CN109371053A

CN109371053A - A kind of High-productive Monascus Pigment Strain construction method

Info

Publication number: CN109371053A
Application number: CN201811581705.1A
Authority: CN
Inventors: 龙传南; 曾斌; 陶琴琴; 刘心怡; 刘梦梦; 彭玲; 程芳婷; 王淑琴; 吾蔚蔚
Original assignee: Jiangxi Science and Technology Normal University
Current assignee: Jiangxi Science and Technology Normal University
Priority date: 2018-12-24
Filing date: 2018-12-24
Publication date: 2019-02-22
Anticipated expiration: 2038-12-24
Also published as: CN109371053B

Abstract

The present invention provides a kind of High-productive Monascus Pigment Strain construction methods.A pair of oligonucleotide sequence successively containing Hind III, Kpn I, Sac I, Pac I, Pme I, Xho I, Xba I, Bgl II restriction enzyme site is connect by the technical solution with plant binary plasmid pCambia0380 first, and building obtains binary plasmid expression vector pCambia0380G；Then, hph expression cassette segment is attached thereto, obtains binary plasmid knockout carrier pHph0380；On this basis, the upstream and downstream homology arm segment for expanding gltp1 gene respectively, is connected on binary plasmid knockout carrier pHph0380, to obtain binary plasmid knockout carrier pHph0380-GLTP.Carrier pHph0380-GLTP is completed into the building of superior strain into parent's red monascus CICC41233 by Agrobacterium tumefaciems EHA105 mediated transformation.Experimental verification shows that High-productive Monascus Pigment Strain constructed by the present invention not only increases the overall productivity of monascorubin, and orientable accumulation alcohol-soluble pigment, meanwhile, shift to an earlier date the integration time of monascorubin in fermentation process significantly.

Description

A kind of High-productive Monascus Pigment Strain construction method

Technical field

The present invention relates to industrial microbial technology fields, further to technique for gene engineering and the fermentation skill of mould A kind of art, and in particular to High-productive Monascus Pigment Strain construction method.

Background technique

Monascorubin is by microorganism monascus ruber (Monasus spp.), using rice as natural colour made of fermenting raw materials Element has more than 1,000 years history in China.As food additives, monascorubin is widely used in food processing and cosmetics system Make equal fields；Because it also has extensive bioactivity such as Adjust-blood lipid, blood pressure lowering, anti-vascular sclerosis, anti-diabetic, inhibition fertilizer Fat, anti-inflammatory, antiallergy, anti-peroxidating, anticancer, antibacterium, antimycotic etc., it is developed and medical field in prebiotic health product Application be also increasingly taken seriously.

Monascorubin is the secondary metabolite of monascus, is completed jointly by fatty acid synthesis pathway and polyketone route of synthesis Anabolism.Its chemical structure is broadly divided into polyketone and fatty acid chain two parts.Before the synthesis of fatty acid chain is with acetyl-CoA Body acts on through fatty acid synthase (Fatty acid synthase, FAS) complex, forms middle length by a series of synthetic reactions Chain fatty acid reacts with acetyl-CoA and generates beta-keto acid；The synthesis of polyketone is equally using acetyl-CoA as precursor, in polyketide synthase Under (Polyketide synthase, PKS) effect, poly ketoboidies compound is successively synthesized, is ultimately formed with chromophore Polyketone.Esterification occurs for the carboxylic group of polyketone and the hydroxyl group of fatty acid chain, to form monascorubin.More than being based on Principle, in order to improve the yield of monascorubin, the prior art generally realizes the metabolism tune of monascus by zymotechnique improvement Control, and then orient accumulation purpose product；Although the optimization of fermentation condition can improve monascorubin yield to a certain extent, by It is limited to the metabolic characteristic of monascus itself, monascorubin accumulation is difficult further to improve.

In addition, the produced monascorubin of monascus is divided into alcohol-soluble pigment and water colo(u)r.Alcohol-soluble pigment is monascus It directly synthesizes, is present in intracellular during the fermentation；Water colo(u)r is ammonia in the pigment and fermentation liquid of monascus itself synthesis The substances such as base acid combine the compound pigment formed, are distributed in extracellular.In the growth course of Natural strains, both monascorubins There is generation, cannot achieve orientation by cultural method merely and produce one of monascorubin.

In addition, course of fermentation is the key factor for influencing integrated artistic duration in the industrialized production of monascin, by Monascin is just accumulated in the secondary metabolite that monascin is monascus, therefore after fermentation starts a period of time.At this In the case of kind, if can be expected to shorten technique duration by genetic recombination or metabolic regulation means by the starting time advance, Improve production efficiency.

Summary of the invention

The present invention is directed to be directed to the technological deficiency of the prior art, a kind of High-productive Monascus Pigment Strain construction method is provided, with Solve the lower technical problem of the monascorubin yield of common red monascus in the prior art.

Another technical problem to be solved by the present invention is that common red monascus is difficult to orient accumulation alcohol-soluble monascorubin.

The invention solves another technical problem be common red monascus during the cultivation process, monascin accumulation It is later to start the time.

To realize the above technical purpose, the invention adopts the following technical scheme:

A kind of High-productive Monascus Pigment Strain construction method, comprising the following steps:

1) plant binary plasmid pCambia0380 is taken, with restriction endonuclease Hind III and Bgl II digestion, and A pair of oligonucleotide sequence as shown in SEQ ID No:1, SEQ ID No:2 is attached thereto by T4DNA ligase afterwards, i.e., Obtain binary plasmid expression vector pCambia0380G；

2) using plasmid pMD19-PgpdA-hph-TtrpC as template, with sequence such as SEQ ID No:3, SEQ ID No:4 institute The pair of primers shown executes PCR amplification, obtains hph expression cassette segment, simultaneously with restriction endonuclease Sac I and Xho I Hph expression cassette segment described in digestion and binary plasmid expression vector pCambia0380G then pass through T4DNA ligase for two Person connects to arrive binary plasmid knockout carrier pHph0380；

3) using red monascus CICC41233 total DNA as template, with sequence such as SEQ ID No:9, SEQ ID No:10 institute The pair of primers shown executes PCR amplification, obtains the upstream homology arm segment of saccharide transporter gltp1 gene；With red monascus CICC41233 total DNA is template, executes PCR with sequence pair of primers as shown in SEQ ID No:11, SEQ ID No:12 and expands Increase, obtains the downstream homology arm segment of saccharide transporter gltp1 gene；The upstream homology arm segment and the downstream is homologous Arm pieces section is respectively connected on the carrier pHph0380 arrive binary plasmid knockout carrier pHph0380-GLTP；

4) the Agrobacterium tumefaciems EHA105 for preparing competence, by frozen-thawed method by the binary plasmid knockout carrier PHph0380-GLTP imports Agrobacterium tumefaciems EHA105, then by the root containing binary plasmid knockout carrier pHph0380-GLTP Cancer Agrobacterium EHA105 is converted into red monascus CICC41233, screening positive clone to get arrive the superior strain.

Preferably, the Agrobacterium tumefaciems EHA105 of step 4) the preparation competence, comprising the following steps: by crown gall agriculture Bacillus EHA105 is inoculated in 5~10mL and contains in the YEP fluid nutrient medium of 50 μ g/mL rifampins, with 28 DEG C of temperature, speed of agitator The CMC model of 200rpm is for 24 hours；The bacterium solution for taking l mL to activate is inoculated in the YEP Liquid Culture that 20mL contains 50 μ g/mL rifampins In base, with 28 DEG C of temperature, speed of agitator 200rpm CMC model to bacterium solution OD₆₀₀Value 0.5；After bacterium solution ice bath 30min, 4 5min is centrifuged with the revolving speed of 5000rpm under the conditions of DEG C, abandons supernatant；Precipitating is resuspended with the sodium chloride solution l0mL of 0.15mmol/L, 5min is centrifuged with the revolving speed of 5000rpm under the conditions of 4 DEG C, supernatant is abandoned, is then suspended in the calcium chloride solution 1mL of 20mmol/L In.

Preferably, described import root for the binary plasmid knockout carrier pHph0380-GLTP by frozen-thawed method Cancer Agrobacterium EHA105, comprising the following steps: binary plasmid knockout carrier pHph0380-GLTP described in 1 μ g is taken to be added to 200 μ L In the Agrobacterium tumefaciems EHA105 of competence, ice bath 30min after mixing；Quick-frozen 1min in liquid nitrogen, 37 DEG C of water-bath 3min, then ice bath 2min；800 μ L YEP fluid nutrient mediums, 28 DEG C of culture 3h are added；3min is centrifuged with the revolving speed of 5000rpm under room temperature, bacterium is concentrated Body；Bacterium solution after taking 200 μ L to be concentrated is coated on the YEP containing 50 μ g/mL rifampins, 50 μ g/mL kanamycins and selectively cultivates On base plate, 2d is cultivated in 28 DEG C of inversions；It selects transformant to cultivate in YEP fluid nutrient medium, and is carried out with primer pair clone Screening, obtains positive clone molecule, as the Agrobacterium tumefaciems EHA105 containing binary plasmid knockout carrier pHph0380-GLTP.

Preferably, described turn the Agrobacterium tumefaciems EHA105 containing binary plasmid knockout carrier pHph0380-GLTP Change into red monascus CICC41233, comprising the following steps:

Red monascus CICC41233 is taken, with MPS solid medium culture 7d, conidium is obtained, with sterile aqueous suspension Spore, vibrating dispersion spore are filtered through 2 layers of lens wiping paper, adjust spore concentration；

The Agrobacterium tumefaciems EHA105 containing binary plasmid knockout carrier pHph0380-GLTP is taken, 3mL is inoculated in and contains 50 μ g/mL rifampin, 50 μ g/mL kanamycins YEP culture medium in, 28 DEG C of culture 48h then transfer and contain 200 μ in 5mL In the AIM induced medium of mol/L acetosyringone, bacterium solution is made to be diluted to OD₆₀₀Value is 0.15, is cultivated for 5~6h, until OD₆₀₀Value is 0.5~0.6；

By red monascus spore liquid obtained as above and crown gall containing binary plasmid knockout carrier pHph0380-GLTP Agrobacterium EHA105 bacterium solution, mixing are coated on the AIM induced medium plate containing 200 μm of ol/L acetosyringones, 25 DEG C, keep away Optical culture 48h.

Preferably, the screening positive clone, comprising the following steps: the AIM induced medium after being protected from light culture 48h One layer of MPS culture containing 100 μ g/mL hph, 200 μm of ol/L cefotaxime, 0.2%Triton X-100 is added on plate again Base, 30 DEG C are continued 5~8d of culture；Picking single colonie is forwarded in the MPS solid medium tablets containing 100 μ g/mL hph, culture The bacterial strain that will be grown after 3d is connected in MPS fluid nutrient medium and cultivates, according to SDS cracking process extract filamentous fungi total DNA into Row analysis of molecules, with sequence be SEQ ID No:15, the pair of primers of SEQ ID No:16 carries out PCR verifying, chooses positive bacteria Strain is the superior strain.

In above technical scheme, the plant binary plasmid pCambia0380, red monascus CICC41233, crown gall Agrobacterium EHA105, the biomaterial for belonging to conventional commercial can be bought from market；Wherein, red monascus CICC41233 Purchased from Chinese industrial microbial strains preservation administrative center.

In above technical scheme, the plasmid pMD19-PgpdA-hph-TtrpC can voluntarily be prepared according to the prior art, Its specific preparation method is referred to bibliography " de Groot MJA, Bundock P, Hooykaas PJJ, et al.1998.Agrobacterium tumefaciens-mediated transformation of filamentous Fungi.Nature Biotechnology, 16:839-842 " implementation.

In above technical scheme, the nucleotide sequence of gltp1 gene is as shown in SEQ ID No:17；GLTP1 amino acid The amino acid sequence of sequence such as SEQ ID No:18.

In above technical scheme, the formula of the YEP culture medium is as follows: 5.0g peptone, 1.0g yeast extract, 5.0g sucrose, 5.0g beef extract, 0.24g magnesium sulfate, pH 7.2；2% agar powder is then separately added if solid medium.

The MPS culture medium prescription is as follows: 10g/L malt extract, 10g/L peptone, 40g/L soluble starch；If 2g/L agar is then separately added for solid medium.

Described to state AIM Fiber differentiation based formulas as follows: (1.25mol/L, pH 4.8, uses phosphoric acid to 0.8mL kaliumphosphate buffer Potassium dihydrogen and dipotassium hydrogen phosphate are prepared), 0.6g MgSO₄.7H₂O, 0.3g NaCL, 1mL CaCL₂(1%), 1mL FeSO₄ (1mg/mL), 1mL (NH₄)₂SO₄(0.33g/L), 10mL glycerol (50%), 40mL MES (pH value 5.5, adjusted with NaOH), 5mL Microelement storing liquid, 1mL CaCL₂(1%), 2g/L glucose (fluid nutrient medium use), 1g/L glucose (solid medium With).PH value 5.4；2% agar powder is then separately added if solid medium.

The present invention provides a kind of High-productive Monascus Pigment Strain construction methods.The technical solution first successively contains a pair Hind III, Kpn I, Sac I, Pac I, Pme I, Xho I, Xba I, the oligonucleotide sequence of Bgl II restriction enzyme site and plant The pCambia0380 connection of object binary plasmid, building obtain binary plasmid expression vector pCambia0380G；Then, hph is expressed Box segment is attached thereto, and obtains binary plasmid knockout carrier pHph0380；On this basis, respectively expand gltp1 gene it is upper, Downstream homology arm segment is connected on binary plasmid knockout carrier pHph0380, to obtain binary plasmid knockout carrier pHph0380-GLTP.By carrier pHph0380-GLTP by Agrobacterium tumefaciems EHA105 mediated transformation to parent's red monascus In CICC41233, the building of superior strain is completed.Experimental verification shows High-productive Monascus Pigment Strain constructed by the present invention not The overall productivity of monascorubin, and orientable accumulation alcohol-soluble pigment are improved only, meanwhile, make monascorubin in fermentation process Integration time significantly shift to an earlier date, production capacity is better than parent's red monascus CICC41233 comprehensively.

Detailed description of the invention

Fig. 1 is the electrophoretogram of gltp1 gene PCR amplified fragments in monascus CICC41233.

Fig. 2 is monascus gltp1 gene knockout schematic diagram.

Fig. 3 is the PCR qualification result electrophoretogram of gene knockout engineered strain；Wherein, swimming lane 1-2: expanded using primers F 3&R3 Increase gltp1 gene；Swimming lane 3-4: selection markers hph gene is expanded using primers F 4&R4；Swimming lane 5-6: it is tested using primers F 5&R5 Card hph is integrated into gltp1 gene loci；Swimming lane 1,3,5: monascus CICC41233 total DNA is template；Swimming lane 2,4,6: it knocks out Bacterial strain monascus GLTP24 total DNA is template.

Fig. 4 is figure compared with red monascus CICC41233 ferments with red monascus GLTP24 and produces monascorubin result；Its In, (a) monascus different fermentations time phenotype；(b) different fermentations time remaining starch content；(c) monascorubin color value；(d) Spectral scan water colo(u)r；(e) spectral scan alcohol-soluble pigment.

Fig. 5 is figure compared with red monascus CICC41233 produces monascorubin result with red monascus covering bacterial strain.

Specific embodiment

Below by specific embodiments of the present invention will be described in detail.In order to avoid excessive unnecessary details, It will not be described in detail in following embodiment to belonging to well known structure or function.Approximation used in following embodiment Language can be used for quantitative expression, show to allow quantity to have certain variation in the case where not changing basic function.It is fixed except having Adopted outer, technical and scientific term used in following embodiment has the phase being commonly understood by with those skilled in the art of the invention Same meaning.

Used partial gene fragments and primer are as shown in table 1 in following embodiment:

The title and sequence of 1 partial gene fragments of table and primer

In table 1, title suffix is the expression forward primer of F, and title suffix is that R is reverse primer.

Embodiment 1

1, binary plasmid knockout carrier pHph0380 is constructed.

It is initial carrier according to commercialized plant binary plasmid pCambia0380.1 couple of oligonucleotide sequence F&R is designed, Sequence has the synthesis of Shanghai Sheng Gong bioengineering Co., Ltd.The sequence successively contains following restriction endonuclease site (Hind III,Kpn I,Sac I,Pac I,Pme I,Xho I,Xba I,Bgl II).Using restriction endonuclease Hind III and Bgl II digestion binary plasmid carrier pCambia0380.By T4DNA ligase, by oligonucleotide sequence with The pCambia0380 connection of digestion carrier obtains binary plasmid carrier pCambia0380G.

Using the plasmid pMD19-PgpdA-hph-TtrpC of laboratory preservation as template, using primer PgpdA-Sac I-F& TtrpC-Xho I-R expands to obtain hph expression cassette.Using restriction endonuclease Sac I and Xho I digestion hph table simultaneously Up to box segment and binary plasmid carrier pCambia0380G.By T4DNA ligase, by hph expression cassette segment and binary plasmid Carrier pCambia0380G connection obtains binary plasmid knockout carrier pHph0380.

2, monascus gltp1 gene cloning, binary plasmid expression vector pNeo0380-GLTP building

According to the gene order (924bp) that transcript profile sequencing provides, design primer Gltp1-JYF-HindIII (contains HindIII) with Gltp1-JYR-SacI (containing SacI) (sequence is shown in Table 1), with red monascus CICC41233, total DNA is mould Plate, PCR amplification obtains genetic fragment (Fig. 1), and is sequenced.Sequencing is correctly met into target fragment, using HindIII and SacI enzyme It cuts PCR fragment and recycles.It digestion pNeo0380 carrier and recycles simultaneously.

Digestion pNeo0380 recycling segment is connect with digestion recycling gltp1 genetic fragment using T4DNA ligase, is converted In E.coli DH5 α competent cell, picked clones is cultivated in LB liquid medium, and is sieved with primer pair clone Choosing.After extraction plasmid and digestion verification, the carrier built are named as pNeo0380-GLTP.

3, monascus gltp1 gene upstream and downstream homology arm fragment amplification and binary plasmid knockout carrier pHph0380- GLTP building.

According to the gltp1 gene order that clone obtains, divide in Monascus ruber NRRL1597 genomic data Downstream sequence thereon is found in analysis.With red monascus CICC41233, total DNA is template, design primer Gltp-QC-UF-Pst I&Gltp-QC-UR-Sac I PCR amplification goes out the upstream 1740bp arm pieces section；Gltp-QC-DF-Bgl II&Gltp-QC-DR-Spe I PCR amplification goes out 1764bp downstream arm segment (sequence is shown in Table 1).Sequencing is correctly met into target fragment, even using digestion enzyme Method is connected respectively on pHph0380 carrier, which is named as pHph0380-GLTP.Monascus gltp1 base It is as shown in Figure 2 because knocking out principle.

4, the building of recombinant bacterial strain red monascus GLTP24

The preparation of 4.1 Agrobacterium tumefaciems competent cells

1. Agrobacterium EHA105 is inoculated in 5~10mL YEP fluid nutrient medium (containing 50 μ g/mL rifampins), 28 DEG C, 200r/min is cultivated for 24 hours.

2. the bacterium solution for taking l mL to activate is inoculated in 20mL and contains in the YEP culture medium of identical antibiotic, trained under similarity condition (about 4h) is supported to OD600 value 0.5.

3. 4 DEG C, being centrifuged (5000r/min, 5min) after bacterium solution ice bath 30min, collecting thallus.

4. abandoning supernatant, it is resuspended and is precipitated with 0.15mmol/L sodium chloride ice cold solution l0mL, is collected by centrifugation under similarity condition Then thallus is suspended in 20mmol/L calcium chloride ice cold solution 1mL.

5. being dispensed by every 200 μ L of pipe, liquid nitrogen flash freezer 1min.

6. bacteria suspension at this time can be converted directly, -70 DEG C of refrigerator-freezers can also be stored in and saved backup.

Above-mentioned YEP culture medium: 5.0g peptone, 1.0g yeast extract, 5.0g sucrose, 5.0g beef extract, 0.24g sulfuric acid Magnesium, pH 7.2.Solid medium adds 2% agar powder again.

4.2 frozen-thawed methods are by binary plasmid vector introduction Agrobacterium tumefaciems

1. 1 μ g binary plasmid carrier pHph0380-GLTP is taken to be added to the Agrobacterium competent cell that 200 μ L dissolve on ice In, light mixed, ice bath 30min.

2. quick-frozen 1min in liquid nitrogen, 37 DEG C of water-bath 3min, then rapid ice bath 2min.

3. 800 μ L YEP fluid nutrient mediums are added, 28 DEG C of culture 3h.

4. being centrifuged (5000r/min, 3min) under room temperature, suitably concentration thallus.

5. take 200 μ L bacterium solutions to be coated on YEP selection plate (containing 50 μ g/mL rifampins, 50 μ g/mL kanamycins), 2d is cultivated in 28 DEG C of inversions.

6. selecting transformant to cultivate in YEP fluid nutrient medium, and screened with primer pair clone, obtains positive gram Longzi.

4.3 Agrobacterium-Mediated Transformation red monascus CICC41233

(1) thallus prepares

Red monascus CICC41233:MPS solid medium culture 7d obtains conidium, with sterile aqueous suspension spore Son, vibrating dispersion spore filter through 2 layers of lens wiping paper, adjust suitable spore concentration.

Agrobacterium tumefaciems: the Agrobacterium inoculation containing binary plasmid carrier (is contained into 50 μ g/ in 3mL YEP culture medium ML rifampin, 50 μ g/mL kanamycins), then 28 DEG C of culture 48h transfer in containing 200 μm of ol/L acetosyringones (AS) In 5mL AIM induced medium, bacterium solution is made to be diluted to OD₆₀₀It is 0.15, is cultivated for 5~6h, until OD₆₀₀It is 0.5~0.6.

Above-mentioned MPS culture medium: 10g/L malt extract, 10g/L peptone, 40g/L soluble starch.Solid medium: Separately add 2g/L agar.

(2) Agrobacterium and red monascus CICC41233 are co-cultured

Prepare AIM induced medium plate (containing 200 μm of ol/L AS).By Agrobacterium and red monascus CICC41233 (pressing equal proportion).Mixture is coated on AIM plate, and 25 DEG C, avoid light place culture 48h.

(3) transformant screening is verified

Adding one layer of screening and culturing medium again on AIM culture medium flat plate, (MPS culture medium contains 100 μ g/mL hph, 200 μ Mol/L cefotaxime, 0.2%Triton X-100).30 DEG C, continue 5~8d of culture.By the single colonie bacterial strain of growth, it is forwarded to Another MPS solid plate (containing hph) is observed after cultivating 3d, and bacterial strain still is able to grow；The bacterial strain that will be grown, is connected to It is cultivated in MPS fluid nutrient medium, extracts filamentous fungi total DNA according to SDS cracking process and carry out analysis of molecules.Using primer pair (figure 3) PCR verifying is carried out.It determines one plant of bacterial strain, is engineered strain red monascus (Monascus ruber) of the invention GLTP24。

Above-mentioned AIM culture medium: 0.8mL kaliumphosphate buffer (1.25mol/L, pH 4.8, with potassium dihydrogen phosphate and phosphoric acid hydrogen Dipotassium is prepared), 0.6g MgSO₄.7H₂O, 0.3g NaCL, 1mL CaCL₂(1%), 1mL FeSO₄(1mg/mL), 1mL (NH₄)₂SO₄(0.33g/L), 10mL glycerol (50%), 40mL MES (pH value 5.5, adjusted with NaOH), 5mL microelement storing liquid, 1mL CaCL₂(1%), 2g/L glucose (fluid nutrient medium use), 1g/L glucose (solid medium use).PH value 5.4.Solid Culture medium adds 2% agar powder again.

5, red monascus GLTP24 and parent plant CICC41233 fermentation produces the comparison of monascorubin ability

5.1 use the culture of MPS solid medium

By red monascus GLTP24 and red monascus CICC41233, after MPS solid medium culture 7d, collect Spore suspension is 1*10 according to spore inoculating amount⁵A/mL.Fermentation condition are as follows: 30 DEG C, 180rpm fermented to the 6th day.

Produce the fermentation medium component of monascorubin are as follows: 9.0% rice meal, 0.2%NaNO₃, 0.1%KH₂PO₄, 0.2%MgSO₄·7H₂O, 0.2% acetic acid.

The measurement of 5.2 monascorubin color values

Extracellular monascorubin (water-soluble) color value measurement: by fermentation liquid, constant volume freezes high speed centrifugation to 25mL in centrifuge tube (10 000rpm, 30min), supernatant is exo-cell pigment.A certain amount of filtrate water dilution suitable multiple is taken, is ginseng with water According to calculating its total color with the absorption angle value of spectrophotometric determination (the absorption main peak of the compound pigment of red yeast rice is in 505nm) dilution Valence.Calculation method are as follows: total color value=extension rate * absorbance.

Monascorubin (alcohol is molten) color value measurement intracellular: precipitating is collected after centrifugation in fermentation liquid, quiet at 60 DEG C with 70% ethyl alcohol Extraction 1h is set, during which vortex oscillation is for several times.It freezes high speed centrifugation (10 000rpm, 20min), supernatant is para chrome.It takes A certain amount of filtrate dilutes suitable multiple with 70% ethyl alcohol, and using 70% ethyl alcohol as reference, with spectrophotometric determination, (red yeast rice is compound The absorption main peak of pigment is in 505nm) the absorption angle value of dilution, calculate its total color value.Calculation method is same as above.

Red monascus GLTP24, ferment 36h, compares parent strain, and visual inspection generates a large amount of monascorubin, shows Produce the time advance (Fig. 4 a) of monascorubin.The molten color value of alcohol is 0.29U, compares parent's red monascus CICC41233, the molten color of alcohol Valence is 0.09U, improves 2 times (Fig. 4 c, 4e).Ferment 144h, and the molten color value of alcohol is 49.46U.Compared to parent's red monascus CICC41233, the molten color value of alcohol are 28.42U, improve 74% (Fig. 4 c, 4e).Water colo(u)r do not have marked difference (Fig. 4 c, 4d)

5.3 remaining starch assays

Configure I₂-KI(2.6g/L I₂, 5.0g/L KI) and solution, OD value is measured at wavelength 600nm, prepares soluble form sediment Powder standard curve.Fermentation liquid room temperature is centrifuged (10000r/min, 20min), measures the content of the starch in supernatant.Fig. 4 b table Bright, engineered strain red monascus GLTP24 can remarkably promote starch degradation metabolism.Ferment 36h, 48h, 144h, engineering bacteria Strain red monascus GLTP24 remaining starch content is respectively 0.56mg/mL, 0.15mg/mL, 0mg/mL；Red monascus CICC41233 residue 44.91mg/mL, 34.26mg/mL, 9.58mg/mL.

6, the building of bacterial strain is covered, and its ferments with parent strain monascus CICC41233 and produces pair of monascorubin ability Than

The acquisition of 6.1 covering bacterial strains

Binary plasmid expression vector pNeo0380-GLTP is passed through mediated by agriculture bacillus by the operating method of reference implementation example 4 Transformation Engineering bacterial strain red monascus GLTP24 (selection markers change G418 into) obtains covering bacterial strain.

6.2 monascorubin fermentabilities compare

By red monascus CICC41233 and 4 plants of covering bacterial strain monascus HU1, monascus HU2, monascus HU3, red yeast rice Mould HU4 collects spore suspension after MPS solid medium culture 7d, is 1*10 according to spore inoculating amount⁵A/mL.Ferment item Part are as follows: 30 DEG C, 180rpm fermented to the 6th day.

The measurement of 6.3 monascorubin color values

Red monascus CICC41233 and 4 plants of covering bacterial strain monascus HU1, monascus HU2, monascus HU3, monascus HU4, while the 6d that ferments.Monascorubin color value is as a result, compared with parent strain, no difference of science of statistics (such as Fig. 5).

Embodiment 2

1, binary plasmid knockout carrier pHph0380 is constructed

It is initial carrier according to commercialized plant binary plasmid pCambia0380.1 couple of oligonucleotide sequence F&R is designed, Successively contain following restriction endonuclease site (Hind III, Kpn I, Sac I, Pac I, Pme I, Xho I, Xba I,Bgl II).Using restriction endonuclease Hind III and Bgl II digestion binary plasmid carrier pCambia0380.It is logical T4DNA ligase is crossed, oligonucleotide sequence is connect with carrier, obtains binary plasmid carrier pCambia0380G.

Using the plasmid pMD19-PgpdA-hph-TtrpC of laboratory preservation as template, using primer PgpdA-Sac I-F& TtrpC-Xho I-R expands to obtain hph expression cassette segment.It is opened using restriction endonuclease Sac I and Xho I digestion simultaneously Promoter fragment and binary plasmid carrier pCambia0380G.By T4DNA ligase, oligonucleotide sequence is connect with carrier, Obtain binary plasmid knockout carrier pHph0380.

2, the building of high yield monascus pigment engineered strain red monascus GLTP24

The engineering strain that the present invention is building up to, classification naming are red monascus (Monascus ruber) GLTP24。

The present invention clones the homologous arm pieces of upstream and downstream of gltp1 gene using red monascus CICC41233 as parent strain Section, is implemented in binary plasmid carrier pHph0380, is mediated by Agrobacterium tumefaciems EHA105, and it is red to obtain engineering strain red Aspergillus GLTP24.

(1) binary plasmid knockout carrier pHph0380-GLTP is constructed

Using monascus CICC41233 total DNA as template, PCR amplification obtains the upstream and downstream homology arm segment of gltp1 gene. Then restriction endonuclease Pst I and SacI digestion upstream homology arm segment simultaneously are used, using restriction enzyme nucleic acid Enzyme Bgl II and Spe I digestion downstream homology arm segment and binary plasmid carrier pHph0380 simultaneously.It is connected by T4DNA Genetic fragment is connect by enzyme with carrier, obtains binary plasmid knockout carrier pHph0380-GLTP.

(2) Agrobacterium-Mediated Transformation red monascus CICC41233

Binary plasmid carrier pHph0380-GLTP is mediated using Agrobacterium tumefaciems EHA105, converts red monascus In CICC41233, engineered strain red monascus GLTP24 is successfully obtained.

3, engineering strain red monascus GLTP24 fermentation improves the yield of monascorubin

Red monascus CICC41233 and red monascus GLTP24, while fermenting and producing monascorubin.Fermentation medium group It is divided into: 9.0% rice meal, 0.2%NaNO₃, 0.1%KH₂PO₄, 0.2%MgSO₄·7H₂O, 0.2% acetic acid.Bacterial strain spore inoculating Amount is 1*10⁵A/mL.Fermentation condition are as follows: 30 DEG C, 180rpm fermented to the 6th day.The result shows that engineered strain fermentation produces red yeast rice color Plain time advance, while significantly promoting starch degradation metabolism.Ferment 36h, and the molten color value of red monascus GLTP24 alcohol is 0.29U compares parent's red monascus CICC41233, and the molten color value of alcohol is 0.09U, improves 2 times.Ferment 144h, the molten color value of alcohol For 49.46U.Compared to parent red monascus CICC41233, the molten color value of alcohol is 28.42U, improves 74%.

4, the building and fermentation of monascus covering bacterial strain produce monascorubin

For the effect for further studying the gene, using the DNA of monascus CICC41233 as template, PCR amplification obtains sugar and turns It transports protein gene (gltp1).Then restriction endonuclease HindIII and SacI digestion genetic fragment simultaneously are used, and Binary plasmid expression vector pNeo0380.By T4DNA ligase, genetic fragment is connect with carrier, obtains binary plasmid table Up to carrier pNeo0380-GLTP.Expression vector, Transformation Engineering bacterial strain red monascus are mediated using Agrobacterium tumefaciems EHA105 In GLTP24, covering bacterial strain red monascus HU1, red monascus HU2, red monascus HU3, red monascus are successfully obtained HU4.4 plants of red monascus cover bacterial strain, and produced monascorubin is compared with parent strain, without statistical difference.

The embodiments of the present invention have been described in detail above, but content is only the preferred embodiment of the present invention, It is not intended to limit the invention.All any modifications, equivalent replacements, and improvements etc. done in application range of the invention, should all It is included within protection scope of the present invention.

Sequence table

<110>Jiangxi Science & Technology Normal University

<120>a kind of High-productive Monascus Pigment Strain construction method

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<210> 2

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<212> DNA

<213>artificial sequence (Artificial sequence)

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gatcttctag actcgaggtt taaacttaat taagagctcg gtacca 46

<210> 3

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<212> DNA

<213>artificial sequence (Artificial sequence)

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gtgacgaact cgtgagctct gtac 24

<210> 4

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<212> DNA

<213>artificial sequence (Artificial sequence)

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ggacttctag actcgagaag aagga 25

<210> 5

<211> 22

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<213>artificial sequence (Artificial sequence)

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ctgcactcga cctgctgagg tc 22

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<213>artificial sequence (Artificial sequence)

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tattcttctc ttcgccggag cc 22

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<213>artificial sequence (Artificial sequence)

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cccaagctta tgtttgtccc cgaatcccca cg 32

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<212> DNA

<213>artificial sequence (Artificial sequence)

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cgagctctta aagggcctcg gccgaactg 29

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<213>artificial sequence (Artificial sequence)

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aactgcagcc atctgtatcc tccgtagtta caga 34

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cgagctcgct tacctgacaa tagcagctct tac 33

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gaagatctca gcttctaatg ttgttttgat attgg 35

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ggactagttc tgaatatgga tttcagtgat tatcc 35

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atgcctgaac tcaccgcgac 20

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<213>artificial sequence (Artificial sequence)

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cggtcggcat ctactctatt 20

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aggaatagag tagatgccga cc 22

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<213>artificial sequence (Artificial sequence)

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tccaagtgga caaacttgtg g 21

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<212> DNA

<213>red monascus (Monascus rubber)

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atgtttgtcc ccgaatcccc acgtttcctg taccgtcaag gcaaagtcga ggaggctcgt 60

caggtcatgg ccaaactggc tggtgtccca cccaaccatc gccagatcgt ctcggagatg 120

caggagatga aagagaagct cgacgaagag aaagccgccg gcaaggcacc ctggtatgag 180

gtgttcacgg gcccagccat gttctggcgg acaacgctcg gaatagtctt gcagtctctg 240

caacagctca ctggcgccaa cttcatcttc ttttacggta acactatctt ctatgccacc 300

ggccttgaga acagctacga aacccagatt attatgggaa ctgtcaattt cgccatgtcc 360

atcgtctccc tgtgggtcgt ccagcgcttc cgccgtcgtc ccatcctaat tattggtggc 420

atcgccatgt tcatctgttt cctgatcttc gcctccgtcg gccacttctc cctcgaccat 480

gaaaaccccc agaatacccc caaagcaggt acggcactta ttgtcttctc ctgcttcttc 540

atcgccgcat acgccgttag ctggggtcca cttatctggg ctatctgcgg tgaactgttc 600

ccatccaaat accgtgacgt ttgcgtcagc ttgagtaccg catcgaactg gacctggaac 660

ttcctactct gcttctttac gcccttcatc tccagcgcta tcgattatcg gtacggttat 720

gtcttcggcg catgttgtgc tgccggatcc atcatcacct acttcttcgt caacgaatcc 780

tacggccgga ccctcgagga aatcgacacc atgtacgtca tgcacgtcaa gccatggcag 840

agcaagaact gggttgctcc tcctgccatc cgaaacggcg agaacagggt ccccaacttg 900

gaaagccacg aagctactcc tgcacacagt tcggccgagg ccctttaa 948

<210> 18

<211> 315

<212> PRT

<213>red monascus (Monascus rubber)

<400> 18

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

1 5 10 15

Glu Glu Ala Arg Gln Val Met Ala Lys Leu Ala Gly Val Pro Pro Asn

20 25 30

His Arg Gln Ile Val Ser Glu Met Gln Glu Met Lys Glu Lys Leu Asp

35 40 45

Glu Glu Lys Ala Ala Gly Lys Ala Pro Trp Tyr Glu Val Phe Thr Gly

50 55 60

Pro Ala Met Phe Trp Arg Thr Thr Leu Gly Ile Val Leu Gln Ser Leu

65 70 75 80

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

85 90 95

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

100 105 110

Gly Thr Val Asn Phe Ala Met Ser Ile Val Ser Leu Trp Val Val Gln

115 120 125

Arg Phe Arg Arg Arg Pro Ile Leu Ile Ile Gly Gly Ile Ala Met Phe

130 135 140

Ile Cys Phe Leu Ile Phe Ala Ser Val Gly His Phe Ser Leu Asp His

145 150 155 160

Glu Asn Pro Gln Asn Thr Pro Lys Ala Gly Thr Ala Leu Ile Val Phe

165 170 175

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

180 185 190

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

195 200 205

Val Ser Leu Ser Thr Ala Ser Asn Trp Thr Trp Asn Phe Leu Leu Cys

210 215 220

Phe Phe Thr Pro Phe Ile Ser Ser Ala Ile Asp Tyr Arg Tyr Gly Tyr

225 230 235 240

Val Phe Gly Ala Cys Cys Ala Ala Gly Ser Ile Ile Thr Tyr Phe Phe

245 250 255

Val Asn Glu Ser Tyr Gly Arg Thr Leu Glu Glu Ile Asp Thr Met Tyr

260 265 270

Val Met His Val Lys Pro Trp Gln Ser Lys Asn Trp Val Ala Pro Pro

275 280 285

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

290 295 300

Ala Thr Pro Ala His Ser Ser Ala Glu Ala Leu

305 310 315

Claims

1. a kind of High-productive Monascus Pigment Strain construction method, it is characterised in that the following steps are included:

1) plant binary plasmid pCambia0380 is taken, with restriction endonuclease Hind III and Bgl II digestion, then will A pair of oligonucleotide sequence as shown in SEQ ID No:1, SEQ ID No:2 by T4 DNA ligase be attached thereto to get To binary plasmid expression vector pCambia0380G；

2) using plasmid pMD19-PgpdA-hph-TtrpC as template, with sequence as shown in SEQ ID No:3, SEQ ID No:4 Pair of primers executes PCR amplification, hph expression cassette segment is obtained, with restriction endonuclease Sac I and Xho I digestion simultaneously The hph expression cassette segment and binary plasmid expression vector pCambia0380G then pass through T4 DNA ligase for the two It connects to get binary plasmid knockout carrier pHph0380 is arrived；

3) using red monascus CICC41233 total DNA as template, with sequence as shown in SEQ ID No:9, SEQ ID No:10 Pair of primers executes PCR amplification, obtains the upstream homology arm segment of saccharide transporter gltp1 gene；With red monascus CICC41233 total DNA is template, executes PCR with sequence pair of primers as shown in SEQ ID No:11, SEQ ID No:12 and expands Increase, obtains the downstream homology arm segment of saccharide transporter gltp1 gene；The upstream homology arm segment and the downstream is homologous Arm pieces section is respectively connected on the carrier pHph0380 arrive binary plasmid knockout carrier pHph0380-GLTP；

2. a kind of High-productive Monascus Pigment Strain construction method according to claim 1, it is characterised in that the step 4) system The Agrobacterium tumefaciems EHA105 of standby competence, comprising the following steps: Agrobacterium tumefaciems EHA105 is inoculated in 5~10mL and contains 50 In the YEP fluid nutrient medium of μ g/mL rifampin, with 28 DEG C of temperature, speed of agitator 200rpm CMC model for 24 hours；LmL is taken to activate Bacterium solution be inoculated in 20mL and contain in the YEP fluid nutrient medium of 50 μ g/mL rifampins, with 28 DEG C of temperature, speed of agitator 200rpm CMC model to bacterium solution OD₆₀₀Value 0.5；After bacterium solution ice bath 30min, it is centrifuged under the conditions of 4 DEG C with the revolving speed of 5000rpm 5min abandons supernatant；Precipitating is resuspended with the sodium chloride solution l0mL of 0.15mmol/L, under the conditions of 4 DEG C with the revolving speed of 5000rpm from Heart 5min abandons supernatant, is then suspended in the calcium chloride solution 1mL of 20mmol/L.

3. a kind of High-productive Monascus Pigment Strain construction method according to claim 1, it is characterised in that described to pass through liquid nitrogen The binary plasmid knockout carrier pHph0380-GLTP is imported Agrobacterium tumefaciems EHA105 by freeze-thaw method, comprising the following steps: is taken Binary plasmid knockout carrier pHph0380-GLTP described in 1 μ g is added in the Agrobacterium tumefaciems EHA105 of 200 μ L competence, is mixed Ice bath 30min after conjunction；Quick-frozen 1min in liquid nitrogen, 37 DEG C of water-bath 3min, then ice bath 2min；800 μ L YEP fluid nutrient mediums are added, 28 DEG C of culture 3h；3min is centrifuged with the revolving speed of 5000rpm under room temperature, thallus is concentrated；Bacterium solution after taking 200 μ L to be concentrated, which is coated on, to be contained On the YEP selective medium plate for having 50 μ g/mL rifampins, 50 μ g/mL kanamycins, 2d is cultivated in 28 DEG C of inversions；It selects and turns Beggar cultivates in YEP fluid nutrient medium, and is screened with primer pair clone, and positive clone molecule is obtained, as containing double The Agrobacterium tumefaciems EHA105 of first plasmid knockout carrier pHph0380-GLTP.

4. a kind of High-productive Monascus Pigment Strain construction method according to claim 1, it is characterised in that it is described will be containing double The Agrobacterium tumefaciems EHA105 of first plasmid knockout carrier pHph0380-GLTP is converted into red monascus CICC41233, including Following steps:

Red monascus CICC41233 is taken, with MPS solid medium culture 7d, conidium is obtained, with sterile aqueous suspension spore Son, vibrating dispersion spore are filtered through 2 layers of lens wiping paper, adjust spore concentration；

The Agrobacterium tumefaciems EHA105 containing binary plasmid knockout carrier pHph0380-GLTP is taken, 3mL is inoculated in and contains 50 μ g/ ML rifampin, 50 μ g/mL kanamycins YEP culture medium in, 28 DEG C of culture 48h then transfer and contain 200 μm of ol/L in 5mL In the AIM induced medium of acetosyringone, bacterium solution is made to be diluted to OD₆₀₀Value is 0.15,5~6h is cultivated for, until OD₆₀₀Value It is 0.5~0.6；

By red monascus spore liquid obtained as above and crown gall agriculture bar containing binary plasmid knockout carrier pHph0380-GLTP Bacterium EHA105 bacterium solution, mixing are coated on the AIM induced medium plate containing 200 μm of ol/L acetosyringones, 25 DEG C, are protected from light training Support 48h.

5. a kind of High-productive Monascus Pigment Strain construction method according to claim 1, it is characterised in that the screening is positive Clone, comprising the following steps: add one layer again on the AIM induced medium plate being protected from light after culture 48h containing 100 μ g/mL Hph, 200 μm of ol/L cefotaxime, 0.2%Triton X-100 MPS culture medium, 30 DEG C are continued 5~8d of culture；Picking single bacterium It falls and is forwarded in the MPS solid medium tablets containing 100 μ g/mL hph, cultivate the bacterial strain that will be grown after 3d, be connected to MPS It is cultivated in fluid nutrient medium, extracts filamentous fungi total DNA according to SDS cracking process and carry out analysis of molecules, be SEQ ID with sequence The pair of primers progress PCR verifying of No:15, SEQ ID No:16, choosing positive strain is the superior strain.