caatgctttc 3360

taccacgagc tggggttgat ggggtacaac tacagtaagg acttccggcg tctccataac 3420

atgcaacgag cagatcttcg agccagcggc accttagact tcattcctct gatggacgag 3480

ggtaatggct gtcctctcct gctgcatcct gcatcattgg acgtcgcctt ccagactgtc 3540

atcggcgcat actcctcccc aggtgatcgg cgtctacgct gtctgtatgt acccactcac 3600

gttgatcgca tcacacttgt cccatccctt tgcctggcaa cggctgagtc cggatgcgag 3660

aaggttgcct tcaatactat caatacgtac gacaagggag actacttgag cggtgacatt 3720

gtggtgtttg acgcggagca gaccaccctg ttccaggttg aaaatattac ttttaagccc 3780

ttttcacccc cggatgcttc aactgaccat gcgatgtttg cccgatggag ctggggtccg 3840

ttgactccgg actcgctgct ggataacccg gagtattggg ccaccgcgca ggacaaggag 3900

gcgattccta ttatcgaacg catcgtctac ttctatatcc gatcgttcct cagtcagctt 3960

acgctggagg agcgccagca ggcagccttc catttgcaga agcagatcga gtggctcgaa 4020

caagtcctgg ccagcgccaa ggagggtcgt cacctatggt acgaccccgg gtgggagaat 4080

gatactgagg cccagattga gcacctttgt actgctaact cctaccaccc tcatgttcgc 4140

ctggttcagc gagtcggcca acacctgctc cccaccgtac gatcgaacgg caacccattc 4200

gaccttctgg accacgatgg gctcctgacg gagttctata ccaacacact cagcttcgga 4260

cccgcactac actacgcccg ggaattggtg gcgcagatcg cccatcgcta tcagtcaatg 4320

gatattctgg agattggagc agggaccggc ggcgctacca agtacgtgtt ggccacgccc 4380

cagctggggt tcaacagcta cacatacacc gatatctcca ccggattctt cgagcaagcg 4440

cgggagcaat ttgccccctt cgaggaccgg atggtgtttg aacccctcga tatccgccgc 4500

agtcccgccg agcagggctt cgagccgcat gcctatgatc tgatcattgc ctccaatgtg 4560

ctacatgcga cacccgacct agagaaaacc atggctcacg cccgctctct gctcaagcct 4620

ggaggccaga tggttattct ggagattacc cacaaagaac acacacggct cgggtttatc 4680

tttggtctgt tcgccgactg gtgggctggg gtggatgatg gtcgctgcac tgagccgttt 4740

gtctcgttcg accgctggga tgcgatccta aagcgtgtcg ggttttccgg tgtggacagt 4800

cgcaccacgg atcgggacgc aaatctattc ccgacctctg tgtttagtac ccatgcaatt 4860

gacgccaccg tggagtactt agacgcgccg cttgccagca gcggcaccgt caaggactct 4920

taccctccct tggtggtggt aggagggcag accccccaat ctcagcgtct cctgaacgat 4980

ataaaagcga tcatgcctcc tcgtccgctc cagacataca agcgcctcgt ggatttgcta 5040

gacgcggagg agctgccgat gaagtccacg tttgtcatgc tcacggagct ggacgaggaa 5100

ttattcgccg ggctcactga agagaccttc gaggcaacca agctgctgct cacgtacgcc 5160

agcaatacgg tctggctgac agaaaatgcc tgggtccaac atcctcacca ggcgagcacg 5220

atcggcatgc tacgctccat ccgccgggag catcctgact tgggagttca tgttctggac 5280

gtcgacgcgg ttgaaacctt cgatgcaacc ttcctggttg aacaggtgct tcggcttgag 5340

gagcatacgg atgagctggc cagttcaact acatggactc aagaacccga ggtctcctgg 5400

tgtaaaggcc gcccgtggat tcctcgtctg aagcgcgatc tggctcgcaa taaccgaatg 5460

aactcctcgc gccgtcccat atacgagatg atcgattcgt cgcgggctcc cgtggcatta 5520

cagacggctc gggattcatc atcctacttc ttggagtccg ctgaaacctg gtttgtgcct 5580

gagagtgttc agcagatgga aacaaagacg atctatgtcc actttagctg tccccatgcg 5640

cttagggtcg gacagctcgg gtttttctat cttgtgcagg gtcacgtcca ggagggcaat 5700

cgcgaagtgc ccgtcgtggc cttagcagag cgtaacgcat ccattgtgca cgttcgtccc 5760

gattatatat atactgaggc agataacaat ctgtctgagg gtggtggcag ccttatggta 5820

accgtcctcg ccgcggcggt gttggcggag acggtgatca gtaccgccaa gtgcctgggg 5880

gtaactgact caatcctcgt tctgaatccc cccagcatat gtgggcagat gttgctccat 5940

gctggtgaag agatcggtct tcaagttcat ctggccacca cttctggcaa caggagttcg 6000

gtttctgctg gagacgccaa gtcctggcta acattgcatg ctcgcgacac ggactggcac 6060

ctgcgacggg tactgccccg gggtgtccag gctttagtcg acttatcagc cgaccagagc 6120

tgtgaaggtt tgactcagag gatgatgaaa gttctgatgc ctggctgtgc ccattaccgt 6180

gcggcagacc tgttcacaga caccgtttcc actgaattgc atagcggatc gcggcatcaa 6240

gcttcactgc ccgccgcata ttgggagcat gtggtatcct tagcccgcca gggacttcct 6300

agtgtcagcg aggggtggga ggtgatgccg tgcactcaat ttgcagcgca tgccgacaag 6360

acgcgcccgg atctctcgac agttatttcc tggccccggg agtcggacga ggctacgctt 6420

cctaccaggg ttcgctccat tgacgctgag accctctttg cggccgacaa aacatatctc 6480

ctggtcggac tgactggaga tcttggacga tcactaggtc gttggatggt ccagcatggg 6540

gcctgccaca ttgtacttac gagcagaaat ccgcaggtga accccaagtg gctggcgcat 6600

gttgaagaac tgggtggtcg agtcactgtt ctttccatgg acgtgacaag ccaaaactca 6660

gtggaagctg gcctggctaa actcaaggat ctgcatctgc caccagtggg gggtattgcc 6720

tttggccctc tggttctgca ggatgtgatg ctaaataata tggaactgcc aatgatggag 6780

atggtgctca accccaaggt cgaaggcgtc cgcatcctgc acgagaagtt ctccgatccg 6840

accagtagca accctctcga cttcttcgtg atgttctcct cgattgtggc cgtcatgggc 6900

aacccgggtc aggctaacta cagtgcggct aactgctacc ttcaagcgct ggcgcagcag 6960

cgagttgcat ccggattagc agcgtccacc atcgacatcg gtgccgtgta cggcgttggg 7020

ttcgtcactc gggcggagct ggaggaggac tttaatgcaa ttcggttcat gttcgattcg 7080

gttgaggaac atgaactgca tacactgttt gctgaggcag tggtggccgg tcgacgagcc 7140

gtgcaccagc aagagcagca gcggaagttc gcgacagtgc tcgacatggc tgatctggaa 7200

ctgacaaccg gaattccgcc cctggatcca gccctcaaag atcggatcac cttcttcgac 7260

gacccccgca taggcaactt aaaaattccg gagtaccgag gggccaaagc aggcgaaggg 7320

gcagccggct ccaagggctc ggtcaaagaa cagctcttgc aggcgacgaa cctggaccag 7380

gtccgtcaga tcgtcatcga tggactctcc gcgaagctgc aggtgaccct gcagatcccc 7440

gatggggaaa gcgtgcatcc caccatccca ctaatcgatc agggggtgga ctctctgggc 7500

gcggtcaccg tgggaacctg gttctccaag cagctgtacc ttgatttgcc actcctgaaa 7560

gtgcttgggg gtgcttcgat caccgatctc gctaatgagg ctgctgcgcg attgccacct 7620

agctccattc ccctcgtcgc agccaccgac gggggtgcag agagcactga caatacttcc 7680

gagaatgaag tttcgggacg cgaggatact gaccttagtg ccgccgccac catcactgag 7740

ccctcgtctg ccgacgaaga cgatacggag ccgggcgacg aggacgtccc gcgttcccac 7800

catccactgt ctctcgggca agaatactcc tggagaatcc agcagggagc cgaagacccc 7860

accgtcttta acaacaccat tggtatgttc atgaagggct ctattgacct taaacggctg 7920

tacaaggcgt tgagagcggt cttgcgccgc cacgagatct tccgcacggg gtttgccaac 7980

gtggatgaga acgggatggc ccagctggtg tttggtcaaa ccaaaaacaa agtccagacc 8040

atccaagtgt ctgaccgagc cggcgccgaa gagggctacc gacaactggt gcagacacgg 8100

tataaccctg ccgcaggaga caccttgcgg ctggtggact tcttctgggg ccaggacgac 8160

catctgctgg ttgtggctta ccaccgactc gtcggggatg gatctactac agagaacatc 8220

ttcgtcgaag cgggccagct ctacgacggc acgtcgctaa gtccacatgt ccctcagttt 8280

gcggacctgg cggcacggca acgcgcaatg ctcgaggatg ggagaatgga ggaggatctc 8340

gcgtactgga agaaaatgca ttaccgaccg tcctcaattc cagtgctccc actgatgcgg 8400

cccctggtag gtaacagtag caggtccgat actccaaatt tccagcactg tggaccctgg 8460

cagcagcacg aagccgtggc gcgacttgat ccgatggtgg ccttccgcat caaggagcgc 8520

agtcgcaagc acaaggcgac gccgatgcag ttctatctgg cggcgtatca ggtgctgttg 8580

gcgcgcctca ccgacagcac cgatctcacc gtgggcctcg ccgacaccaa ccgtgcgact 8640

gtcgacgaga tggcggccat ggggttcttc gccaacctcc ttcccctgcg cttccgggat 8700

ttccgccccc atataacgtt tggcgagcac cttatcgcca cccgtgacct ggtgcgtgag 8760

gccttgcagc acgcccgcgt gccctacggc gtcctcctcg atcaactggg gctggaggtc 8820

ccggtcccga ccagcaatca acctgcgcct ttgttccagg ccgtcttcga ttacaagcag 8880

ggccaggcgg aaagtggaac gattgggggt gccaagataa ccgaggtgat tgccacgcgc 8940

gagcgcaccc cttacgatgt cgtgctggag atgtcggatg atcccaccaa ggatccgctg 9000

ctcacggcca agttacagag ttcccgctac gaggctcacc accctcaagc cttcttggag 9060

agctacatgt cccttctctc tatgttctcg atgaatcccg ccctgaagct ggcatga 9117

<210> 2

<211> 1092

<212> DNA

<213> Aspergillus terreus (Aspergillus terreus)

<400> 2

atgggcgacc agccattcat tccaccaccg cagcaaacag cgctgacggt aaatgaccat 60

gatgaagtca ccgtctggaa tgccgcaccc tgccccatgc tgccccgcga ccaggtatac 120

gtccgcgtcg aggccgtggc gatcaatccc agtgacacga agatgcgcgg acagtttgcc 180

acgccctggg cgtttctcgg aacggactat gccggcacgg tcgtcgcagt gggttcggac 240

gtgactcata tccaagtggg tgaccgggtc tacggggcac agaacgagat gtgcccacgc 300

accccggatc agggggcatt ctcgcagtac acggtcacgc gaggccgtgt ttgggccaag 360

atccccaagg gcttgtcgtt cgagcaggct gccgcgctac ctgcgggcat cagtaccgct 420

ggattggcga tgaagttgct tgggctgcct ttgccatcgc cttcggcaga ccagccaccc 480

acccactcca agccggtgta tgtgttggtc tatgggggca gtacggccac tgccactgtc 540

actatgcaaa tgctccgcct gtccggatat attccaattg caacatgctc cccccacaat 600

ttcgacctgg ccaaatcgcg cggcgcagag gaggtctttg actatcgggc cccgaatctc 660

gcgcagacga tccgtaccta caccaagaac aatctccgct atgctctcga ctgtatcacc 720

aacgtcgagt ccaccacatt ctgcttcgca gccatcggcc gcgcgggggg gcactacgtc 780

tccctgaacc cgttccctga acacgcggcc acgcgcaaga tggtcacgac cgactggacc 840

ctggggccga ccatctttgg cgagggatca acctggcccg ccccctatgg gcgtcccggc 900

agtgaggaag agcggcagtt cggcgaggat ctgtggcgca tcgcggggca gctcgtcgaa 960

gatggacgcc tcgtccatca tccgttgcgc gtggtgcagg gcggcttcga tcacattaag 1020

caaggcatgg agctcgtccg gaagggagag ctgtcggggg agaaactcgt ggttcggctc 1080

gaggggccgt aa 1092

<210> 3

<211> 771

<212> DNA

<213> Aspergillus terreus (Aspergillus terreus)

<400> 3

atgcgttacc aagcatctcc agcgctggtg aaggcgcctc gagcgcttct ttgcatccat 60

ggggctggct gctctcccgc catcttccgc gtgcaattgt ctaagctccg ggctgcgctg 120

cgcgaaaact ttgaattcgt ctacgtgaca gctccgttcc cttcctctgc agggcctggg 180

attctccccg tcttcgccga cctagggcca tattactcct ggtttgaaag cagcagcgac 240

aacaatcata atggaccctc cgtgagcgaa cgcctcgccg ccgtccacga ccccatccgc 300

cgcaccattg tcgactggca gactcaacac ccccacatcc ctatcgtggg tgctatcggt 360

ttctccgaag gtgccctggt gacgaccttg ctcctctggc agcagcagat gggtcacctg 420

ccctggttgc cccggatgag tgttgcgctg ttgatctgtc cctggtatca agacgaggca 480

agccagtata tgaggaacga agtgatgaag aaccatgacg acgacaacga cagcaaagat 540

accgagtggc aggaggaact ggtcattcgg ataccgacat tacatctgca gggtcgcgat 600

gattttgcgc tcgcaggatc gaagatgctg gtggcgcgcc atttctcccc ccgagaggcg 660

caggtattgg agtttgctgg gcagcatcag tttcccaatc gaccgcgcga cgtgttggag 720

gtcattaatc gttttcgtaa gctgtgtgtg acggcccaga cattggagta g 771

<210> 4

<211> 1035

<212> DNA

<213> Aspergillus terreus (Aspergillus terreus)

<400> 4

atggtgcaag acacatcaag cgcaagcact tcgccaattt taacaagatg gtacatcgac 60

acccgccctc taaccgcctc aacagcagcc cttcctctcc ttgaaaccct ccagcccgct 120

gatcaaatct ccgtccaaaa atactaccat ctgaaggata aacacatgtc tctcgcctct 180

aatctgctca aatacctctt cgtccaccga aactgtcgca tcccctggtc ttcaatcgtg 240

atctctcgaa ccccagatcc gcacagacga ccatgctata ttccaccctc aggctcacag 300

gaagacagct tcaaagacgg atataccggc atcaacgttg agttcaacgt cagccaccaa 360

gcctcaatgg tcgcgatcgc gggaacagct tttactccca atagtggtgg ggacagcaaa 420

ctcaaacccg aagtcggaat tgatattacg tgcgtaaacg agcggcaggg acggaacggg 480

gaagagcgga gcctggaatc gctacgtcaa tatattgata tattctcgga agtgttttcc 540

actgcagaga tggccaatat aaggaggtta gatggagtct catcatcctc actgtctgct 600

gatcgtcttg tggactacgg gtacagactc ttctacactt actgggcgct caaagaggcg 660

tatataaaaa tgactgggga ggccctctta gcaccgtggt tacgggaact ggaattcagt 720

aatgtcgtcg ccccggccgc tgttgcggag agtggggatt cggctgggga tttcggggag 780

ccgtatacgg gtgtcaggac gactttatat aaaaatctcg ttgaggatgt gaggattgaa 840

gttgctgctc tgggcggtga ttacctattt gcaacggctg cgaggggtgg tgggattgga 900

gctagttcta gaccaggagg tggtccagac ggaagtggca tccgaagcca ggatccctgg 960

aggcctttca agaagttaga tatagagcga gatatccagc cctgtgcgac tggggtgtgt 1020

aattgcctat cctaa 1035

<210> 5

<211> 1587

<212> DNA

<213> Aspergillus terreus (Aspergillus terreus)

<400> 5

atgactgtcg acgcgctcac acagccgcac caccttctgt cgctggcttg gaatgacacg 60

cagcaacatg gctcgtggtt tgcgcccttg gtcactacca gtgcggggct actatgcctt 120

cttctttacc tgtgctcgag tggccggaga tctgatctgc cggtgttcaa tccgaaaaca 180

tggtgggaac tgacgaccat gagggccaaa cgggattttg atgcgaatgc accgtcatgg 240

attgagagct ggttctcgca aaatgataag cccattcggt tcatagtcga ctctgggtac 300

tgcaccattc ttccctcctc tatggccgat gagtttcgca agatgaaaga gctctgtatg 360

tacaagttct tgggcacgga ctttcactct catcttcccg gattcgatgg attcaaggaa 420

gtcacgaggg atgcacatct catcaccaag gtggttatga accagttcca gacccaagct 480

cccaagtacg tcaagcctct tgccaatgaa gccagcggga ttatcacgga tatttttggc 540

gacagcaatg aatggcacac agtgcctgtc tataaccagt gtctggactt agtgacccga 600

acagtgactt ttattatggt cgggagcaag ttagcccata atgaggagtg gcttgacatc 660

gccaagcacc acgcggtgac gatggcaatt caagcgcgcc agctgcgcct ctggcccgtc 720

attctgcgcc cccttgtaca ttggctcgag ccccagggag ccaaactccg ggcgcaggtt 780

cgacgagccc ggcaacttct cgatcccatt atccaggagc gacgtgcgga aagagatgcc 840

tgccgggcaa agggcattga gccgcctcgc tacgtagact cgatccagtg gttcgaggat 900

actgccaagg ggaaatggta cgatgcagcc ggggcgcaac tggccatgga ctttgctggt 960

atctacggaa cctccgacct gctgatcggt gggttggtgg acatcgtccg acatccccat 1020

ctccttgagc ccctccgtga tgagatccgg acggtcatcg gccaaggggg ttggacacct 1080

gcctcgctgt acaagctcaa actgctggat agttgtctca aggagtcaca gcgcgtcaag 1140

cccgtcgaat gtgccaccat gcgcagctat gcattgcagg atgtgacttt ctccaatgga 1200

acctttatcc caaaaggaga gctggtggcg gtagctgccg accgcatgag caaccccgag 1260

gtctggccag agccggcaaa atacgatcct taccggtata tgcgcctgcg agaggacccg 1320

gctaaagcgt tcagtgccca actggagaac accaacgggg accacatcgg cttcggttgg 1380

catccacggg cttgccccgg ccggttcttt gcctctaagg agatcaagat gatgttagcc 1440

tacttgctca tacgatacga ctggaaggtg gtccccgacg aaccgttgca gtactaccgc 1500

cattctttca gcgtgcgcat tcatcccacc acgaagctca tgatgcgccg gcgcgacgag 1560

gatatccgcc ttcctggttc actatag 1587

<210> 6

<211> 2088

<212> DNA

<213> Aspergillus terreus (Aspergillus terreus)

<400> 6

atggctcaac tcgacactct cgacctggtg gtcctggtgg tgcttttggt gggtagcgcc 60

gcctacttca ccaagggcac ctactgggcc gttcccaagg acccgtatgc cgcctccggt 120

cccgccatga atggtggcgc caaggcgggc aaatccaggg acatcattga gaaaatggaa 180

gagactggca agaactgtgt gattttctac ggctcgcaga ccggtaccgc cgaggattat 240

gcgtcgcgcc tggccaagga aggctcccag cgtttcggcc tcaagaccat ggtcgcagat 300

ctggaagact acgattatga gaacctggac aagttccccg aggacaaggt tgccttcttc 360

gtcatggcca cctatggtga gggtgaaccc accgacaacg ccgtcgagtt ctaccagttc 420

atctcgggtg aggacgtcgc gttcgagagc ggcgcctctg ccgacgacaa gcccctgtcc 480

tccctcaagt atgtcacttt cggtctcggt aacaacacct atgagcacta tcaggctatg 540

gttcgcaatc tggatgccgc tctcaccaag ctgggtgcgc agcgcattgg agatgctggt 600

gaaggtgatg acggcgctgg caccatggaa gaagatttcc tggcctggaa agagcccatg 660

tggactgccc tgtccgaggc catgaacctt caggagcgcg aagccgtcta tgagccggtg 720

ttctcggtca cggaagatga atccctgtcc cccgaagacg aagccgtcta cctcggtgag 780

ccgaccaagg gtcaccgtga cggcaccccc agtggcccgt attccgctca caaccccttc 840

atcgccccca tcgtcgagtc tcgtgaactg ttcaacgtca aggaccgtaa ttgtctgcac 900

atggagatca gcatcgctgg tagcaacctt tcttaccaga ctggtgatca catcgcgatt 960

tggcccacga acgctggtgc cgaggtggac cggttcctcc aggtgtttgg tcttgagaac 1020

aagcgtcatt ccgtcatcaa catcaagggt atcgatgtga ccgccaaggt tcccattccg 1080

actcccacca cgtatgatgc tgctgttcgc tactatatgg aaattgctgc gcccgtctcc 1140

cgtcagtttg tggctaccct ggctgcgttt gctcccgatg aggagactaa ggcggaaatc 1200

gtgcgtttgg gtagcgacaa ggactacttc cacgagaaaa tcagcaacca gtgcttcacc 1260

atcgctcagg ctcttcagag tgtcacctcc aagcccttct cggctgtccc gttctctctg 1320

cttatcgagg gtctcaataa gctccagccc cgttactact ccatctcttc ctcctccatg 1380

gtccagaagg ataagatcag cattactgcc gtcgtggaat ccactcgctt gcctggtgcc 1440

gcccaccttg tcaagggtgt cacgaccaac tatctccttg ccctgaagca aaagcagaat 1500

ggggatccgt ctcccgaccc tcacggctta acttatacta tcactgggcc ccgtaacaag 1560

tacgacggaa tccacgttcc cgttcacgtc cgccactcca atttcaagct cccctctgat 1620

ccctctcggc ccattatcat ggttggccct ggtaccggtg tggctccctt ccgtggattc 1680

atccaggagc gtgccgcctt ggccgccaag ggtgagaacg tcggtcccac cgtgttgttc 1740

tttggatgcc gcaggcgcga tgaggacttt atgtacgcag atgaattcaa gacctaccag 1800

gaacagcttg gggacaagct tcagatcatt actgcgtttt ctcgtgaaac ttcccagaag 1860

gtgtatgttc agcacagact gcgtgaacac tccgatctgg tgagcagcct cctgaagcag 1920

aaggctaact tttacgtctg cggtgacgcc gccaacatgg cgcgtgaagt caaccttgtg 1980

cttggccaga tcatcgcgca acagcggggt ctcccggctg aacgggccga ggaaatggtg 2040

aagcacatgc gcagcagcgg cagctaccag gaggacgtgt ggtcatga 2088

<210> 7

<211> 3456

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atgggtgtta agccagttac tttgtatgac gttgctgaat acgctggagt ttcctaccaa 60

actgtctcta gagttgttaa tcaagcttct catgtctccg ctaagactag agagaaggtt 120

gaggctgcta tggctgaatt gaactatatt ccaaatagag ttgctcagca gttggctgga 180

aagcaatctt tgttgattgg agtcgctact tcttctttgg ctttgcatgc tccatctcag 240

attgttgctg ctattaagtc cagagctgac cagttgggag cttctgttgt tgtttctatg 300

gttgagagat ctggagttga ggcttgcaag gctgctgttc ataacttgtt ggctcagaga 360

gtttctggat tgattattaa ttacccattg gacgatcaag acgctattgc cgttgaggcc 420

gcttgtacca acgtcccagc tttgttcttg gacgtttccg atcaaactcc aattaattct 480

attatttttt ctcacgagga tggaactaga ttgggagttg aacacttggt tgctttggga 540

catcaacaga ttgctttgtt ggctggacca ttgtcttccg tttctgctag attgagattg 600

gccggatggc acaagtactt gaccagaaac cagattcaac caattgctga gagagaggga 660

gattggtctg ctatgtctgg attccagcag actatgcaga tgttgaacga aggaattgtc 720

ccaaccgcta tgttggtcgc taatgaccaa atggctttgg gagctatgag agctattact 780

gaatctggat tgagagtcgg agctgacatt tctgttgttg gatatgatga cactgaggat 840

tcttcttgct acattccacc attgactact attaagcaag acttcagatt gttgggacag 900

acttctgttg atagattgtt gcagttgtcc caaggacaag ctgttaaagg aaaccaattg 960

ttgccagttt ctttggttaa gagaaagact actttggctc caaacactca gactgcttcc 1020

ccaagagctt tggctgactc tttgatgcaa ttggctagac aagtctctag attggagtct 1080

ggacaaggtg gcggcggctc tgttaacaac tccatgaagg atttcttagg caagaaaacg 1140

gtggatggag ctgatagtct caatttggcc gtgaatctgc aacaacagca gagttcaaac 1200

acaattgcca atcaatcgct ttcctcaatt ggattggaaa gttttggtta cggctctggt 1260

atcaaaaacg agtttaactt ccaagacttg ataggttcaa actctggcag ttcagatccg 1320

acattttcag tagacgctga cgaggcccaa aaactcgaca tttccaacaa gaacagtcgt 1380

aagagacaga aactaggttt gctgccggtc agcaatgcaa cttcccattt gaacggtttc 1440

aatggaatgt ccaatggaaa gtcacactct ttctcttcac cgtctgggac taatgacgat 1500

gaactaagtg gcttgatgtt caactcacca agcttcaacc ccctcacagt taacgattct 1560

accaacaaca gcaaccacaa tataggtttg tctccgatgt catgcttatt ttctacagtt 1620

caagaagcat ctcaaaaaaa gcatggaaat tccagtagac acttttcata cccatctggg 1680

ccggaggacc tttggttcaa tgagttccaa aaacaggccc tcacagccaa tggagaaaat 1740

gctgtccaac agggagatga tgcttctaag aacaacacag ccattcctaa ggaccagtct 1800

tcgaactcat cgattttcag ttcacgttct agtgcagctt ctagcaactc aggagacgat 1860

attggaagga tgggcccatt ctccaaagga ccagagattg agttcaacta cgattctttt 1920

ttggaatcgt tgaaggcaga gtcaccctct tcttcaaagt acaatctgcc ggaaactttg 1980

aaagagtaca tgacccttag ttcgtctcat ctgaatagtc aacactccga cactttggca 2040

aatggcacta acggtaacta ttctagcacc gtttccaaca acttgagctt aagtttgaac 2100

tccttctctt tctctgacaa gttctcattg agtccaccaa caatcactga cgccgaaaag 2160

ttttcattga tgagaaactt cattgacaac atctcgccat ggtttgacac ttttgacaat 2220

accaaacagt ttggaacaaa aattccagtt ctggccaaaa aatgttcttc attgtactat 2280

gccattctgg ctatatcttc tcgtcaaaga gaaaggataa agaaagagca caatgaaaaa 2340

acattgcaat gctaccaata ctcactacaa cagctcatcc ctactgttca aagctcaaat 2400

aatattgagt acattatcac atgtattctc ctgagtgtgt tccacatcat gtctagtgaa 2460

ccttcaaccc agagggacat cattgtgtca ttggcaaaat acattcaagc atgcaacata 2520

aacggattta catctaatga caaactggaa aagagtattt tctggaacta tgtcaatttg 2580

gatttggcta cttgtgcaat cggtgaagag tcaatggtca ttccttttag ctactgggtt 2640

aaagagacaa ctgactacaa gaccattcaa gatgtgaagc catttttcac caagaagact 2700

agcacgacaa ctgacgatga cttggacgat atgtatgcca tctacatgct gtacattagt 2760

ggtagaatca ttaacctgtt gaactgcaga gatgcgaagc tcaattttga gcccaagtgg 2820

gagtttttgt ggaatgaact caatgaatgg gaattgaaca aacccttgac ctttcaaagt 2880

attgttcagt tcaaggccaa tgacgaatcg cagggcggat caacttttcc aactgttcta 2940

ttctccaact ctcgaagctg ttacagtaac cagctgtatc atatgagcta catcatctta 3000

gtgcagaata aaccacgatt atacaaaatc ccctttacta cagtttctgc ttcaatgtca 3060

tctccatcgg acaacaaagc tgggatgtct gcttccagca cacctgcttc agaccaccac 3120

gcttctggtg atcatttgtc tccaagaagt gtagagccct ctctttcgac aacgttgagc 3180

cctccgccta atgcaaacgg tgcaggtaac aagttccgct ctacgctctg gcatgccaag 3240

cagatctgtg ggatttctat caacaacaac cacaacagca atctagcagc caaagtgaac 3300

tcattgcaac cattgtggca cgctggaaag ctaattagtt ccaagtctga acatacacag 3360

ttgctgaaac tgttgaacaa ccttgagtgt gcaacaggct ggcctatgaa ctggaagggc 3420

aaggagttaa ttgactactg gaatgttgaa gaataa 3456

<210> 8

<211> 214

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

tgtgtggaat tgtgagcgga taacaatttc acacactaac ccctacttga cagcaatata 60

taaacagaag gaagctgccc tgtcttaaac cttttttttt atcatcatta ttagcttact 120

ttcataattg cgactggttc caattgacaa gcttttgatt ttaacgactt ttaacgacaa 180

cttgagaaga tcaaaaaaca actaattatt cgaa 214

<210> 9

<211> 1002

<212> DNA

<213> Pichia pastoris (Pichia pastoris)

<400> 9

tctaacactt tgtatagcac atcgtaccag tttatcaaaa tccaacaagt tttcctctgg 60

gtactgaaat tcgattagta ataaacgggc gcacttggtg atctcctcat cctgttcact 120

agccttcttt tcgcttaact tcatcaacga gatcaactcg agtctgcgag cagagaactg 180

tttccaaaga gtttcatcgc tgaaattcag cttctttcga ataaaatgtg tcactcccac 240

tttattactc gtggtgttat ttgttttcca cgaactagtt gaatcgccat ctttactacc 300

gtcctgggtg acatccggcg aattcgggac aaatgtgctg ttccggtagc ttgtaggaag 360

cggcatccgt agggcaatat acgactatag cttctaaagc gtagtacaat gaaatgttcg 420

aaggaacaac aaacggattt gtttttcgta ggctcaaccc gttgaggtgt aactctttag 480

cgaaagggta agattgattg ttcgaagtag ggcctcaaag ggaaagagaa aaaaaaaata 540

acaccaagag ttacgtaagc atatattttt tacgtaaagc atgattgaat ttcagcagta 600

ttgtttaaca aggctgatgt cgtgtgccaa tcaaaacaaa agagattcgc ataatgccat 660

aattggggtg tgtgggcgcc ccctaaaacg tctttctcat catcatctgc aacccccatc 720

gaacctcatt aaatcacatg acttgtgcga tcctcggtca actcgttccg tgcacccatt 780

ccaccccggg ctgaccaacg caaggttctc cgagagtccg ctaccccaga tttatatcag 840

caaccagtca cctttttccg ggcacgactc tatatgccct ggaaaaccgg agacgatgag 900

cctgactgta aaaggtgaca gaacccccaa ctctggttaa tctcttcaac aaatacttta 960

ttttctttca attcaaagaa cacagtatca agtatatcaa ga 1002

<210> 10

<211> 44

<212> DNA

<213> primers (Primer)

<400> 10

ttaagtgaga ccttcgtttg tgcagatctt gtgtggaatt gtga 44

<210> 11

<211> 40

<212> DNA

<213> primers (Primer)

<400> 11

aagctatggt gtgtggggga tccgcacaaa cgaaggtctc 40

<210> 12

<211> 46

<212> DNA

<213> primers (Primer)

<400> 12

agtgagacct tcgtttgtgc agatcttcat ctaacacttt gtatag 46

<210> 13

<211> 40

<212> DNA

<213> primers (Primer)

<400> 13

cagaagatta agtgagaact agtagttcgt ttgtgcaagc 40

<210> 14

<211> 26

<212> DNA

<213> primers (Primer)

<400> 14

tcagagaaat ttaccatgaa atctcc 26

<210> 15

<211> 26

<212> DNA

<213> primers (Primer)

<400> 15

ggagatttca tggtaaattt ctctga 26

<210> 16

<211> 40

<212> DNA

<213> primers (Primer)

<400> 16

ccacacagag ctccgaataa taactgttat ttttcagtgt 40

<210> 17

<211> 40

<212> DNA

<213> primers (Primer)

<400> 17

taacagttat tattcggagc tctgtgtgga attgtgagcg 40

<210> 18

<211> 46

<212> DNA

<213> primers (Primer)

<400> 18

gcttgcacaa acgaactact agttctcact taatcttctg tactct 46

<210> 19

<211> 45

<212> DNA

<213> primers (Primer)

<400> 19

agtgagacct tcgtttgtgc agatcttttt tgtagaaatg tcttg 45

<210> 20

<211> 1047

<212> DNA

<213> Saccharomyces cerevisiae

<400> 20

atgtctattc cagaaactca aaaagccatt atcttctacg aatccaacgg caagttggag 60

cataaggata tcccagttcc aaagccaaag cccaacgaat tgttaatcaa cgtcaagtac 120

tctggtgtct gccacaccga tttgcacgct tggcatggtg actggccatt gccaactaag 180

ttaccattag ttggtggtca cgaaggtgcc ggtgtcgttg tcggcatggg tgaaaacgtt 240

aagggctgga agatcggtga ctacgccggt atcaaatggt tgaacggttc ttgtatggcc 300

tgtgaatact gtgaattggg taacgaatcc aactgtcctc acgctgactt gtctggttac 360

acccacgacg gttctttcca agaatacgct accgctgacg ctgttcaagc cgctcacatt 420

cctcaaggta ctgacttggc tgaagtcgcg ccaatcttgt gtgctggtat caccgtatac 480

aaggctttga agtctgccaa cttgagagca ggccactggg cggccatttc tggtgctgct 540

ggtggtctag gttctttggc tgttcaatat gctaaggcga tgggttacag agtcttaggt 600

attgatggtg gtccaggaaa ggaagaattg tttacctcgc tcggtggtga agtattcatc 660

gacttcacca aagagaagga cattgttagc gcagtcgtta aggctaccaa cggcggtgcc 720

cacggtatca tcaatgtttc cgtttccgaa gccgctatcg aagcttctac cagatactgt 780

agggcgaacg gtactgttgt cttggttggt ttgccagccg gtgcaaagtg ctcctctgat 840

gtcttcaacc acgttgtcaa gtctatctcc attgtcggct cttacgtggg gaacagagct 900

gataccagag aagccttaga tttctttgcc agaggtctag tcaagtctcc aataaaggta 960

gttggcttat ccagtttacc agaaatttac gaaaagatgg agaagggcca aattgctggt 1020

agatacgttg ttgacacttc taaataa 1047

<210> 21

<211> 2139

<212> DNA

<213> Saccharomyces cerevisiae

<400> 21

atgtcgccct ctgccgtaca atcatcaaaa ctagaagaac agtcaagtga aattgacaag 60

ttgaaagcaa aaatgtccca gtctgccgcc actgcgcagc agaagaagga acatgagtat 120

gaacatttga cttcggtcaa gatcgtgcca caacggccca tctcagatag actgcagccc 180

gcaattgcta cccactattc tccacacttg gacgggttgc aggactatca gcgcttgcac 240

aaggagtcta ttgaagaccc tgctaagttc ttcggttcta aagctaccca atttttaaac 300

tggtctaagc cattcgataa ggtgttcatc ccagacccta aaacgggcag gccctccttc 360

cagaacaatg catggttcct caacggccaa ttaaacgcct gttacaactg tgttgacaga 420

catgccttga agactcctaa caagaaagcc attattttcg aaggtgacga gcctggccaa 480

ggctattcca ttacctacaa ggaactactt gaagaagttt gtcaagtggc acaagtgctg 540

acttactcta tgggcgttcg caagggcgat actgttgccg tgtacatgcc tatggtccca 600

gaagcaatca taaccttgtt ggccatttcc cgtatcggtg ccattcactc cgtagtcttt 660

gccgggtttt cttccaactc cttgagagat cgtatcaacg atggggactc taaagttgtc 720

atcactacag atgaatccaa cagaggtggt aaagtcattg agactaaaag aattgttgat 780

gacgcgctaa gagagacccc aggcgtgaga cacgtcttgg tttatagaaa gaccaacaat 840

ccatctgttg ctttccatgc ccccagagat ttggattggg caacagaaaa gaagaaatac 900

aagacctact atccatgcac acccgttgat tctgaggatc cattattctt gttgtatacg 960

tctggttcta ctggtgcccc caagggtgtt caacattcta ccgcaggtta cttgctggga 1020

gctttgttga ccatgcgcta cacttttgac actcaccaag aagacgtttt cttcacagct 1080

ggagacattg gctggattac aggccacact tatgtggttt atggtccctt actatatggt 1140

tgtgccactt tggtctttga agggactcct gcgtacccaa attactcccg ttattgggat 1200

attattgatg aacacaaagt cacccaattt tatgttgcgc caactgcttt gcgtttgttg 1260

aaaagagctg gtgattccta catcgaaaat cattccttaa aatctttgcg ttgcttgggt 1320

tcggtcggtg agccaattgc tgctgaagtt tgggagtggt actctgaaaa aataggtaaa 1380

aatgaaatcc ccattgtaga cacctactgg caaacagaat ctggttcgca tctggtcacc 1440

ccgctggctg gtggtgttac accaatgaaa ccgggttctg cctcattccc cttcttcggt 1500

attgatgcag ttgttcttga ccctaacact ggtgaagaac ttaacaccag ccacgcagag 1560

ggtgtccttg ccgtcaaagc tgcatggcca tcatttgcaa gaactatttg gaaaaatcat 1620

gataggtatc tagacactta tttgaaccct taccctggct actatttcac tggtgatggt 1680

gctgcaaagg ataaggatgg ttatatctgg attttgggtc gtgtagacga tgtggtgaac 1740

gtctctggtc accgtctgtc taccgctgaa attgaggctg ctattatcga agatccaatt 1800

gtggccgagt gtgctgttgt cggattcaac gatgacttga ctggtcaagc agttgctgca 1860

tttgtggtgt tgaaaaacaa atctagttgg tccaccgcaa cagatgatga attacaagat 1920

atcaagaagc atttggtctt tactgttaga aaagacatcg ggccatttgc cgcaccaaaa 1980

ttgatcattt tagtggatga cttgcccaag acaagatccg gcaaaattat gagacgtatt 2040

ttaagaaaaa tcctagcagg agaaagtgac caactaggcg acgtttctac attgtcaaac 2100

cctggcattg ttagacatcc aattgattcg gtcaagttg 2139

<210> 22

<211> 6648

<212> DNA

<213> Saccharomyces cerevisiae

<400> 22

atgagtagtg ttaaccactc tctccgtcat tcaaagctac cgccgcattt ccttggtctc 60

aactcggttg aagtcgctgc tccctccaag gtcagagact ttgtcaggga ccatggtggc 120

cactcggtca tcacgagagt gctgatcgca aacaacggta tagctgccgt gaaagaaatt 180

cgttccgtca ggaaatgggc gtatgaaacg tttggtaacg atagagccat tcaatttatt 240

gttatggcta ccccagagga tcttgaagct aatgctgaat atattcgaat ggctgaccag 300

tatgtcatgg tcccaggagg aactgcaaac aacaactatg cgaacgtcga cctcattgta 360

gaaatagcag aatctactga tgctcatgct gtttgggctg gttggggttt tgcctccgaa 420

aatccccatt tgcctgagca actggccgct tctcctaaga agattatctt cattggccct 480

ccgggctctg ccatgcgatc tcttggtgac aagatttcct ctactattgt cgcacaacat 540

gctaaagtcc catgtattcc ttggtcagga actggtgtcg atcaggttat aatcgacccc 600

gtaagcaatt tggtttccgt tgatgaagaa acgtacgcca aaggatgctg ttccgatcca 660

caggacggtt tggcaaaagc caaggctatt ggtttccctg tgatgattaa agcttccgaa 720

ggtggtggtg gtaaaggaat tagaaaagtt gacagggagg aagattttct ttctctttat 780

gatcaagctg ctaatgaaat tccaggttcc ccaattttta tcatgaagct tgctggagat 840

gccaggcatt tggaagttca attacttgct gatcaatatg gaaccaacat ctcccttttt 900

ggaagagatt gttccgttca aagaagacac caaaagatca tagaagaggc accagttacc 960

attgccaaac aagacacttt caggcaaatg gaacaagccg ctgtcagact gggtcaattg 1020

gttggatacg tttctgccgg taccgttgag tatctatatt cacacgctga ggacaagttc 1080

tacttcttgg aactgaaccc tcgtcttcaa gttgagcatc caaccacaga aatggccaca 1140

ggtgtcaatc ttccagttgc ccagttgcta attgcaatgg gtattccttt gaatagaatc 1200

agagatatca gggtacttta cggacttgaa ccaaatggcg ctacagaaat tgactttgaa 1260

ttcaaaactg aagaaagctt gaagagtcaa agaaaaccca ttccaaaggg tcacactatt 1320

gcatgtcgta tcacatctga agatcctggt gaaggtttta agccttctgg tggtgctcta 1380

tatgagctaa atttcagatc ttcttctagc gtttggggtt acttcagtgt aggaaacaaa 1440

tcctcaattc attctttcag tgactctcaa tttggtcata tattctcgtt tggcgaaaac 1500

cgtcaaatcg ccagaaaaaa tatggtcgtc gccttgaaag agctttctat tcgtggtgac 1560

tttagaacta caattgagta cttaataaaa ctgttggaaa cagctgattt cgagaacaac 1620

accatcacta ctggttggtt ggacgaactg atctcgaaga agctgactgc tgaaagacct 1680

gatgaaacca cagcaatttt atgtggtgct gaaaaaggtc aaatcccagg caaagaactt 1740

cttcgtacta ttttcccaat tgaatttatt tatgaaggaa agaagtacaa gtttactgtg 1800

gttcaggctg catttgacaa atacaacgtc tttgtcaacg gatgtatgat tactgtaagt 1860

gtaacccatt tgaaggatgg cagtttattg gtagcacttg atggtaaatc ccattctgtc 1920

tattacttgc aggaagaagt cggaaatact aggttgtcgg tggatggtaa atcttgcatt 1980

ttagaagttg agcatgagcc aactgaactt cgtactccat ctccaggtaa acttatcaaa 2040

tatcttgtgg aacacggtga tcacgtcaaa attggacaac cttacgctga agttgaagta 2100

atgaagatgt gtatgccttt ggtcagtcag gagaatggaa ctatcaggtt attgaagcag 2160

ccaggatctt cggttgccgc tggagacatc cttgctattc ttgcattgga tgatcccagc 2220

aaggtgaagc atgctttgcc attcgatggt acaatccctg atatgaaaca gccatttatc 2280

catagcaaca aaccagttta taagttcatt tctcttctct ccgtgctgaa aaacatttta 2340

gcagggtatg ataatcaagt tgtgatgaac gatactctgc agagtctatt ggatgtgttg 2400

aagaaccctg aacttcctta ttcggaatgg aatcattcga tatctgcact tcattcaagg 2460

ttaccaattc atttggacga acaattgacc agtttgattg agagatcgca tcaacgtggt 2520

gcagactttc cagctaagca cttgctcaag cttttggaca aggagcaggc tgttaatcct 2580

gatccacttt tctcccaggt cattgcgcct cttactgctg ttgccaaaag ctacgaacat 2640

ggacttgaag ttcatgaaca caatgtattc gccgatttga tcacccaata ctacgacata 2700

gagagcttgt ttgccgataa aagggaggaa gatgttattt tacagctacg tgatgagaac 2760

aaatcgtccc ttgacaaggt catcgatgtc gtcttgtcac attccagagt tggagctaag 2820

aaccatttaa tcagagctat tctggaaatt tatcaaacta tctgccaaaa tgatctccaa 2880

gctgcaacca ttttgaagaa acctttgaaa aagattgttg agctagattc tagatttaca 2940

gcaaaggttt cgttaaaagc tagagagatt ttgattcaat gttcccttcc ctctatcaaa 3000

gaacgttcag accagctcga gcatatcctt cgatcttcag ttgtacaaac tcagtacgga 3060

gagagcttca atggaaacta caaactgcct aacttggacg ttatacaaga cgtaattgat 3120

tccaagtaca ttgtattcga tgttttgaca caatttgttg ttagcccaaa caagtatata 3180

tttgcagcag cagccgaggt gtatctgcga agagcttaca gggcttactc ggtgagagaa 3240

gttaaacatc atttcgtagg tgattctgct ctcccaattg tggaatggaa gttccaattg 3300

ccgctgttat caacagctgc ttacaattcc gtgcctgaag ctatgagaaa ctcctccagt 3360

aaccgatcct ctatttcaat ggatagagca gttgctgtct ccgatttgac cttcatgatc 3420

aacaagaatg attctcaacc tttgagaaca ggtatcataa ttcccacaaa ccacttagat 3480

gacattgagg agtccttgtc atctgccatt gatgtcttcc ctaaacgtcc acgtaacaat 3540

ggaccagctc ctgacagaac taatgtggct cctgagcaac ctactaacgt atgcaatgtt 3600

ttcattgcca atgtttctgg ctacaacagt gaggctgaga tcgttgacaa gattagcagc 3660

gttctttctg agttgaaaga cgacctcagg gctagtggcg ttcgaagagt tacctttgtc 3720

ttgggagaca aggttggaac ttatccaaaa tactatacct tcaaatttcc agactatttt 3780

gaagacgaga caatccgtca catagagcct gctcttgcgt tccagctgga actaagaaga 3840

ttgtccaatt tcaatattaa acctgttcca actgagaata gaaatattca tgtgtatgag 3900

gcagttgcca aaaatacttc atgcattgac aggagatttt ttactagggg tatcatcaga 3960

acaagcagaa tcagagagga tgtgactatc tctgaatacc ttatcagcga agctaatcgt 4020

cttatgagtg acattttgga cgctcttgag attattgata cctccaacac tgatttgaac 4080

catatattca tcaatttctc tgctgttttc aatgtcacgc cagatgacgt tgaagcagcg 4140

ttcggtggtt tcttagaaag gtttggacgt aggctgtgga gactacgtgt ttctgctgct 4200

gaaatccgta ttatgtgcac ggaccctgag actggtatcc cattcccact tcgtgcttta 4260

attaacaacg tttcaggata cgttgtgaaa tctgaaatgt atcaagaggt gaaaaatgat 4320

catggggaat gggttttcaa aagtcttggt cctacaccag gttcaatgca ccttagacca 4380

atttcaacac catacccaac caaagaatgg cttcaaccaa aacgttacaa agctcatctt 4440

atgggtacta cttacgtgta tgatttccct gaattattcc gtcaagctac gctctcccaa 4500

tggaaaaaat actctcctac tgcgagagtt ccttctgatg tgtttgtggc caatgaattg 4560

atcgtcgatg attcaggtga actaactgaa gtaagcagag aacccggcgc caacgttgtg 4620

ggtatggtgg ccttcaaggt aaccgcaaaa actcctgagt atccacgcgg tcgccatttc 4680

atcataattg ctaatgatat caccttcaag atcggatcct ttggccctca agaagatgaa 4740

tatttcaaca aggccacaca acttgcaaga aaattgggca ttcctcgaat ttatctgtca 4800

gccaactcgg gtgctagaat tggagttgct gaagaacttc ttccattatt caaagtagcc 4860

tggaaggaag aaggtaaacc aagcaaggga tttgaatact tatacctcac atcggaagat 4920

cttactctat tggaaaagtc cggaaagtct aacagcgtta ccactcaaag aatagttgaa 4980

gaaggcgaag aacgccacgt tataactgcc atcattggag ctagtgatgg actgggtgtt 5040

gaatgtctaa gaggttccgg tttgatcgct ggtgctacat ctcgggcgta caaggacatc 5100

ttcactatca cattggtcac ctgtagatct gttggtattg gtgcttactt ggtcagattg 5160

ggtcaacgag ccattcaaat tgaaggacaa ccaataattt tgactggtgc ccctgctatt 5220

aataagttgt tgggtaggga agtgtactct tccaacctgc aacttggtgg tacccagatt 5280

atgtacaaga acggtgtttc acacttaacc gccaatgatg atctcgcagg tgtcgaaaag 5340

attatggatt ggttagctta tgtgcctgct aagagaaaca tgcctgttcc tattttagaa 5400

tcacttcatg acaaatggga cagagatgtg gactataagc ctacaagaaa tgagccgtac 5460

gacgtcagat ggatgatcag tggacgtgaa actcctgatg gtgagttcga atctggattg 5520

tttgactctg ggtccttcac tgaaactttg agtggatggg ctaaaggtgt agtcgtcgga 5580

agagcccgtt taggtggtat tcctatggga gtcattggtg ttgaaactag agtcacagaa 5640

aacctgattc cagctgatcc cgccaatcca gactcaaccg aaatgatgat tcaagaagct 5700

ggtcaagtct ggtaccctaa cagtgccttc aagactgcac aagctatcaa cgatttcaac 5760

aatggtgaac agctaccctt gatgattttg gccaactgga gaggtttctc tggtggtcaa 5820

agagacatgt acaatgaagt tttgaaatac ggttctttca ttgtggatgc tttagtcgac 5880

ttcaagcagc ctatcttcac ttacattcct cccactgctg agttgagagg tggatcttgg 5940

gttgttgtag accctaccat caatgaagac atgatggaaa tgtatgcaga cgtcgaatca 6000

agagcaggtg ttttggaacc agaaggtatg gtaggtatca aataccgtaa ggacaaactc 6060

cttgctacta tggaacgatt ggatgccaaa tatgctgagc ttaaatccaa ggttagcgat 6120

actagtcttt cagaaaagga tgtttccgag atcaagaaac aaattgagca gagagagaag 6180

caattgttgc caatttatgc acaaatctct attcaatttg ctgatcttca tgacagatct 6240

ggtcgtatgt tggccaaggg tgtcattaaa aaggaactgg aatgggttaa ttctcgtcgt 6300

ttcttcttct ggagagtccg tcgtcgtttg aacgaggaat acctcattaa gcgtattacc 6360

gaattcctat ctgcttctgc taccagattg gacaagatct cgaggatcaa ttcttggttg 6420

ccaacatcga ttgatttgga agatgaccag aaggttgcca tttggttgga agaaaaccgt 6480

aaagctcttg acgccaatat caaggagctc agggctgagc atgttagaag aactctggct 6540

actcttgtca gaactgatat ggatactact tccaagagtt tggctgaatt gatcaacctt 6600

cttcctgaaa ccgaaaagga atcaatttta tctaagatca agtcatga 6648

Claims (14)

1. A method for heterologous production of dihydromonacolin L comprising:

(1) providing engineering yeast bacteria, and using a promoter to drive and express the following exogenous genes:lovB，lovC，lovG，npgA(ii) a And driving the expression of the transcription activator by an ethanol inducible promoterutaSaidutaThe nucleotide sequence of (A) is shown as SEQ ID NO. 7; with P _AOX1R As a drivelovB，lovC，lovG，npgAPromoter of expression, P _AOX1R The nucleotide sequence of (A) is shown as SEQ ID NO. 8; the yeast is pichia pastoris;

(2) and (2) culturing the yeast engineering bacteria in the step (1) by taking ethanol as a carbon source, a precursor and/or an inducer, thereby generating a product, namely the dihydromonacolin L.

2. A method for heterologous production of monacolin J comprising:

(a) providing engineering yeast bacteria, and using a promoter to drive and express the following exogenous genes:lovB，lovC，lovG，npgA(ii) a And driving the expression of the transcription activator by an ethanol inducible promoterutaSaidutaThe nucleotide sequence of (A) is shown as SEQ ID NO. 7; the yeast is pichia pastoris; with P _AOX1R As a drivelovB，lovC，lovG，npgAPromoter of expression, P _AOX1R The nucleotide sequence of (A) is shown as SEQ ID NO. 8;

(b) providing engineering yeast bacteria, and using a promoter to drive and express the following exogenous genes:lovA，cpr(ii) a And driving the expression of the transcription activator by an ethanol inducible promoteruta；

(c) And (b) culturing the mixed bacteria of the engineering yeast bacteria in the step (a) and the step (b) by using ethanol as a carbon source, a precursor and/or an inducer, thereby generating the product monacolin J.

3. The method of claim 1 or 2, wherein the engineered yeast strain of (1) or (a) further expresses exogenous alcohol dehydrogenase ADH, acetyl-coa synthetase ACS and acetyl-coa decarboxylase ACC.

4. The method of claim 1 or 2, wherein in (1) or (a) or (b), P is _ICL1 Is an ethanol inducible promoter.

5. The method according to claim 2, wherein the ratio of the bacteria (a) to the bacteria (b) in the mixture of the engineered yeast bacteria (a) and (b) in (c) is 1: 0.1-1.

6. The method according to claim 5, wherein the ratio of the bacteria (a) to the bacteria (b) is 1:0.2 to 0.5.

7. The method of claim 1 or 2, wherein in step (2) or (c), the culture is performed in a yeast basic nitrogen source medium to which ethanol is added, and the feeding is performed using glucose as a carbon source in the early stage of fermentation; and after the wet weight of the thalli reaches 200-400 g/L, switching a carbon source to ethanol for feeding.

8. The yeast engineering bacteria for producing the dihydromonacolin L is characterized by comprising the following exogenous genes driven by a promoter to express:lovB，lovC，lovG，npgAand a transcription activator expressed by an alcohol-inducible promoterutaSaidutaThe nucleotide sequence of (A) is shown as SEQ ID NO. 7; with P _AOX1R As a drivelovB，lovC，lovG，npgAPromoter of expression, P _AOX1R The nucleotide sequence of (A) is shown as SEQ ID NO. 8; the yeast is pichia pastoris.

9. The engineered yeast strain for producing dihydromonacolin L according to claim 8, further comprising the genes encoding exogenous alcohol dehydrogenase ADH, acetyl-CoA synthetase ACS and acetyl-CoA decarboxylase ACC.

10. The yeast engineering bacteria for producing monacolin J is characterized by comprising the following exogenous genes expressed by a promoter:lovA，cpr(ii) a And a transcriptional activator expressed by an ethanol-inducible promoterutaSaidutaThe nucleotide sequence of (A) is shown as SEQ ID NO. 7; with P _AOX1R As a drivelovA，cprPromoter of expression, P _AOX1R The nucleotide sequence of (A) is shown as SEQ ID NO. 8; the yeast is pichia pastoris.

11. Use of the engineered yeast strain of any one of claims 8 to 10 for the production of dihydromonacolin L or monacolin J using ethanol as a carbon source, precursor and/or inducer.

12. A kit for producing monacolin J or an intermediate thereof, wherein the kit comprises the engineered yeast strain of any one of claims 8 to 10.

13. A kit for producing monacolin J or an intermediate thereof, comprising a construct comprising a promoter and the following genes:lovB、lovC、lovG、npgAtranscriptional activator expressed by ethanol-inducible promoteruta(ii) a And/or another construct comprising a promoter and a gene of the group consisting of:lovA、cpra transcriptional activator uta expressed from an ethanol inducible promoter; the nucleotide sequence of uta is shown as SEQ ID NO. 7; with P_AOX1RAs a drivelovB、lovC、lovG、npgA、lovA、cprPromoter of expression, P_AOX1RThe nucleotide sequence of (A) is shown as SEQ ID NO. 8.

14. The kit of claim 12 or 13, further comprising a culture medium comprising ethanol as a carbon source, precursor and/or inducer.

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