阅读说明：本技术 一种甲羟戊酸激酶基因rkmk及其应用 (Mevalonate kinase gene RKMK and application thereof ) 是由 张琦 郭彩娜 魏云林 季秀玲 于 2021-08-11 设计创作，主要内容包括：本发明公开了一种甲羟戊酸激酶基因RKMK及其用途,其核苷酸序列如SEQ ID NO:1所示,该基因编码的氨基酸序列如SEQ ID NO:2所示；该基因分离自红冬孢酵母(Rhodosporidium kratochvilovae)YM25235,将该基因与载体连接,转入红冬孢酵母细胞中,实验结果显示RKMK基因的过表达能够促进红冬孢酵母合成类胡萝卜素；本发明通过基因工程手段对微生物进行改造,来提高微生物体内类胡萝卜素的产量,为大规模商业化生产类胡萝卜素奠定基础。(The invention discloses a mevalonate kinase gene RKMK The nucleotide sequence is shown as SEQ ID NO. 1, and the amino acid sequence coded by the gene is shown as SEQ ID NO. 2; the gene is separated from red winter spore yeast ( Rhodosporidium kratochvilovae ) YM25235, the gene is connected with a vector and transferred into an rhodosporidium cell, and the experimental result shows that RKMK The overexpression of the gene can promote the rhodosporidium toruloides to synthesize the carotenoid; the invention improves the yield of carotenoid in the microorganism by modifying the microorganism by means of genetic engineering, and is a large-scale commercial productLays a foundation for the chemical production of carotenoid.)

1. Mevalonate kinase geneRKMKThe nucleotide sequence is shown in SEQ ID NO. 1.

2. The mevalonate kinase gene according to claim 1RKMKApplication in promoting microbial production of carotenoid is provided.

Technical Field

The invention belongs to the technical field of biology and genetic engineering, and relates to a mevalonate kinase geneRKMKAnd its use in increasing Rhodosporidium toruloidesRhodosporidium kratochvilovae) In carotenoid productionApplication is carried out.

Background

Mevalonate kinase (MVK), the first of three consecutive ATP-dependent enzymes in the Mevalonate (MVA) pathway, is one of the rate-limiting enzymes controlling the entire metabolic pathway, and the N-terminus of the Mevalonate kinase protein contains a conserved region rich in Gly/Ser, which is involved in binding ATP (adenosine triphosphate). Mevalonate kinase is responsible for the transfer of one phosphate group at position ATP-gamma to the hydroxy group at position 5 of mevalonate to form mevalonate-5-phosphate and release ADP. Mevalonate kinases of different biological origin are usually monomeric or dimeric, composed of identical subunits, with molecular weights of 70-105 kDa. All mevalonate kinases have 3 conserved regions constituting the active center of the enzyme, wherein conserved region 1 is involved in binding and catalyzing reactions of mevalonate substrates, while conserved region 2 and conserved region 3 are involved in binding and catalyzing reactions of ATP, thus indicating that their enzymatic reaction mechanisms are similar; mevalonate kinase catalyzes the sequential phosphorylation of mevalonate, i.e., mevalonate is the first substrate to bind to the enzyme and then Mg. ATP, and mevalonate-5-phosphate is the first substrate to be released before ADP is released (Rongbao lotus et al. mevalonate kinase gene research progress [ J ]. Chinese agro-scientific report 2011,13(3):17-25. DOI:10.3969/J. issn.1008-0864.2011.03.03.).

In plants, the enzyme is mainly involved in the synthesis of isoprenoid derivatives, and many plant isoprenoids have important commercial values, such as food flavors, beverages, vitamin A, vitamin D, vitamin E, natural pesticides (such as pyrethrin), rubber and the like (Rongbao lotus and the like, plant mevalonate kinase gene research advances [ J ]. Shandong agricultural science, 2011(4):12-16. DOI: 10.3969/j.issn.1001-4942.2011.04.004.). Research shows that the regulation of mevalonate kinase activity can affect the regeneration and growth of plant and participate in the maintenance of basic life activity and special organ in plant cell. In animals, this enzyme plays an important role in the control of cholesterol biosynthesis, is involved in human genetic diseases including mevalonic aciduria, hyperimmunoglobulinemia D, and periodic exothermic syndrome, is also involved in the synthesis of larval hormones in insects, and is a target enzyme for the action of pesticides. In microorganisms, the enzyme is also a key enzyme in an isoprenoid synthesis way, and a new way is opened for screening effective enzyme inhibitors, exploring effective biological pesticides and effectively solving animal and plant diseases and fungal diseases.

The carotenoid has strong oxidation resistance, provitamin A activity, coloring function and anticancer capability. Due to these diverse biological activities and functions, they are very important for human health, and have been widely studied and applied in the fields of foods, nutritional foods, animal feeds, cosmetics, and the like.

No report on the mevalonate kinase gene in promoting microbial carotenoid production has been found at present.

Disclosure of Invention

The invention aims to provide a mevalonate kinase geneRKMKThe gene of the invention is derived from Rhodosporidium toruloides (A)Rhodosporidium kratochvilovae) YM25235, its nucleotide sequence is shown in SEQ ID NO:1, the gene sequence is 2679bp long, and the coded amino acid sequence is polypeptide shown in SEQ ID NO:2, and the gene is connected with carrier and transferred into Rhodosporidium toruloides cell, and the improvement of the gene expression level promotes the synthesis of carotenoid.

The purpose of the invention is realized by the following technical scheme:

1. extracting total RNA from Rhodosporidium toruloides YM25235, reverse transcribing to synthesize cDNA, and amplifying with the synthesized cDNA as templateRKMKThe specific primer of (1) is amplified by a polymerase chain reaction to obtain a target sequence, the vector pRH2034 is subjected to double digestion and recovery, a target fragment is connected with the vector by a one-step cloning method to obtain a connection product recombinant plasmid pRHRKMK, the recombinant plasmid pRHRKMK is transferred into escherichia coli, a positive single clone is screened out by PCR, and the recombinant plasmid pRHRKMK is used for PCRBamHⅠ、EcoPerforming enzyme digestion verification on the two restriction endonucleases of RV, extracting plasmids after verifying that positive clones are cultured, sequencing to obtain mevalonate kinase gene with the fragment size of 2679bpRKMK；

2. Transforming the recombinant vector pRHRKMK into Rhodosporidium toruloides YM25235 by a PEG-mediated protoplast method, screening transformants to obtain an overexpression strain containing pRHRKMK, culturing the overexpression strain containing pRHRKMK, extracting pigments, and measuring the content of total carotenoids by an ultraviolet-visible spectrophotometer.

The Rhodosporidium toruloides (A), (B) and (C)Rhodosporidium kratochvilovae) YM25235 has the advantages of short production cycle, stable heredity, safe production, etc.

The invention provides a novel method for producing carotenoids, which improves the yield of carotenoids in microorganisms by modifying the microorganisms by means of genetic engineering, and separates mevalonate kinase gene from cDNA reverse transcription of total RNA extracted from Rhodosporidium toruloides YM25235RKMKRhodosporidium toruloides YM25235RKMKThe overexpression of the gene causes the increase of the transcription level of the gene in the cell, and then the gene is translated into corresponding protein, thereby causing the increase of the expression level of enzymes related to the synthesis of the carotenoid in the cell; the research result is helpful for clarifying the carotenoid production mechanism in the rhodosporidium toruloides YM25235, provides reference for disclosing the mechanism of improving the carotenoid yield by microorganisms, provides good application prospect and economic benefit for industrial production of the carotenoid, and lays a foundation for large-scale commercial production of the carotenoid; the method is simple, easy to operate and suitable for industrial production and market popularization and application.

Drawings

FIG. 1 shows a scheme for producing Rhodosporidium toruloides YM25235 of the present inventionRKMKPCR amplification of the gene; DNA molecular weight marker DL 5000; 2. negative control; 3. geneRKMKA cDNA fragment of (1);

FIG. 2 is a plasmid map of recombinant plasmid pRHRKMK;

FIG. 3 is a PCR-verified electrophoretogram of colonies; DNA molecular weight marker DL 5000; 2. geneRKMKA cDNA fragment of (1); 3-7 is a transformant;

FIG. 4 shows restriction analysis of the recombinant plasmid pRHRKMK; wherein: DNA molecular weight marker DL 10000; 2. negative control 3 of plasmid pRH2034BamH I andEcor V double enzyme digestion; 4. recombinant plasmid pRHRKMKBamH I、EcoR V double enzyme digestion; 5. geneRKMKA cDNA fragment of (1); molecular weight of DNALabel DL 5000;

FIG. 5 shows the positive clone validation of recombinant plasmid pRHRKMK transformed Rhodosporidium toruloides YM 25235; DNA molecular scalar DL 5000; 2. negative control; 3. PCR products amplified with YM25235 genome; 4. PCR products amplified with plasmid pRHRKMK; 5. PCR products amplified with YM25235/pRHRKMK strain genome;

FIG. 6 comparison of carotenoid content of the over-expressed strain YM25235/pRHRKMK with that of the control strain YM 25235.

Detailed Description

The present invention is further illustrated in detail below with reference to the drawings and examples, but the scope of the present invention is not limited to the above description, and reagents and methods used in the examples are, unless otherwise specified, conventional reagents and methods are used.

Example 1: isolation of mevalonate kinase Gene from Rhodosporidium toruloides YM25235RKMKAnd construction of overexpression vector pRHRKMK

Extracting total RNA of Rhodosporidium toruloides YM25235 with UNlQ-10 column type Trizol total RNA extraction Kit (product number: SK 1321) from Biotechnology engineering (Shanghai) Ltd, performing reverse transcription according to the instructions in the Kit (product number: R212-02) from Vazyme corporation HiScript II 1st Strand cDNA Synthesis Kit (+ gDNA wiper) to synthesize cDNA, performing polymerase chain reaction with 1. mu.L cDNA as template, and sequencing according to transcriptomeRKMKDesigning specific primers RKMK-F and RKMK-R (primer 1 and primer 2), carrying out PCR amplification on the cDNA template obtained by the above by using the primers RKMK-F and RKMK-R on a PCR instrument (Beijing six Biotech limited), wherein the primers, the amplification system and the amplification conditions used in the reaction are as follows:

primer 1: RKMK-F: 5' -ATCACTCACCATGGCGGATCCTATGACCTCGACCGCGGG-3’

(SEQ ID NO: 3) (double underlined is the homologous sequence at the end of the upstream vector and single underlined isBamHI cleavage site)

Primer 2: RKMK-R: 5' -CCGGTCGGCATCTACGATATCCTACGCCCTGACCCAGC-3' (SEQ ID NO: 4) (double underlined is the downstream vector end homologous sequence and single underlined isEcoRV enzyme cleavage site);

the PCR amplification system was as follows (50. mu.L):

amplification conditions: pre-denaturation at 95 deg.C for 3min, denaturation at 95 deg.C for 15s, annealing at 64 deg.C for 15s, and extension at 72 deg.C for 2min30s for 30 cycles, and final extension at 72 deg.C for 5 min; after the reaction, 2. mu.L of the product was taken and subjected to electrophoresis analysis in 0.8% agarose gel, the results of which are shown in FIG. 1; the amplified fragment was designated as 2700bpRKMK(ii) a pRH2034 throughBamHⅠ、EcoRV, carrying out double enzyme digestion by two restriction enzymes; the two fragments were recovered with a multifunctional DNA recovery Kit (Beijing Baitaike Biotechnology Co., Ltd., product number: DP 1502), and the two recovered fragments were ligated with a seamless Cloning Kit (Clonexpress II One Step Cloning Kit C112, Nanjing Nodezaar Biotechnology Co., Ltd.) to obtain a recombinant plasmid pRHRKMK in the following ligation system (20. mu.L):

gently beating and mixing by using a pipette, centrifuging for a short time to collect reaction liquid to the bottom of the tube, and then reacting for 30min at 37 ℃ in a PCR instrument (Beijing Liu Biotechnology Co., Ltd.); cooled to 4 ℃ or immediately placed on ice to cool.

Adding 10 muL of obtained ligation products into 100 muL DH5 alpha competent cells, flicking the walls of the tubes uniformly, carrying out ice bath for 30min, immediately placing the cells on ice after carrying out heat shock on water bath at 42 ℃ for 90s for cooling for 90s, adding 900 muL of LB liquid culture medium into a connecting system, carrying out oscillation incubation for 1h at 37 ℃ and 100rpm, centrifuging the supernatant at 5000rpm for 10min, then carrying out supernatant at 900 muL, slightly blowing and beating suspended bacteria by the residual LB culture medium about 100 muL, coating an LB solid plate (containing 100 mug/mL spectinomycin) on the supernatant, carrying out inverted culture at 37 ℃ for 12-16 h, picking white colonies growing on the plate, verifying positive clones by colony PCR, and inoculating the clones verified to the LB liquid culture plateCulturing overnight in a nutrient medium (containing 100 mug/mL spectinomycin), randomly selecting 5 white colonies growing on a plate and numbering as No. 1-5, carrying out positive clone verification through colony PCR, and obtaining a result shown in figure 3, wherein the selected five monoclonal strains are amplified to form specific strips with the same size as a target fragment through colony PCR, which indicates that the selected five DH5 alpha strains are successfully transferred into recombinant plasmids; extracting Plasmid (OMEGA Plasmid Mini Kit I, OMEGA USA) withBamHⅠ、EcoPerforming double digestion verification on pRHRKMK by RV; the results are shown in FIG. 4, which shows that the recombinant plasmid pRHRKMK produced two bands of about 2.7kb and about 10kb by double digestion (lane 4 in FIG. 4), which were associated with the two bandsRKMKThe size of the fragment is consistent with that of the fragment of the pRH2034 vector after double digestion, and the success of the construction of the recombinant plasmid pRHRKMK is preliminarily shown; the plasmid with the correct restriction enzyme digestion verification is sent out for further verification, and the sequencing result shows that the amplified fragment has the size of 2679bp, the sequence composition is the nucleotide sequence shown in SEQ ID NO. 1 and is named asRKMKAnd is andRKMKthe sizes of cDNA fragments of the genes are consistent, which indicates that the expression vector pRHRKMK is successfully constructed, and the plasmid map of the recombinant vector pRHRKMK is shown in figure 2.

Example 2:RKMKanalysis of Carotenoid content in Gene-overexpressed Rhodosporidium toruloides YM25235

1. Transformed Rhodosporidium toruloides YM25235

Selecting a DH5 alpha strain which is successfully transferred into a correct recombinant vector pRHRKMK, carrying out monoclonal inoculation on an LB liquid culture medium (containing 100 microgram/mL spectinomycin) for overnight culture, extracting a Plasmid (OMEGA Plasmid Mini Kit I, OMEGA corporation, USA), measuring the concentration, and storing at-20 ℃ for later use; selecting single colony of Rhodosporidium toruloides YM25235, inoculating to 5mL YPD liquid culture medium, and shake culturing at 30 deg.C and 200rpm overnight; transferring the overnight cultured bacterial liquid into 50mL YPD liquid culture medium at 30 deg.C and 200rpm, and performing shaking culture to OD₆₀₀At 0.5, the culture was centrifuged at 4500 rpm for 5min at 4 ℃ to collect the cells; prepared citric acid buffer (30 mM citric acid, 83mM sodium citrate, 600mM mannitol, NaOH) was usedpH value is 5.4), the thalli is washed twice, the thalli is collected by centrifugation at 4 ℃ and 4000 rpm for 5min and is suspended by 1mL of citric acid buffer solution, the thalli is collected by centrifugation at 4 ℃ and 4000 rpm for 5min and is placed on ice for standby; preparing lyase solution (0.156 g snailase, 0.08g lywallzyme, ddH₂O to 5 mL), filtering the enzyme solution by using a sterile filter membrane with the diameter of 0.22 mu m, and placing the enzyme solution in a sterile centrifuge tube with the volume of 50mL for later use; mixing 4mL of enzyme solution with the bacterial solution, placing at 30 ℃, performing shaking culture and enzymolysis at 90rpm for 2.5h, centrifuging the culture at 4 ℃ and 1300rpm for 10min, and collecting thalli; with STC (1.2M sorbitol, 10mM Tris-HCl, 100mM CaCl)₂) Washing the collected thallus twice on ice to prepare yeast competent cells; subpackaging yeast competent cells into 5mL sterile centrifuge tubes for later use according to 100 mu L per tube; add 2-5. mu.g pRHRKMK recombinant plasmid to 100. mu.L of competent cells and mix gently (usually the fragment volume should not exceed 10. mu.L), incubate on ice for 10min, add 200. mu.L of precooled PTC (50% PEG, 10mM Tris-HCl, 100mM CaCl)₂) Ice-bath for 10min, adding 800 μ L precooled PTC, mixing gently, ice-bath for 10min, centrifuging at 4 deg.C and 1500rpm for 10min, and collecting thallus; adding 1.6mL of 0.4M sucrose YPD liquid culture medium for suspension, and performing shaking culture at 30 ℃ and 90rpm for 12h to recover the thallus; centrifuging the recovered thallus at 1300rpm for 10min to collect thallus, discarding supernatant, and suspending thallus in 100 μ L culture medium, spreading on 0.4M sucrose YPD solid culture medium (containing 130 μ g/mL hygromycin B), and performing inverted culture at 30 deg.C for 2-3 d; numbering the transformants obtained after coating, transferring the transformants to a YPD solid culture medium (containing 150. mu.g/mL hygromycin), and performing inverted culture at 30 ℃ for 2 days; selecting transformants by color according to the known functions of the gene, specifically, inoculating the obtained transformants into 5mL YPD medium, carrying out shake culture at 30 ℃ and 200rpm for 120h, observing the color by using YM25235 wild strain as a control, and selecting the transformants with the color being redder than that of YM 25235; the selected transformant was selected, the genomic DNA of the yeast transformant was extracted according to the procedures of the DNA extraction kit of Shanghai Biotechnology engineering Co., Ltd, and then PCR verification was carried out, the results are shown in FIG. 5, from which it can be seen that the genomic DNA of the yeast transformant was amplified by PCR using the genome of the yeast transformant as a templateRKMKThe cDNA fragments of (1) are identical in size, and the recombinant DNA is transformedThe correct gene verification of the chemostat indicatesRKMKThe fragment has been successfully ligated into the genome of the yeast transformant.

2、RKMKAnalysis of Carotenoid content in Gene-overexpressed Rhodosporidium toruloides YM25235

Culturing overexpression strain containing pRHRKMK at 28 deg.C for 168 hr, extracting carotenoid, and determining total carotenoid content (mg/g dry thallus) at 445nm with ultraviolet-visible spectrophotometer by using original Rhodosporidium toruloides YM25235 strain as control, as shown in FIG. 6; as can be seen from the figure, the total carotenoid synthesis amount of the over-expression strain YM25235/pRHRKMK is obviously improved compared with that of the wild type Rhodosporidium toruloides YM25235 strain, the carotenoid synthesis amount of the wild type Rhodosporidium toruloides YM25235 strain is 5.41 +/-0.02 mg/g, and the carotenoid synthesis amount of the over-expression strain YM25235/pRHRKMK is 6.65 +/-0.11 mg/g, namely the carotenoid synthesis amount of the over-expression strain YM25235/pRHRKMK is 1.23 times that of the control strain; the results showed that mevalonate kinase geneRKMKThe overexpression of (a) can cause the increase of the total carotenoid content in the rhodosporidium toruloides YM25235 strain,RKMKthe gene can promote the synthesis of total carotenoid.