CN113652440B - 3-ketoacyl coenzyme A thiolase gene RKACAA1-2 and application thereof - Google Patents

Abstract

The invention discloses a 3-ketoacyl coenzyme A thiolase geneRKACAA12, the nucleotide sequence of the gene is shown as SEQ ID NO. 1, and the amino acid sequence of the gene is shown as SEQ ID NO. 2; the gene is derived from rhodosporidiumRhodosporidium kratochvilovae) YM25235, and the gene is transformed into rhodosporidium YM25235, and the experimental result showsRKACAA1Overexpression of the-2 gene resulted in increased carotenoid synthesis levels in YM25235 strain; the invention uses genetic engineering handThe section reforms the microorganism to improve the yield of carotenoid in the microorganism, and lays a foundation for large-scale commercial production of carotenoid.

Description

3-ketoacyl-CoA thiolase geneRKACAA1-2And applications thereof

Technical Field

The invention belongs to the field of biotechnology and genetic engineering, and relates to a 3-ketoacyl coenzyme A thiolase geneRKACAA1-2And applications thereof.

Background

Carotenoids (carotenoids) are a general term for a class of important natural pigments, belonging to a class of terpenes, having multiple conjugated double bonds, and generally being yellow, orange or red in color. The structure of carotenoids is based on isoprenoids, and most typical carotenoid chemical structures contain 40 carbon atoms and are polymerized from 8 isoprenoids. Carotenoids can be divided into two broad categories depending on the type of carbon skeleton terminal chemical groups. One class is the closed-loop carotenoids containing a cyclic structure, such as alpha-carotene, beta-carotene, lutein, anthocyanidin, and the like. The other group is open-loop carotenoids not containing a cyclic structure, such as phytoene, lycopene, etc. Currently, more than 800 natural carotenoids have been found in organisms such as higher plants, animals, fungi, etc., of which about 50 are very important as provitamin a for humans, which are capable of being converted to vitamin a, α -carotene, β -lutein and β -carotene.

Research shows that carotenoid is a fat-soluble substance with various functions, has special nutritive value and capability of being converted into essential vitamins, is an important natural pigment capable of providing natural colors for animals and plants, and is also an important antioxidant. Typical carotenoids include beta-carotene, lycopene, lutein, etc., wherein the beta-carotene has important physiological health care functions of resisting oxidation, resisting cancer, provitamin A, preventing senile macular degeneration of eyes, delaying aging, improving immunity, etc., the lycopene can improve the antioxidation of self tissues and cells, enhance the immune system capacity and effectively maintain the health of human bodies, and the lutein is an important component of macular pigment in retina, can filter blue light, prevent retina damage, etc. Carotenoids are various, and different carotenoids have different physiological functions and are widely used in various aspects of nutrition fortification and coloring of medicines, health care, foods and feeds.

Carotenoids can be obtained by biological (plant, microbial) extraction and chemical synthesis. Among them, the safety of chemically synthesized pigments is questioned, and some chemically synthesized carotenoids (e.g., astaxanthin) have been found to be harmful and banned from pharmacy sales. With the development of biotechnology and the increasing awareness of food safety, the demand for carotenoids of natural origin obtained by biological extraction is increasing. Natural carotenoids of plant origin are relatively high in production cost and affected by seasons, while natural carotenoids of microbial origin are favored by researchers because of their natural, efficient, low-cost production and easy industrialization.

Disclosure of Invention

The invention provides a 3-ketoacyl-CoA thiolase geneRKACAA1The gene is derived from rhodosporidiumRhodosporidium kratochvilovae) Isolated from YM 25235; the nucleotide sequence of the gene is shown as SEQ ID NO. 1 or the nucleotide sequence complementary to the SEQ ID NO. 1, the length of the gene is 1263bp (basic group), and the amino acid sequence of the gene codes for polypeptide shown as SEQ ID NO. 2.

Another object of the present invention is to provide the 3-ketoacyl-CoA thiolase geneRKACAA1-2 for use in promoting the production of carotenoids by microorganisms.

The rhodosporidium has the advantages thatRhodosporidium kratochvilovae) YM25235 has the advantages of short production period, stable heredity, safe production and the like.

The above objective is accomplished by the following technical scheme that total RNA is extracted from rhodosporidium YM25235, then cDNA is synthesized by reverse transcription, cDNA is used as a template, a target fragment is obtained by PCR amplification, a vector pRH2034 is subjected to double digestion and recovery, the target fragment and the vector are connected by a one-step cloning method, a recombinant plasmid pRHRKACAA1-2 is obtained, the connection product is transferred into escherichia coli, positive monoclonal is selected by PCR, and the recombinant plasmid pRHRKACAA1-2 is used forBamHⅠ、EcoPerforming enzyme digestion verification on the R V two restriction enzymes, and extracting positive clone after culturingPlasmid, sequencing to obtain 3-ketoacyl-CoA thiolase gene with fragment size of 1263bpRKACAA1-2; transforming recombinant vector pRHRKACAA1-2 into rhodosporidium YM25235 by PEG-mediated protoplast method, and screening the transformant to obtainRKACAA1-2The over-expression strain is cultured, and the content of total carotenoid is measured by using an ultraviolet-visible spectrophotometer.

The invention uses rhodosporidium yeast to produce the rhodosporidium extractRhodosporidium kratochvilovae) Separating the total RNA gene of YM25235 to obtain the 3-ketoacyl-CoA thiolase geneRKACAA1-2, the full length of the gene is 1263bp; the gene is transferred into rhodosporidium YM25235, and the experimental result shows thatRKACAA1The overexpression of the-2 gene can cause the transcription level of the gene in cells to be improved to a certain extent, which indicates that the exogenous gene is transcribed in the bacterial body;RKACAA1overexpression of the-2 gene can promote the synthesis of total carotenoids; the research result provides a reference for revealing a mechanism of improving the carotenoid yield by microorganisms, is helpful for improving the carotenoid content by modifying the mechanism by a genetic engineering means, provides good application prospect and economic benefit for the industrialized production of the carotenoid, and lays a foundation for large-scale commercial production of the carotenoid.

Drawings

FIG. 1 shows rhodosporidium YM25235 of the present inventionRKACAA1-2Gene PCR amplification map; DNA molecular weight marker DL2000;2. a negative control; cDNA fragment of RKACAA1-2;

FIG. 2 is a plasmid map of recombinant plasmid pRHRKACAA 1-2;

FIG. 3 is a colony PCR verification electrophoresis pattern; DNA molecular weight marker DL2000;2. a negative control; 3. RKACAA1-2is a cDNA fragment of (C); 4-8 are transformants;

FIG. 4 restriction analysis of recombinant plasmid pRHRKACAA 1-2; dna molecule scalar DL10000;2. a negative control; 3. empty plasmid pRH2034BamHⅠ、EcoR V double enzyme cutting; 4. recombinant plasmid pRHRKACAA1-2BamHⅠ、EcoR V double enzyme cutting; 5.RKACAA1-2cDNA fragments of the genes; 6. DNA molecular scalar DL2000;

FIG. 5 is a heavy weightPositive cloning verification of the group plasmid pRHRKACAA1-2 transformed YM25235 strain; the figure 1.DNA molecular scalar DL2000;2. a negative control; 3. wild strain-specific gene bands; 4.RKACAA1-2; 5. verifying the transformant;

FIG. 6 is a comparison of total carotenoid content of over-expressed strain YM25235/pRHRKACAA1-2 and 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, all employ conventional reagents and methods.

Example 1: from rhodosporidium yeastRhodosporidium kratochvilovae) Isolation of 3-Keto acyl-CoA thiolase Gene in YM25235RKACAA1Construction of-2 and its overexpression vector pRHRKACAA1-2

Total RNA of rhodosporidium YM25235 was extracted using UNlQ-10 column Trizol Total RNA extraction kit (product number: SK 1321) from Biotechnology (Shanghai) Co., ltd, and then reverse transcribed into cDNA according to Vazyme Co., kit (product number: R212-02) HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wind), and polymerase chain reaction was performed with 1. Mu.L as a template, based on the findings found in transcriptome sequencingRKACAA12, designing specific primers RKACAA1-2-F and RKACAA1-2-R, and carrying out PCR amplification on a PCR instrument (Beijing Liuzhihai Biotechnology Co., ltd.) by using the obtained cDNA template, wherein the primers, components and amplification conditions are as follows:

RKACAA1-2-F: 5’-ATCACTCACCATGGCGGATCCTATGGCCAGCCTCATTTCG-3' (single underlined sections areBamII site, double underlined part is homology arm);

RKACAA1-2-R：5’-CCGGTCGGCATCTACGATATCCTACTCAGCGACGAAGAC-3' (single underlined sections areEcoR v site, double underlined is homology arm);

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

；

amplification conditions: pre-denaturing at 95 ℃ for 5min, denaturing at 95 ℃ for 15s, annealing at 60 ℃ for 30s, extending at 72 ℃ for 1min for 15s, performing 30 cycles, and finally extending at 72 ℃ thoroughly for 5min, taking 2 mu L of a product after the reaction, and performing electrophoresis analysis in agarose gel with the concentration of 1%, wherein the result is shown in FIG. 1, and amplifying to obtain a fragment with the size of 1263bp; pRH2034 was passed throughBamHⅠ、EcoPerforming double enzyme digestion by using two restriction enzymes R V; the above two fragments were recovered with a multifunctional DNA recovery kit (Beijing Baitaike Biotechnology Co., ltd., product number: DP 1502) and the fragments were ligated into vector pRH2034 using a seamless cloning kit (Vazyme ClonExpress II One Step Cloning Kit, nanjinouzan Biotechnology Co., ltd.) and the recombinant vector ligation system was as follows (20. Mu.L):

；

gently blowing 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 ℃; cooling to 4deg.C or immediately cooling on ice;

adding 10 mu L of the obtained connection product into 100 mu L DH5 alpha competent cells, lightly mixing, carrying out ice bath for 30min, immediately placing on ice for cooling for 90s after heat shock at 42 ℃, adding 900 mu L of LB liquid medium into the connection system, carrying out shake culture at 37 ℃ and 100rpm for 1h, centrifuging at 5000rpm for 10min, discarding 900 mu L of supernatant, leaving about 100 mu L of medium to suspend thalli, coating LB solid plates containing 100 mu g/mL of spectinomycin (Spe+) and carrying out inversion culture at 37 ℃ for overnight, randomly picking white colonies growing on the 5 plates, carrying out number 1-5, verifying positive clones through colony PCR, and obtaining a result shown in figure 3, wherein the five picked monoclonal strains are amplified into specific strips with the same size as the target fragments through PCR, and the five DH5 alpha strains are successfully transferred into recombinant plasmids; inoculating the clone with positive verification into LB liquid medium (containing 100 mug/mL spectinomycin)During overnight culture, plasmid extraction (OMEGA Plasmid Mini Kit I, OMEGA Co., USA) was performed usingBamHⅠ、EcoPerforming enzyme digestion verification on the R V two restriction enzymes; the results of the enzyme digestion are shown in FIG. 4, from which it can be seen that the recombinant plasmid pRHRKACAA1-2 was subjected toBamHⅠ、EcoAfter double cleavage of R V, the plasmid pRH2034 appearsBamHⅠ、EcoR V double enzyme cutting obtained linear carrier fragment size same strip, andRKACAA1-2the cDNA fragments of the genes are the same in size, which indicates that the recombinant plasmid is successfully transferred into the escherichia coli DH5 alpha strain; after cleavage verification, plasmids were extracted and sequenced (Kunming, biotechnology Co., ltd.) in the same manner. Sequencing results show that the amplified fragment has 1263bp, and the nucleotide sequence shown as SEQ ID NO. 1 is namedRKACAA1-2, withRKACAA1-2The cDNA fragments of the genes are consistent in size and sequence, which indicates that the expression vector pRHRKACAA1-2 is successfully constructed, and the plasmid map of the recombinant vector pRHRKACAA1-2 is shown in FIG. 2.

Example 2: analysis of the Synthesis relationship of carotenoids in the RKACAA1-2 Gene and rhodosporidium

1. Transformed rhodosporidium YM25235

Selecting DH5 alpha strain which is successfully transferred into a correct recombinant vector pRHRKACAA1-2, inoculating the DH5 alpha strain into an LB liquid medium (containing 100 mug/mL spectinomycin) for overnight culture, extracting plasmids (OMEGA Plasmid Mini Kit I, OMEGA company in America), measuring the concentration, and storing at-20 ℃ for later use; selecting rhodosporidium YM25235 single colony, inoculating into 5mL YPD liquid culture medium, and shaking culturing at 30deg.C and 200rpm for overnight; the overnight cultured bacterial liquid was transferred to 50mL of YPD liquid medium at 30℃and 200rpm in an inoculum size of 1% and cultured with shaking until the bacterial liquid had reached OD ₆₀₀ The culture was centrifuged at 4500 rpm at 4℃for 5min to collect the cells; washing (30 mM citric acid, 83mM sodium citrate, 600mM mannitol, naOH to adjust pH to 5.4) the thallus twice with a pre-prepared citric acid buffer solution, suspending the thallus with 1mL citric acid buffer solution, centrifuging at 4 ℃ and 4000 rpm for 5min to collect the thallus, and placing on ice for later use; preparation of a lyase solution (0.156 g of snailase, 0.08g of muramidase, ddH) ₂ O constant volume to 5 mL) with 0.22. Mu.mm sterile filter membrane filtering enzyme solution, and placing in a 50mL sterile centrifuge tube for standby; mixing 4mL of enzyme solution with bacterial solution, placing at 30deg.C, shaking at 90rpm for enzymolysis for 2.5 hr, centrifuging at 1300rpm for 10min at 4deg.C, and collecting bacterial cells; with STC (1.2M sorbitol, 10mM Tris-HCl, 100mM NaCl) ₂ ) Washing the collected thalli twice on ice to prepare yeast competent cells; subpackaging the yeast competent cells into 5mL sterile centrifuge tubes for standby according to 100 mu L of each tube; mu.L of 2-5. Mu.g pRHRKACAA1-2 recombinant plasmid was added to 100. Mu.L of competent cells and gently mixed (usually the fragment volume should not exceed 10. Mu.L), incubated on ice for 10min, and 200. Mu.L of pre-chilled PTC (50% PEG, 10mM Tris-HCl, 100mM CACl) was added ₂ ) Ice-bath for 10min, adding 800 μl of precooled PTC, mixing gently, ice-bath for 10min, centrifuging at 4deg.C at 1500rpm for 10min, and collecting thallus; adding 1.6mL of 0.4M sucrose YPD liquid culture medium to suspend, and carrying out shaking culture at 30 ℃ and 90rpm for 12h to recover thalli; centrifuging the recovered thallus at 1300rpm for 10min, collecting thallus, discarding supernatant to obtain 100 μl of culture medium suspension thallus, finally coating on 0.4M sucrose YPD solid culture medium containing 130ug/mL hygromycin B (HygB+), and culturing at 30deg.C for 2-3d; the transformants obtained after the coating were numbered and transferred onto a solid medium containing 150. Mu.g/mL hygromycin (HygB+) YPD and cultured upside down at 30℃for 2d; according to the known function of the gene, screening transformants by color, specifically operating to insert the obtained transformants into 5mL YPD medium, culturing for 120h at 30 ℃ under shaking at 200rpm, observing the color by using YM25235 wild strain as a control, and screening transformants which are redder than YM 25235; the selected transformant was selected, then genomic DNA of the yeast transformant was extracted according to the procedure in the Shanghai Biotechnology Co., ltd DNA extraction kit specification, and PCR was performed, and the result was shown in FIG. 5, in which it was seen that the genome of the yeast transformant was amplified by PCR using the genome of the yeast transformant as a templateRKACAA1-2, the gene verification of the recombinant transformant is correct, indicating that the cDNA fragments of 1-2 are identical in sizeRKACAAThe 1-2cDNA fragment has been successfully ligated into the genome of a yeast transformant.

2、RKACAA1-2Analysis of carotenoid content in rhodosporidium YM25235 with Gene overexpression

Culturing the overexpression strain containing pRHRKACAA1-2 at 28deg.C for 168 hr, extracting carotenoid, and measuring total carotenoid content (mg/g dry thallus) at 445nm with ultraviolet-visible spectrophotometer with wild rhodosporidium YM25235 strain as control, as shown in figure 6; as shown in the figure, the total carotenoid synthesis amount of the over-expression strain YM25235/pRHRKACAA1-2 is obviously improved compared with that of the wild rhodosporidium strain YM25235, the carotenoid synthesis amount of the wild rhodosporidium strain YM25235 is 5.53+/-0.07 mg/g, and the carotenoid synthesis amount of the over-expression strain YM25235/pRHRKACAA1-2 is 8.38+/-0.06 mg/g, namely, the carotenoid synthesis amount of the over-expression strain YM25235/pRHRKACAA1-2 is 1.51 times that of the control strain; the results show that 3-ketoacyl-CoA thiolase geneRKACAA1Overexpression of-2 leads to an increase in the total carotenoid content in the rhodosporidium YM25235 strain,RKACAA1-2the gene can promote the synthesis of total carotenoid.

Sequence listing

<110> university of Kunming engineering

<120> 3-ketoacyl-CoA thiolase gene RKACAA1-2 and use thereof

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1263

<212> DNA

<213> rhodosporidium YM25235 (Rhodosporidium kratochvilovae YM 25235)

<400> 1

atggccagcc tcatttcgtc caccctcgac tcgatccgcg gctccggcaa gagcaagctc 60

ctgcgcaaga gccccgacga tgtcgtcatc accatggcgg tccgtacgcc cctccagcgc 120

gcgcgcaagg gcggcttcaa ggacatgagc gtccaggagc tcctcattgc cttcttcaag 180

acctccatcc ccgagatgaa gatcgacccg gccctcattg gcgacatctg cgtcggcact 240

gtcctcccgc ccaaggcgcc gtacgacgcc cgtgctgccg ccctcgcgtc cggcattccc 300

gaaaccgtcc ctcttcagat catcaaccgc ttctgctctt cgggcctcat ggccgtcgcc 360

aacattgcaa accagatccg caacggcgag atcgagattg gtcttgcggt cggctttgag 420

agcatgagcg cgactccgga caggggcgcg gacagctttg cggaggaggt cctcgcgcac 480

ccggtcgcga aggacacgca gttccccatg ggctggacct cggagaacgt ttcgagtgac 540

tttaacatca cccgtgagcg catggacgag ttggcggcga tttcgcagca gcgcgcggcg 600

caggcgcagg cggctggcct tttcgacaag gagattatcc ccatcgaggc tctccaggcg 660

ccgtccaccc cggcggcggc gggcgcgccc gcgccgcagc gcgtcaaggt gctcgtcacc 720

aaggacgacg gcatccgccc ggggacgacc aaggagggcc tcggcaagat tcgccaggcg 780

ttcccgcagt ggggcaacgg cacgacgacg ggcggcaatg cgagccagat caccgacggc 840

gcggccggtg tcctcctcat gacccgtcgc aaggcggagg agctcggcct cgagatcctc 900

ggcaagcacg tcgcgacctc ggtcgcgggt cttccccctc gcattatggg catcggcccc 960

gccttcgcca tcccgaaggt ccttgcccgc tgcggcatct ccaaggagga cgtcgacctc 1020

tgggaggtca acgaggcttt tgcgtcgatg ctgagttact gcatcgacat cttcggcctt 1080

ccccatgaca aggtcaacgt caagggtggt gccatcgctc tcggccaccc tcttggctgc 1140

actggtgccc gccagatcgc gacgggcctg cacgagatcc gccgccgcga gggcaagatc 1200

ctcgttacct ccatgtgcat tggtctcggc atgggcgcag cgtcggtctt cgtcgctgag 1260

tag 1263

<210> 2

<211> 420

<212> PRT

<213> rhodosporidium YM25235 (Rhodosporidium kratochvilovae YM 25235)

<400> 2

Met Ala Ser Leu Ile Ser Ser Thr Leu Asp Ser Ile Arg Gly Ser Gly

1 5 10 15

Lys Ser Lys Leu Leu Arg Lys Ser Pro Asp Asp Val Val Ile Thr Met

20 25 30

Ala Val Arg Thr Pro Leu Gln Arg Ala Arg Lys Gly Gly Phe Lys Asp

35 40 45

Met Ser Val Gln Glu Leu Leu Ile Ala Phe Phe Lys Thr Ser Ile Pro

50 55 60

Glu Met Lys Ile Asp Pro Ala Leu Ile Gly Asp Ile Cys Val Gly Thr

65 70 75 80

Val Leu Pro Pro Lys Ala Pro Tyr Asp Ala Arg Ala Ala Ala Leu Ala

85 90 95

Ser Gly Ile Pro Glu Thr Val Pro Leu Gln Ile Ile Asn Arg Phe Cys

100 105 110

Ser Ser Gly Leu Met Ala Val Ala Asn Ile Ala Asn Gln Ile Arg Asn

115 120 125

Gly Glu Ile Glu Ile Gly Leu Ala Val Gly Phe Glu Ser Met Ser Ala

130 135 140

Thr Pro Asp Arg Gly Ala Asp Ser Phe Ala Glu Glu Val Leu Ala His

145 150 155 160

Pro Val Ala Lys Asp Thr Gln Phe Pro Met Gly Trp Thr Ser Glu Asn

165 170 175

Val Ser Ser Asp Phe Asn Ile Thr Arg Glu Arg Met Asp Glu Leu Ala

180 185 190

Ala Ile Ser Gln Gln Arg Ala Ala Gln Ala Gln Ala Ala Gly Leu Phe

195 200 205

Asp Lys Glu Ile Ile Pro Ile Glu Ala Leu Gln Ala Pro Ser Thr Pro

210 215 220

Ala Ala Ala Gly Ala Pro Ala Pro Gln Arg Val Lys Val Leu Val Thr

225 230 235 240

Lys Asp Asp Gly Ile Arg Pro Gly Thr Thr Lys Glu Gly Leu Gly Lys

245 250 255

Ile Arg Gln Ala Phe Pro Gln Trp Gly Asn Gly Thr Thr Thr Gly Gly

260 265 270

Asn Ala Ser Gln Ile Thr Asp Gly Ala Ala Gly Val Leu Leu Met Thr

275 280 285

Arg Arg Lys Ala Glu Glu Leu Gly Leu Glu Ile Leu Gly Lys His Val

290 295 300

Ala Thr Ser Val Ala Gly Leu Pro Pro Arg Ile Met Gly Ile Gly Pro

305 310 315 320

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

325 330 335

Asp Val Asp Leu Trp Glu Val Asn Glu Ala Phe Ala Ser Met Leu Ser

340 345 350

Tyr Cys Ile Asp Ile Phe Gly Leu Pro His Asp Lys Val Asn Val Lys

355 360 365

Gly Gly Ala Ile Ala Leu Gly His Pro Leu Gly Cys Thr Gly Ala Arg

370 375 380

Gln Ile Ala Thr Gly Leu His Glu Ile Arg Arg Arg Glu Gly Lys Ile

385 390 395 400

Leu Val Thr Ser Met Cys Ile Gly Leu Gly Met Gly Ala Ala Ser Val

405 410 415

Phe Val Ala Glu

420

<210> 3

<211> 40

<212> DNA

<213> Artificial sequence (Artifical)

<400> 3

atcactcacc atggcggatc ctatggccag cctcatttcg 40

<210> 4

<211> 39

<212> DNA

<213> Artificial sequence (Artifical)

<400> 4

ccggtcggca tctacgatat cctactcagc gacgaagac 39

Claims (2)

1. 3-ketoacyl coenzyme A thiolase geneRKACAA12, the nucleotide sequence of which is shown as SEQ ID NO. 1.

2. The 3-ketoacyl-CoA thiolase gene of claim 1RKACAA1-2 promoting rhodosporidium yeastRhodosporidium kratochvilovae) Use in the production of carotenoids.

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