CN118222528A - Glutamate dehydrogenase gene RkGDH and application thereof - Google Patents

Abstract

The invention discloses a glutamate dehydrogenase gene RkGDH which is separated from rhodosporidium (Rhodosporidium kratochvilovae) YM25235, the nucleotide sequence of which is shown as SEQ ID NO. 1, and the amino acid sequence of which is shown as SEQ ID NO. 2; the gene is connected with a vector and transferred into rhodosporidium cell, and the experimental result shows that the overexpression of RkGDH gene can promote rhodosporidium to synthesize carotenoid; the invention provides a reference for large-scale commercial production of carotenoids.

Description

Glutamate dehydrogenase gene RkGDH and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering, and relates to a glutamate dehydrogenase gene RkGDH, in particular to a glutamate dehydrogenase gene RkGDH cloned from rhodosporidium (Rhodosporidium kratochvilovae) YM25235, and the gene is connected with a vector and transferred into a yeast cell to improve the expression level of the gene and finally promote the synthesis of carotenoid.

Background

Carotenoids are naturally occurring fat-soluble pigments that are commonly found in photosynthetic systems of higher plants, algae and phototrophic bacteria. Carotenoids are a terpenoid compound consisting of an isoprene backbone, mostly a forty-carbon molecular backbone formed by the joining of two eicosanoid tails and tails, from which a variety of different compounds can be derived. Natural carotenoid has strong oxidation resistance, and can remove free radical, prevent aging, and prevent cancer, so it can be widely used in various fields such as food, pharmacy, feed, and cosmetics.

Currently, commercial carotenoid sources are mainly obtained by chemical synthesis, and small amounts are natural carotenoids extracted from plants and produced by microbial fermentation. Chemically synthesized carotenoids, although having a molecular structure similar to that of natural carotenoids, are more healthy. The advantage of using microorganisms for the production of carotenoids is that low cost substrates are used, which can reduce the production costs. Thus, the development of metabolic engineering has led to the efficient and low cost production of carotenoids by microorganisms as a promising alternative.

The carotenoid is prepared by using acetyl CoA as a substrate, synthesizing isopentenyl pyrophosphate (isopentenylpyrophosphate, IPP) and dimethylallyl pyrophosphate (DIMETHYLALLYL DIPHOSPHATE, DMAPP) through a mevalonic acid (mevalonate, MVA) way, concentrating the two, and further converting the two into lycopene, beta-carotene and other different carotenes through different reaction processes.

The GDH2 gene encodes an NAD-linked glutamate dehydrogenase that catalyzes oxidative deamination of glutamate to alpha-ketoglutarate and ammonium using NADH as a cofactor involved in glutamate production when overexpressed or when NH ^4＋ is abundant. The GDH2 gene encodes a 1092 amino acid protein on chromosome XII that has a high degree of sequence similarity to the crossed glutamate dehydrogenase of Neurospora (Neurosporu). Alpha-ketoglutarate is an important compound in the TCA cycle, and can effectively promote the synthesis of acetyl CoA, a substrate for carotenoid synthesis, through the TCA cycle, so that the synthesis content of the carotenoid is increased. GDH2 plays an important role in glutamate metabolism, mainly participates in biosynthesis and decomposition of glutamate, and under the condition of glucose growth, the concentration of NAD cofactor in cytoplasm is higher, so that it forces the reaction to degrade towards glutamate to obtain NAD-NADH balance, and the response of cells under environmental change is influenced by regulating NAD-NADH balance. GDH2 also interacts with GDH1 and GDH3 genes, together regulating glutamate homeostasis and ammonia uptake. Therefore, the glutamic acid dehydrogenase gene GDH2 is researched, the influence of the glutamic acid dehydrogenase gene GDH2 on the synthesis mechanism of carotenoid produced by rhodosporidium (Rhodosporidium kratochvilovae) YM25235 is analyzed, and a foundation is laid for the large-scale production of the carotenoid.

Disclosure of Invention

The invention provides a glutamate dehydrogenase gene RkGDH which is separated from rhodosporidium (Rhodosporidium kratochvilovae) YM25235, the nucleotide sequence of the gene is shown as SEQ ID NO.1, the length of the gene is 3039bp (basic group), and the amino acid sequence coded by the gene is shown as SEQ ID NO. 2. The gene is connected with a vector and transferred into rhodosporidium cell, and the improvement of the expression level of the gene promotes the synthesis of carotenoid.

The invention aims at realizing the following technical scheme:

1. Extracting total RNA from rhodosporidium YM25235, performing reverse transcription to synthesize cDNA, taking the synthesized cDNA as a template, adopting a specific primer for amplifying RkGDH gene, performing polymerase chain reaction to amplify to obtain a target sequence, performing double digestion and recovery on a vector pRH2034, connecting a target fragment and the vector by a one-step cloning method to obtain a connection product recombinant plasmid pRHRkGDH2, transferring the recombinant plasmid pRHRkGDH2 into escherichia coli, screening out positive monoclonal by PCR, performing enzyme digestion verification on the recombinant plasmid pRHRkGDH by using two restriction enzymes BamHI and EcoRV, extracting plasmid after culturing positive clones, and sequencing to obtain a glutamate dehydrogenase gene RkGDH with the fragment size of 3039 bp;

2. The recombinant vector pRHRkGDH is transformed into rhodosporidium YM25235 by using a PEG-mediated protoplast method, transformants are screened to obtain an over-expression strain containing pRHRkGDH2, the over-expression strain containing pRHRkGDH2 is cultured, pigments are extracted, and the content of total carotenoid is measured by using an ultraviolet-visible spectrophotometer.

The rhodosporidium YM25235 strain has the advantages of low raw material cost, short production period, stable heredity, high safety and the like, the glutamic acid dehydrogenase gene RkGDH2 is cloned from the cDNA of the total RNA reverse transcription extracted from the strain, the rhodosporidium is modified by a genetic engineering means, and the overexpression of RkGDH genes in the rhodosporidium YM25235 causes the increase of the content of carotenoid in cells; the invention provides a novel method for producing carotenoid, which provides good application prospect and economic benefit for the industrialized production of carotenoid; the method is simple, is easy and convenient to operate, and is suitable for industrial production and market popularization and application.

Drawings

FIG. 1 is a diagram showing PCR amplification of RkGDH gene of rhodosporidium YM25235 of the present invention; 1. DNA molecular weight marker DL5000; 2. a negative control; 3. a cDNA fragment of gene RkGDH;

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

FIG. 3 is a colony PCR verification electrophoresis pattern; DNA molecular weight marker DL5000;2. a negative control; 3. a cDNA fragment of gene RkGDH; the rest is transformant;

FIG. 4 is a restriction analysis of recombinant plasmid pRHRkGDH 2; wherein: DNA molecular weight marker DL10000;2. a negative control; thirdly, carrying out double digestion on BamHI and EcoRV of the plasmid pRH 2034; 4. BamHI and EcoRV of the recombinant plasmid pRHRkGDH are digested simultaneously; 5. a cDNA fragment of gene RkGDH; DNA molecular weight marker DL5000;

FIG. 5 positive cloning verification of transformed rhodosporidium YM25235 with recombinant plasmid pRHRkGDH 2; dna molecule scalar DL5000;2. a negative control; 3. PCR products amplified with YM25235 genome; 4. PCR products amplified with plasmid pRHRkGDH; 5. PCR products amplified with YM25235/pRHRkGDH strain genome;

FIG. 6 results of carotenoid content comparison of overexpressing strain YM25235/pRHRkGDH2 with control strain YM 25235.

Detailed Description

The present invention will be described in further detail with reference to the accompanying drawings and examples, but the scope of the present invention is not limited to the above, and the reagents and methods used in the examples, unless otherwise specified, employ conventional reagents and methods.

Example 1

1. Total RNA of rhodosporidium YM25235 is extracted by using a UNlQ-10 column Trizol total RNA extraction kit (product number: SK 1321) of biological engineering (Shanghai) Co., ltd.), then cDNA is synthesized by reverse transcription according to the operation instruction in a Vazyme company kit (product number: R212-02) HISCRIPT II 1st Strand cDNA Synthesis Kit (+ GDNA WIPER), 1 mu L of cDNA is taken as a template for polymerase chain reaction, specific primers RkGDH-F and RkGDH-R are designed according to RkGDH sequences obtained in transcriptome sequencing, PCR amplification is performed on a PCR instrument (Beijing Liuzhuzhu biological technology Co., ltd.) by using primers RkGDH-F and RkGDH-R according to the cDNA template obtained in the above, and the primers, amplification system and amplification conditions used for the reaction are as follows:

RkGDH2-F:5'-ATCACTCACCATGGCGGATCCGATGATTGCCCCGCCGTC-3', (double underlined as upstream vector end homologous sequence, single underlined as bamhi cleavage site);

RkGDH2-R: 5'-CCGGTCGGCATCTACGATATCTCACGCCTCGGCATCAG-3', (double underlined is the downstream vector end homologous sequence, single underlined is the EcoRV cleavage site);

The PCR amplification system was added to 50. Mu.L as follows （50μL）：Template cDNA1μL、RkGDH2-F 2μL、RkGDH2-R 2μL、dNTPs Mix(10mM each) 1μL、Vazyme 2×Phanta Max Buffer 25μL、Vazyme Phanta Max Super-Fidelity DNA Polymerase 1μL,ddH₂O;

Amplification conditions: pre-denaturation at 95℃for 3min; denaturation at 95℃for 15s, annealing at 68℃for 15s, extension at 72℃for 3min for 5s for 30 cycles; finally, thoroughly extending for 5min at 72 ℃; taking 2 mu L of the product after the reaction, and performing electrophoresis analysis in agarose gel with the concentration of 1%, wherein the result is shown in figure 1; amplifying to obtain a fragment with the size of 3039bp, which is named RkGDH < 2 >;

pRH2034 is subjected to double digestion by two restriction enzymes of BamHI and EcoRV; the above 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 connected with a seamless cloning kit (LIGHTNING ™ DNA Assembly Mix Plus, jiangsu Bai Shi Mei Biotechnology Co., ltd.) to obtain a recombinant plasmid pRHRkGDH, the connection system was as follows (10. Mu.L): LIGHTNING ™ DNA Assembly Mix Plus. Mu.L of linearized vector pRH 2034. Mu. L, rkGDH2 fragment 2. Mu.L; gently stirring and mixing by using a pipette, collecting the reaction liquid to the bottom of a tube by short centrifugation, and then reacting for 30min at 37 ℃ in a PCR (Beijing Liu Biotechnology Co., ltd.); cooling to4 ℃ or immediately cooling on ice.

2. Adding 10 mu L of obtained connection product into 100 mu L DH5 alpha competent cells, uniformly mixing the walls of a flick tube, carrying out ice bath for 30min, immediately placing on ice for cooling for 90s after carrying out water bath heat shock for 90s at 42 ℃, adding 900 mu L of LB liquid medium into the connection system, carrying out shaking incubation for 1h at 37 ℃ and 100rpm, centrifuging for 10min at 5000rpm, discarding 900 mu L of supernatant, slightly blowing about 100 mu L of LB medium to obtain suspension thalli, coating the suspension thalli on an LB solid plate (containing 100 mu g/mL of spectinomycin), carrying out inversion culture for 12-16h at 37 ℃, randomly picking up 5 white colonies growing on the plate, carrying out positive verification through colony PCR, and obtaining a result as shown in figure 3, wherein the picked monoclonal strains can be seen to amplify specific strips with the same size as the target fragments through colony PCR, and the fact that the picked DH5 alpha strains are successfully transferred into recombinant plasmids; inoculating positive clones into LB liquid medium (containing 100 mug/mL spectinomycin) for overnight culture, collecting thalli and extracting plasmids (OMEGA PLASMID MINI KIT I kit, OMEGA Co., USA), carrying out double digestion verification on pRHRkGDH2 by BamHI and EcoRV, and obtaining a result shown in figure 4, wherein the result shows that recombinant plasmid pRHRKGDH2 generates two bands of about 3kb and about 10kb after double digestion, the two bands are respectively consistent with the sizes of RkGDH fragments and pRH2034 fragments after double digestion, and the plasmid map of recombinant vector pRHRkGDH2 is shown in figure 2, and the preliminary shows that recombinant plasmid pRHRkGDH2 is successfully constructed; sequencing by using a sequencing primer, and sending out the plasmid with correct enzyme digestion verification for further verification. Sequencing results show that the sequence obtained by sequencing is completely consistent with the target sequence, and no base mutation, deletion and the like are generated.

3. The DH5 alpha strain successfully transferred into the correct recombinant vector pRHRkGDH is selected and inoculated into LB liquid medium (containing 100 mu g/mL spectinomycin) for overnight culture, plasmids (OMEGA PLASMID MINI KIT I kit, OMEGA company, america) are extracted, the concentration is measured, and the obtained product is stored at-20 ℃ for standby. The recombinant vector pRHRkGDH was transformed into rhodosporidium YM25235 by PEG-mediated protoplast transformation, as follows: selecting single colony of rhodosporidium YM25235, inoculating into 5mL YPD liquid culture medium, and shake culturing at 28deg.C and 160rpm overnight to obtain seed solution; Transferring the seed solution into 50mL of YPD liquid culture medium according to the inoculation amount of 1%, culturing at 28 ℃ and 160rpm until the bacterial solution OD ₆₀₀ is 0.45-0.5, centrifuging the bacterial solution at 4 ℃ and 4500rcf for 5min, and collecting bacterial cells; the collected thalli are washed twice by a prepared citric acid buffer solution (30 mmol/L citric acid, 83mmol/L sodium citrate, 600mmol/L mannitol and NaOH for adjusting the pH value to 5.4), and then are suspended by 800-1000 mu L of the citric acid buffer solution and placed on ice for standby; preparing enzymolysis solution (0.075 g snailase, 0.03g Sigma muramidase, constant volume to 5mL with citric acid buffer solution), filtering with 0.22 μm sterile filter membrane, and placing into 5mL sterile centrifuge tube; mixing 4mL of enzyme solution with 800-1000 mu L of thallus suspended by citric acid buffer solution, placing the mixture at 28 ℃ for shaking culture at 90rpm for 2.5-3 hours for enzymolysis, observing enzymolysis efficiency by a microscope, and centrifuging the culture at 1300rpm for 10 minutes at 4 ℃ under the condition that the enzymolysis quantity is confirmed to be enough for collecting the thallus; adding 10mL of STC buffer (1.2 mol/L sorbitol, 10mmol/L Tris-HCl, 100mmol/L CaCl ₂) to wash the collected thalli twice on ice to prepare yeast competent cells; Suspending thalli by using 800-1000 mu L of STC buffer solution, and sub-packaging 100 mu L of thalli into a 5mL sterile centrifuge tube for standby application; Adding 10 mu L PRHRKGDH of recombinant plasmid into 100 mu L competent cells, gently mixing, incubating on ice for 10min, adding 200 mu L of pre-cooled PTC buffer (50% PEG, 10mmol/L Tris-HCl, 100mmol/L CaCl ₂), ice-bathing for 10min, adding 200 mu L of pre-cooled PTC buffer again, ice-bathing for 10min, finally adding 800 mu L of pre-cooled PTC buffer, mixing gently, centrifuging at 28deg.C for 10min at 150deg.C, collecting thallus, discarding 1mL supernatant, adding 1mL of 0.4mol/L sucrose YPD liquid medium, suspending, shake culturing at 28deg.C at 90rpm for 24 hr to recover thallus; Centrifuging the recovered thallus at 1300rpm for 10min, collecting thallus, discarding the supernatant to obtain 100 μl of suspended thallus, coating on 0.4mol/L sucrose YPD solid medium (containing 40 μg/mL hygromycin B), culturing at 28deg.C for 3-4 days in an inverted manner until the solid medium grows out, numbering the transformant, transferring to YPD solid medium containing 150 μg/mL hygromycin B, and culturing at 28deg.C for two days in an inverted manner; according to the known function of the gene and the characteristic of rhodosporidium YM25235, taking color as the basis of screening an over-expression strain, specifically performing grafting on the obtained transformant in a 5mL test tube containing YPD liquid culture medium, culturing for 120h at 28 ℃ under shaking at 160rpm, and observing the color by taking YM25235 wild strain as a control to screen out a transformant with redder color than YM 25235; The selected transformants were picked, genomic DNA of the yeast transformants was extracted according to the procedure in the DNA extraction kit (purchased from Shanghai Biotechnology Co., ltd.) and subjected to PCR verification, and the results are shown in FIG. 5, in which it was seen that a band having the same size as the cDNA fragment of RkGDH2 was amplified by PCR using the genome of the transformants as a template, and the gene verification of the recombinant transformants was correct, indicating that the RkGDH fragment was successfully introduced into the genome of the yeast transformants.

Example 2: analysis of carotenoid content of overexpressing Strain YM25235/pRHRkGDH2

Inoculating positive transformant into 50mL YPD liquid medium, fermenting and culturing at 28deg.C and 160rpm for 168h, centrifuging at 4500rpm for 6min, collecting thalli in 50mL centrifuge tube, washing twice with precooled ddH ₂ O, centrifuging at 4500rpm for 8min, thoroughly removing supernatant, lightly knocking tube wall to make thalli uniformly adhere on tube inner wall, oven drying at 55deg.C, grinding thalli into powder, extracting total carotenoid with acetone-methanol mixed solution (V _{（ Acetone (acetone) ）}：V_{（ Methanol ）} =4:1) 0.4g of the powder, measuring total carotenoid in sea buckthorn oil by ultraviolet spectrophotometry with reference to Yang Mozheng et al, improving [ J ]. Central national university (Nature science edition), 2009,18 (03): 5-8 degrees by ultraviolet-visible spectrophotometry, measuring absorbance at 450nm with wild rhodosporidium YM25235 strain as control, and calculating total carotenoid content (mg/g dry thalli), wherein total carotenoid expression amount of wild rhodosporidium strain 62 is 5.08.+ -. 35.45 mg/35.858 total carotenoid is synthesized by YM 6.07 mg/35.35 mg of YM 2.858 strain. The total carotenoid synthesis amount of the over-expression strain YM25235/pRHRkGDH is obviously improved compared with that of the wild rhodosporidium strain YM25235 (shown in figure 6); the results show that overexpression of glutamate dehydrogenase gene RkGDH can cause increase of total carotenoid content in rhodosporidium YM25235 strain, and RkGDH gene can promote synthesis of total carotenoids.

Claims (2)

1. A glutamate dehydrogenase gene RkGDH with a nucleotide sequence shown in SEQ ID NO. 1.

2. Use of the glutamate dehydrogenase gene RkGDH according to claim 1 for promoting the production of carotenoids by rhodosporidium (Rhodosporidium kratochvilovae).