CN105385604A - Winter 4 monascus strain with low-yielding citrinin and high-yielding pigment - Google Patents

Abstract

The invention relates to a winter 4 monascus strain with low-yielding citrinin and high-yielding pigment. The class name of the strain is Monascus purpureus, the preservation number is CGMCC No.11010, the preservation date is July 15th, 2015. Genetic engineering utilizes a hygromycin resistance gene in the Winter 4 strain to replace a functional gene of oxygenase gene ORF3 in a citrinin synthetic gene cluster, and a monascus Winter 4 gene engineering strain with the low-yielding citrinin and the high-yielding pigment is obtained. It is found through comparison of a genetically modified strain and an original wild strain that for the monascus Winter 4 strain, the citrinin yield is decreased by 96.48 percent, the yellow pigment yield of a mutant strain is increased by 89.9 percent, the orange pigment yield is increased by 172.1%, and the red pigment yield is increased by 140.6 percent.

Description

The Winter4 monascus strain of low-yield citrinin high yield pigment

Technical field

The invention belongs to technical field of food biotechnology, be specifically related to a strain and there is low-yield citrinin and the monascus Winter4 bacterial strain of high yield pigment ability.

Technical background

Late Cambrian Citrinin in monascus to be extracted be in 1981, professor HinchungWong of Hong Kong Chinese University waits product separation from monascus to go out a kind of material with bacteriostatic activity of yellow, by its called after monascidinA.To nineteen ninety-five, French professor Blnac etc. use the multinomial detection meanss such as nucleus magnetic resonance, mass spectrum, ultraviolet and fluorometric analysis to carry out qualitative analysis and structure determination to this material, confirm that monascidinA is exactly Citrinin (citrinin).Citrinin is a kind of mycotoxins, and in the kind of monascus, monascus parpureus Went and red monascus are the bacterial classifications that can produce Citrinin.The target organ of Citrinin is kidney, probes into through zooperal, shows the symptoms such as Citrinin can cause kidney enlargement, urine volume increases, and tubular ectasia and kidney epithelia cytopathy are even downright bad, causes death once excessive.Per os is taken in, and the medial lethal dose of mouse is 110mg/kg.Also studies have found that, Citrinin can damage the metabolic function of liver.In vivo test shows, Citrinin energy and serum protein combine, and have Mutation induction effect.Separately have research to confirm, Citrinin also has toxic effect to gene.In addition, Citrinin can accumulate in cell mitochondrial, interference electron transfer system, cause DNA biosynthesis block, so make RNA and protein synthesis suppressed, cause plastosome enlargement, cause necrocytosis.

Gene knockout is a kind of technology making the specific gene inactivation of body or disappearance.In general, what gene knockout was mainly applied is that ((homologousrecombination) principle is replaced target fragment to be knocked out with the homologous fragment of design, thus reached the object knocking out target gene in DNA homology restructuring.Primary process is that the recombinant vectors built is imported recipient cell, by the method displacement target gene of homologous recombination, infers the function of target gene with this.

Therefore knock out Citrinin synthesis related gene in monascus genome by the technology fixed point of gene knockout, the object blocking Citrinin route of synthesis, reduce Citrinin output can be reached.

Summary of the invention

The object of the invention is to the citrinin biosynthesis gene screening the Winter4 monascus strain of the high yield pigment obtained by destroying contriver, block Citrinin route of synthesis thus the Citrinin output of reduction monascus strain, obtain the Winter4 monascus genetic modification bacterial strain of a strain low-yield citrinin high yield pigment.

The object of the invention is to be achieved through the following technical solutions:

One plant height produces the monascus Winter4 bacterial strain of pigment ability, this strain classification name is called: monascus parpureus Went Monascuspurpureus, preserving number is: CGMCCNo.11010 preservation date: on July 15th, 2015, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.

And cultivate in liquid medium within, compared with parent strain, Citrinin output is reduced to 3.52% of parent strain, and yellow pigment, citraurin, haematochrome improve 89.9%-172.1%.

And XhoI restriction enzyme site is contained in described hygromycin gene upstream, SmaI restriction enzyme site is contained in hygromycin gene downstream, the distance between two restriction enzyme sites is 2513bp.

The monascus Winter4 bacterial strain of high yield pigment ability preparing Red kojic rice, slightly carry monascorubin, essence proposes application in monascorubin.

Advantage of the present invention and positively effect:

1, monascus Winter4 mutant strain provided by the invention is replaced by goal gene ORF3 by the method hygromycin gene of homologous recombination, belong to the rite-directed mutagenesis of gene, this method eliminates the impact of the uncertain factor that random mutation brings, and can have further understanding by the changing conditions of Citrinin and pigment to Citrinin route of synthesis.

2, find by genetic modification bacterial strain and original wild strain being contrasted, monascus strain Winter4 bacterial strain Citrinin output provided by the invention is reduced to 3.52% of parent strain; And the yellow pigment output increased of mutant strain 89.9%, citraurin improves 172.1%, and haematochrome improves 140.6%.For monascus provides microorganism resource in the safety applications of field of food.

3, the genetic modification bacterial strain that provides of the application produce pigment method is saved time, technology is easy, be easy to that development downstream produces, genetic information can operate and stable.

Accompanying drawing explanation

Fig. 1 is with wild strain monascus DNA for template, pcr amplification ORF3 fragment upstream figure.In figure: M:DL2000 nucleic acid molecular weight Marker, 1: upstream sequence.

Fig. 2 is with wild strain monascus DNA for template, pcr amplification ORF3 segments downstream figure.In figure: M:DL2000 nucleic acid molecular weight Marker, 1: downstream sequence.

Fig. 3 is ORF3 upstream recombinant plasmid double digestion and PCR proof diagram.In figure: M1.DNA molecular weight marker DL10000; M2.DNA molecular weight marker DL2000; 1. recombinant plasmid; 2. double digestion checking recombinant plasmid.

Fig. 4 is ORF3 upstream and downstream recombinant plasmid double digestion and PCR proof diagram.In figure: M.DNA molecular weight marker DL10000; 1. recombinant plasmid; 2. double digestion checking recombinant plasmid; 3.PCR verifies recombinant plasmid.

Fig. 5 verifies for utilizing primer ORF3-1 and hph-R, and this is ORF3 fragment upstream, Totomycin upstream sequence and a part of Totomycin sequence to primer amplification sequence, and object stripe size is about 2500bp.In figure: CK.FM-46 wild strain contrasts; M.DNA molecular weight marker DL10000; 1. mutant strain Winter4.

Fig. 6 verifies for utilizing primer ORF3-4 and hph-F, and this is ORF3 segments downstream, Totomycin downstream sequence and a part of Totomycin sequence to primer amplification sequence, and object stripe size is about 2500bp.CK.FM-46 wild strain contrasts; M.DNA molecular weight marker DL10000; 1. mutant strain Winter4.

Fig. 7 verifies for utilizing primer ORF3-1 and ORF3-4, and this is ORF3 upstream and downstream fragment, Totomycin upstream and downstream sequence and Totomycin sequence to primer amplification sequence, and object stripe size is about 4500bp; For wild strain, extension increasing sequence comprises ORF3 upstream and downstream fragment and ORF3 sequence, and object stripe size is about 3200bp.CK.FM-46 wild strain contrasts; M.DNA molecular weight marker DL10000; 1. mutant strain Winter4.

Fig. 8 is for utilizing summer-up, summer-down and winter-up, winter-down two to verifying that primer is verified, this is ORF3 upstream and downstream fragment, Totomycin upstream and downstream sequence and upstream and downstream homology arm the preceding paragraph sequence to primer amplification sequence, and object stripe size is about 2500bp and 1500bp respectively.1,3. mutant strain Winter4 upstream; 2,4. mutant strain Winter4 downstream; 5. wild strain upstream control; 6. wild strain downstream contrast.

Fig. 9 is mutant strain and wild strain Citrinin yield comparison.

Figure 10 wild strain pigment production compares with other bacterial strain.In figure, FM-46 is the parent strain of Winter4 bacterial strain, and FM-23, M2, FJ-1, ZS are other monascus strain.

Figure 11 is that mutant strain and wild strain 3 kinds of pigment productions contrast.

Concrete embodiment

In order to understand the present invention, below in conjunction with embodiment, the invention will be further described: following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.

Winter4 monascus strain used in this application is the bacterial strain of applicant oneself screening, and be now preserved in University Of Science and Technology Of Tianjin's food nutrition and key lab of safety education portion, preserving number is: FM-46.

The application utilizes homologous recombination technique to carry out fixed point to wild-type monascus strain by construction recombination plasmid and knocks out, that the functional gene ORF3 (monooxygenase gene) utilizing hygromycin gene to substitute in citrinin biosynthesis gene bunch realizes, XhoI restriction enzyme site is contained in hygromycin gene upstream, SmaI restriction enzyme site is contained in hygromycin gene downstream, distance between two restriction enzyme sites is 2513bp, and concrete steps are as follows:

(1) increase for needing the goal gene ORF3 knocked out to design primer respectively with downstream at its upstream.

(2) then utilize identical restriction enzyme to carry out enzyme cut to the upstream and downstream fragment of ORFs with the pUCH2-8 plasmid of hygromycin gene mark, then connect, by this process, the upstream and downstream of goal gene are cloned on vector plasmid, construct recombinant plasmid.

(3) then recombinant plasmid is used for the conversion to wild red aspergillus protoplastis, the ORF3 gene order of monascus and recombinant plasmid generation homologous recombination, there are 2 homologous recombination respectively in 2 homology arms of recombinant plasmid with the homologous sequence on genome, thus make hph fragment displace ORF3 fragment, finally make gene inactivation.

(4) by screening and pcr amplification, mutant strain is verified.Then detect the situation of the pigment generation of mutant strain and the synthesis of Citrinin, finally obtain the monascus Winter4 mutant strain of ORF3 gene successful knockout.

Present invention also offers the primer needed for amplification ORF3 upstream and downstream and restriction enzyme site thereof.

Present invention also offers the host cell of carrier containing said gene and carrier.Present invention also offers ORF3 gene upstream and downstream sequence, the gene of ORF3, the carrier containing this gene, the host cell containing this carrier knocking out the effect in ORF3.

Experimental technique in following examples, if no special instructions, is ordinary method.

The reagent material used in following examples, if no special instructions, is the purchase of routine biochemistry reagent suppliers and obtains.

Embodiment 1

Containing the structure of gene ORF3 recombinant plasmid

(1) extraction of pUCH2-8 plasmid

Be inoculated in the 5mLLB liquid nutrient medium containing 100 μ g/mLAmp by the frozen intestinal bacteria containing pUCH2-8 plasmid, 37 DEG C of shaken overnight are cultivated.Within second day, take out bacterium liquid, extract pUCH2-8 plasmid with the little extraction reagent kit of plasmid, step is as follows:

A. get the bacterium liquid of 1-5mL incubated overnight, add in centrifuge tube, the centrifugal 3min of 12000rpm, exhausts supernatant as far as possible;

B. in the centrifuge tube of collecting precipitation, add 250 μ LBufferP1, use liquid-transfering gun or turbula shaker to suspend evenly by bacterial precipitation;

C. in centrifuge tube, add 250 μ LBufferP2, leniently put upside down mixing 5-6 time, make the abundant cracking of thalline.This step bacterium liquid should limpid thickness.Time used must not more than 5min, in order to avoid damage plasmid;

D. in centrifuge tube, adding 350 μ LBufferN3, now there is white flock precipitate in mixing 4-6 time of leniently turning upside down immediately;

E.13000rpm centrifugal 10min, draws supernatant, joins and load in the SpinColumnCM of CollectionTube, notes not sucking-off precipitation;

F.13000rpm centrifugal 30-60s, outwells the waste liquid in CollectionTube, is put back to by SpinColumnCM in CollectionTube;

G. in SpinColumnCM, add 700 μ LBufferPW, the centrifugal 30-60s of 12000rpm, outwells the waste liquid in CollectionTube, is placed back in by SpinColumnCM in CollectionTube;

H. in SpinColumnCM, add 700 μ LBufferPW, the centrifugal 1min of 12000rpm, outwells the waste liquid in CollectionTube, is placed back in by SpinColumnCM in CollectionTube;

I.13000rpm centrifugal 1min, outwells waste liquid.SpinColumnCM is uncapped and is placed in room temperature number minute, thoroughly to dry BufferPW remaining on adsorption film;

J. SpinColumnCM is placed in a new centrifuge tube, the unsettled dropping in the middle part to adsorption film 50-100 μ L sterilized water, room temperature places the centrifugal 2min of 2min, 14000rpm, is collected by plasmid solution in centrifuge tube, preserves plasmids for-20 DEG C.

(2) amplification of goal gene upstream and downstream fragment

According to ORF3 upstream and downstream gene order design of amplification primers, primer sequence is as follows:

The monascus wild strain genomic dna obtained with extraction is for template, and with the upstream gene fragment of primer ORF3-1 and ORF3-2 amplification ORF3, with the downstream gene fragment of primer ORF3-3 and ORF3-4 amplification ORF3, PCR system is as follows:

Wherein, during amplification fragment upstream: F, R are respectively ORF3-1, ORF3-2.During amplification segments downstream, F, R refer to ORF3-3, ORF3-4. respectively

The reaction conditions of PCR is:

Wherein, annealing temperature is different according to the difference of amplimer, and actual temp is:

(3) the enzyme of carrier pUCH2-8 and upstream and downstream gene fragment is cut, and it is as follows that carrier and fragment enzyme cut system, 37 DEG C of incubation 3h.

Wherein, the restriction endonuclease kind used is different, specific as follows because fragment is different:

ORF3 upstream ApaIXhoI

ORF3 downstream SmaIXbaI

(4) DNA purifying recovery test kit reclaims, and concrete steps are:

A. column equilibration step: the centrifugal 60s of the balance liquid BL adding 500 μ L to (adsorption column puts into collection tube) in adsorption column CB2,12000rpm, outwells the waste liquid in collection tube, is placed back in by adsorption column in collection tube;

B. according to the volume of PCR reaction solution or endonuclease reaction liquid, add equimultiple bulk solution PC wherein, fully mix;

C. add in an adsorption column CB2 (adsorption column puts into collection tube) by previous step gained solution, room temperature places the centrifugal 60s of 2min, 12000rpm, outwells the waste liquid in collection tube, adsorption column is put into collection tube;

D. in adsorption column CB2, add 700 μ L rinsing liquid PW, the centrifugal 1min of 12000rpm, outwells the waste liquid in collection tube, and adsorption column CB2 is put into collection tube;

E. repeating step d once;

F. put back in collection tube by adsorption column CB2, the centrifugal 2min of 12000rpm, removes rinsing liquid as far as possible.Adsorption column CB2 is placed in room temperature and places several minutes, dry up hill and dale;

G. adsorption column CB2 is put into a clean centrifuge tube, to the sterilized water that the unsettled dropping in adsorption film mid-way is appropriate, room temperature places 5min.The centrifugal 2min of 13000rpm collects DNA solution.

(5) connection of carrier pUCH2-8 and goal gene

Linked system is as follows, overnight incubation under 16 DEG C of conditions.

Embodiment 2

The qualification of ORF3-PCUH2-8 recombinant plasmid

(1) recombinant plasmid transformed competence colibacillus cell DH5 α

A. DH5 α competent escherichia coli cell 100 μ L is melted in ice bath, then be connected product with 20 μ L carriers and mix.

B. ice bath 30min.

C.42 DEG C heat shock 60s, leaves standstill centrifuge tube rapidly and is placed on ice, ice bath 2min.

D. 800 μ LLB liquid nutrient mediums are added, mixing, 37 DEG C, 250rpm shaking culture 1h.

E. by cultured bacterium liquid under room temperature, the centrifugal 5min of 5000rpm, removes section top substratum, leave 100 μ about L substratum, again hanged cell, be uniformly coated on the LB flat board containing Amp, just be put in 37 DEG C of constant incubators after 1h, be inverted overnight incubation.

(2) bacterium colony PCR screens recombinant plasmid

After the competent cell transformed is longer, the single bacterium colony of picking, is dissolved in 10 μ L sterilized waters and mixes, and getting 1 μ L is that template carries out PCR, and PCR system is as follows:

Wherein, when screening fragment upstream: F, R are respectively ORF3-1, ORF3-2.During screening segments downstream, F, R refer to ORF3-3, ORF3-4. respectively

PCR primer loading is carried out agarose gel electrophoresis detection.

Contained in 5ml in the LB liquid nutrient medium of 100 μ g/mLAmp by the colony inoculation filtered out, 37 DEG C of overnight shakings are divided into two parts after cultivating: a extraction recombinant plasmid, detect and double digestion checking for follow-up PCR; Another part is stored in-80 DEG C of conservations (80% glycerine of 800 μ L bacterium liquid+200 μ L sterilizings, mixing).

(3) PCR verifies recombinant plasmid

Extraction plasmid is carried out PCR detection as template, and PCR condition is with Rib gene amplification, and PCR reaction system is composed as follows:

Wherein, when verifying fragment upstream: F, R are respectively ORF3-1, ORF3-2.During checking segments downstream, F, R refer to ORF3-3, ORF3-4. respectively

(4) double digestion checking recombinant plasmid

Double digestion checking system:

Wherein, the use of restriction endonuclease is consistent with restriction endonuclease used when endonuclease bamhi during structure plasmid and plasmid.The result of PCR and double digestion is detected by agarose electrophoresis.

Embodiment 3

The recombinant plasmid transformed of monascus

(1) preparation of monascus protoplastis

A. monascus is seeded to slant medium, cultivates 7 days for 30 DEG C;

B. get two pipe inclined-planes, add 5ml aqua sterilisa respectively, gently scrape phage surface with disinfection inoculation ring, release spore;

C.10ml spore liquid is seeded in 100ml Spore cultivation base, 30 DEG C, and 170rpm cultivates 40h;

D. filter with individual layer miracloth and obtain mycelium, with enzymolysis buffer solution mycelium 2 times;

E. mycelium is joined in enzymolysis solution, 30 DEG C, 100rpm, concussion digestion 4h;

F. mycelia liquid filter 23 layer miracloth, filtrate 1000g after enzymolysis, 10 DEG C, centrifugal 20min, collects protoplastis;

G. remove supernatant liquor, carefully hanged precipitation with 10mlSTCbuffer, 1000g, 10 DEG C, centrifugal 10min;

H. repeat 7 once, remove supernatant;

I. the STCbuffer adding about 0.5ml has hanged precipitation, microscopy.

(2) conversion of protoplastis

A. the concentration adjusting protoplastis is 6 × 10 ⁷individual/ml, removes 400 μ L protoplastiss, adds 10 μ g plasmid DNA, ice bath 20min after mixing;

B. the PEG8000 solution of 100 μ L is added, ice bath 20min after mixing;

C. add 2mlPEG8000 solution, after mixing, room temperature (about 20 DEG C) places more than 5min;

D. use 4ml sorbyl alcohol (1M) solution dilution, the centrifugal 10min of 3000r/min, precipitation 2ml Sorbitol Solution USP suspends;

E. getting 200 μ L coats on the regeneration culture medium of 15ml, cultivates 12h for 30 DEG C;

F. cover the soft agar that 10ml contains 100 μ g/ml Totomycin on upper strata, cultivate 6-7 days observationss for 30 DEG C.

Embodiment 4

Transform the checking of mutant strain

Being forwarded on the malt extract medium containing 100 μ g/ml Totomycin by the mutant strain that grows out after transforming, can the judgement of continued growth be mutant strain.

Adopt CTAB method to extract mutant strain genome, it is verified then to utilize PCR primer pair.

Checking primer comprises the cross primer that two groups of amplified fragments primers form with amplification Totomycin primer, the checking primer that the front primer of amplification fragment upstream and the rear primer of amplification segments downstream form, and a pair checking primer according to the design of monascus insertion point context.

Verify that the specific name of correct bacterial strain is: monascus parpureus Went Monascuspurpureus, preserving number is: CGMCCNo.11010 preservation date: on July 15th, 2015, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.

Embodiment 5

Mutant strain produces the detection of Citrinin

Scraping spore from monascus inclined-plane, in access Spore cultivation base, 40h is cultivated in 30 DEG C of concussions, and then get in 10ml spore liquid access thalline enlarged culturing base, 30 DEG C of concussions are cultivated.The sampling in 6th day of cultivating.

Accurately take solid sample (mycelium after oven dry) Mg, add 20mlTEF solution (toluene: ethyl acetate: formic acid=7:3:1) (thalline and TEF solution proportion are about 1:10), supersound process 10min (working strength 40%, work 5s, interval 5s), then the centrifugal 20min of 3000r/min, residue 15mlTEF solution extracts 2 times as stated above, supernatant liquor is merged, after 40 DEG C of vacuum rotating evaporates to dryness, chromatogram methanol constant volume is to Vml, and after 0.22 μ L membrane filtration, high performance liquid chromatography detects.

The working method that chromatogram detects is with reference to standard GB/T/T5009.222-2008.

Chromatographic condition is: C18 chromatographic column (250mm × 4.6mm, granularity 5 μ L); Column temperature 28 DEG C; Moving phase is acetonitrile+water=35+65, water phosphoric acid adjust pH to 2.5; Flow velocity 1.5ml/min; Fluorimetric detector, excitation wavelength 331nm, emission wavelength 500nm; Sample size 20 μ L.

In sample, Citrinin calculation formula is

$C1=C\×\frac{V}{M}$

Wherein, citrinin content (mg/kg) in C1-solid sample;

The Citrinin concentration (mg/L) that C – instrument detects;

The final constant volume of V-(mL).

Embodiment 6

Mutant strain produces the detection of pigment

Scraping spore from monascus inclined-plane, in access Spore cultivation base, 40h is cultivated in 30 DEG C of concussions, and then get in 10ml spore liquid access thalline enlarged culturing base, 30 DEG C of concussions are cultivated.The sampling in 6th day of cultivating.Remove supernatant by after centrifugal for fermented liquid 5000r/min 10min, thalline is dried to constant weight with 60 DEG C, accurately takes Mg mycelium, adds 70% ethanolic soln of Vml, 60 DEG C of water-bath 1h, filters, and filtrate detects OD respectively with after 70% ethanolic soln dilution certain multiple ₅₀₅, OD ₄₆₅, OD ₄₁₀, obtain redness, orange, the look valency of xanthein according to formulae discovery.

In sample, look valency calculation formula is

Wherein, the absorbancy of OD-diluent;

V1-soaks mycelial 70% ethanol contend;

M-detects and weighs mycelial quality (g)

By contrasting the cultivation of bacterial strain after original strain and transformation: cultivate in 2% wort, 2% glucose, 0.1% protein culture medium, compared with parent strain, Citrinin output reduces 96.48%, yellow pigment output increased 89.9%; Citraurin output increased 172.1%; Haematochrome output increased 140.6%.

Claims (4)

1. a plant height produces the monascus Winter4 bacterial strain of pigment ability, this strain classification name is called: monascus parpureus Went Monascuspurpureus, preserving number is: CGMCCNo.11010 preservation date: on July 15th, 2015, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City Institute of Microorganism, Academia Sinica.

2. the monascus Winter4 bacterial strain of high yield pigment ability according to claim 1, it is characterized in that: cultivate in liquid medium within, compared with parent strain, Citrinin output is reduced to 3.52% of parent strain, and yellow pigment, citraurin, haematochrome improve 89.9%-172.1%.

3. the monascus Winter4 bacterial strain of high yield pigment ability according to claim 1, it is characterized in that: XhoI restriction enzyme site is contained in described hygromycin gene upstream, SmaI restriction enzyme site is contained in hygromycin gene downstream, the distance between two restriction enzyme sites is 2513bp.

4. according to bacterial strain according to claim 1 preparing Red kojic rice, slightly carry monascorubin, essence proposes application in monascorubin.