CN109371053B - Construction method of monascus pigment producing strain - Google Patents
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Abstract
The invention provides a construction method of a monascus pigment high-yield strain. Firstly, connecting a pair of oligonucleotide sequences sequentially containing Hind III, Kpn I, Sac I, Pac I, Pme I, Xho I, Xba I and Bgl II enzyme cutting sites with a plant binary plasmid pCambia0380 to construct a binary plasmid expression vector pCambia 0380G; then, connecting the hph expression cassette fragment with the hph expression cassette fragment to obtain a binary plasmid knockout vector pHph 0380; on the basis, the upstream and downstream homologous arm segments of the GLTP1 gene are amplified respectively and connected to a binary plasmid knockout vector pHph0380, so that the binary plasmid knockout vector pHph0380-GLTP is obtained. The vector pHph0380-GLTP is mediated and transformed into a parent monascus ruber CICC41233 by agrobacterium tumefaciens EHA105, and the construction of a high-yield strain is completed. Experimental verification shows that the high-yield monascus pigment strain constructed by the invention not only improves the overall yield of monascus pigment, but also can directionally accumulate alcohol-soluble pigment, and meanwhile, the accumulation time of the monascus pigment in the fermentation process is obviously advanced.
Description
Technical Field
The invention relates to the technical field of industrial microorganisms, further relates to a genetic engineering technology and a mould fermentation technology, and particularly relates to a monascus pigment producing strain construction method.
Background
The monascus pigment is a natural pigment prepared by fermenting monascus spp and rice serving as a raw material, and has a history of more than one thousand years in China. As a food additive, monascus pigment is widely applied to the fields of food processing, cosmetic manufacturing and the like; because it also has a wide range of biological activities such as regulating blood lipid, lowering blood pressure, preventing angiosclerosis, resisting diabetes, inhibiting obesity, resisting inflammation, resisting allergy, preventing peroxidation, resisting cancer, resisting bacteria, resisting fungi, etc., its application in the development of health care products and medical fields is also receiving more and more attention.
The monascus pigment is a secondary metabolite of monascus, and the anabolism is completed by a fatty acid synthesis pathway and a polyketone synthesis pathway together. The chemical structure of the compound is mainly divided into two parts of polyketone and fatty acid chain. The synthesis of Fatty acid chain takes acetyl CoA as a precursor, and medium-long chain Fatty acid is formed through a series of synthesis reactions under the action of a Fatty Acid Synthase (FAS) complex, and reacts with the acetyl CoA to generate beta-keto acid; polyketone synthesis also takes acetyl CoA as a precursor, and polyketone body compounds are sequentially synthesized under the action of Polyketide synthase (PKS), so that polyketone with a chromogenic group is finally formed. The carboxyl groups of the polyketone undergo an esterification reaction with the hydroxyl groups of the fatty acid chain, thereby forming the monascus pigment. Based on the principle, in order to improve the yield of monascus pigment, the prior art generally realizes the metabolic regulation of monascus through fermentation process improvement, and further directionally accumulates target products; although the optimization of the fermentation conditions can improve the yield of the monascus pigment to a certain extent, the monascus pigment accumulation is difficult to further improve due to the metabolic characteristics of the monascus.
In addition, monascus pigments produced by monascus are classified into alcohol-soluble pigments and water-soluble pigments. The alcohol-soluble pigment is directly synthesized by monascus in the fermentation process and exists in cells; the water-soluble pigment is a composite pigment formed by combining a pigment synthesized by monascus and amino acids and the like in fermentation liquor and is distributed outside cells. In the growth process of natural strains, both monascus pigments are produced, and one monascus pigment cannot be produced directionally by only depending on a culture method.
In addition, in the industrial production of monascus, the fermentation process is a key factor affecting the overall process duration, and since monascus is a secondary metabolite of monascus, monascus is accumulated only after a period of time when fermentation starts. In this case, if the initiation time can be advanced by means of gene recombination or metabolic regulation, it is expected to shorten the process time and improve the production efficiency.
Disclosure of Invention
The invention aims to provide a monascus pigment production strain construction method aiming at the technical defects of the prior art, so as to solve the technical problem of low monascus pigment yield of common monascus ruber in the prior art.
The invention also aims to solve the technical problem that the common monascus ruber is difficult to directionally accumulate the alcohol-soluble monascus pigment.
The invention also aims to solve the technical problem that the starting time of monascus accumulation is late in the culture process of the common monascus ruber.
In order to achieve the technical purpose, the invention adopts the following technical scheme:
a method for constructing monascus pigment production strains comprises the following steps:
1) taking plant binary plasmid pCambia0380, carrying out enzyme digestion by using restriction endonucleases Hind III and Bgl II, and then connecting a pair of oligonucleotide sequences shown as SEQ ID No. 1 and SEQ ID No. 2 with the oligonucleotide sequences through T4DNA ligase to obtain a binary plasmid expression vector pCambia 0380G;
2) carrying out PCR amplification by using plasmid pMD19-PgpdA-hph-TtrPC as a template and a pair of primers with sequences shown as SEQ ID No. 3 and SEQ ID No. 4 to obtain a hph expression cassette fragment, simultaneously carrying out enzyme digestion on the hph expression cassette fragment and a binary plasmid expression vector pCambia0380G by using restriction endonucleases Sac I and Xho I, and then connecting the two through T4DNA ligase to obtain a binary plasmid knockout vector pHph 0380;
3) performing PCR amplification by using the total DNA of the monascus ruber CICC41233 as a template and a pair of primers with sequences shown as SEQ ID No. 9 and SEQ ID No. 10 to obtain an upstream homologous arm fragment of the sugar transporter gltp1 gene; performing PCR amplification by using the total DNA of the monascus ruber CICC41233 as a template and a pair of primers with sequences shown as SEQ ID No. 11 and SEQ ID No. 12 to obtain a downstream homologous arm fragment of the sugar transporter gltp1 gene; respectively connecting the upstream homology arm segment and the downstream homology arm segment to the vector pHph0380 to obtain a binary plasmid knockout vector pHph 0380-GLTP;
4) preparing competent agrobacterium tumefaciens EHA105, introducing the binary plasmid knockout vector pHph0380-GLTP into the agrobacterium tumefaciens EHA105 by a liquid nitrogen freeze-thaw method, then transforming the agrobacterium tumefaciens EHA105 containing the binary plasmid knockout vector pHph0380-GLTP into monascus ruber CICC41233, and screening positive clones to obtain the production strain.
Preferably, the preparation of competent agrobacterium tumefaciens EHA105 in step 4) comprises the following steps: inoculating agrobacterium tumefaciens EHA105 into 5-10 mL YEP liquid culture medium containing 50 mu g/mL rifampicin, and culturing for 24h under the conditions of 28 ℃ and 200rpm of stirring speed; inoculating lmL activated bacteria liquid into 20mL YEP liquid culture medium containing 50 μ g/mL rifampicin, and culturing at 28 deg.C and stirring speed of 200rpm until bacteria liquid OD600 value is 0.5; carrying out ice-bath on the bacterial liquid for 30min, centrifuging at the rotation speed of 5000rpm for 5min at the temperature of 4 ℃, and removing the supernatant; the pellet was resuspended in 0.15mmol/L NaCl solution L0mL, centrifuged at 5000rpm for 5min at 4 deg.C, the supernatant was discarded, and suspended in 1mL of 20mmol/L calcium chloride solution.
Preferably, the method for introducing the binary plasmid knockout vector pHph0380-GLTP into the Agrobacterium tumefaciens EHA105 by a liquid nitrogen freeze-thaw method comprises the following steps: adding 1 mu g of the binary plasmid knockout vector pHph0380-GLTP into 200 mu L of competent Agrobacterium tumefaciens EHA105, mixing and carrying out ice bath for 30 min; quickly freezing in liquid nitrogen for 1min, water bathing at 37 deg.C for 3min, and ice bathing for 2 min; adding 800 μ L YEP liquid culture medium, and culturing at 28 deg.C for 3 hr; centrifuging at the rotation speed of 5000rpm for 3min at normal temperature, and concentrating thallus; spreading 200 μ L of concentrated bacterial liquid on YEP selective culture medium plate containing 50 μ g/mL rifampicin and 50 μ g/mL kanamycin, and performing inversion culture at 28 deg.C for 2 d; and selecting a transformant to culture in a YEP liquid culture medium, and screening clones by using a primer to obtain a positive clone, namely the agrobacterium tumefaciens EHA105 containing the binary plasmid knockout vector pHph 0380-GLTP.
Preferably, the transformation of agrobacterium tumefaciens EHA105 containing the binary plasmid knockout vector pHph0380-GLTP into monascus ruber CICC41233 comprises the following steps:
taking monascus ruber CICC41233, culturing for 7 days with MPS solid culture medium to obtain conidia, suspending the conidia with sterile water, shaking to disperse the conidia, filtering with 2 layers of mirror paper, and adjusting the concentration of the conidia;
taking Agrobacterium tumefaciens EHA105 containing a binary plasmid knockout vector pHph0380-GLTP, inoculating the Agrobacterium tumefaciens EHA105 into 3mL of YEP culture medium containing 50 mu g/mL rifampicin and 50 mu g/mL kanamycin, culturing at 28 ℃ for 48h, then transferring into 5mL of AIM induction culture medium containing 200 mu mol/L acetosyringone, diluting the bacterial liquid to OD600 value of 0.15, and continuing culturing for 5-6 h until OD600 value is 0.15600The value is 0.5 to 0.6;
the monascus ruber spore liquid and agrobacterium tumefaciens EHA105 bacterial liquid containing binary plasmid knockout vector pHph0380-GLTP obtained above are mixed and coated on an AIM induction culture medium plate containing 200 mu mol/L acetosyringone, and the mixture is cultured for 48 hours at 25 ℃ in the dark.
Preferably, the screening of positive clones comprises the following steps: adding a layer of MPS culture medium containing 100 mu g/mL hph, 200 mu mol/L cefotaxime and 0.2% Triton X-100 on the AIM induction culture medium plate after being cultured for 48 hours in a dark place, and continuously culturing for 5-8 days at 30 ℃; selecting a single colony, transferring the single colony to an MPS solid culture medium plate containing 100 mu g/mL hph, culturing for 3d, inoculating a strain capable of growing to an MPS liquid culture medium, culturing, extracting total DNA of the filamentous fungi according to an SDS cracking method, carrying out molecular analysis, carrying out PCR verification by using a pair of primers with the sequences of SEQ ID No. 15 and SEQ ID No. 16, and selecting a positive strain as the production strain.
In the technical scheme, the plant binary plasmid pCambia0380, the monascus ruber CICC41233 and the agrobacterium tumefaciens EHA105 belong to conventional commercial biological materials and can be purchased from the market; wherein, the monascus ruber CICC41233 is purchased from China center for Industrial culture Collection of microorganisms.
In the above technical scheme, the plasmid pMD19-PgpdA-hph-TtrpC can be prepared by itself according to the prior art, and its specific preparation method can be referred to references "d e G roo t MJ A, Bund ock P, Hooykaa s PJJ, et al 1998.Agrobacterium tumefaciens-mediated transformation of filing funngium. Nature Biotechnology, 16: 839-842 ".
In the technical scheme, the nucleotide sequence of the gltp1 gene is shown as SEQ ID No. 17; the amino acid sequence of the GLTP1 amino acid sequence is shown in SEQ ID No. 18.
In the above technical scheme, the formulation of the YEP 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; if the culture medium is a solid culture medium, 2% agar powder is added.
The MPS culture medium comprises the following components: 10g/L malt extract, 10g/L peptone, 40g/L soluble starch; in the case of the solid medium, agar was added thereto at a concentration of 2 g/L.
The formula of the AIM induction culture medium is as follows: 0.8mL of potassium phosphate buffer (1.25mol/L, pH 4.8, prepared from potassium dihydrogen phosphate and dipotassium hydrogen phosphate), 0.6g of MgSO 24.7H2O，0.3g NaCL，1mL CaCL2(1％)，1mL FeSO4(1mg/mL)，1mL(NH4)2SO4(0.33g/L), 10mL of glycerol (50%), 40mL of MES (pH 5.5 adjusted with NaOH), 5mL of storage solution of trace elements, 1mL of CaCl2(1%), 2g/L glucose (for liquid medium), 1g/L glucose (for solid medium). The pH value is 5.4; if the culture medium is a solid culture medium, 2% agar powder is added.
The invention provides a construction method of a monascus pigment producing strain. Firstly, connecting a pair of oligonucleotide sequences sequentially containing Hind III, Kpn I, Sac I, Pac I, Pme I, Xho I, Xba I and Bgl II enzyme cutting sites with a plant binary plasmid pCambia0380 to construct a binary plasmid expression vector pCambia 0380G; then, connecting the hph expression cassette fragment with the hph expression cassette fragment to obtain a binary plasmid knockout vector pHph 0380; on the basis, the upstream and downstream homologous arm segments of the GLTP1 gene are amplified respectively and connected to a binary plasmid knockout vector pHph0380, so that the binary plasmid knockout vector pHph0380-GLTP is obtained. The vector pHph0380-GLTP is mediated and transformed into a parent monascus ruber CICC41233 by agrobacterium tumefaciens EHA105, and the construction of a production strain is completed. Experimental verification shows that the monascus pigment producing strain constructed by the invention not only improves the overall yield of monascus pigment, but also can directionally accumulate alcohol-soluble pigment, and meanwhile, the accumulation time of monascus pigment in the fermentation process is obviously advanced, and the production capacity is comprehensively superior to that of the parent monascus ruber CICC 41233.
Drawings
FIG. 1 is an electrophoretogram of PCR amplified fragment of gltp1 gene in Monascus CICC 41233.
FIG. 2 is a schematic diagram of the knockout of the gltp1 gene of Monascus purpureus.
FIG. 3 is an electrophoresis chart of the PCR identification result of the gene knockout engineering strain; wherein, lanes 1-2: amplifying a gltp1 gene by using a primer F3& R3; lanes 3-4: adopting a primer F4& R4 to amplify and screen the hph gene; lanes 5-6: verifying that the hph is integrated to the gltp1 gene site by using a primer F5& R5; lanes 1,3, 5: the monascus CICC41233 total DNA is used as a template; lanes 2,4, 6: the total DNA of the knockout strain monascus GLTP24 is taken as a template.
FIG. 4 is a graph comparing the results of fermentation of Monascus ruber CICC41233 with Monascus ruber GLTP24 to produce monascus pigment; wherein (a) the monascus has different fermentation time phenotypes; (b) residual starch content in different fermentation times; (c) the color value of the monascus pigment; (d) spectrally scanning the water-soluble pigment; (e) the alcohol soluble pigment was spectrally scanned.
FIG. 5 is a graph comparing the results of Monascus ruber CICC41233 and Monascus ruber anaplerotis producing monascorubin.
Detailed Description
Hereinafter, specific embodiments of the present invention will be described in detail. Well-known structures or functions may not be described in detail in the following embodiments in order to avoid unnecessarily obscuring the details. Approximating language, as used herein in the following examples, may be applied to identify quantitative representations that could permissibly vary in number without resulting in a change in the basic function. Unless defined otherwise, technical and scientific terms used in the following examples have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The partial gene fragments and primers used in the following examples are shown in Table 1:
table 1 partial Gene fragment and primer names and sequences
In Table 1, the suffix name is F for the forward primer and R for the reverse primer.
Example 1
1. Binary plasmid knockout vector pHph0380 was constructed.
The original vector was the commercial plant binary plasmid pCambia 0380. Design 1 pairs of oligonucleotide sequences F & R, the sequences were synthesized by Shanghai Bioengineering, Inc. This sequence contains the following restriction endonuclease sites (Hind III, Kpn I, Sac I, Pac I, Pme I, Xho I, Xba I, Bgl II) in that order. The binary plasmid vector pCambia0380 was digested with the restriction endonucleases Hind III and Bgl II. The oligonucleotide sequence was ligated to the digested vector pCambia0380 by T4DNA ligase to obtain the binary plasmid vector pCambia 0380G.
The hph expression cassette is obtained by taking the plasmid pMD19-PgpdA-hph-TtrpC preserved in a laboratory as a template and adopting a primer PgpdA-Sac I-F and TtrpC-Xho I-R for amplification. The hph expression cassette fragment and the binary plasmid vector pCambia0380G were digested simultaneously with restriction endonucleases Sac I and Xho I. The hph expression cassette fragment and binary plasmid vector pCambia0380G were ligated by T4DNA ligase to obtain the binary plasmid knockout vector pHph 0380.
2. Monascus GLTP1 gene cloning, and construction of binary plasmid expression vector pNeo0380-GLTP
Primers Gltp1-JYF-HindIII (containing HindIII) and Gltp1-JYR-SacI (containing SacI) are designed according to a gene sequence (924bp) provided by transcriptome sequencing (the sequences are shown in a table 1), and a gene fragment is obtained by PCR amplification by taking red aspergillus rubrus CICC41233 and total DNA as a template (figure 1) and sequencing. And (3) digesting the PCR fragment by adopting HindIII and SacI and recovering the fragment which is correctly sequenced and accords with the target fragment. Meanwhile, the pNeo0380 vector is cleaved and recovered.
Connecting the recovered fragment of enzyme-digested pNeo0380 with the recovered fragment of enzyme-digested gltp1 gene by using T4DNA ligase, transforming E.coli DH5 alpha competent cells, selecting clones to culture in an LB liquid culture medium, and screening the clones by using primers. After plasmid extraction and enzyme digestion verification, the constructed vector is named pNeo 0380-GLTP.
3. The method comprises the steps of amplification of homologous arm fragments of the upstream and downstream of monascus GLTP1 gene, and construction of binary plasmid knockout vector pHph 0380-GLTP.
According to the sequence of the cloned gltp1 gene, the sequence is analyzed in Monascus ruber NRRL1597 genome data to find the sequence of the upstream and the downstream. Using monascus ruber CICC41233 and total DNA as a template, designing a primer Gltp-QC-UF-PstI and Gltp-QC-UR-Sac I, and carrying out PCR amplification to obtain an upstream arm fragment of 1740 bp; the 1764bp downstream arm fragment was amplified by Gltp-QC-DF-Bgl II & Gltp-QC-DR-SpeI PCR (see Table 1 for sequence). And (3) connecting the fragments which are correctly sequenced and meet the target with a pHph0380 vector by adopting a method of enzyme digestion and enzyme ligation, wherein the constructed vector is named as pHph 0380-GLTP. The principle of the Monascus gltp1 gene knockout is shown in FIG. 2.
4. Construction of recombinant Strain Monascus ruber GLTP24
4.1 preparation of Agrobacterium tumefaciens competent cells
Firstly, inoculating agrobacterium tumefaciens EHA105 into 5-10 mL YEP liquid culture medium (containing 50 mu g/mL rifampicin), and culturing at 28 ℃ and 200r/min for 24 h.
② one mL of the activated bacterial suspension was inoculated into 20mL of YEP medium containing the same antibiotic and cultured under the same conditions (about 4 hours) to an OD600 value of 0.5.
③ carrying out ice bath on the bacterial liquid for 30min, centrifuging at 4 ℃ (5000r/min, 5min), and collecting thalli.
Fourthly, the supernatant was discarded, the pellet was resuspended in 0.15mmol/L sodium chloride ice-cold solution L0mL, and the cells were collected by centrifugation under the same conditions and suspended in 1mL of 20mmol/L calcium chloride ice-cold solution.
Fifthly, subpackaging according to 200 mu L per tube, and quickly freezing for 1min by liquid nitrogen.
Sixthly, the bacterial suspension can be directly transformed and can also be stored in a freezer at the temperature of 70 ℃ below zero for later use.
The YEP medium described above: 5.0g peptone, 1.0g yeast extract, 5.0g sucrose, 5.0g beef extract, 0.24g magnesium sulfate, pH 7.2. And 2% of agar powder is added into the solid culture medium.
4.2 liquid nitrogen Freeze thawing method for introducing binary plasmid vector into Agrobacterium tumefaciens
Adding 1 microgram of binary plasmid vector pHph0380-GLTP into 200 microliter of ice-dissolved agrobacterium-infected cells, gently mixing, and carrying out ice bath for 30 min.
② quick freezing in liquid nitrogen for 1min, water bathing at 37 ℃ for 3min, and then rapidly ice-bathing for 2 min.
③ adding 800 mu L YEP liquid culture medium, and culturing for 3h at 28 ℃.
Fourthly, centrifuging at normal temperature (5000r/min, 3min), and properly concentrating the thalli.
Fifthly, 200 mu L of the bacterial liquid is taken and coated on a YEP selection plate (containing 50 mu g/mL rifampicin and 50 mu g/mL kanamycin), and the bacterial liquid is subjected to inversion culture at 28 ℃ for 2 d.
Sixthly, selecting the transformant to culture in YEP liquid culture medium, and screening the clone by using the primer to obtain a positive clone.
4.3 Agrobacterium tumefaciens-mediated transformation of Monascus ruber CICC41233
(1) Preparation of cells
Monascus ruber CICC 41233: and (5) culturing in an MPS solid culture medium for 7d to obtain conidia, suspending the conidia in sterile water, shaking to disperse the conidia, filtering by 2 layers of mirror paper, and adjusting the proper concentration of the conidia.
Agrobacterium tumefaciens: the agrobacterium containing the binary plasmid vector is inoculated in 3mL YEP culture medium (containing 50 mu g/mL rifampicin and 50 mu g/mL kanamycin), cultured for 48h at 28 ℃, then transferred into 5mL AIM induction culture medium containing 200 mu mol/L Acetosyringone (AS), so that the bacterial liquid is diluted to OD600 of 0.15, and further cultured for 5-6 h until OD600 is 0.5-0.6.
The MPS medium described above: 10g/L malt extract, 10g/L peptone and 40g/L soluble starch. Solid medium: an additional 2g/L agar was added.
(2) Co-culture of Agrobacterium and Monascus ruber CICC41233
AIM induction medium plates (containing 200. mu. mol/L AS) were prepared. Agrobacterium was mixed with Monascus ruber CICC41233 (in equal proportions). The mixture was spread on an AIM plate and incubated at 25 ℃ in the dark for 48 h.
(3) Transformant screening and validation
An additional layer of screening medium (MPS medium, containing 100. mu.g/mL hph, 200. mu. mol/L cefotaxime, 0.2% Triton X-100) was added to the AIM medium plates. And continuing culturing for 5-8 days at 30 ℃. Transferring the grown single colony strain to another MPS solid plate (containing hph), and observing after culturing for 3d, wherein the strain can still grow; inoculating the strain capable of growing into MPS liquid culture medium, culturing, and extracting filamentous fungus total DNA according to SDS lysis method for molecular analysis. PCR validation was performed using primer pairs (fig. 3). A strain is determined, and is an engineering strain of Monascus ruber (Monascus ruber) GLTP 24.
The above AIM medium: 0.8mL of potassium phosphate buffer (1.25mol/L, pH 4.8, prepared with potassium dihydrogen phosphate and dipotassium hydrogen phosphate), 0.6g of MgSO4.7H2O, 0.3g of NaCL, 1mL of CaCl2 (1%), 1mL of FeSO4(1mg/mL), 1mL (NH4) of 2SO4(0.33g/L), 10mL of glycerol (50%), 40mL of MES (pH 5.5, adjusted with NaOH), 5mL of trace element stock, 1mL of CaCl2 (1%), 2g/L of glucose (for liquid medium), 1g/L of glucose (for solid medium). The pH value is 5.4. And 2% of agar powder is added into the solid culture medium.
5. Comparison of Monascus ruber GLTP24 and parent strain CICC41233 capability of producing monascus pigment by fermentation
5.1 cultivation with MPS solid Medium
The monascus ruber GLTP24 and monascus ruber CICC41233 were cultured in MPS solid medium for 7 days, and spore suspension was collected at spore inoculum size of 1 × 105/mL. The fermentation conditions were: fermentation was carried out at 30 ℃ and 180rpm until day 6.
The fermentation medium for producing the monascus pigment comprises the following components: rice flour 9.0%, NaNO3 0.2%, KH2PO4 0.1%, MgSO 4& 7H2O 0.2%, acetic acid 0.2%.
5.2 Red Rice pigment color number determination
And (3) measuring the color value of the extracellular monascus pigment (water soluble): and (3) metering the fermentation liquor to 25mL in a centrifuge tube, freezing and centrifuging at a high speed (10000 rpm for 30min), and obtaining the supernatant as the extracellular pigment. Diluting a certain amount of filtrate with water by a proper amount, measuring the absorbance value of the diluted solution (the main absorption peak of the red yeast rice composite pigment is 505nm) by using a spectrophotometer by taking water as a reference, and calculating the total color value. The calculation method comprises the following steps: total color number is dilution times absorbance.
And (3) measuring the color value of the intracellular monascus pigment (alcohol soluble): centrifuging the fermentation liquid, collecting precipitate, standing and extracting with 70% ethanol at 60 deg.C for 1 hr, and performing vortex oscillation for several times. Freezing and high-speed centrifuging (10000 rpm, 20min), and obtaining the supernatant as the intracellular pigment. Diluting a certain amount of filtrate with 70% ethanol by a proper amount, measuring the absorbance value of the diluted solution (the main absorption peak of red rice composite pigment is 505nm) with spectrophotometer by taking 70% ethanol as reference, and calculating the total color value. The calculation method is the same as above.
Monascus ruber GLTP24, fermented for 36h, produced significant amounts of monascin compared to the parental strain as observed by appearance, indicating that the time to produce monascin was advanced (fig. 4 a). The alcohol soluble color value is 0.29U, and compared with the parent monascus ruber CICC41233, the alcohol soluble color value is 0.09U, which is improved by 2 times (fig. 4c, 4 e). Fermenting for 144h, and the alcohol-soluble color value is 49.46U. Compared with the parent monascus ruber CICC41233, the alcohol soluble color value is 28.42U, which is increased by 74% (fig. 4c, 4 e). The water-soluble pigments did not differ significantly (FIGS. 4c, 4d)
5.3 residual starch content determination
A standard curve of soluble starch was prepared by preparing a solution of I2-KI (2.6g/L I2,5.0g/L KI) and measuring the OD at a wavelength of 600 nm. Centrifuging the fermentation liquid at normal temperature (10000r/min, 20min), and measuring the content of starch in the supernatant. FIG. 4b shows that the engineering strain monascus ruber GLTP24 can significantly promote starch degradation and metabolism. Fermenting for 36h, 48h and 144h, wherein the residual starch contents of the engineering strain monascus ruber GLTP24 are respectively 0.56mg/mL, 0.15mg/mL and 0 mg/mL; the monascus ruber CICC41233 was 44.91mg/mL, 34.26mg/mL, 9.58mg/mL remaining.
6. Construction of anaplerotic strain, and comparison of anaplerotic strain with parent strain monascus CICC41233 for producing monascus pigment by fermentation
6.1 obtaining of the complementing Strain
Referring to the procedure of example 4, a complementation strain was obtained by transforming the engineering strain monascus ruber GLTP24 (selection marker was changed to G418) with agrobacterium-mediated transformation of the binary plasmid expression vector pNeo 0380-GLTP.
6.2 Monascus pigment fermentation Capacity comparison
The monascus ruber CICC41233 and the 4 anaplerotic strains monascus HU1, monascus HU2, monascus HU3 and monascus HU4 were cultured in MPS solid medium for 7 days, and spore suspension was collected according to the spore inoculation amount of 1 × 105/mL. The fermentation conditions were: fermentation was carried out at 30 ℃ and 180rpm until day 6.
The fermentation medium for producing the monascus pigment comprises the following components: rice flour 9.0%, NaNO3 0.2%, KH2PO4 0.1%, MgSO 4& 7H2O 0.2%, acetic acid 0.2%.
6.3 Red Rice pigment color number determination
And (3) measuring the color value of the extracellular monascus pigment (water soluble): and (3) metering the fermentation liquor to 25mL in a centrifuge tube, freezing and centrifuging at a high speed (10000 rpm for 30min), and obtaining the supernatant as the extracellular pigment. Diluting a certain amount of filtrate with water by a proper amount, measuring the absorbance value of the diluted solution (the main absorption peak of the red yeast rice composite pigment is 505nm) by using a spectrophotometer by taking water as a reference, and calculating the total color value. The calculation method comprises the following steps: total color number is dilution times absorbance.
And (3) measuring the color value of the intracellular monascus pigment (alcohol soluble): centrifuging the fermentation liquid, collecting precipitate, standing and extracting with 70% ethanol at 60 deg.C for 1 hr, and performing vortex oscillation for several times. Freezing and high-speed centrifuging (10000 rpm, 20min), and obtaining the supernatant as the intracellular pigment. Diluting a certain amount of filtrate with 70% ethanol by a proper amount, measuring the absorbance value of the diluted solution (the main absorption peak of red rice composite pigment is 505nm) with spectrophotometer by taking 70% ethanol as reference, and calculating the total color value. The calculation method is the same as above.
Monascus ruber CICC41233 and 4 anaplerotic strains of Monascus ruber HU1, Monascus ruber HU2, Monascus ruber HU3 and Monascus ruber HU4, and fermenting for 6 d. The monascus pigment color number results were not statistically different from the parental strain (see fig. 5).
Example 2
1. Construction of binary plasmid knockout vector pHph0380
The original vector was the commercial plant binary plasmid pCambia 0380. Design 1 pairs of oligonucleotide sequences F & R, containing the following restriction endonuclease sites (Hind III, Kpn I, Sac I, Pac I, Pme I, Xho I, XbaI, Bgl II) in order. The binary plasmid vector pCambia0380 was digested with the restriction endonucleases Hind III and Bgl II. The oligonucleotide sequence was ligated to the vector by T4DNA ligase to obtain binary plasmid vector pCambia 0380G.
The hph expression cassette fragment is obtained by taking the plasmid pMD19-PgpdA-hph-TtrpC preserved in a laboratory as a template and adopting a primer PgpdA-Sac I-F and TtrpC-Xho I-R for amplification. The promoter fragment and the binary plasmid vector pCambia0380G were digested simultaneously with the restriction endonucleases Sac I and Xho I. The oligonucleotide sequence is connected with the vector through T4DNA ligase to obtain a binary plasmid knockout vector pHph 0380.
2. Construction of engineering strain of monascus ruber GLTP24 for producing monascus pigment
The genetic engineering strain constructed by the invention is classified and named as monascus ruber (Mo na c us rube r) GLTP 24.
The invention uses monascus ruber CICC41233 as parent strain, clones the upstream and downstream homologous arm segments of GLTP1 gene, constructs in binary plasmid vector pHph0380, and obtains gene engineering strain monascus ruber GLTP24 through agrobacterium tumefaciens EHA105 mediation.
(1) Construction of binary plasmid knockout vector pHph0380-GLTP
And (3) performing PCR amplification by using the total DNA of the monascus CICC41233 as a template to obtain the upstream and downstream homologous arm fragments of the gltp1 gene. Then, restriction endonucleases Pst I and SacI are used for simultaneously digesting the upstream homologous arm fragment, restriction endonucleases Bgl II and Spe I are used for simultaneously digesting the downstream homologous arm fragment and the binary plasmid vector pHph 0380. The gene fragment is connected with the vector through T4DNA ligase to obtain a binary plasmid knockout vector pHph 0380-GLTP.
(2) Agrobacterium tumefaciens mediated transformation of monascus ruber CICC41233
The engineering strain monascus ruber GLTP24 is successfully obtained by transforming monascus ruber CICC41233 by adopting agrobacterium tumefaciens EHA105 mediated binary plasmid vector pHph 0380-GLTP.
3. The gene engineering strain monascus ruber GLTP24 is fermented to improve the yield of monascus pigment
The monascus ruber CICC41233 and the monascus ruber GLTP24 are fermented simultaneously to produce monascus pigment. The fermentation medium comprises the following components: rice flour 9.0%, NaNO3 0.2%, KH2PO4 0.1%, MgSO 4& 7H2O 0.2%, acetic acid 0.2%. The inoculation amount of the strain spores is 1 × 105/mL. The fermentation conditions were: fermentation was carried out at 30 ℃ and 180rpm until day 6. The results show that the time for producing the monascus pigment by the fermentation of the engineering strain is advanced, and the starch degradation and metabolism are obviously promoted. After fermentation for 36h, the alcohol soluble color value of the monascus ruber GLTP24 is 0.29U, and compared with that of the parent monascus ruber CICC41233, the alcohol soluble color value is 0.09U, which is improved by 2 times. Fermenting for 144h, and the alcohol-soluble color value is 49.46U. Compared with parent monascus ruber CICC41233, the alcohol soluble color value is 28.42U, which is improved by 74 percent.
4. Construction of monascus anaplerosis strain and fermentation for producing monascus pigment
To further study the effect of the gene, the sugar transporter gene (gltp1) was obtained by PCR amplification using the DNA of Monascus purpureus CICC41233 as a template. Then, restriction endonucleases HindIII and SacI are adopted to simultaneously cut the gene fragment and a binary plasmid expression vector pNeo 0380. The gene fragment is connected with the vector through T4DNA ligase to obtain a binary plasmid expression vector pNeo 0380-GLTP. Agrobacterium tumefaciens EHA105 mediated expression vector is adopted to transform the engineering strain monascus ruber GLTP24, and anaplerotic strains monascus ruber HU1, monascus ruber HU2, monascus ruber HU3 and monascus ruber HU4 are successfully obtained. Compared with the parent strain, the monascus pigment produced by the 4 monascus ruber anaplerosis strains has no statistical difference.
The embodiments of the present invention have been described in detail, but the description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention. Any modification, equivalent replacement, and improvement made within the scope of the application of the present invention should be included in the protection scope of the present invention.
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Claims (5)
1. A method for constructing a monascus pigment production strain is characterized by comprising the following steps:
1) taking plant binary plasmid pCambia0380, carrying out enzyme digestion by using restriction endonucleases Hind III and Bgl II, and then connecting a pair of oligonucleotide sequences shown as SEQ ID No. 1 and SEQ ID No. 2 with the plant binary plasmid pCambia0380 and Bgl II through T4DNA ligase to obtain a binary plasmid expression vector pCambia 0380G;
2) taking a plasmid pMD19-PgpdA-hph-TtrPC as a template, performing PCR amplification by using a pair of primers with sequences shown as SEQ ID No. 3 and SEQ ID No. 4 to obtain a hph expression cassette fragment, simultaneously digesting the hph expression cassette fragment and a binary plasmid expression vector pCambia0380G by using restriction endonucleases Sac I and Xho I, and then connecting the two through T4DNA ligase to obtain a binary plasmid knockout vector pHph 0380;
3) performing PCR amplification by using the total DNA of the monascus ruber CICC41233 as a template and a pair of primers with sequences shown as SEQ ID No. 9 and SEQ ID No. 10 to obtain an upstream homologous arm fragment of the sugar transporter gltp1 gene; performing PCR amplification by using the total DNA of the monascus ruber CICC41233 as a template and a pair of primers with sequences shown as SEQ ID No. 11 and SEQ ID No. 12 to obtain a downstream homologous arm fragment of the sugar transporter gltp1 gene; respectively connecting the upstream homology arm segment and the downstream homology arm segment to the vector pHph0380 to obtain a binary plasmid knockout vector pHph 0380-GLTP;
4) preparing competent agrobacterium tumefaciens E H A105, introducing the binary plasmid knockout vector pHph0380-GLTP into the agrobacterium tumefaciens EHA105 by a liquid nitrogen freeze-thaw method, then transforming the agrobacterium tumefaciens EHA105 containing the binary plasmid knockout vector pHph0380-GLTP into monascus ruber CICC41233, and screening positive clones to obtain the production strain.
2. The method for constructing monascus pigment producing strain according to claim 1, wherein the step 4) of preparing competent agrobacterium tumefaciens EHA105 comprises the following steps: inoculating agrobacterium tumefaciens EHA105 into 5-10 mL YEP liquid culture medium containing 50 mu g/mL rifampicin, and culturing for 24h under the conditions of 28 ℃ and 200rpm of stirring speed; inoculating lmL activated bacteria solution into 20mL YEP liquid culture medium containing 50 μ g/mL rifampicin, and culturing at 28 deg.C and stirring speed of 200rpm until bacteria solution OD600A value of 0.5; carrying out ice-bath on the bacterial liquid for 30min, centrifuging at the rotation speed of 5000rpm for 5min at the temperature of 4 ℃, and removing the supernatant; the pellet was resuspended in 0.15mmol/L NaCl solution L0mL, centrifuged at 5000rpm for 5min at 4 deg.C, the supernatant was discarded, and suspended in 1mL of 20mmol/L calcium chloride solution.
3. The method for constructing a monascus pigment producing strain according to claim 1, wherein the binary plasmid knockout vector pHph0380-GLTP is introduced into agrobacterium tumefaciens EHA105 by a liquid nitrogen freeze-thaw method, comprising the following steps: adding 1 mu g of the binary plasmid knockout vector pHph0380-GLTP into 200 mu L of competent Agrobacterium tumefaciens EHA105, mixing and carrying out ice bath for 30 min; quickly freezing in liquid nitrogen for 1min, water bathing at 37 deg.C for 3min, and ice bathing for 2 min; adding 800 μ L YEP liquid culture medium, and culturing at 28 deg.C for 3 hr; centrifuging at the rotation speed of 5000rpm for 3min at normal temperature, and concentrating thallus; spreading 200 μ L of concentrated bacterial liquid on YEP selective culture medium plate containing 50 μ g/mL rifampicin and 50 μ g/mL kanamycin, and performing inversion culture at 28 deg.C for 2 d; and selecting a transformant to culture in a YEP liquid culture medium, and screening clones by using a primer to obtain a positive clone, namely the agrobacterium tumefaciens EHA105 containing the binary plasmid knockout vector pHph 0380-GLTP.
4. The method for constructing a monascus pigment producing strain according to claim 1, wherein the agrobacterium tumefaciens EHA105 containing the binary plasmid knockout vector pHph0380-GLTP is transformed into monascus ruber CICC41233, comprising the following steps: taking monascus ruber CICC41233, culturing for 7 days with MPS solid culture medium to obtain conidia, suspending the conidia with sterile water, shaking to disperse the conidia, filtering with 2 layers of mirror paper, and adjusting the concentration of the conidia; taking Agrobacterium tumefaciens EHA105 containing a binary plasmid knockout vector pHph0380-GLTP, inoculating the Agrobacterium tumefaciens EHA105 into 3mL of YEP culture medium containing 50 mu g/mL of rifampicin and 50 mu g/mL of kanamycin, culturing at 28 ℃ for 48h, then transferring the Agrobacterium tumefaciens EHA into 5mL of AIM induction culture medium containing 200 mu mol/L of acetosyringone, and diluting the bacterium solution to OD600The value is 0.15, and the cultivation is continued for 5 to 6 hours until the OD is reached600The value is 0.5 to 0.6; the monascus ruber spore liquid and agrobacterium tumefaciens EHA105 bacterial liquid containing binary plasmid knockout vector pHph0380-GLTP obtained above are mixed and coated on an AIM induction culture medium plate containing 200 mu mol/L acetosyringone, and the mixture is cultured for 48 hours at 25 ℃ in the dark.
5. The method for constructing a monascus pigment producing strain according to claim 1, wherein the screening of the positive clones comprises the following steps: adding a layer of MPS culture medium containing 100 mu g/mL hph, 200 mu mol/L cefotaxime and 0.2% Triton X-100 on the AIM induction culture medium plate after being cultured for 48 hours in a dark place, and continuously culturing for 5-8 days at 30 ℃; selecting a single colony, transferring the single colony to an MPS solid culture medium plate containing 100 mu g/mL hph, culturing for 3d, inoculating a strain capable of growing to an MPS liquid culture medium, culturing, extracting total DNA of the filamentous fungi according to an SDS cracking method, carrying out molecular analysis, carrying out PCR verification by using a pair of primers with the sequences of SEQ ID No. 15 and SEQ ID No. 16, and selecting a positive strain as the production strain.
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