CN112410355A - Acyl-coenzyme A oxidase 2 gene RKACOX2 and application thereof - Google Patents

Acyl-coenzyme A oxidase 2 gene RKACOX2 and application thereof Download PDF

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CN112410355A
CN112410355A CN202011322537.1A CN202011322537A CN112410355A CN 112410355 A CN112410355 A CN 112410355A CN 202011322537 A CN202011322537 A CN 202011322537A CN 112410355 A CN112410355 A CN 112410355A
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张琦
刘韬
和美霞
魏云林
季秀玲
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Kunming University of Science and Technology
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Abstract

The invention discloses an acyl coenzyme A oxidase 2 geneRKACOX2The 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 derived from Rhodosporidium toruloides (Rhodosporidium kratochvilovae) YM25235, transferring the gene into Rhodosporidium toruloides YM25235, and experimental results show thatRKACOX2Overexpression of a gene leads to a certain increase in the transcription level of this gene in the cellAt the same timeRKACOX2The over-expression of the gene can promote the synthesis of total carotenoid and each component of the carotenoid; the invention improves the microorganism by means of gene engineering, can improve the yield of the microbial carotenoid, and lays a foundation for large-scale commercial production of the carotenoid.

Description

Acyl-coenzyme A oxidase 2 gene RKACOX2 and application thereof
Technical Field
The invention belongs to the field of biotechnology and genetic engineering, and relates to an acyl-CoA oxidase 2 geneRKACOX2And the application thereof; in particular to a method for preparing a compound preparation from a yeast (rhodosporidium toruloides)Rhodosporidiumkratochvilovae) The gene is cloned in YM25235, linked with a vector and transferred into yeast cells, so that the expression level of the gene is increased and the synthesis of carotenoid is finally promoted.
Background
Carotenoids (carotenoids) are a generic name for terpenoids and their derivatives, which are composed of isoprenoids as basic units. To date, humans have found over 600 natural carotenoids in nature; studies have shown that most carotenoids are compounds containing forty carbons, whose main chain is composed of 4 isoprene units, and are divided into two main groups according to the elements constituting the molecular structure: the molecular structure only contains two elements of carbon and hydrogen, such as beta-carotene, lycopene and the like, which are taken as the first class and are called as carotene family; besides two elements, namely carbon and hydrogen, the molecular structure also contains one or more oxygen atoms to form hydroxyl, methoxy, carboxyl, epoxy compounds and the like, and astaxanthin, lutein, zeaxanthin, canthaxanthin and the like are taken as the second class and are called as the lutein family.
Carotenoids have a variety of physiological functions, and it has been demonstrated that carotenoids have functions of preventing cancer, delaying the progression of cancer, regulating cell growth, and resisting oxidation. The carotenoid molecules have a large number of double bonds, which can quench singlet oxygen, clear free radicals, enhance the resistance of human bodies to diseases, promote the connection and communication among cells, reduce the occurrence of chronic diseases and delay aging; however, the human body cannot synthesize carotenoids by itself and can only ingest them by external sources. Therefore, carotenoids are receiving great attention in the fields of medicine, health care, food and the like.
In addition, carotenoids are also widely used as a feed additive. Research shows that a certain amount of carotenoid is added into animal feed, which is beneficial to improving animal body color and feather color and increasing ornamental value; is also beneficial to improving the production performance, the organism immunity, the breeding efficiency and the like of animals, and has certain promotion effect on increasing the economic benefit of the breeding industry.
Common carotenoid production processes include microbial synthesis, plant extraction and chemical synthesis. The plant extraction method has complex process and high production cost. Meanwhile, the development of plant extraction methods is also limited by various factors (regions, climates, transportation conditions and the like), and large-scale production still has great difficulty. The chemical synthesis method has stronger production capacity and lower production cost compared with the plant extraction method. However, because the activity of the synthesized product is low, the product generally has solvent and raw material residues, and the safety problem possibly exists, so the use is limited to a certain extent; the physiological function of the carotenoid synthesized by the chemical synthesis is poorer than that of the natural carotenoid, and the carotenoid is not easy to be absorbed by a human body; the extraction of carotenoid by biological synthesis is the extraction method with the most development potential at present. The carotenoid produced by microbial fermentation only needs a low-cost natural substrate as a carbon source, and has the advantages of simple process, high production efficiency and consistent biological activity with natural plant extracts. In addition, the production of carotenoid by microbial fermentation is not influenced by environmental factors such as regions, climate and the like.
Disclosure of Invention
The invention aims to provide an acyl-CoA oxidase 2 geneRKACOX2The gene is derived from Rhodosporidium toruloides (Rhodosporidium kratochvilovae) YM25235 by separation; the nucleotide sequence of the gene is shown as SEQ ID NO. 1 or a fragment of the nucleotide sequence, or the nucleotide sequence complementary with the SEQ ID NO. 1, the length of the gene sequence is 2220bp (basic group), and the amino acid sequence coded by the gene is polypeptide shown as SEQ ID NO. 2 or a fragment thereof.
Another object of the present invention is to provide an acyl-CoA oxidase 2 geneRKACOX2The recombinant expression vector of (1) is constructed by directly connecting the gene shown in SEQ ID NO. 1 with different expression vectors (plasmids, viruses or carriers); can be constructed by methods well known to those skilled in the artAcyl-coa containing oxidase 2 geneRKACOX2And suitable transcription/translation regulatory elements. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombinant techniques, and the like. The acyl-CoA oxidase 2 geneRKACOX2Can be operably linked to an appropriate promoter of an expression vector to direct mRNA synthesis. Representative examples of such promoters are: lac or trp promoter of E.coli; the PL promoter of lambda phage; eukaryotic promoters include CMV early promoter, HSV thymidine kinase promoter, early and late SV40 promoter, LTRs of retrovirus, and other known promoters capable of controlling the expression of genes in prokaryotic or eukaryotic cells or viruses. The expression vector also includes a ribosome binding site for translation initiation, a transcription terminator, and the like. The insertion of enhancer sequences into vectors will enhance transcription in higher eukaryotic cells. Enhancers are cis-acting elements of DNA expression, usually about 10-300bp, that act on a promoter to enhance gene transcription, such as adenovirus enhancers.
Another object of the present invention is to provide an acyl-CoA oxidase 2 geneRKACOX2Or a host cell of the above recombinant expression vector.
Another object of the present invention is to provide the above-mentioned acyl-CoA oxidase 2 geneRKACOX2Application to the promotion of microbial production of carotenoids.
The acyl-CoA oxidase 2 geneRKACOX2Is red winter spore yeast (Rhodosporidium kratochvilovae) The important gene in the YM25235 fatty acid metabolic process and the coded enzyme are responsible for the degradation of branched-chain fatty acid, can regulate and control the fatty acid metabolism and further influence the rhodosporidium toruloides (R) ((R))Rhodosporidium kratochvilovae) Synthesis of carotenoids from YM25235 strain.
The Rhodosporidium toruloides (A), (B) and (C)Rhodosporidium kratochvilovae) YM25235 has the advantages of short production cycle, stable heredity, safe production, etc.
The invention relates to a method for preparing a red wintergreen spore yeast (Rhodosporidium toruloides)Rhodosporidium kratochvilovae) acyl-CoA oxidase 2 group isolated from YM25235 Total RNA GeneDue to the fact thatRKACOX2The total length of the gene is 2220 bp; the gene is transferred into Rhodosporidium toruloides YM25235, and the experimental result shows thatRKACOX2The overexpression of the gene can cause the transcription level of the gene in the cell to be improved to a certain extent, which indicates that the exogenous gene is transcribed in the thalli;RKACOX2the over-expression of the gene can promote the synthesis of total carotenoid and each component of the carotenoid; the research result provides reference for disclosing a mechanism of increasing the yield of the carotenoid by the microorganism, is beneficial to improving the carotenoid content by modifying the microorganism by a genetic engineering means, has good application prospect and economic benefit for industrial production of the carotenoid, and lays a foundation for large-scale commercial production of the carotenoid.
Drawings
FIG. 1 shows a scheme for producing Rhodosporidium toruloides YM25235 of the present inventionRKACOX2A gene PCR amplification map;
FIG. 2 is a plasmid map of recombinant plasmid pRHRKACOX 2;
FIG. 3 shows the PCR identification of YM25235 strain transformed with recombinant plasmid pRHRKACOX2, FIG. 1. DNA molecular scalar DL 5000; 2. PCR products with primers RKACOX2-F and RKACOX2-R using YM25235 genome as template; 3. PCR products of primers RKACOX2-F and RKACOX2-R with plasmid pRHRKACOX2 as template; 4. PCR products of primers RKACOX2-F and RKACOX2-R with YM25235/pRHRKACOX2 strain genome as template;
FIG. 4 shows a comparison of the total carotenoid content of the over-expressed strain YM25235/pRHRKACOX2 with the control strain YM25235/pRH 2034;
FIG. 5 shows the comparison of the carotenoid content of each component in the overexpressed strain YM25235/pRHRKACOX2 with that of the control strain YM25235/pRH 2034;
FIG. 6 is a relative quantitative analysis of carotenoid synthesis related genes of overexpression strain YM25235/pRHRKACOX2 and control strain YM25235/pRH 2034.
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 conventional methods.
Example 1: from Rhodosporidium toruloides (A)Rhodosporidium kratochvilovae) Isolation of acyl-CoA oxidase 2 Gene from YM25235RKACOX2The nucleotide sequence of (A) and the construction of an overexpression vector pRHRKACOX2 thereof
Extracting total RNA of Rhodosporidium toruloides YM25235 by using UNlQ-10 column type Trizol total RNA extraction Kit (product number: SK 1321) of bio-engineering (Shanghai) Co., Ltd, performing reverse transcription according to PrimeScript RT reagent Kit With gDNA Eraser (Perfect Real Time) of TaKaRa Co., Ltd to synthesize cDNA, performing polymerase chain reaction by using 0.5 mu L as a template, and sequencing according to the transcription setRKACOX2Designing specific primers RKACOX2-F and RKACOX2-R (primer 1 and primer 2), carrying out PCR amplification on the cDNA template obtained in the way on a PCR instrument (BIOER company), wherein the primers, components and amplification conditions used in the reaction are as follows:
Figure 711071DEST_PATH_IMAGE002
(the double underlined section is the homology arm);
RKACOX2-R：
Figure DEST_PATH_IMAGE004
(underlined part isEcoR V site, double underlined part is homology arm);
Figure DEST_PATH_IMAGE005
amplification conditions: pre-denaturing at 95 ℃ for 5 min, further denaturing at 95 ℃ for 30s, annealing at 62 ℃ for 30s, extending at 72 ℃ for 2min for 30s, performing 30 cycles, finally completely extending at 72 ℃ for 10min, taking 2. mu.L of the product after the reaction, performing electrophoresis analysis in 1% agarose gel to obtain a fragment of about 2200bp, recovering the fragment by ethanol precipitation, and using a seamless Cloning Kit (Vazyme Clon express II One Step Cloning Kit, Nanjing Nuo Tou Zan Biotech Limited public Key)And) connecting the recovered fragment to a vector pRH2034 to obtain a recombinant vector; transformation of the recombinant vector into the vector with CaCl2Carrying out overnight culture on Escherichia coli DH5 alpha treated by the method on an LB solid plate containing spectinomycin (100 mug/mL), selecting a white colony growing on the plate, and verifying positive clones by colony PCR; inoculating positive clones into LB liquid culture medium (containing 100 mug/mL spectinomycin) for overnight culture, extracting plasmids (OMEGA Plasmid Mini Kit I, American OMEGA company) for sequencing (Kunming engine science and technology, Inc.), wherein the result shows that the size of the amplified fragment is 2220bp, which is named as RKACOX2, and the sequence composition is a nucleotide sequence shown as SEQ ID NO: 1; the recombinant vector was pRHRKACOX2 (FIG. 2).
Example 2: analysis of the relationship between RKACOX2 gene and carotenoid synthesis in Rhodosporidium toruloides
1. Transformed Rhodosporidium toruloides YM25235
The recombinant vector pRHRKACOX2 is transformed into Rhodosporidium toruloides YM25235 by a PEG-mediated protoplast method, transformants are screened by a YPD culture medium containing hygromycin B (hygromycin B) with the final concentration of 150 mug/mL, then genomic DNA of the yeast transformants is extracted according to the steps in the DNA extraction kit specification of Shanghai biological engineering Co., Ltd, and then PCR verification is carried out, wherein the result is shown in FIG. 3.
2. Analysis of changes in the content of RKACOX2 Gene and Total carotenoids in Rhodosporidium toruloides
Culturing overexpression strain containing pRHRKACOX2 at 28 deg.C, extracting carotenoid, and determining total carotenoid content (mg/g dry thallus) at 445nm with ultraviolet-visible spectrophotometer using Rhodosporidium toruloides strain transferred to empty plasmid pRH2034 as control, the result is shown in FIG. 4; as can be seen from the graphs, the total carotenoid synthesis of the overexpressed strain YM25235/pRHRKACOX2 was significantly improved as compared with the control strain containing the empty plasmid pRH2034, the carotenoid synthesis of the control strain containing the empty plasmid pRH2034 was 5.68mg/g, and the carotenoid synthesis of the overexpressed strain YM25235/pRHRKACOX2 was 7.59mg/g, i.e., the carotenoid synthesis of the overexpressed strain YM25235/pRHRKACOX2 was 1.34 times that of the control strain, indicating that RKACOX2 gene was able to promote the synthesis of total carotenoids.
3. Analysis of content variation of components of RKACOX2 gene and rhodosporidium toruloides carotenoid
After culturing the overexpression strain containing pRHRKACOX2 at 28 ℃ for 168h, extracting carotenoid, and performing high performance liquid chromatography analysis by using original Rhodosporidium toruloides YM25235 strain as a control, the result is shown in FIG. 5, and it can be seen from the figure that the content of each component of carotenoid of the overexpression strain YM25235/pRHRKACOX2 is improved to different degrees compared with the original strain; wherein, the content of the over-expression strain YM25235/pRHRKACOX2 rhodotorula rubra erythrogenin (torularhodin) is increased by 6.5 percent, the content of beta-carotene (beta-carotene) is increased by 60.7 percent, the content of the round yeast extract (torulone) is increased by 164.6 percent, and the content of gamma-carotene (gamma-carotene) is increased by 176.4 percent; the results show thatRKACOX2The gene can promote the synthesis of each component of carotenoid.
Example 3: analysis of relationship between RKACOX2 gene and carotenoid synthesis related gene in Rhodosporidium toruloides
Culturing the transgenic strain YM25235/pRHRKACOX2 and the control strain YM25235/pRH2304 on a constant temperature shaker at 30 ℃ and 220rpm for 120h by shaking, extracting total RNA of Rhodosporidium toruloides YM25235 by using UNlQ-10 column type Trizol total RNA extraction Kit (product number: SK 1321) of Shanghai bioengineering (Shanghai) Co., Ltd, performing reverse transcription according to PrimeScript RT reagent Kit With gDNA Eraser (Perfect Time) of TaKaRa Co., synthesizing related genes by the reverse transcription of the transgenic strain YM 25235/pRKAHRCOX 2 and the control strain YM25235/pRH2304 carotenoidRkCrtI、RkCrtYBIs analyzed for the transcript level of (2). 5.8S rRNA of Rhodosporidium toruloides YM25235 is used as an internal reference gene, and carotenoid is used for synthesizing related genesRkCrtI、RkCrtYBThe transcription level of the corresponding gene of the control strain YM25235/pRH2304 is used as a control, and the 2-delta-Delta CT value is 1; the relative quantitative (Bestar SybrGreen qPCR MasterMix kit, DBI, Germany) analysis results are shown in FIG. 6, which shows that the transgenic strain YM25235/pRHRKACOX2RKCrtYBTransformation of genesThe recording level is the control strain YM25235/pRH2304RKCrtYB1.9 times the level of gene transcription. Transgenic strain YM25235/pRHRKACOX2RKCrtIThe transcription level of the gene was that of the control strain YM25235/pRH2304RKCrtI10.1 fold higher gene transcript levels. These results indicate that overexpression of RKACOX2 gene in Rhodosporidium toruloides YM25235 results in an increase in the transcription level of mRNA of genes involved in carotenoid synthesis in cells.
Sequence listing
<110> university of Kunming science
<120> acyl-CoA oxidase 2 gene RKACOX2 and application thereof
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<213> Rhodosporidium toruloides YM25235(Rhodosporidium kratochvilovae YM25235)
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ggcgagtaca acgtcaagct gaagcgcaag ttcatgttcg agctcgagcg cgacccgctc 180
ttcaagctct ccgacattca cgacctcacc aaggccgaga ttcgcgagcg gaccatggcc 240
aagtttgcgt cgatggtcca ctacgtcacg aacgagccta tcgcccagtt ccagcagcgc 300
atgaacgtcg tttctaccgc cgacccggct ttctggacgc gatatggagt gcactatggc 360
ctgttcctcg gcgcgctccg ctctggcgca acgcccaacc agatgagcta ctggatgtcg 420
cgcggcgtca tcaccctgca gggcatggtc ggctgcttcg cgatgactga gctcgctcac 480
ggttcgaacg tcgcgggcct cgagacgacc gcaaccttcg acgagacggc ggaccagttc 540
gtcatccaca cgccgtccgt ctcggcgacc aagtggtgga tcggcggcgc ggcgcactcg 600
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acgttcgtcg tcccgcttcg caaccccaag acgtacgagc tcctgcccgg catcgcaatc 720
ggcgacattg gcaagaagat ggggcgcgac ggcatcgaca acggctggat ccagttcacc 780
aacgtccgca tcccgcgcgc atacatgctc atgaagcaca ccaaggtgac gcgcgagggc 840
gaggtgcgcg agccgccgct cgcgcagctg acatacggcg cgctgctgca aggccgcacg 900
gcgatggtcg ccgacgcggc gtcgacggcg aagaaggcgc tgacgatcgc gctccggtac 960
gcggcggtgc ggcggcagtt caagacgggc gacaacgcgc tcgagacgca gctcctcgac 1020
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agcgactcgg acgccgacga gaccaagtcg gtcctcgaga agctcaagga gacgcacgcg 1200
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cgctcgctcg tctcgtccta cgccgacgcg gtcaagggcg cgaagctccc gggcgggacg 1440
gcgtacctca acgcgctccc ctccgtgctc acgacgtcct gcccgtcgga cgcggcgacg 1500
ctcgagctcg acacgctcca ggccgcgtgg gactgcgtct cggcgaacgt ggtcaagcag 1560
gcgcacgaca agttcgaggg cgcgctcaag agcggcgagg ccaagggccg cgaggaggcg 1620
ctcgagatgt gctcgcagga gcggttcatc gccgccaagg tgcacactgc cggctacctc 1680
ttccgcatgt tccgcgaggc gctcgtcgag ctcgccaaga ctgagccggc ggacaacggc 1740
gtcgtcgcga cgctggagca gatctgcacg ctgtacgggt gctgggcgat cgaggagaac 1800
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gacctctcgg accacatcat caactcgccg ctcggacggt acgacggcga catctacaac 1980
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Met Pro Thr Pro Val Asp Ala Gln Pro Arg Pro Pro Ala Pro Arg Glu
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Arg Lys Phe Met Phe Glu Leu Glu Arg Asp Pro Leu Phe Lys Leu Ser
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Asp Ile His Asp Leu Thr Lys Ala Glu Ile Arg Glu Arg Thr Met Ala
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Lys Phe Ala Ser Met Val His Tyr Val Thr Asn Glu Pro Ile Ala Gln
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Phe Gln Gln Arg Met Asn Val Val Ser Thr Ala Asp Pro Ala Phe Trp
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Thr Arg Tyr Gly Val His Tyr Gly Leu Phe Leu Gly Ala Leu Arg Ser
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Gly Ala Thr Pro Asn Gln Met Ser Tyr Trp Met Ser Arg Gly Val Ile
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Thr Leu Gln Gly Met Val Gly Cys Phe Ala Met Thr Glu Leu Ala His
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Gly Ser Asn Val Ala Gly Leu Glu Thr Thr Ala Thr Phe Asp Glu Thr
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Ala Asp Gln Phe Val Ile His Thr Pro Ser Val Ser Ala Thr Lys Trp
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Trp Ile Gly Gly Ala Ala His Ser Ala Thr His Ser Ala Val Phe Ala
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Arg Leu Ile Val Lys Gly Met Asp Tyr Gly Thr Lys Thr Phe Val Val
210 215 220
Pro Leu Arg Asn Pro Lys Thr Tyr Glu Leu Leu Pro Gly Ile Ala Ile
225 230 235 240
Gly Asp Ile Gly Lys Lys Met Gly Arg Asp Gly Ile Asp Asn Gly Trp
245 250 255
Ile Gln Phe Thr Asn Val Arg Ile Pro Arg Ala Tyr Met Leu Met Lys
260 265 270
His Thr Lys Val Thr Arg Glu Gly Glu Val Arg Glu Pro Pro Leu Ala
275 280 285
Gln Leu Thr Tyr Gly Ala Leu Leu Gln Gly Arg Thr Ala Met Val Ala
290 295 300
Asp Ala Ala Ser Thr Ala Lys Lys Ala Leu Thr Ile Ala Leu Arg Tyr
305 310 315 320
Ala Ala Val Arg Arg Gln Phe Lys Thr Gly Asp Asn Ala Leu Glu Thr
325 330 335
Gln Leu Leu Asp Tyr Pro Ile His Gln Arg Arg Leu Leu Pro Leu Leu
340 345 350
Ala Gln Ala Val Ala Met Gly Phe Thr Ser Phe Arg Met Thr Ala Leu
355 360 365
Phe Glu Glu Met Ser Glu Glu Leu Glu Ser Leu Gly Ser Asp Ser Asp
370 375 380
Ala Asp Glu Thr Lys Ser Val Leu Glu Lys Leu Lys Glu Thr His Ala
385 390 395 400
Thr Ser Ala Gly Leu Lys Ala Phe Cys Thr Trp Asn Ala Leu Glu Thr
405 410 415
Ile Glu Lys Cys Arg Ala Ser Leu Gly Gly His Gly Tyr Ser Ala Tyr
420 425 430
Ser Ala Leu Pro Ser Met Tyr Asn Asp Gln Ala Val Gln Cys Thr Trp
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Glu Gly Asp Asn Thr Ile Leu Thr Leu Gln Ser Gly Arg Ser Leu Val
450 455 460
Ser Ser Tyr Ala Asp Ala Val Lys Gly Ala Lys Leu Pro Gly Gly Thr
465 470 475 480
Ala Tyr Leu Asn Ala Leu Pro Ser Val Leu Thr Thr Ser Cys Pro Ser
485 490 495
Asp Ala Ala Thr Leu Glu Leu Asp Thr Leu Gln Ala Ala Trp Asp Cys
500 505 510
Val Ser Ala Asn Val Val Lys Gln Ala His Asp Lys Phe Glu Gly Ala
515 520 525
Leu Lys Ser Gly Glu Ala Lys Gly Arg Glu Glu Ala Leu Glu Met Cys
530 535 540
Ser Gln Glu Arg Phe Ile Ala Ala Lys Val His Thr Ala Gly Tyr Leu
545 550 555 560
Phe Arg Met Phe Arg Glu Ala Leu Val Glu Leu Ala Lys Thr Glu Pro
565 570 575
Ala Asp Asn Gly Val Val Ala Thr Leu Glu Gln Ile Cys Thr Leu Tyr
580 585 590
Gly Cys Trp Ala Ile Glu Glu Asn Ala Gln His Phe Leu Lys Tyr Arg
595 600 605
Phe Phe Thr Pro Ser Gln Met Asp Ala Ile Thr Ala Glu Val Thr Arg
610 615 620
Leu Cys Ala Val Leu Arg Lys Ser Ala Val Leu Leu Thr Asp Ser Phe
625 630 635 640
Asp Leu Ser Asp His Ile Ile Asn Ser Pro Leu Gly Arg Tyr Asp Gly
645 650 655
Asp Ile Tyr Asn His Tyr Phe Ala His Val Lys Ala Ala Asn Pro His
660 665 670
Glu Pro Val Ala Pro Tyr Phe Ala Arg Val Ile Lys Pro Leu Ile Glu
675 680 685
Arg Glu Asp Leu Ser Phe Asp Asp Ala Asp Glu Ile Asp Leu Asp Ala
690 695 700
Glu Leu Gln Glu Leu Glu Glu Glu Glu Ala Asp Asp Glu Glu Val Glu
705 710 715 720
Asp Glu Ala Asp Lys Thr Ala Gln Ala Glu Lys Glu Leu Glu Lys Pro
725 730 735
Ala Lys Glu
<210> 3
<211> 40
<212> DNA
<213> Artificial sequence (Artificial)
<400> 3
accagatcac tcaccatggc gatgccgacg cccgtcgatg 40
<210> 4
<211> 39
<212> DNA
<213> Artificial sequence (Artificial)
<400> 4
ccggtcggca tctacgatat cctactcctt cgccggctt 39

Claims (2)

1. acyl-CoA oxidase 2 geneRKACOX2The 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.
2. The acyl-CoA oxidase 2 gene according to claim 1RKACOX2Application in promoting microbial production of carotenoid is provided.
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CN108624600A (en) * 2018-05-22 2018-10-09 昆明理工大学 The purposes of zinc finger transcriptional factor genes RkMsn4
CN110747206A (en) * 2019-11-05 2020-02-04 昆明理工大学 3-hydroxy-3-methylglutaryl coenzyme A reductase gene RKHMGR and application thereof
CN110747206B (en) * 2019-11-05 2021-11-23 昆明理工大学 3-hydroxy-3-methylglutaryl coenzyme A reductase gene RKHMGR and application thereof
CN113621630A (en) * 2021-08-05 2021-11-09 昆明理工大学 3-ketoacyl-CoA thiolase gene RkACAA1-1 and application thereof
CN113652440A (en) * 2021-08-05 2021-11-16 昆明理工大学 3-ketoacyl-coenzyme A thiolase gene RKACAA1-2 and application thereof
CN113621630B (en) * 2021-08-05 2023-03-24 昆明理工大学 3-ketoacyl-CoA thiolase gene RkACAA1-1 and application thereof
CN113652440B (en) * 2021-08-05 2023-04-21 昆明理工大学 3-ketoacyl coenzyme A thiolase gene RKACAA1-2 and application thereof
CN114107340A (en) * 2021-08-11 2022-03-01 昆明理工大学 Mevalonate kinase gene RKMK and application thereof
CN115011616A (en) * 2022-01-26 2022-09-06 昆明理工大学 Acetaldehyde dehydrogenase gene RKALDH and application thereof
CN114540356A (en) * 2022-02-25 2022-05-27 昆明理工大学 Rhodosporidium toruloides promoter and application thereof
CN114540356B (en) * 2022-02-25 2023-09-01 昆明理工大学 Rhodosporidium toruloides promoter and application thereof

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