CN108913732A - A kind of method and application of citrinin J heterologous production - Google Patents

Abstract

The present invention relates to the transformations and application of the Pichia yeast engineering of heterologous production citrinin J a kind of.In the present invention, transformation is optimized to Yeast engineering bacterium strain by gene recombination technology, obtains the Yeast engineering bacteria that can produce citrinin J or in which product DihydromonacolinL L.The purpose product yield of the Yeast engineering bacteria dramatically increases, and provides new approach for industrial production citrinin J and its downstream product.

Description

A kind of method and application of citrinin J heterologous production

Technical field

The invention belongs to bioengineering fields；More specifically, the present invention relates to a kind of finishing for heterologous production citrinin J is red The transformation and application of Yeast engineering bacteria.

Background technique

Simvastatin is artificial semisynthetic drug, due to it has obvious inhibiting effect to cholesterol in human body biosynthesis by It is widely used as blood lipid-lowering medicine.Currently, industrial production Simvastatin is mostly derived from bioanalysis or the change of precursor citrinin J Method conversion, and the industry of citrinin J obtains the hydrolysis for being derived from Aspergillus terreus tunning Lovastatin.It is raw in the Simvastatin During production, citrinin J can be converted into Simvastatin through one step of enzymatic, and hydrolyze Lovastatin and obtain citrinin J Process be full chemistry method hydrolysis, step is more complex, by-product is more and needs to introduce catalyst.Using microorganism chassis cell into The heterologous synthesis citrinin J of row one-step method, can substitute chemical hydrolysis, avoid problem above, for citrinin J and pungent cut down him The industrial production in spit of fland provides new method.

Pichia pastoris (Pichia pastoris) is excellent microorganism chassis cell, because it can express extensive weight Histone and receive significant attention, the microorganism as a generally recognized as safe is already used to successful expression more than 1000 Recombinant protein.Pichia pastoris can have the complicated albumen of progress to turn over by fast-growth in the culture medium that ingredient determines to high density Rear rhetorical function is translated to instruct albumen correctly to fold.In addition to this, Pichia pastoris is natural methylotrophic microorganisms, its energy It is enough strong to grow into high density, methanol metabolic capability under conditions of sole carbon source in methanol.Methanol is the by-product of coal chemical industry Object can be synthesized by natural gas, can also carbon dioxide through the air restored and obtained, and methanol is compared to common hair Ferment type carbohydrate, reducing power is stronger, can provide more driving forces for the synthesis of heterologous product.It is this big scale of construction of methanol, low Price, reproducible feature promote it gradually to develop into the very good material of biochemical derivatization product.While Pichia pastoris is There is mature matched heterologous expression vector, the research that the conversion, screening and scale for Pichia pastoris are amplified has been carried out more Year, Pichia pastoris can be used as a good chassis cell and be used to be synthetically produced high added value biological products.

Summary of the invention

It is co-cultured the purpose of the present invention is to provide a kind of by being mixed by recombination yeast engineering bacteria for substrate of ethyl alcohol, with The method of heterologous synthesis citrinin J.

In the first aspect of the present invention, the heterologous production method of DihydromonacolinL L a kind of is provided, including：(1) ferment is provided Female engineering bacteria makes it express the expression cassette of the following group gene of external source：LovB, lovC, lovG, npgA, and opened with ethanol inducible Mover driving expression activating transcription factor uta；(2) using ethyl alcohol as carbon source, precursor and/or inducer, the yeast work of (1) is cultivated Journey bacterium, to generate product DihydromonacolinL L.

In another aspect of this invention, the heterologous production method of citrinin J a kind of is provided, including：(a) yeast work is provided Journey bacterium makes it express the expression cassette of the following group gene of external source：LovB, lovC, lovG, npgA, and with ethanol-inducible promoter Driving expression activating transcription factor uta；(b) Yeast engineering bacteria is provided, it is made to express the expression cassette of the following group gene of external source： LovA, cpr, and expression activating transcription factor uta is driven with ethanol-inducible promoter；(c) using ethyl alcohol as carbon source, precursor And/or inducer, the Mixed Microbes of culture (a) and the Yeast engineering bacteria of (b), to generate product citrinin J.

In a preferred embodiment, in (1) or (a), the Yeast engineering bacteria also express external source alcohol dehydrogenase ADH, Acetyl-CoA-synthetase ACS and acetyl coenzyme A decarboxylase ACC.

In another preferred example, in (1) or (a), with P_AOX1RAs driving lovB, lovC, lovG, the starting of npgA expression Son；Or

(b) in, with P_AOX1RPromoter as driving lovA, cpr expression；Or

(1) or (a) or (b) in, with P_ICL1For ethanol-inducible promoter.

In another preferred example, in (c), in the Mixed Microbes of the Yeast engineering bacteria of (a) and (b), (a) bacterium:(b) ratio of bacterium It is 1:0.1~1；Preferably 1:0.2~0.5 (ratio of number of two primary yeast cells in flora)；Or, step (2) or (c) In, to add ethyl alcohol (addition 0.2~1%；Preferably 0.3~0.8%；More preferably 0.4~0.6%) yeast basic nitrogen source training Feeding base is cultivated, and uses glucose to carry out feed supplement as carbon source in earlier fermentation；Reach 200~400g/L in thallus weight in wet base (preferably 250~350g/L；More preferably 280~320g/L) after, switch carbon source later to ethyl alcohol feed supplement.

In another preferred example, the gene lovB, lovC, lovG, npgA, lovA, cpr is from Aspergillus terreus.

In another preferred example, the lovB gene has SEQ ID NO:Nucleotide sequence shown in 1 or its degeneracy Sequence, or with SEQ ID NO:1 sequence has 70% or more (preferably 80% or more；More preferably 90% or more；More preferably 93% with On；More preferably 95% or more；More preferably 97% or more, such as 98%~99%) the phase same sex coding congenerous albumen nucleotide Sequence.

In another preferred example, the lovC gene has SEQ ID NO:Nucleotide sequence shown in 2 or its degeneracy Sequence, or with SEQ ID NO:2 sequences have 70% or more (preferably 80% or more；More preferably 90% or more；More preferably 93% with On；More preferably 95% or more；More preferably 97% or more, such as 98%~99%) the phase same sex coding congenerous albumen nucleotide Sequence.

In another preferred example, the lovG gene has SEQ ID NO:Nucleotide sequence shown in 3 or its degeneracy Sequence, or with SEQ ID NO:3 sequences have 70% or more (preferably 80% or more；More preferably 90% or more；More preferably 93% with On；More preferably 95% or more；More preferably 97% or more, such as 98%~99%) the phase same sex coding congenerous albumen nucleotide Sequence.

In another preferred example, the npgA gene has SEQ ID NO:Nucleotide sequence shown in 4 or its degeneracy Sequence, or with SEQ ID NO:4 sequences have 70% or more (preferably 80% or more；More preferably 90% or more；More preferably 93% with On；More preferably 95% or more；More preferably 97% or more, such as 98%~99%) the phase same sex coding congenerous albumen nucleotide Sequence.

In another preferred example, the lovA gene has SEQ ID NO:Nucleotide sequence shown in 5 or its degeneracy Sequence, or with SEQ ID NO:5 sequences have 70% or more (preferably 80% or more；More preferably 90% or more；More preferably 93% with On；More preferably 95% or more；More preferably 97% or more, such as 98%~99%) the phase same sex coding congenerous albumen nucleotide Sequence.

In another preferred example, the cpr gene has SEQ ID NO:Nucleotide sequence shown in 6 or its degeneracy Sequence, or with SEQ ID NO:6 sequences have 70% or more (preferably 80% or more；More preferably 90% or more；More preferably 93% with On；More preferably 95% or more；More preferably 97% or more, such as 98%~99%) the phase same sex coding congenerous albumen nucleotide Sequence.

In another preferred example, the uta gene has SEQ ID NO:Nucleotide sequence shown in 7 or its degeneracy Sequence, or with SEQ ID NO:6 sequences have 70% or more (preferably 80% or more；More preferably 90% or more；More preferably 93% with On；More preferably 95% or more；More preferably 97% or more, such as 98%~99%) the phase same sex coding congenerous albumen nucleotide Sequence.

In another preferred example, the encoding gene of the alcohol dehydrogenase ADH has SEQ ID NO:Core shown in 20 Nucleotide sequence or its degenerate sequence, or with SEQ ID NO:20 sequences have 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, such as 98%~99%) volume of the phase same sex The nucleotide sequence of code congenerous albumen.

In another preferred example, the encoding gene of the acetyl-CoA-synthetase ACS has SEQ ID NO:Shown in 21 Nucleotide sequence or its degenerate sequence, or with SEQ ID NO:21 sequences have 70% or more (preferably 80% or more；More preferably 90% or more ground；More preferably 93% or more；More preferably 95% or more；More preferably 97% or more, such as 98%~99%) the phase same sex Encode the nucleotide sequence of congenerous albumen.

In another preferred example, the encoding gene of the acetyl coenzyme A decarboxylase ACC has SEQ ID NO:Shown in 22 Nucleotide sequence or its degenerate sequence, or with SEQ ID NO:22 sequences have 70% or more (preferably 80% or more；More preferably 90% or more ground；More preferably 93% or more；More preferably 95% or more；More preferably 97% or more, such as 98%~99%) the phase same sex Encode the nucleotide sequence of congenerous albumen.

In another preferred example, promoter P_AOX1RGene order have SEQ ID NO:Nucleotide sequence shown in 8.

In another preferred example, promoter P_ICL1Gene order have SEQ ID NO:Nucleotide sequence shown in 9.

In another aspect of this invention, it provides a kind of for producing the Yeast engineering bacteria of DihydromonacolinL L, the yeast The expression cassette of the following group gene in engineering bacteria comprising external source：LovB, lovC, lovG, npgA and activating transcription factor uta；Compared with It goodly, also include the coding of the alcohol dehydrogenase ADH of external source, acetyl-CoA-synthetase ACS and acetyl coenzyme A decarboxylase ACC Gene.

In another aspect of this invention, it provides a kind of for producing the Yeast engineering bacteria of citrinin J, the yeast engineering The expression cassette of the following group gene in bacterium comprising external source：LovA, cpr and activating transcription factor uta.

In a preferred embodiment, the Yeast engineering bacteria is methanotrophic yeast；Preferably, the methanol nutrition Type yeast includes Pichia pastoris.

In another preferred example, the Pichia pastoris is Pichia pastoris GS115.

In another preferred example, the Yeast engineering bacteria is used for using ethyl alcohol as carbon source, precursor and/or inducer, raw Produce DihydromonacolinL L or citrinin J.

In another aspect of this invention, provide it is a kind of for producing the kit of citrinin J or in which mesosome, it is described It include the Yeast engineering bacteria in kit.

In another aspect of this invention, it provides a kind of for producing the kit of citrinin J or in which mesosome, the examination It include a construction in agent box comprising the expression cassette of the following group gene：LovB, lovC, lovG, npgA, activating transcription factor Uta, and/or, another construction comprising the expression cassette of the following group gene：LovA, cpr, activating transcription factor uta.

It in another preferred example, further include the training for containing ethyl alcohol as carbon source, precursor and/or inducer in the kit Support base.

Other aspects of the invention are apparent to those skilled in the art due to this disclosure 's.

Detailed description of the invention

Fig. 1, manual transcription system P_ICL1-UTA_P_AOX1RAnd to the GFP expression intensity impinged upon under the conditions of ethyl alcohol or methanol.

The schematic diagram of Fig. 2, pichia pastoris engineered strain fermenting and producing DihydromonacolinL L；Wherein, DML, dihydro is not received can Woods L；WT, Pichia pastoris wild-type strain.

The schematic diagram of Fig. 3, double bacterial strains mixed culture production citrinin J；Wherein, MJ, citrinin J；WT, Pichia pastoris Wild type.

The fermentation yield figure of Fig. 4, double bacterial strains difference initial inoculation ratio mixed culture production citrinin J；Wherein, MJ, Citrinin J.

The 5L reactor leaven line chart of the optimal initial inoculation ratio mixed culture production citrinin J of Fig. 5, double bacterial strains； Wherein, MJ, citrinin J.

Specific embodiment

The present inventor passes through in-depth study, optimizes transformation to Yeast engineering bacterium strain by gene recombination technology, obtains Obtain the Yeast engineering bacteria that can produce citrinin J or in which product DihydromonacolinL L.The purpose of the Yeast engineering bacteria produces Produce amount dramatically increases, and provides new approach for industrial production citrinin J and its downstream product.

Term

As used herein, " expression cassette " or " expression casette " refers to include institute needed for expression desired polypeptides It is necessary to the gene expression systems of element, and usually it includes following elements：Promoter, the gene order for encoding polypeptide, terminator； Additionally alternative is including signal coding sequence etc.；These elements are operatively connected.

As used herein, " expression construct " refers to recombinant DNA molecules, it includes expected nucleic acid encode sequence Column, may include one or more expression casettes." construction " is commonly included in expression vector.

As used herein, " external source " or " heterologous " refers to two or more pieces nucleic acid or albumen from separate sources Relationship between matter sequence, or the albumen (or nucleic acid) from separate sources and the relationship between host cell.For example, if The combination of nucleic acid and host cell is not usually naturally occurring, then nucleic acid is external source for the host cell.It is specific It is " external source " for cell that sequence is inserted into it or organism.

Gene and its expression system

In the present invention, citrinin J or in which mesosome are realized by being transformed into polygenic combination in Yeast engineering bacteria Efficient production of the DihydromonacolinL L in Yeast engineering bacteria.Include for gene used in production DihydromonacolinL L： LovB, lovC, lovG, npgA；Include for gene used in production citrinin J：LovB, lovC, lovG, npgA, lovA, cpr.In a preferred embodiment of the invention, from the lovB of Aspergillus terreus, lovC, lovG, npgA, lovA, cpr gene difference With SEQ ID NO:1,SEQ ID NO:2,SEQ ID NO:3,SEQ ID NO:4,SEQ ID NO:5,SEQ ID NO:6 institutes The nucleotide sequence shown.

In order to optimize the yield of citrinin J or in which mesosome DihydromonacolinL L, present inventor has performed be directed to engineering The further optimization of bacterium and fermentation medium is realized using ethyl alcohol as substrate high yield DihydromonacolinL L or citrinin J. In a preferred embodiment of the present invention, using ethanol-inducible promoter (preferably P_ICL1) drive activating transcription factor UTA Expression, while driving the expression of citrinin J synthesis related gene.Rna polymerase activity, while energy are recruited since UTA has Enough and citrinin J synthesis related gene promoter (such as P_AOX1R) combine, therefore ethanol-inducible promoter is (preferably P_ICL1) alcohol induced signal will be amplified by the artificial rerecording device, and then reach enhancing citrinin J synthesis related gene The purpose of expression.In a preferred embodiment of the invention, promoter P_ICL1Nucleotide sequence see SEQ ID NO:9, transcription The nucleotide sequence of activity factor uta is shown in SEQ ID NO:7, promoter P_AOX1RNucleotide sequence see SEQ ID NO:8.

In a preferred embodiment of the present invention, the generation of acetyl coenzyme A and malonyl coenzyme A is converted into for further promotion ethyl alcohol Thanking to flux, the present inventor has been overexpressed alcohol dehydrogenase ADH, acetyl-CoA-synthetase ACS and acetyl coenzyme A decarboxylase ACC, Further promote the yield of DihydromonacolinL L.

Above-mentioned gene can be naturally occurring, for example it can be by isolated or purified from animals and plants or microorganism.In addition, What the gene was also possible to manually to prepare, for example the base can be obtained according to conventional genetic engineering recombinant technique Cause, or the gene is obtained by artificial synthesized method.In a preferred embodiment of the invention, adh has SEQ ID NO:Nucleotide sequence shown in 20, acs have SEQ ID NO:Nucleotide sequence shown in 21, acc have SEQ ID NO:Nucleotide sequence shown in 22.

The nucleotide sequence of the above-mentioned gene referred to can be with SEQ ID NO:Column shown in 1~9,20~22 are identical, It can be the variant of their degeneracy.As used herein, " variant of degeneracy " refers to that coding has same function in the present invention Can protein, but be selected from SEQ ID NO:The differentiated nucleic acid sequence of sequence shown in 1~9,20~22.

The gene may include：The coded sequence of encoding mature polypeptide；The coded sequence of mature polypeptide and various Additional coding sequence；The coded sequence (and optional additional coding sequence) and non-coding sequence of mature polypeptide.

The invention further relates to the variants of the gene, and its corresponding wild polypeptide of polypeptide of coding is in amino It is different on acid sequence, it is segment, analog or the derivative of wild polypeptide.The variant of this polynucleotides can be natural hair The variant that raw allelic variant or non-natural occur.These nucleotide variants include substitution variants, Deletion variants With insertion variant.As known in the art, allelic variant is the alternative forms of a polynucleotides, it may be one or Substitution, missing or the insertion of multiple nucleotide, but not from substantially change its encode polypeptide function.

The invention further relates to hybridize with above-mentioned sequence and have at least 70%, preferably at least 80% between two sequences The polynucleotides of the phase same sex, more preferably at least polynucleotides of the 80% phase same sex.The present invention is more particularly directed under strict conditions with The interfertile polynucleotides of polynucleotides of the present invention.In the present invention, " stringent condition " refers to：(1) strong in lower ion Hybridization and elution under degree and higher temperature, such as 0.2 × SSC, 0.1%SDS, 60 DEG C；Or added with denaturant when (2) hybridization, such as 50% (v/v) formamide, 0.1% calf serum/0.1%Ficoll, 42 DEG C etc.；Or (3) are only identical between two sequences Property at least just hybridizes at 90% or more, more preferably 95% or more.Also, the polypeptide of interfertile polynucleotide encoding with Corresponding wild polypeptide has identical biological function and activity.

Although being obtained from other species them it should be understood that multiple genes of the invention are preferably obtained from specific species Homologous gene (as have 80% or more, such as 90%, 95%, even 98%, 99% sequence identity) also the present invention consider Within the scope of.The Method and kit for of the aligned sequences phase same sex is also well known in the art, such as BLAST.

The full length sequence of each gene of the invention or its segment can usually use PCR amplification method, recombination method or artificial synthesized Method obtain.It, can disclosed related nucleotide sequence, especially open reading frame according to the present invention for PCR amplification method Sequence carrys out design primer, expands and obtains related sequence.When sequence is longer, twice or repeatedly PCR amplification can be carried out, then again The segment that each time amplifies is stitched together by proper order.

The present invention also relates to the carriers comprising the polynucleotides, and genetically engineered with the carrier Host cell.

In the present invention, the sequence of each gene be can be plugged into recombinant expression carrier.Term " recombinant expression carrier " refers to ability Bacterial plasmid, bacteriophage known to domain, yeast plasmid, plant cell virus, mammalian cell virus or other carriers.Always It, as long as can replicate and stablize in host, any plasmid and carrier can be used.One important feature of expression vector is Usually contain replication orgin, promoter, marker gene and translation control element.

The sequence of each gene can be inserted respectively into recombinant expression carrier, and multiple recombinant expression carrier corotation hosts are thin Born of the same parents；The expression cassette of multiple genes can also be inserted into series in same recombinant expression carrier, be transferred to host cell.Institute The recombinant expression carrier stated also may include the expression regulation sequence being connected with the series of operations of the gene, in order to albumen Expression.It should be understood that constructing recombinant expression carrier with can be convenient after having understood technology contents of the invention in those skilled in the art. The recombinant expression carrier of acquisition is also included in the present invention.

In expression regulation sequence or expression cassette, according to different needs, the promoter of induction type or composing type can be applied, The promoter of induction type can realize more controllable protein expression and production of chemicals, be conducive to industrial applications.

Carrier comprising above-mentioned appropriate gene order and appropriate promoter or control sequence, can be used for converting suitable When host cell, allow it to expression protein.In the present invention, the host cell is preferably Yeast engineering bacteria, More preferably methanotrophic yeast cells, such as Pichia pastoris.

Synthetic method

The invention proposes a kind of using ethyl alcohol as carbon source, the citrinin J heterologous production method of precursor, inducer, passes through The growth and determining alcohol metabolism access flow direction of Pichia pastoris in ethanol are detected, demonstrates Pichia pastoris using ethyl alcohol as substrate It is capable of supply that enough precursor acetyl coenzyme As.Through the transcriptional control heredity route in artificial reconstructed methanotrophic yeast cells, mention The Intensity of Transcription of Endothelial of high ethano evoked promoter enhances the expression of heterologous pathway enzyme.Yeast expression system and fermentation of the invention Method, so that heterologous synthetic quantity of the citrinin J in Pichia pastoris reaches 2.2g/L.

Further to promote output of the citrinin J in Pichia pastoris, the present invention by substrate of ethyl alcohol by being promoted Cytosolic acetyl coacetylase metabolic flux in Pichia pastoris, while alcohol induced rerecording device is constructed by engineer, it is promoted The transcriptional expression of heterologous enzyme gene.Under the premise of guaranteeing precursor abundance and enzyme high efficient expression, product citrinin J is promoted to close At.

DihydromonacolinL L synthesizes citrinin J through the catalysis oxidation of enzyme LovA, in order to avoid introducing in unicellular More foreign genes lead to Metabolic stress, and present inventors have proposed the strategies that building double bacterial strains co-culture.The present inventors have additionally discovered that In co-culture system, the original mixture proportion of two bacterial strains can influence the growth of bacterial strain and the synthesis of product to a certain extent, because Initial inoculation ratio is optimized in this present inventor, obtains preferably initial inoculation ratio.

Acetyl coenzyme A is the core mesostate of cell base metabolism, it is led to by a molecular acid and coacetylase It crosses thioester bond to be formed by connecting, carboxyl groups can be provided for intracellular various biochemical reactions, be the substrate that albumen is acylated, participate in Enzyme transcription and function controlling.Meanwhile a series of precursor molecule of acetyl coenzyme A or bioactive substances, including fatty acid, terpene Class compound, polyketides etc..With going deep into the research of microbiological plants concept synthesising target compound, people couple Precursor supply problem in microbial hosts is also paid attention to further, by carrying out rationality transformation for acetyl coenzyme A metabolism network, The matter energy stream for promoting carbon metabolism to generate more flows to acetyl coenzyme A, and is promoted eventually as the substrate of target compound Product formation.Ethyl alcohol is the precursor that can be used for improving acetyl coenzyme A supply, is able to ascend acetyl coenzyme A by adding ethyl alcohol and spreads out The synthesis of biology.

In single host cell carry out heterologous pathway building and metabolic engineering often to the growth of host cell, Metabolism brings burden, causes the decline of final product synthetic quantity.In the present invention, flora body is co-cultured by design and rational many cells Complicated biosynthesis pathway is assigned to independent microbial cell by system, each cell inoculation ratio of adjusting can be total in flora Optimize heterologous pathway in cultivating system, alleviate Metabolic stress, promotes Product formation.Synthetic biology element and traditional molecule are grasped Make the combined use of technology, can engineer construct promoter transcription regulated and control network, enhance the Intensity of Transcription of Endothelial of promoter, then Improve the expression of heterologous protein.

In a specific embodiment of the present invention, it constructs first using ethyl alcohol as the recombination of substrate synthesizing dihydro citrinin L Pichi strain.Use the promoter P of engineer_AOX1RDrive gene lovB, lovC, lovG, npgA expression, using opening Mover P_ICL1Activating transcription factor UTA expression is driven, is able to produce DihydromonacolinL L through cultivation and fermentation using ethyl alcohol as substrate. Further to promote the metabolic flux that ethyl alcohol is converted into acetyl coenzyme A and malonyl coenzyme A, the present invention has been overexpressed alcohol dehydrogenase Enzyme ADH, acetyl-CoA-synthetase ACS and acetyl coenzyme A decarboxylase ACC further promote the synthesis of DihydromonacolinL L, obtain To the superior strain P.p/DML of DihydromonacolinL L.Recombinant bacterium P.p/DML is fermented can to produce DihydromonacolinL L, and two Hydrogen citrinin L enters recombinant bacterium P.p/sAR and synthesizes citrinin J through catalysis oxidation.It include composition wherein in recombinant bacterial strain Type promoter P_GAPDrive the expression cassette and artificial synthesized promoter P of activating transcription factor UTA expression_AOX1RDrive lovA and The expression cassette of cpr expression.

In a specific embodiment of the present invention, the more excellent fermentation strategies cultivated through shaking flask level are amplified to 5L reaction Technique is carried out in device and amplifies lab scale, and the large-scale production ability of citrinin J is evaluated.Culture co-cultures bacterial strain P.p/ respectively DML and P.p/sAR carries out combined inoculation according to the optimal initial inoculation ratio of shaking flask to logarithmic growth phase.In earlier fermentation, make It is that carbon source maintains bacterial strain fast-growth, while inhibiting promoter P with glucose_ICL1Heterologous pathway expression is checked in starting.In thallus When weight in wet base grows to 300g/L, switching carbon source arrives ethyl alcohol, opens heterologous pathway expression, interval 8h sampling progress thallus weight in wet base and not It receives Kelin J determination of yield, finally obtains citrinin J yield 2.2g/L in 96h.

Present invention will be further explained below with reference to specific examples.It should be understood that these embodiments are merely to illustrate the present invention Rather than it limits the scope of the invention.In the following examples, the experimental methods for specific conditions are not specified, usually according to conventional strip Part such as J. Pehanorm Brooker etc. is write, Molecular Cloning:A Laboratory guide, the third edition, Science Press, condition described in 2002, or According to the normal condition proposed by manufacturer.

Material

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

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

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

Pichia pastoris produces the alcohol fermentation of bacterial strain, is using YNE culture medium：The training of 1.34% (w/v) yeast basic nitrogen source Base (YNB), 0.5% (v/v) ethyl alcohol are supported, per ethyl alcohol of addition for 24 hours.

In 121 DEG C, high pressure sterilization 20min, the 50% independent wiring solution-forming of (w/v) glucose makes for solid, fluid nutrient medium With 0.22 μm of aperture sterilised membrane filter filtration sterilization, according to the final grape that corresponding volume is added in super-clean bench using concentration when use Sugared mother liquor.

Antibiotic usage amount：Ampicillin, 50 μ g/ml；Bleomycin, Escherichia coli culture use 50 μ g/ml；Bi Chi Yeast Cultivation uses 100 μ g/ml；Geneticin, 250 μ g/ml.

Peptone, yeast powder and malt extract are purchased from Beijing Baeyer enlightening Bioisystech Co., Ltd.

Basic nitrogen source medium YNB is purchased from Sigma company.

Glucose, glycerol, methanol, ethyl alcohol, ethyl acetate, ammonium hydroxide, potassium dihydrogen phosphate, potassium phosphate,monobasic, phosphoric acid and chlorination Sodium etc. is purchased from Sinopharm Chemical Reagent Co., Ltd..

DNA purification and recovery kit, the small extraction reagent kit of plasmid and Yeast genome extracts kit are purchased from Tiangeng biochemistry section Skill (Beijing) Co., Ltd.

PCR primer, total gene synthesis and plasmid order-checking etc. are completed by Suzhou Jin Weizhi Biotechnology Co., Ltd, PCR high Fidelity enzyme and restriction enzyme are purchased from TaKaRa.

Single slice is seamless Cloning Kit and multiple clips seamless integration kit are purchased from Nanjing Nuo Weizan company.

The building of embodiment 1, citrinin J biosynthesis gene heterogenous expression plasmid

1.pZ_UTA_P_AOX1RThe building of-BCGN plasmid

It is starting with Pichia pastoris commercialization plasmid pPIC ZB (Invitrogen), according to promoter P_AOX1RSequence it is complete Synthetic promoter segment is removed original AOX1 through agarose electrophoresis and is started using I digested plasmid pPIC ZB of Bgl II and Xho Sub-piece, by promoter P_AOX1RPlasmid fragments after segment and digestion carry out seamless clone, through monoclonal screening and sequence verification, Plasmid pZ_P can be obtained_AOX1R。

According to the fully synthetic uta segment of UTA nucleotide sequence, while design primer is expanded by template of Pichia pastoris genome Available promoter P_ICL1Segment, by OverlapPCR technology by uta segment and P_ICL1Segment carries out Ligation in vitro, will even P after connecing_ICL1PPIC ZB segment after-UTA segment and digestion carries out seamless clone, available plasmid pZ_P_ICL1-UTA。

PCR amplification lovB (SEQ ID NO:1),lovC(SEQ ID NO:2),lovG(SEQ ID NO:And npgA 3) (SEQ ID NO:4) genetic fragment, by each genetic fragment respectively with linearization plasmid pZ_P_AOX1RSeamless clone is carried out, respectively Obtain plasmid pZ_P_AOX1R-LovB、pZ_P_AOX1R-LovC、pZ_P_AOX1R- LovG and pZ_P_AOX1R-NpgA。

Use restriction endonuclease BamHI single endonuclease digestion plasmid pZ_P_AOX1R- LovB, design primer is to TT~lacOcAOX-F (SEQ ID NO:And BamH~TT-R (SEQ ID NO 10):11), with plasmid pZ_P_AOX1R- LovC is template, can obtain expression cassette through PCR amplification Segment, by the segment and linearization plasmid pZ_P_AOX1R- LovB carries out seamless clone, can obtain plasmid through bacterium colony PCR verifying pZ_P_AOX1R-BC.Same method is continued to use, the expression cassette of lovG and npgA can be continued on the basis of this plasmid Segment obtains plasmid pZ_P_AOX1R-BCGN。

In plasmid pZ_P_ICL1On the basis of-UTA plasmid, primer pair TT~pICL1-F (SEQ ID NO is used:12) and BamH~TT-R (SEQ ID NO:11), through the available P of PCR amplification_ICL1The expression cassette segment of driving expression UTA.It will be linear The plasmid pZ_P of change_AOX1R- BCGN and expression cassette segment carry out seamless Cloning Transformation Escherichia coli, and expression plasmid pZ_ can be obtained UTA_P_AOX1R-BCGN。

2.pK_UTA_P_AOX1RThe building of-sAR plasmid

Use XhoI and SalI double digestion plasmid pZ_P_AOX1R；It expands to obtain lovA (SEQ ID NO with primer PCR:5) and Genetic fragment (the SEQ ID NO of cpr:6), by each genetic fragment respectively with linearization plasmid pZ_P_AOX1RSeamless clone is carried out, Obtain plasmid pZ_P_AOX1R-LovA、pZ_P_AOX1R-CPR。

Design primer is to TT~KanaHis-F (SEQ ID NO:And Amp~KanaHis-R (SEQ ID NO 13):14), KanaHis~Amp-F (SEQ ID NO:And lacOcAOX~Amp-R (SEQ ID NO 15):16), it is with plasmid pPIC3.5K Template carries out PCR amplification.Design primer is to Amp~lacOcAOX-F (SEQ ID NO simultaneously:And KanaHis~TTSpe-R 17) (SEQ ID NO:18), with plasmid pZ_P_AOX1R- LovA is that template carries out PCR amplification, obtains expression cassette segment and linearization plasmid The seamless clone of multiple clips is carried out, plasmid pK_P is obtained_AOX1R-LovA。

Use SpeI linearization plasmid pK_P_AOX1R- LovA uses primer pair TT~lacOcAOX-F (SEQ ID NO:10) With KanaHis~TTSpe-R (SEQ ID NO:18) with plasmid pZ_P_AOX1R- CPR is template, obtains segment and line through PCR amplification Property plasmid carry out seamless clone, available plasmid pK_P_AOX1R-sAR.Continue to use restriction endonuclease SpeI linearization for enzyme restriction plasmid pK_P_AOX1R- sA uses primer TT~pGAP-F (SEQ ID NO:And BamH~TT-R (SEQ ID NO 19):11) with plasmid pZ-P_GAP- UTA is that template carries out linearization plasmid with expression cassette segment through the available expression cassette segment of PCR amplification respectively Single slice is seamless clone, can obtain plasmid pK_UTA_P_AOX1R-sAR。

Embodiment 2, P_ICL1The promotion that driving expression UTA expresses ethyl alcohol

The plasmid pZ_P of aforementioned preparation_AOX1R, with Xho and Sal double digestion linearization plasmid, design primer expands eGFP report Genetic fragment, and obtain P_ICL1Plasmid pZ_P can be obtained through colony screening by seamless integration kit in-UTA_ICL1-UTA_ P_AOX1R-eGFP.Meanwhile with P_ICL1Driving expression eGFP, P_AOX1Driving expression eGFP is control.Linearization plasmid, and electrotransformation Pichia pastoris wild strain screens transformant.Gained monoclonal transformant carries out genotype identification, carries out ethyl alcohol or methanol is carbon source Fermentation.

As shown in Figure 1, using artificial re-recording system P_ICL1-UTA_P_AOX1RGFP expression intensity under the conditions of ethyl alcohol is compared Original P_ICL1Promoter intensity is substantially improved, and is much higher than original P_AOX1GFP expression intensity under the conditions of methanol and ethyl alcohol.Card Manual transcription device in the bright present invention can be obviously improved the expression of heterologous protein.

Embodiment 3, production citrinin J co-culture the acquisition of bacterial strain

1, the building of DihydromonacolinL L bacterial strain is produced

The plasmid pZ_UTA_P of aforementioned preparation_AOX1R- BCGN, using SpeI single endonuclease digestion, electrotransformation finishes red after purified recycling Yeast wild strain carries out transformant screening using the YPD solid plate of blasticidin resistance.Gained monoclonal transformant carries out base Because type is identified, after alcohol induced fermentation, the metabolite extracted in fermentation liquid carries out efficient liquid phase chromatographic analysis, synthesizing dihydro The recombinant bacterium of citrinin L is the Pichi strain for producing DihydromonacolinL L, is named as P.p/DML.

2, metabolic engineering optimization promotes DihydromonacolinL L production

To further enhance the supply of acetyl coenzyme A and the synthesis of DihydromonacolinL L, produced in DihydromonacolinL L On the basis of bacterial strain, it is further overexpressed alcohol dehydrogenase ADH (SEQ ID NO:20), acetyl-CoA-synthetase ACS (SEQ ID NO:And acetyl coenzyme A decarboxylase ACC (SEQ ID NO 21):22).

Using saccharomyces cerevisiae genome as template, design primer can get alcohol dehydrogenase gene adh and second through PCR amplification Acyl-CoA synthetase gene acs.Using Pichia pastoris genome as template, design primer is through the available acetylcoenzyme of PCR amplification A decarboxylase acc.Using restriction endonuclease BspT104I and KpnI double digestion plasmid pGAPZ α, signal peptide piece is removed through DNA gel electrophoresis Section obtains linearization plasmid, and linearization plasmid is carried out seamless clone respectively with three genetic fragments respectively, converts Escherichia coli, warp Bacterium colony PCR verifying and sequencing result compare available P_GAPExpress the plasmid pZ-P of each enzyme gene_GAP-ADH、pZ-P_GAP- ACS and pZ-P_GAP-ACC.Design primer removes wherein HIS4 through PCR amplification to using Pichia pastoris commercial carrier pPIC3.5K as template The plasmid pKdH of removal HIS4 riddled basins can be obtained in gene.Simultaneously respectively with plasmid pZ-P_GAP- ADH and pZ-P_GAP- ACS is template, carries out PCR amplification and obtains expression casette.Expression casette and the plasmid pKdH of linearisation are carried out respectively more Segment is seamless clone, can be obtained plasmid pKdH-P_GAP- ADH and pKdH-P_GAP-ACS.Use SpeI single endonuclease digestion plasmid pKdH-P_GAP- ADH linearizes it, linearization plasmid and expression cassette segment is carried out seamless clone and conversion, through screening available plasmid pKdH-P_GAP-ADH+P_GAP-ACS.The plasmid is linearized using BlnI, the competent cell of electrotransformation Pichia pastoris P.p/DML, Using the YPD Screening of Media transformant for adding Geneticin and genotype verifying is carried out, available overexpression ADH and ACS Finish red Pichi strain.

Using BamHI and BlnI double digestion plasmid pPIC3.5K, design primer and with pZ-P_GAP- ACC is template, through PCR Amplification obtains P_GAP- ACC-TT segment, carries out seamless clone for the two, screens and verify available plasmid pK- through bacterium colony PCR P_GAP-ACC.The plasmid is linearized, electrotransformation has been overexpressed the Pichia pastoris competent cell of ADH and ACS, with MGY plate screening Transformant and genotype verifying, while each expression plasmid list copy integration is screened, available ADH, ACS and ACC are overexpressed DihydromonacolinL L produce bacterial strain bacterial strain P.p/DML_OE, gained Pichia pastoris transformant extracts after alcohol induced fermentation Tunning carries out quantitative analysis through high performance liquid chromatography, as shown in Fig. 2, the DihydromonacolinL L compared to bacterial strain before optimizing is produced Amount promotes 195%, and the Strain Designation after the optimization is P.p/DML_OE (P.p/DML with overexpression).

3, the building of catalysis DihydromonacolinL L synthesis citrinin J bacterial strain

Use BlnI single endonuclease digestion plasmid pK_UTA_P_AOX1R- sAR, electrotransformation Pichia pastoris wild strain, makes after purified recycling Transformant screening is carried out with the YPD solid plate of geneticin resistant.Gained monoclonal transformant carries out genotype identification.

Above-mentioned acquisition pK_UTA_P has been converted into_AOX1RThe bacterial strain of-sAR is co-cultured with bacterial strain P.p/DML_OE and is fermented.It Afterwards, the metabolite extracted in fermentation liquid carries out efficient liquid phase chromatographic analysis, as shown in figure 3, determining that tunning is that Mo Na can Woods J.

The above-mentioned Pichia pastoris recombinant bacterial strain for being converted into citrinin J to be catalyzed DihydromonacolinL L, is named as P.p/ sAR。

Embodiment 4 co-cultures recombinant bacterial strain production citrinin J

1. producing the determination of the co-cultivation optimal initial inoculation ratio of bacterial strain of citrinin J

The present inventor has found after studying repeatedly, different in P.p/DML and P.p/sAR double bacterial strains co-culture system Initial inoculation ratio makes co-culture system be in different stable states.Therefore, inventors believe that, it is necessary to co-culture body The initial inoculation ratio of two bacterial strains optimizes in system.

It is 1 that the present inventor, which sets gradually the initial inoculation ratio of bacterial strain P.p/DML and bacterial strain P.p/sAR,:0.1,1: 0.2,1:0.5,1；1,1:1.5,1:2, observe influence of the different vaccination ratio for output.

The output of the citrinin J of each group is as shown in Figure 4, it is seen then that bacterial strain P.p/DML and bacterial strain P.p/sAR are 1:0.2 When~0.5 initial inoculation ratio, the output of citrinin J is significantly ideal；It and is 1 in initial inoculation ratio:When 0.2, not The output of Kelin J received reaches highest, is 156mg/L.

2. recombination engineering 5L reactor high cell density fermentation

The initial inoculation ratio of bacterial strain P.p/DML and bacterial strain P.p/sAR is 1 by the present inventor:0.2 co-culture system amplification Into 5L reactor, fermentation lab scale is carried out.Bacterial strain P.p/DML and bacterial strain P.p/sAR are cultivated in shaking flask respectively to logarithmic growth Phase, measurement bacterium is dense, carries out 1:It is accessed in 5L reactor after 0.2 mixing, control pH is 4.5, and dissolved oxygen is between 30~70%.? Earlier fermentation uses glucose to add as carbon source progress feed supplement stream, controls glucose feed rate from low to high, gradually from 24g/h It is promoted to 40g/h, while detecting the glucose residual concentration in culture medium, avoids glucose feed supplement is too fast from causing glucose a large amount of Accumulation.After maintaining cell high-speed rapid growth to reach 300g/L to thallus weight in wet base, switch carbon source to ethyl alcohol feed supplement, ethyl alcohol feed rate is same Sample from slow to fast, steps up to 24g/h from 0g/h, during which monitors the ethyl alcohol residual concentration in fermentation liquid, avoid alcohol accumulation It is excessively high, feed rate 24g/h is maintained until during which fermentation maintains pH to exist in 4.5, dissolved oxygen to 110h after thallus adapts to ethyl alcohol Between 30~70%.Measure the yield in fermentation process.As shown in figure 5, co-culture system can produce not when fermenting 96h The amount of Kelin J received reaches 2.2g/L.

All references mentioned in the present invention is incorporated herein by reference, independent just as each document It is incorporated as with reference to such.In addition, it should also be understood that, after reading the above teachings of the present invention, those skilled in the art can To make various changes or modifications to the present invention, such equivalent forms equally fall within model defined by the application the appended claims It encloses.

Sequence table

<110>East China University of Science

<120>A kind of method and application of citrinin J heterologous production

<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>Primer (Primer)

<400> 10

ttaagtgaga ccttcgtttg tgcagatctt gtgtggaatt gtga 44

<210> 11

<211> 40

<212> DNA

<213>Primer (Primer)

<400> 11

aagctatggt gtgtggggga tccgcacaaa cgaaggtctc 40

<210> 12

<211> 46

<212> DNA

<213>Primer (Primer)

<400> 12

agtgagacct tcgtttgtgc agatcttcat ctaacacttt gtatag 46

<210> 13

<211> 40

<212> DNA

<213>Primer (Primer)

<400> 13

cagaagatta agtgagaact agtagttcgt ttgtgcaagc 40

<210> 14

<211> 26

<212> DNA

<213>Primer (Primer)

<400> 14

tcagagaaat ttaccatgaa atctcc 26

<210> 15

<211> 26

<212> DNA

<213>Primer (Primer)

<400> 15

ggagatttca tggtaaattt ctctga 26

<210> 16

<211> 40

<212> DNA

<213>Primer (Primer)

<400> 16

ccacacagag ctccgaataa taactgttat ttttcagtgt 40

<210> 17

<211> 40

<212> DNA

<213>Primer (Primer)

<400> 17

taacagttat tattcggagc tctgtgtgga attgtgagcg 40

<210> 18

<211> 46

<212> DNA

<213>Primer (Primer)

<400> 18

gcttgcacaa acgaactact agttctcact taatcttctg tactct 46

<210> 19

<211> 45

<212> DNA

<213>Primer (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 (10)

1. a kind of heterologous production method of DihydromonacolinL L, including：

(1) Yeast engineering bacteria is provided, it is made to express the expression cassette of the following group gene of external source：LovB, lovC, lovG, npgA, and with Ethanol-inducible promoter driving expression activating transcription factor uta；

(2) using ethyl alcohol as carbon source, precursor and/or inducer, the Yeast engineering bacteria of (1) is cultivated, to generate product dihydro not Receive Kelin L.

2. a kind of heterologous production method of citrinin J, including：

(a) Yeast engineering bacteria is provided, it is made to express the expression cassette of the following group gene of external source：LovB, lovC, lovG, npgA, and with Ethanol-inducible promoter driving expression activating transcription factor uta；

(b) Yeast engineering bacteria is provided, it is made to express the expression cassette of the following group gene of external source：LovA, cpr, and with ethanol inducible Promoter driving expression activating transcription factor uta；

(c) using ethyl alcohol as carbon source, precursor and/or inducer, the Mixed Microbes of the Yeast engineering bacteria of culture (a) and (b), thus raw At product citrinin J.

3. method according to claim 1 or 2, which is characterized in that (1) or in (a), the Yeast engineering bacteria is also expressed outer Alcohol dehydrogenase ADH, acetyl-CoA-synthetase ACS and the acetyl coenzyme A decarboxylase ACC in source.

4. method according to claim 1 or 2, which is characterized in that (1) or in (a), with P_AOX1RAs driving lovB, lovC, The promoter of lovG, npgA expression；Or

(b) in, with P_AOX1RPromoter as driving lovA, cpr expression；Or

(1) or (a) or (b) in, with P_ICL1For ethanol-inducible promoter.

5. method according to claim 2, which is characterized in that in (c), in the Mixed Microbes of the Yeast engineering bacteria of (a) and (b), (a) bacterium:(b) ratio of bacterium is 1:0.1~1；Preferably 1:0.2~0.5；Or

Step (2) or (c) in, cultivated with adding the yeast basic nitrogen source medium of ethyl alcohol, earlier fermentation use grape Sugar carries out feed supplement as carbon source；After thallus weight in wet base reaches 200~400g/L, switch carbon source later to ethyl alcohol feed supplement.

6. a kind of for producing the Yeast engineering bacteria of DihydromonacolinL L, which is characterized in that comprising outer in the Yeast engineering bacteria The expression cassette of the following group gene in source：LovB, lovC, lovG, npgA and activating transcription factor uta；Preferably, it also includes The encoding gene of the alcohol dehydrogenase ADH of external source, acetyl-CoA-synthetase ACS and acetyl coenzyme A decarboxylase ACC.

7. a kind of for producing the Yeast engineering bacteria of citrinin J, which is characterized in that comprising external source in the Yeast engineering bacteria The expression cassette of the following group gene：LovA, cpr and activating transcription factor uta.

8. Yeast engineering bacteria as claimed in claims 6 or 7, which is characterized in that the Yeast engineering bacteria is methanotrophic Yeast；Preferably, the methanotrophic yeast includes Pichia pastoris.

9. the purposes of Yeast engineering bacteria described in claim 6 or 7 is used for using ethyl alcohol as carbon source, precursor and/or inducer, Produce DihydromonacolinL L or citrinin J.

10. a kind of for producing the kit of citrinin J or in which mesosome, which is characterized in that include in the kit Any Yeast engineering bacteria of claim 6~8；Or

It include a construction in the kit comprising the expression cassette of the following group gene：LovB, lovC, lovG, npgA, transcription Activity factor uta and/or another construction comprising the expression cassette of the following group gene：LovA, cpr, activating transcription factor uta； And/or

It further include the culture medium for containing ethyl alcohol as carbon source, precursor and/or inducer in the kit.