CN1283780C - Method for constructing genetic engineering fungus of monascus with no citrinin - Google Patents

Abstract

The present invention relates to a method for constructing gene engineering fungi of monascus without citrinin, which belongs to the technical field of microorganism gene engineering. The present invention provides citrinin coding gene pksCT sequences deriving from CICC5006, a cloning vehicle pBpksCT and a knockout vehicle pBpksCTHyg for constructing CICC5006 citrinin genes, and a method for knocking out the CICC5006 citrinin genes by inducing REMI. The method of the present invention can be used for blocking the metabolic pathway of citrinin in monascus so as to further solve the problems of associated citrinin and pollution in the process of producing pigment, guarantee the security of monascus products, and reach the purposes of improving the product quality of the monascus and further developing.

Description

A kind of construction process of genetic engineering fungus of monascus with no citrinin

Technical field

A kind of construction process of genetic engineering fungus of monascus with no citrinin the invention belongs to microbiological genetic engineering (or molecular breeding) field, relates to the engineering bacteria that does not produce Citrinin monascus pigment high yield by engineered method structure and seed selection.

Technical background

Monascus (Monascus) is the distinctive bacterial classification in east, is the important Microbial resources of China, can produce the natural product of biologically active widely, has very high commercial value.The history of the existing more than one thousand years of the application of red colouring agent for food, also used as a Chinese medicine, its medical functions are early on the books in Chinese Ancient Books.Previous studies show that, red colouring agent for food, also used as a Chinese medicine has multi-efficiencies such as reducing blood-fat, hypotensive, anti-oxidant, anticancer, antibiotic, antifatigue, prevention senile dementia.Monascus ruber meta-bolites such as cholesterol enzyme inhibitors monacolinK, pigment, mycotoxins Citrinin etc. all are polyketide class materials.Show the Monascus ruber polyketide synthase gene (PKSs) that is richly stored with.

Wherein most important monascus product is a pigment, is widely used in food, beverage and cosmetic industry.Current research shows have a kind of colour component to have antineoplastic action.Pigment molecular is by a short chain fatty acid (C ₆Or C ₈) and one form through esterification condensation through polyketide approach synthetic six ketoboidies.Supervene but in the pigment production process, usually have Citrinin (mycotoxins), cause the insecurity of monascorubin product.The seed selection of no citrinin red colouring agent for food, also used as a Chinese medicine bacterial classification has become the focus of red colouring agent for food, also used as a Chinese medicine circle concern and has pressed for one of significant problem of solution.

France investigator Hajjaj H etc. tentatively illustrates the relation of monascus pigment and Citrinin with isotope tracer technique, and the two has common tetraketone body precursor, may be at the shared same PKS of biosynthesizing initial period.Because the existence of this metabolism relation, the application traditional breeding way obtains no citrinin pigment high-yield strains and seems very difficult.During the pigment high yield, citrinin content also improves often; Or blocked Citrinin, the pigment approach also is blocked.In addition, also there is the high shortcoming of recovery mutation rate in traditional method for mutation breeding.Therefore, the problem that thoroughly solve Citrinin also need be carried out directional transformation by means of approach engineering and genetic engineering technique to bacterial classification on the basis of understanding its pathways metabolism and key enzyme encoding gene.

Summary of the invention

The construction process that the purpose of this invention is to provide a kind of genetic engineering fungus of monascus with no citrinin, by cloning red monascus (Monascus ruber) CICC5006 (available from Chinese industrial microbial strains preservation center, be numbered CICC5006) the Citrinin encoding gene, and make up and the monascus engineering bacteria of the pigment high yield of Citrinin is not produced in seed selection with the method for REMI mediation.

The invention provides the Citrinin coding gene sequence pksCT and the amino acid sequence corresponding thereof that derive from CICC5006.The cloning vector pBpksCT and the knockout carrier pBpksCTHyg that contain CICC5006 Citrinin gene are provided, and the gene knockout method of REMI mediation.Utilize achievement of the present invention, can be used for blocking the pathways metabolism of monascus Citrinin, and then fundamentally solve Citrinin association and pollution problem in the pigment production process, and guarantee the security of monascus product, reach the purpose of improving monascus product quality and deep development.This method also can be widely used in other filamentous fungus and fungus harmful product encoding gene knock out structure with the important products engineering bacteria.

Technical scheme of the present invention: the gene order of the M.purpureus Citrinin of announcing with GeneBank is the basic design primer,

pksCT-F：5′-GGCCGCGGCCGC?GCTTCTTACCAACTTCCCTT-3′，

pksCT-R：5′-GGCCGTTAACACCTTCAGTCCGCTATCTATC-3′。Pass through round pcr, with the CICC5006 genomic dna is template, with pksCT-F, pksCT-R is the Citrinin encoding gene fragment pksCT that primer amplification obtains 4.1kb, it comprises 5 ' end non-translated sequence, with three functional structure domain encoding sequences, be respectively ketone acetyl synthetic enzyme N-terminal structural domain (ketoacyl-synt-N terminaldomain) encoding sequence, ketone acetyl synthetic enzyme C-terminal structural domain (ketoacyl-synt-C terminal domain) encoding sequence, Transacetylase structural domain (Acyl transferase domain) encoding sequence.

Not I and Hpa I restriction enzyme site have been introduced respectively with the pksCT fragment two ends that primer pksCT-F, pksCT-R amplification obtains, cut with Not I and Hpa I enzyme respectively after the purified recovery, insert NotI and the Sma I site of pBluescript II (-), make up and obtain the pBpksCT carrier.Will be from the Hpa I endonuclease bamhi (this fragment contains from the promotor of Aspergillus nidulans trpC gene and hygromycin B resistant gene hph sequence) of carrier pCB1003 1.4kb, the Sma I site (Sma I site is positioned at pksCT gene order 2253bp place) of inserting carrier pBpksCT makes up and obtains monascus Citrinin knockout carrier pBpksCTHyg.Carrier pBpksCT is used for sequencing, carrier pBpksCTHyg knocks out CICC5006 Citrinin gene by REMI (restricted enzyme-mediated integration) mediation, structure obtains genetic engineering fungus of monascus with no citrinin, and it is a kind of pigment high yield engineering bacteria.

The nucleotides sequence of gene pksCT is classified as shown in the SEQ ID NO:1.

1-1075 is 5 ' non-coding region in the pksCT nucleotide sequence; 1076-1078 is initiator codon ATG; Two exons are arranged, be respectively 1076-1656 and 1740-4111; An introne 1 657-1739.

According to above nucleotide sequence, its amino acid consists of shown in the SEQ ID NO:2.

Wherein 1-28 may be a signal peptide; 390-631 is a ketone acetyl synthetic enzyme N-terminal structural domain (ketoacyl-synt-N terminal domain); 639-806 is a ketone acetyl synthetic enzyme C-terminal structural domain (ketoacyl-synt-C terminal domain); 910-991 is Transacetylase structural domain (Acyltransferase domain).

Beneficial effect of the present invention

1.PCR method is separated, clone CICC5006 Citrinin encoding gene pksCT, length contains three functional domains for 4.1kb.Make up knockout carrier pBpksCTHyg, knock out the Citrinin gene of CICC5006 by the mediation of REMI method.Made up the monascus pigment production engineering bacterium of no citrinin simultaneously.This project bacterium can be stablized and gone down to posterity, and can be used for the production bacterium of monascorubin.And also can make up new knockout carrier with any or two the structural domain sequences in three structural domains of pksCT, also can knock out the Citrinin gene of CICC5006 with the mediation of REMI method.Also can make up simultaneously the monascus pigment production engineering bacterium of no citrinin.

2.REMI method can not only mediate conversion and the gene knockout of CICC5006, can also be applied to the gene transformation of other monascus kind and other filamentous fungus such as aspergillus (Aspergillus), sickle-like bacteria (Fusarium), wood mould (Trichoderma) etc. and catching of gene knockout and gene and promotor.

3. utilize the inventive method, can be used for blocking the pathways metabolism of monascus Citrinin, and then fundamentally solve Citrinin association and pollution problem in the pigment production process, and guarantee the security of monascus product, reach the purpose of improving monascus product quality and deep development.

Description of drawings

Fig. 1 carrier pBpksCT collection of illustrative plates;

Fig. 2 carrier pBpksCTHyg collection of illustrative plates;

The HPLC collection of illustrative plates of Fig. 3 Citrinin standard specimen;

Fig. 4 wild-type CICC5006 fermented liquid HPLC collection of illustrative plates;

Fig. 5 CICC5006 knocks out back transformant fermented liquid HPLC collection of illustrative plates by the REMI mediation.

Embodiment

The clone of embodiment 1:CICC 5006 Citrinin encoding genes

Design of primers: the gene order of the M.purpureus Citrinin of announcing with GeneBank is two pairs of primers of basic design, pksCT-F:5 '-GGCCGCGGCCGCGCTTCTTACCAACTTCCCTT-3 ', pksCT-R:5 '-GGCCGTTAACACCTTCAGTCCGCTATCTATC-3 '.

The extracting method of CICC5006 chromosomal DNA

Get 1g CICC5006 fresh mycelia and place the mortar of precooling, add appropriate amount of quartz sand, add the 1mLCTAB extraction buffer, add Proteinase K final concentration 0.1mg/mL, grind to form uniform muddy liquid on ice, change the 5mL centrifuge tube over to.The 10%SDS that adds 1/10 volume, 65 ℃ of water-bath 25min～30min are cooled to room temperature then.With equal-volume mixed solvent phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extracting 2 times, the upper strata water changes new centrifuge tube over to, adds the active RNA enzyme of no DNA enzyme (DNase) I (RNase I) to final concentration 20 μ g/mL, and 37 ℃ of temperature are bathed 30min.With equal-volume mixed solvent chloroform: primary isoamyl alcohol (24: 1) extracting 1 time, the 3mol/L NaAc of upper water addition 1/10 volume, mixing gently, the Virahol (or 2 times of volume dehydrated alcohols) that adds 0.6～0.8 volume again, choose chromosomal DNA with little glass stick, (or room temperature is placed 30min to volatilize residual ethanol after washing with 70% ethanol, the centrifugal 10min of 6000r/min, precipitation is washed once with 70% ethanol, drying at room temperature), add TE (pH 8.0) damping fluids (or aseptic deionized water) dissolving of proper volume, 4 ℃ of preservations, or-20 ℃ of preservations after the packing.

The pksCT gene clone

Adopting round pcr, is template with the CICC5006 genomic dna, carries out pcr amplification with primer pksCT-F, pksCT-R.The PCR reaction conditions is: 1.5mM MgCl ₂10mM Tris-HCl, pH 8.8; 50mMKCl; 0.1% (w/v) Triton X-100; 0.2-0.25mM dNTP; 0.4 every primer of μ M and 1-3U PfuDNA polysaccharase, the 10-100ng genomic dna.PCR is reflected on MJ (PTC-200) the PCR instrument and carries out.The PCR program is: 95 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 63 ℃ of annealing 30s, 72 ℃ of extensions 4min30s, totally 35 circulations; Extend 8min after 72 ℃.

With pksCT-F, pksCT-R is the Citrinin coding gene sequence called after pksCT that primer amplification obtains 4.1kb, comprise 5 ' end non-translated sequence, with three functional structure domain encoding sequences, be respectively ketone acetyl synthetic enzyme N-terminal structural domain encoding sequence, ketone acetyl synthetic enzyme C-terminal structural domain encoding sequence, Transacetylase structural domain encoding sequence.

Introduce Not I and Hpa I restriction enzyme site respectively with the pksCT two ends that primer pksCT-F, pksCT-R amplification obtains, cut with Not I and Hpa I enzyme respectively after the purified recovery, insert Not I and the Sma I site of pBluescript II (-), make up and obtain the pBpksCT carrier.The pBpksCT carrier is used to check order and knocks out the structure of Citrinin genophore.PksCT to its insertion adopts the Sanger chain termination method to carry out nucleotide sequencing.

Embodiment 2: the structure of knockout carrier

Will be from the Hpa I endonuclease bamhi (this fragment contains from the promotor of Aspergillusnidulans trpC gene and hygromycin B resistant gene hph sequence) of carrier pBC10031.4kb, the Sma I site (Sma I site is positioned at pksCT gene order 2253bp place) of inserting carrier pBpksCT makes up and obtains monascus Citrinin knockout carrier pBpksCTHyg.Carrier pBpksCTHyg knocks out CICC5006 Citrinin gene by REMI (restrictedenzyme-mediated integration) mediation, makes up no citrinin pigment high yield engineering bacteria.

The gene knockout method of embodiment 3:REMI mediation

The preparation of protoplastis

Bacterial strain CICC5006 cultivates 7d～10d producing on the spore substratum 30 ℃, collects spore and is prepared into the spore suspension (1 * 10 of homogeneous ⁸Individual/ml).Get the potato agar sugar solid plate that 200 μ l coating is covered with glassine paper, cultivate 30h for 30 ℃, collect mycelium, 1mol/L MgSO ₄Solution is washed once.The mycelium of each dull and stereotyped results adds 30ml lyase liquid (0.3% lyase lysing enzyme, 0.1% cellulase cellulase and 1% helicase snailase), 30 ℃, 60r/min enzymolysis.Protoplasma body fluid is filtered, and the centrifugal 10min of filtrate 3200r/min abandons supernatant, and the resuspended precipitation of precooling 1.0mol/L Sorbitol Solution USP is centrifugal.The protoplastis precipitation is resuspended in the 1.0mol/L sorbyl alcohol, and it is standby to put ice bath.

The gene knockout method of REMI mediation

Method 1. above-mentioned protoplasma body fluid are centrifugal, precipitation precooling STC solution (1mol/L sorbyl alcohol, 50mmol/L TrisHCl (pH 8.0), 50mmol/L CaCl ₂) wash 1-2 time, be resuspended in precooling STC solution again and wash, and adjust concentration to 4 * 10 ⁷-1 * 10 ⁸Individual/ml.Get 100 μ l protoplasma body fluid, add linearizing or not linearizing knockout carrier pBpksCTHyg 1-20 μ g, pressure-vaccum mixing gently.Place on ice, add restriction enzyme Hind III (or Sma I, Sal I, Sac I) 80-130U, pressure-vaccum mixing gently, ice bath 30-60min.Add 1mL PTC solution (40%PEG4000,50mmol/L TrisHCl (pH 8.0), 50mmol/L CaCl ₂), room temperature 40～120min.Coating contains the regeneration agarose solid medium flat board of 100-150 μ g/ml hygromycin B.30 ℃ of dark 4d～5d that cultivate, meter transforms subnumber, and the transformant of growth separates through monospore, and switching contains the PDA agarose solid medium flat board of 150 μ g/ml hygromycin B, and the transformant of growth was transferred for 5 generations more continuously, to obtain stable transformant.

Or method 2. above-mentioned protoplasma body fluid are centrifugal, precipitation be resuspended in electroporation transform damping fluid (TrisHClpH 7.510mmol/L, sucrose 600mmol/L, LiAc 1mmol/L, PEG400030%), concentration all is adjusted to 4 * 10 ⁷-1 * 10 ⁸Individual/ml.Get 70 μ l protoplasma body fluid, add linearizing or not linearizing knockout carrier pBpksCTHyg 1-20 μ g, pressure-vaccum mixing gently.Place on ice, add restriction enzyme Hind III (or Sma I, Sal I, Sac I) 80-130U, pressure-vaccum mixing gently, ice bath 15-20min.Change in the 0.1cm cuvette cup of precooling, 200-400V, 200ohms shocks by electricity, and adds the recovery liquid nutrient medium (the potato glucose liquid nutrient medium that contains 0.6mol/L sucrose) of 900 μ l precoolings immediately, change the aseptic centrifuge tube of 1.5ml over to, ice bath 10-20min.30 ℃, 60-100r/min cultivates 2-3h.Coating contains the regeneration agarose solid medium flat board of 100-150 μ g/mlhygromycin B.30 ℃ of dark 4d～5d that cultivate, meter transforms subnumber, and the transformant of growth separates through monospore, and switching contains the PDA agarose solid medium flat board of 150 μ g/ml hygromycin B, and the transformant of growth was transferred for 5 generations more continuously, to obtain stable transformant.

The protoplast regeneration substratum: add sucrose respectively in CM (0.6% caseinic acid hydrolyzate, 0.5% sucrose, 0.6% yeast extract), potato agar sugar culture-medium, Sha Shi, Vogel ' s agarose solid medium, final concentration is 0.6mol/L.

Embodiment 4: the screening of transformant

The screening of stable conversion

Transformant in the dull and stereotyped growth of the regeneration agarose solid medium that contains 120 μ g/ml hygromycin B separates through monospore, and switching contains the PDA agarose solid medium flat board of 150 μ g/ml hygromycin B.The transformant of growth was transferred for 5 generations more continuously, to obtain stable transformant.

The PCR method checking

With pksT-F (5 '-GGCCGAATTCATGCGACGAAGATGTTACTGC-3 ') and pksT-R (5 '-GGCCGAATTCAGGTCCTGTCCAGCCGAAAT-3 ') is primer, is that template is carried out pcr amplification with transformant and wild type strain CICC5006 genomic dna respectively.The PCR reaction conditions is: 1.5mM MgCl ₂10mM Tris-HCl, pH 8.8; 50mM KCl; 0.1% (w/v) Triton X-100; 0.2-0.25mM dNTP; 0.4 every primer of μ M and 1-3U Pfu archaeal dna polymerase, the 10-100ng genomic dna.PCR is reflected on MJ (PTC-200) the PCR instrument and carries out.The PCR program is: 95 ℃ of pre-sex change 5min; 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ of extensions 2.5min, totally 32 circulations; Extend 8min after 72 ℃.As a result, be that template can increase and obtains the fragment of 0.8bp with the wild type strain genomic dna, and when being template with the transformant genomic dna, can increase obtain the fragment of 2.2kb.Illustrate that hygromycin gene hph fragment inserted in the Citrinin gene of CICC5006, show that Citrinin gene among the CICC5006 is destroyed and knock out.

Citrinin detection-HPLC

The mensuration that to carry out Citrinin through stable conversion and the wild-type CICC5006 of PCR checking.

Fermentation culture

30 ℃ on the PDA inclined-plane of bacterial strains to be measured are cultivated 7d～10d, and the spore suspension concentration of collecting spore and being prepared into homogeneous is 1 * 10 ⁷～1 * 10 ⁸Individual/ml.The 250ml that 30ml potato glucose liquid nutrient medium is equipped with in 10% inoculum size inoculation shakes bottle, and 200r/min cultivated 2 days for 30 ℃.The 500ml that 70ml YES liquid nutrient medium (4% sucrose, 2% yeast extractive substance) is housed with the switching of 15% inoculum size shakes bottle, 28 ℃ of static cultivations 14 days.

HPLC detects

With fermented liquid and mycelium mixed grinding, get 10ml and grind liquid, add ethanol and be settled to 30ml, mixing, 60 ℃ of water-bath 1h.The centrifugal 15min of 3000r/min.With the aperture is to be directly used in HPLC behind the inclined to one side fluorine millipore filtration of the 0.45 μ m micro-filtration to analyze.

The HPLC testing conditions

Fluoroscopic examination wavelength: λ ex=331nm, λ em=500nm;

Moving phase: V _{Second cyanogen}: V _Water=35: 65 (transferring pH 2.5) with phosphoric acid;

Appearance time: 12.324min;

Flow velocity: 1.0mL/min, column temperature: 28 ℃.

The HPLC detected result

Wild-type CICC5006 fermented liquid has the peak at 12.885min, and proving conclusively with Citrinin standard specimen (available from sigma company) contrast is the Citrinin peak.And the transformant of wild-type CICC5006 after knocking out by REMI mediation when 12.885min for not observing the peak, in the transformant that conclusive evidence screens the Citrinin gene destroyed with knock out.

Sequence table

<210>SEQ?ID?NO：1

<211>4111

<212>DNA

＜213〉monascus (Monascus ruber) CICC5006

<214>

gcttcttacc?aacttccctt?ttttcttcag?ccagtgcttt?tgtactttct?cttctccagc 60

gattccttcg?tatacataag?tgccacccaa?ttgaacaaag?ttgcgtccaa?cctcttacat 120

gatccagtat?ccacttggca?agtgattcct?cttcaaattc?tgaaagtttg?agtccatttg 180

caaaaggaat?tttgcagttg?cagcatgatt?gatatgggga?ttttcccatt?ttttaaatca 240

gaaatggcta?ataagactcg?accctcgtgc?tcagcgagat?tatttaaagg?tccatcgcga 300

attggtggca?ttatggcgcg?tcgaattgta?tggaatcaat?atagtggacg?ttttttccga 360

gtatggtata?tccggcgacc?actatgcacc?atgaatagat?gcaaccttac?acccttgtca 420

gatgcaagat?tactctatat?aagctggcat?gccgatgtga?ttcgcaacca?ctcatttgaa 480

cttggctaaa?gatcggcaaa?cagtattctt?gaagaaagga?cgtgcgccga?cacgcattct 540

ggtccaagct?cgtacagcag?ggttggggac?cgtccgtact?tctacacgct?agcctacatc 600

tcatcaacgc?ctgttgttgg?aggcagaagc?taggcgctga?ggtgcaagct?ttcgggatta 660

tttatcggag?taacttcatc?cttgcgaagt?aaatccttca?ggatctgtac?ttccgcagcc 720

catatcatct?atgaagcccg?tatagcggaa?gcacaagcga?gcgggacggc?tggcgaatac 780

caacatctga?gaatatccga?cacaagcaag?aagagtgggg?aggggcgcga?ggtaactgtc 840

cggaataatg?ttctgctgtg?gattttcagt?tcagcctcac?aggcaggtgc?agtgggcact 900

ccatcagact?catatatagt?tgctactatc?cacgagatcg?gcaaacggtc?tcgcatggca 960

gtatataccg?tgtttgtgtg?atatacctac?cagtacccta?tcattttcaa?tacgatttcc 1020

catcggtcag?cttcaacgtg?accaaggacg?gcagtttcaa?ttgcggctat?aagttatgat 1080

tgactcaact?tcgcactcaa?atctgagatc?aaaggcgttt?atattcggcc?cgcaggattt 1140

gtctttcgat?gtcaggtcct?tcaacaagct?tcacagccag?ctccaaaatc?accaatgggt 1200

tcttgatgcg?ctcgccagcc?tgccaaagct?ttgggacaac?tttgccgcca?gtgaccaaaa 1260

ggtacagcaa?tccaacactg?ggaagctgct?tgagaacttg?aatgcatgga?tttccagtgg 1320

ggtagcccca?gaggaggcat?ttcctttgcc?aaatgtgctt?ctttccccgc?tcgtggtcat 1380

aggacagttg?gttgaataca?tgacttttct?caaagcggca?ttcccagacc?taggaaagaa 1440

acatgacctg?cccatatcaa?ttaaggaaga?tacggagacc?ttcggcctct?gcaccggcac 1500

cctgtgtgct?tttgcagtag?cctgctcttc?caacatagcc?gatattcaac?attacggcgc 1560

ggtagctgcg?agactggcga?tgttggttgg?agccatcgtc?gataccgaag?aggtactatc 1620

tgatccggaa?ggaaagtcag?tgagtttttc?agcgtcgtgg?aatagcgccg?aattcagtga 1680

ctcattcacc?cacgtcctcg?agacattccc?tgatgtacgt?cctcacaatt?cgcaccaaga 1740

ccagagcaaa?cgctaaccat?cgttctacag?gcatatgtgt?ctgtcatcgt?tgaccagaga 1800

cgtgcgacgt?tgacggcgtc?gaagaagacg?gcgccagcta?ttattgagcg?gttgaaacaa 1860

gaaggcgctc?atgtcacgtc?aattgcgctt?tcgggacgat?tccactggaa?gaagcatcag 1920

gatgccgtat?cgagtctcat?tcagttttgt?ggacttgatc?caggcttgca?attggctgat 1980

gcgacgaaga?tgttactgcc?tagtcgatcc?agctccgatg?ggcaatatat?cactactgga 2040

aaactccacg?agctagcttt?gcgggcaatt?ctcttggaac?agtctgagtg?gtacaagaca 2100

tgccgcattt?cctacttgtc?aaagttcatc?atggacgatg?cagcggtgat?ttgtttcggg 2160

ccggagaggt?gcatgccacc?aacacttgca?cgaaaacttg?gtccacgcct?gacatacgta 2220

tccgagattg?atatctcatc?gtcacgagta?cccgggcagc?ttctcggtgg?aacgcaaaag 2280

ctgaacctca?cagacctacc?cgatgagcgc?atcgctgtta?ttggaatggc?gtgtcggcta 2340

ccgggtgccg?aagaccacga?gggattctgg?gagatcctga?aaacgggtca?gtcgcagcat 2400

agagaggtgc?ccgaagatcg?gttcggcatg?gcgacggcct?ggcgagaagc?ggacaaacgc 2460

aaatggtacg?ggaatttcat?cgacaattac?gacaccttcg?atcataaatt?cttcaagaag 2520

agtccccgag?aaatggcatc?cacggaccct?caacaccgcc?tgatgttgca?ggtagcatat 2580

caggccgtcg?aacaatccgg?gtacttcaga?aataatggta?cgaatcggcg?cattggatgc 2640

ttcatgggtg?tcggcaatgt?cgattacgag?gacaacattg?cgtgttaccc?tgccaatgcg 2700

tattcggcta?ccggcaacct?gaagagtttt?cttgctggca?agattagcca?ccatttcggc 2760

tggacaggac?ctagtctgac?acttgacacg?gcatgctcgt?catccagtgt?ggctattcac 2820

caggcatgcc?gttccatccc?tagcggggaa?tgtaacgggg?cactcgccgg?aggtgtcaac 2880

gttataacga?gtcccaactg?gtatcataac?ctagccggcg?catctttcct?cagccctact 2940

ggtcaatgca?agccgtttga?cgctaagggg?gatgggtact?gcagaggtga?aggtgttggt 3000

gccgtgtttt?tgaaacggct?ttcctctgca?atcgcagatg?gtgaccaggt?attcggtgtg 3060

atcgcgagta?cgaaggttta?tcaaaatcag?aattgtactg?caatcactgt?tcctaacgca 3120

atatctctgt?cggagctctt?tacggatgtc?gtgcgtcaag?caaggcttga?gcctaaggac 3180

attactcttg?ttgaggcaca?cggaacagga?actgcagtcg?gggacccagc?tgaatacgat 3240

ggcattcgag?ctgtctttgg?aggccctata?cgctccgatg?ttctgtcgct?aggatccgtg 3300

aagggccttg?tcggtcacac?cgaatgcgct?tcaggtgtgg?tctctttgat?aaagaccctc 3360

cttatgatcc?agcaaggctt?tatcccaccc?caggccagct?tttcaagcat?caacccttca 3420

ctcaatgcaa?aggctgagga?aaaaatagag?atctcgaccc?gtttaaagcc?ttgggatgca 3480

cctttcagag?ctgcactcat?caacaactac?ggtgcttccg?ggtccaatgc?ctccatggtt 3540

gttacgcaac?caccaaatct?gacggagacc?ccctcgacac?cgttgccagg?gaaaagctat 3600

ccattctgga?tcagtgcatt?tgaccaacag?agccttcaaa?gttacgttcg?gaggttgcgc 3660

caatttctcg?aaaaacatgc?ggcagacaaa?aacctaagcg?tcgcaaactt?gtctttccaa 3720

gttgcttgtc?aatcaaactg?gtctcttccc?caagccctgg?tattcagtgc?cagcacaaag 3780

gaggagctga?atagggccct?tgcatctttt?gagaaaggca?gcacggattt?cccatctgtc 3840

cagcttccgg?atccgaagcc?cgtcatccta?tgctttggag?ggcaagtttc?cacctatgtt 3900

ggtttggatc?aagaggtcta?taacagcact?gcgatcttga?gccattactt?agatcagtgc 3960

gatgccatgt?gcctttcgct?aggcctgcaa?agtatctacc?cggctatttt?ccaacggtcc 4020

ccaatcgagg?atattgctca?gcttcaaaca?gcgctgtttg?cgatgcagta?ttcctgcgcc 4080

aaggcatgga?tagatagcgg?actgaaggtg?t 4111

<210>SEQ?ID?NO：2

<211>991

<212>PRT

＜213〉monascus (Monascus ruber) CICC5006

<214>

Met?Ile?Asp?Ser?Thr?Ser?His?Ser?Asn?Leu?Arg?Ser?Lys?Ala?Phe

1 5 10 15

Ile?Phe?Gly?Pro?Gln?Asp?Leu?Ser?Phe?Asp?Val?Arg?Ser?Phe?Asn

20 25 30

Lys?Leu?His?Ser?Gln?Leu?Gln?Asn?His?Gln?Trp?Val?Leu?Asp?Ala

35 40 45

Leu?Ala?Ser?Leu?Pro?Lys?Leu?Trp?Asp?Asn?Phe?Ala?Ala?Ser?Asp

50 55 60

Gln?Lys?Val?Gln?Gln?Ser?Asn?Thr?Gly?Lys?Leu?Leu?Glu?Asn?Leu

65 70 75

Asn?Ala?Trp?Ile?Ser?Ser?Gly?Val?Ala?Pro?Glu?Glu?Ala?Phe?Pro

80 85 90

Leu?Pro?Asn?Val?Leu?Leu?Ser?Pro?Leu?Val?Val?Ile?Gly?Gln?Leu

95 100 105

Val?Glu?Tyr?Met?Thr?Phe?Leu?Lys?Ala?Ala?Phe?Pro?Asp?Leu?Gly

110 115 120

Lys?Lys?His?Asp?Leu?Pro?Ile?Ser?Ile?Lys?Glu?Asp?Thr?Glu?Thr

125 130 135

Phe?Gly?Leu?Cys?Thr?Gly?Thr?Leu?Cys?Ala?Phe?Ala?Val?Ala?Cys

140 145 150

Ser?Ser?Asn?Ile?Ala?Asp?Ile?Gln?His?Tyr?Gly?Ala?Val?Ala?Ala

155 160 165

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

170 175 180

Leu?Ser?Asp?Pro?Glu?Gly?Lys?Ser?Val?Ser?Phe?Ser?Ala?Ser?Ser

185 190 195

Ser?Gln?Phe?Ala?Pro?Arg?Pro?Glu?Gln?Thr?Leu?Thr?Ile?Val?Leu

200 205 210

Gln?Ala?Tyr?Val?Ser?Val?Ile?Val?Asp?Gln?Arg?Arg?Ala?Thr?Leu

215 220 225

Thr?Ala?Ser?Lys?Lys?Thr?Ala?Pro?Ala?Ile?Ile?Glu?Arg?Leu?Lys

230 235 240

Gln?Glu?Gly?Ala?His?Val?Thr?Ser?Ile?Ala?Leu?Ser?Gly?Arg?Phe

245 250 255

His?Trp?Lys?Lys?His?Gln?Asp?Ala?Val?Ser?Ser?Leu?Ile?Gln?Phe

260 265 270

Cys?Gly?Leu?Asp?Pro?Gly?Leu?Gln?Leu?Ala?Asp?Ala?Thr?Lys?Met

275 280 285

Leu?Leu?Pro?Ser?Arg?Ser?Ser?Ser?Asp?Gly?Gln?Tyr?Ile?Thr?Thr

290 295 300

Gly?Lys?Leu?His?Glu?Leu?Ala?Leu?Arg?Ala?Ile?Leu?Leu?Glu?Gln

305 310 315

Ser?Glu?Trp?Tyr?Lys?Thr?Cys?Arg?Ile?Ser?Tyr?Leu?Ser?Lys?Phe

320 325 330

Ile?Met?Asp?Asp?Ala?Ala?Val?Ile?Cys?Phe?Gly?Pro?Glu?Arg?Cys

335 340 345

Met?Pro?Pro?Thr?Leu?Ala?Arg?Lys?Leu?Gly?Pro?Arg?Leu?Thr?Tyr

350 355 360

Val?Ser?Glu?Ile?Asp?Ile?Ser?Ser?Ser?Arg?Val?Pro?Gly?Gln?Leu

365 370 375

Leu?Gly?Gly?Thr?Gln?Lys?Leu?Asn?Leu?Thr?Asp?Leu?Pro?Asp?Glu

380 385 390

Arg?Ile?Ala?Val?Ile?Gly?Met?Ala?Cys?Arg?Leu?Pro?Gly?Ala?Glu

395 400 405

Asp?His?Glu?Gly?Phe?Trp?Glu?Ile?Leu?Lys?Thr?Gly?Gln?Ser?Gln

410 415 420

His?Arg?Glu?Val?Pro?Glu?Asp?Phg?Phe?Gly?Met?Ala?Thr?Ala?Trp

425 430 435

Arg?Glu?Ala?Asp?Lys?Arg?Lys?Trp?Tyr?Gly?Asn?Phe?Ile?Asp?Asn

440 445 450

Tyr?Asp?Thr?Phe?Asp?His?Lys?Phe?Phe?Lys?Lys?Ser?Pro?Arg?Glu

455 460 465

Met?Ala?Ser?Thr?Asp?Pro?Gln?His?Arg?Leu?Met?Leu?Gln?Val?Ala

470 475 480

Tyr?Gln?Ala?Val?Glu?Gln?Ser?Gly?Tyr?Phe?Arg?Asn?Asn?Gly?Thr

485 490 495

Asn?Arg?Arg?Ile?Gly?Cys?Phe?Met?Gly?Val?Gly?Asn?Val?Asp?Tyr

500 505 510

Glu?Asp?Asn?Ile?Ala?Cys?Tyr?Pro?Ala?Asn?Ala?Tyr?Ser?Ala?Thr

515 520 525

Gly?Asn?Leu?Lys?Ser?Phe?Leu?Ala?Gly?Lys?Ile?Ser?His?His?Phe

530 535 540

Gly?Trp?Thr?Gly?Pro?Ser?Leu?Thr?Leu?Asp?Thr?Ala?Cys?Ser?Ser

545 550 555

Ser?Ser?Val?Ala?Ile?His?Gln?Ala?Cys?Arg?Ser?Ile?Pro?Ser?Gly

560 565 570

Glu?Cys?Asn?Gly?Ala?Leu?Ala?Gly?Gly?Val?Asn?Val?Ile?Thr?Ser

575 580 585

Pro?Asn?Trp?Tyr?His?Asn?Leu?Ala?Gly?Ala?Ser?Phe?Leu?Ser?Pro

590 595 600

Thr?Gly?Gln?Cys?Lys?Pro?Phe?Asp?Ala?Lys?Gly?Asp?Gly?Tyr?Cys

605 610 615

Arg?Gly?Glu?Gly?Val?Gly?Ala?Val?Phe?Leu?Lys?Arg?Leu?Ser?Ser

620 625 630

Ala?Ile?Ala?Asp?Gly?Asp?Gln?Val?Phe?Gly?Val?Ile?Ala?Ser?Thr

635 640 645

Lys?Val?Tyr?Gln?Asn?Gln?Asn?Cys?Thr?Ala?Ile?Thr?Val?Pro?Asn

650 655 660

Ala?Ile?Ser?Leu?Ser?Glu?Leu?Phe?Thr?Asp?Val?Val?Arg?Gln?Ala

665 670 675

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

680 685 690

Gly?Thr?Ala?Val?Gly?Asp?Pro?Ala?Glu?Tyr?Asp?Gly?Ile?Arg?Ala

695 700 705

Val?Phe?Gly?Gly?Pro?Ile?Arg?Ser?Asp?Val?Leu?Ser?Leu?Gly?Ser

710 715 720

Val?Lys?Gly?Leu?Val?Gly?His?Thr?Glu?Cys?Ala?Ser?Gly?Val?Val

725 730 735

Ser?Leu?Ile?Lys?Thr?Leu?Leu?Met?Ile?Gln?Gln?Gly?Phe?Ile?Pro

740 745 750

Pro?Gln?Ala?Ser?Phe?Ser?Ser?Ile?Asn?Pro?Ser?Leu?Asn?Ala?Lys

755 760 765

Ala?Glu?Glu?Lys?Ile?Glu?Ile?Ser?Thr?Arg?Leu?Prs?Pro?Trp?Asp

770 775 780

Ala?Pro?Phe?Arg?Ala?Ala?Leu?Ile?Asn?Asn?Tyr?Gly?Ala?Ser?Gly

785 790 795

Ser?Asn?Ala?Ser?Met?Val?Val?Thr?Gln?Pro?Pro?Asn?Leu?Thr?Glu

800 805 810

Thr?Pro?Ser?Thr?Pro?Leu?Pro?Gly?Lys?Ser?Tyr?Pro?Phe?Trp?Ile

815 820 825

Ser?Ala?Phe?Asp?Gln?Gln?Ser?Leu?Gln?Ser?Tyr?Val?Arg?Arg?Leu

830 835 840

Arg?Gln?Phe?Leu?Glu?Lys?His?Ala?Ala?Asp?Lys?Asn?Leu?Ser?Val

845 850 855

Ala?Asn?Leu?Ser?Phe?Gln?Val?Ala?Cys?Gln?Ser?Asn?Trp?Ser?Leu

860 865 870

Pro?Gln?Ala?Leu?Val?Phe?Ser?Ala?Ser?Thr?Lys?Glu?Glu?Leu?Asn

875 880 885

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

890 895 900

Val?Gln?Leu?Pro?Asp?Pro?Lys?Pro?Val?Ile?Leu?Cys?Phe?Gly?Gly

905 910 915

Gln?Val?Ser?Thr?Tyr?Val?Gly?Leu?Asp?Gln?Glu?Val?Tyr?Asn?Ser

920 925 930

Thr?Ala?Ile?Leu?Ser?His?Tyr?Leu?Asp?Gln?Cys?Asp?Ala?Met?Cys

935 940 945

Leu?Ser?Leu?Gly?Leu?Gln?Ser?Ile?Tyr?Pro?Ala?Ile?Phe?Gln?Arg

950 955 960

Ser?Pro?Ile?Glu?Asp?Ile?Ala?Gln?Leu?Gln?Thr?Ala?Leu?Phe?Ala

965 970 975

Met?Gln?Tyr?Ser?Cys?Ala?Lys?Ala?Trp?Ile?Asp?Ser?Gly?Leu?Lys

980 985 990

Val

991

Claims (7)

1. a construction method without citrinin Monascus genetically engineered bacteria, characterized in that: (1) Primer design: primers were designed based on the M. purpureus citrinin gene published by GeneBank, pksCT-F: 5′-GGCCGCGGCCGCGCTTCTTACCAACTTCCCTT-3′, pksCT-R: 5'-GGCCGTTAACACCTTCAGTCCGCTATCTATC-3'; (2) pksCT gene cloning: the 4.1 kb citrinin gene fragment was amplified by PCR technology using CICC5006 genomic DNA as a template and primers pksCT-F and pksCT-R, named pksCT; (3) Construction of pBpksCT vector: Not I and Hpa I restriction sites were introduced into both ends of pksCT respectively, after purification and recovery, they were digested with Not I and Hpa I respectively, and Not I and Sma I of pBluescript II(-) were inserted site, construct the pBpksCT vector; (4) Construction of the citrinin gene knockout vector pBpksCTHyg: the Hpa I fragment from the vector pCB1003 1.4kb, which contains the promoter from the Aspergillus nidulans trpC gene and the hygromycin B resistance gene hph sequence, is inserted into the vector pBpksCT The Sma I site, the Sma I site is located at 2253bp in the pksCT gene sequence, and the Monascus citrinin knockout vector pBpksCTHyg was constructed; (5) REMI-mediated knockout: the vector pBpksCTHyg knocked out the citrinin gene of the host strain CICC5006 through REMI-mediated knockout, and a citrinin-free Monascus genetically engineered strain was constructed. 2. The method according to claim 1, characterized in that the citrinin gene pksCT has a nucleotide sequence of: SEQ ID NO: 1. 3. The method according to claim 2, characterized in that the citrinin gene pksCT has an amino acid composition of: SEQ ID NO:2. 4. method according to claim 1, it is characterized in that pksCT gene clone: adopt PCR technology, take CICC5006 genomic DNA as template, carry out PCR amplification with primer pksCT-F, pksCT-R, PCR reaction condition is: 1.5mM _MgCl2 ; 10mM Tris-HCl, pH 8.8; 50mM KCl; 0.1% Triton X-100; 0.2-0.25mM dNTP; 0.4μM each primer and 1-3U Pfu DNA polymerase; 10-100ng genomic DNA; Performed on a PCR machine, the PCR program was: 95°C pre-denaturation for 5 minutes, 94°C denaturation for 30 s, 63°C annealing for 30 s, 72°C extension for 4 min 30 s, a total of 35 cycles, and 72°C post-extension for 8 min. 5. The method according to claim 1, characterized in that 1-1075 is the 5' non-coding region in the citrinin gene pksCT nucleotide sequence, 1076-1078 is the initiation codon ATG, and there are two exons , respectively 1076-1656 and 1740-4111, an intron 1657-1739; the amino acid sequence includes three functional domains: 390-631 is the N-terminal domain of ketoacetyl synthase, and 639-806 is the ketoacetyl synthase The C-terminal domain, 910-991 is the acetyltransferase domain. 6. The method according to claim 1, characterized in that the REMI-mediated knockout method: Method 1. Take 100 μl CICC5006 protoplast fluid, add linearized or non-linearized knockout vector pBpksCTHyg 1-20 μg, place on ice, add restriction endonuclease Hind III or Sma I or Sal I or SacI 80-130 U, gently Gently blow and aspirate to mix, put in ice bath for 30-60min, add 1mL of PTC solution, room temperature for 40-120min, spread the regenerated agarose solid medium plate containing 100-150μg/ml hygromycin B, culture in dark at 30℃ for 4d-5d, count The number of transformants, the growing transformants were separated by single spores, transferred to the PDA agarose solid medium plate containing 150 μg/ml hygromycin B, and the grown transformants were continuously transferred for 5 generations to obtain stable transformants; Or method 2. Take 70μl CICC5006 protoplast body fluid, add linearized or non-linearized knockout vector pBpksCTHyg 1-20μg, place on ice, add restriction endonuclease Hind III or Sma I or Sal I or SacI 80-130U, Gently inhale and mix, put in ice bath for 15-20min, transfer to pre-cooled 0.1cm cuvette cup, 200-400V, 200ohms for electric shock, immediately add 900μl pre-cooled potato dextrose liquid medium containing 0.6mol/L sucrose , transferred to a 1.5ml sterile centrifuge tube, ice-bathed for 10-20min, 30°C, 60-100r/min cultured for 2-3h, coated with a regenerated agarose solid medium plate containing 100-150μg/ml hygromycin B, 30°C Culture in dark for 4-5 days, count the number of transformants, separate the growing transformants from single spores, transfer to PDA agarose solid medium plate containing 150 μg/ml hygromycin B, and transfer the growing transformants for 5 generations continuously to obtain stable transformants. 7. The method according to claim 1, characterized in that three functional domains are used in pksCT: any one of the N-terminal domain of ketoacetyl synthase, the C-terminal domain of ketoacetyl synthase, and the acetyltransferase domain or two coding sequences to construct a citrinin gene knockout vector, and the REMI method is mediated to knock out the citrinin gene of CICC5006 to construct a citrinin-free Monascus genetically engineered bacterium.

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