CN113430215B - Acetyl CoA synthetase gene RKACS1 and application thereof - Google Patents

Abstract

The invention discloses an acetyl CoA synthetase geneRKACS1The 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 red winter cell yeast (Rhodosporidium kratochvilovae) YM25235 (fraction)The obtained acetyl CoA synthetase gene is separated and transformed into rhodosporidium toruloides YM25235, and the experimental result shows that the acetyl CoA synthetase geneRKACS1The overexpression of the gene can cause the increase of the content of the total carotenoid and the oil in the rhodosporidium toruloides YM25235 strain, the invention improves the yield of the carotenoid and the oil in the microorganism by modifying the microorganism by a gene engineering means, and lays a foundation for the large-scale commercial production of the carotenoid and the oil.

Description

acetyl-CoA synthetase gene RKACS1 and its application

technical field

The invention belongs to the technical field of biology and genetic engineering, and relates to an acetyl-CoA synthetase gene RKACS1 , in particular to the acetyl-CoA synthetase gene RKACS1 cloned from Rhodosporidium kratochvilovae YM25235 and connecting the gene with a carrier, transfecting Into yeast cells to increase the expression level of the gene and promote the synthesis of carotenoids and oils.

Background technique

Carotenoids (carotenoids) are a class of natural pigments that widely exist in nature. They generally appear yellow, red or orange-red. Currently, there are more than 800 natural carotenoids found in higher plants, animals, fungi and other organisms. Common vegetables and fruits are rich in carotenoids, such as citrus, mango, pumpkin and so on. Most carotenoids are a class of C ₄₀ terpenoids and their derivatives composed of 8 end-to-end isoprenoids, and some carotenoids contain C ₄₅ or C ₅₀ skeletons, which are called high carotenoids , there are also a small number of carotenoids with a carbon skeleton less than C ₄₀ , which are called apocarotenoids; all carotenoids contain polyisoprene structures.

According to the chemical structure of carotenoids, carotenoids can be divided into two categories: carotene and xanthophyll. Among them, carotene is α-carotene containing only C and H. β-carotene, γ-carotene, and lycopene, etc.; lutein is a kind of oxidized carotene, which contains three elements of C, H, and O, and can form hydroxyl, ketone, carboxyl, methoxy, etc. Oxygen-containing functional groups, such as lutein, zeaxanthin, astaxanthin, etc.; oxygen-containing groups cause complex and diverse changes in the molecular structure of carotenoids, and changes in polarity make carotenoids easy to interact with fatty acids, sugars and proteins in the body and other combinations to form a variety of active molecules with different functions. Carotenoids have strong light absorption, and usually have absorption peaks between 430 and 570 nm in wavelength. At present, reversed-phase high-performance liquid chromatography is generally used to detect samples with ultraviolet-visible light detectors, mass spectrometers, nuclear magnetic resonance or diode array detectors. Carotenoids in the qualitative and quantitative analysis.

Carotenoids play a very important role in the field of human nutrition and health. α-carotene, β-carotene, γ-carotene and β-cryptoxanthin can form vitamin A under the action of dioxygenase , They are therefore called provitamin A active substances and are the main source of vitamin A. As an essential micronutrient for the human body, carotenoids have multiple functions, such as anti-oxidation, inhibiting and eliminating free radicals in the body, and slowing down aging. Medical research shows that, in addition to the above functions, carotenoids also have functions such as anti-cancer, prevention of macular degeneration and cataracts, prevention of cardiovascular diseases, prevention of non-alcoholic fatty liver, and strengthening of the body's immunity. In addition, carotenoids can also be combined with animal protein to make organisms present body color and have a certain protective effect. At present, carotenoids have been designated as A-type nutrients by international organizations such as the Food and Agriculture Organization of the United Nations (FAO) and the World Health Organization (WHO), and have been approved as food additives with dual functions of nutrition and pigments in more than 50 countries. Used in medicine, food, health care, beauty and other industries. So far, the production processes of carotenoids that have been reported include natural extraction, chemical synthesis and microbial fermentation, among which the production of carotenoids by microbial fermentation has high biological activity, easy production process, product safety and short production cycle, etc. advantages, has received extensive attention in recent years. Although many strategies have been used to promote the synthesis of carotenoids in microorganisms, due to the limitations of technical issues such as cost-effectiveness, pigment yield and separation and extraction, the application of traditional microbial fermentation technology to produce various carotenoids in industrial production A corresponding strategy is still lacking. Therefore, we hope that from the perspective of bioengineering, we can use genetic technology, species discovery, hybridization and other technologies to obtain high-content raw materials, so as to alleviate the current limitations of the production and development of natural carotenoids. In recent years, metabolic engineering that combines classical genetics and modern molecular biology methods to optimize the original metabolic pathways and regulatory networks of strains or assemble heterologous metabolic pathways to enable microorganisms to produce carotenoids with high efficiency and low cost provides a potential alternative pathway.

Lipids are key substances for organisms to carry out many life activities. Common lipids include linoleic acid (LA), α-linolenic acid (ALA), γ-linolenic acid (GLA), arachidonic acid (ARA) , eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) and other functional oils. Studies have shown that omega-3 fatty acids such as ALA, DHA, and EPA, as important life active substances in the human body, can effectively promote human growth and development. Physiological functions, long-term lack of omega-3 fatty acids and omega-6 fatty acids can easily lead to hereditary obesity, metabolic disorders, and then affect human health; in addition, LA, ARA, DHA, etc. can be used as drug additives to lower blood sugar and blood pressure and prevent pregnancy , Relieve respiratory diseases, prevent arteriosclerosis and digestive system ulcers. Most functional oils are necessary for the human body but cannot be synthesized by themselves. ALA can be obtained from some vegetable oils, and DHA and EPA are mainly derived from fish oil and microbial oils. However, the cost of fish oil is high, it is easily polluted, the quality is unstable, and the output cannot meet the global demand.

Oleaginous microorganisms have a high oil content and a fatty acid composition similar to that of vegetable oils. Compared with vegetable oil, oil-producing microorganisms have the advantages of not competing with food for land, not affected by climate and environment, short growth cycle, and easy to achieve large-scale production. Therefore, the use of microbial fermentation to produce oil has attracted more and more attention. At present, more than 30 kinds of yeasts have been identified as oleaginous yeasts, and most of these oleaginous yeasts can increase the accumulation of intracellular oil by culturing under specific conditions or genetic modification. Therefore, we hope that from the perspective of bioengineering, we can use genetic technology, synthetic biology and other technologies to purposefully transform strains and improve the oil conversion rate of existing strains, thereby alleviating the current pressure on oil resources. In recent years, the combination of classical genetics and modern molecular biology methods to optimize the original metabolic pathways and regulatory networks of strains or to assemble heterologous metabolic pathways provides a potential alternative for the metabolic engineering of microbial high-efficiency and low-cost oil production. .

Contents of the invention

The object of the present invention is to provide an acetyl-CoA synthetase gene RKACS1 , which is isolated from Rhodosporidium kratochvilovae YM25235, and its nucleotide sequence is as shown in SEQ ID NO: 1 or is the nucleotide sequence A fragment, or a nucleotide sequence complementary to SEQ ID NO:1, the gene sequence is 1977bp (base), and the encoded amino acid sequence is the polypeptide or fragment shown in SEQ ID NO:2.

Another object of the present invention is to provide a recombinant expression vector of the acetyl CoA synthetase gene RKACS1 , which is a recombinant vector constructed by directly linking the gene shown in SEQ ID NO: 1 with different expression vectors (plasmids, viruses or carriers). Methods well known to those skilled in the art can be used to construct the expression vector of the nucleotide sequence of the acetyl CoA synthetase gene RKACS1 and suitable transcription/translation regulatory elements; these methods include in vitro recombinant DNA technology, DNA synthesis technology, in vivo recombination technology, etc.; The nucleotide sequence of the acetyl CoA synthetase gene RKACS1 can be effectively connected to the appropriate promoter of the expression vector to guide mRNA synthesis. Representative examples of these promoters are: E. coli lac or trp promoter; lambda phage PL promoter; eukaryotic promoters include CMV early promoter, HSV thymidine kinase promoter, early and late SV40 Transcription virus LTRs and other known promoters that can control the expression of genes in prokaryotic cells or eukaryotic cells or their viruses; expression vectors also include ribosome binding sites for translation initiation and transcription terminators; Inserting an enhancer sequence in the vector will enhance its transcription in higher eukaryotic cells; the enhancer is a cis-acting factor of DNA expression, usually about 10-300bp, acting on the promoter to enhance the transcription of the gene, Such as adenovirus enhancer.

Another object of the present invention is to provide a host cell containing the acetyl-CoA synthetase gene RKACS1 or the above-mentioned recombinant expression vector.

Optimizing host cells with the nucleotide sequence of the present invention or the recombinant vector containing the nucleotide sequence can be carried out by methods well known to those skilled in the art.

Another object of the present invention is to apply the above-mentioned acetyl CoA synthetase gene RKACS1 in carotenoid production.

Another object of the present invention is to apply the above-mentioned acetyl-CoA synthetase gene RKACS1 to oil production.

The present invention isolates the acetyl CoA synthetase gene RKACS1 from the total RNA of Rhodosporidium kratochvilovae YM25235, and the full length of the gene is 1977bp; the overexpression of the RKACS1 gene in Rhodosporidium kratochvilovae will cause the transcription of this gene in the cell The level has increased to a certain extent, indicating that the exogenous gene is transcribed in the bacteria, and then translated into the corresponding protein, resulting in an increase in the expression of enzymes related to carotenoid or lipid synthesis in the cell. The results of this study will help To clarify the mechanism of carotenoid and oil production in Rhodosporidium yeast YM25235, to provide a reference for revealing the mechanism of microorganisms to increase the production of carotenoids and oil, and to improve the content of carotenoids and oil through genetic engineering. It provides good application prospects and economic benefits for the industrial production of carotenoids and oils, and lays the foundation for large-scale commercial production of carotenoids and oils.

Description of drawings

Fig. 1 is the PCR amplification figure of the RKACS1 gene of Rhodosporidium yeast YM25235 of the present invention; 1. DNA molecular weight marker DL2000; 2. Negative control; 3. The cDNA fragment of RKACS1 ;

Fig. 2 is the plasmid map of recombinant plasmid pRHRKACS1;

Figure 3 is the electrophoresis diagram of colony PCR verification; 1. DNA molecular weight marker DL2000; 2. cDNA fragment of RKACS1 ; 3-7 are transformants;

Figure 4 Verification of recombinant plasmid pRHRKACS1 transformed into Rhodosporidium YM25235 positive clone; 1. DNA molecular weight marker DL2000; 2. Wild-type strain-specific gene band; 3. cDNA fragment of RKACS 1; 4. Transformant verification;

The carotenoid content comparison result of Fig. 5 overexpression strain YM25235/pRHRKACS1 and control strain YM25235;

Fig. 6 Comparison results of oil content between the overexpression strain YM25235/pRRKACS1 and the control strain YM25235.

Detailed ways

Below in conjunction with accompanying drawing and embodiment the present invention is described in further detail, but protection scope of the present invention is not limited to described content, reagent and method used in the embodiment, if no special instructions, all adopt conventional reagent, use conventional method.

Example 1: Isolation of the nucleotide sequence of the acetyl-CoA synthetase gene RKACS1 from Rhodosporidium kratochvilovae YM25235

The total RNA of Rhodosporidium YM25235 was extracted using the UNlQ-10 Column Trizol Total RNA Extraction Kit (Product No.: SK1321) of Sangon Bioengineering (Shanghai) Co., Ltd., and then followed the TaKaRa company kit PrimeScript ® RT reagent KitWith gDNA Eraser (Perfect Real Time) was used to synthesize cDNA by reverse transcription, and 1 μL of cDNA was used as a template for polymerase chain reaction. According to the sequence of RKACS1 found in transcriptome sequencing, specific primers RKACS1-F and RKACS1-R (primer 1 and primer 2), the cDNA template obtained above was used for PCR amplification on a PCR instrument (BIOER company), and the primers, components and amplification conditions used in the reaction were as follows:

Primer 1: RKACS1-F: 5'- GATCACTCACCATGG TGACCGAACACACCTACGAC -3' (SEQ ID NO: 3) (underlined is the homologous sequence at the end of the upstream vector)

Primer 2: RKACS1-R: 5'- CCGGTCGGCATCTAC CTACGCCTTGGCGAACTT -3' (SEQ ID NO: 4) (the underline is the homologous sequence at the end of the downstream vector);

The PCR amplification system is as follows (50 μL):

Template cDNA 1µL Foward Primer (RKACS1-F) 2µL Reverse Primer (RKACS1-R) 2µL dNTPs Mix (10 mM) 1µL 2×phanta Max Buffer 25µL Phanta Max Super-Fidelity Polymerase 1µL <![CDATA[ddH₂O]]> Add to 50µL

Amplification conditions: pre-denaturation at 94°C for 5 minutes, followed by denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 2 minutes, a total of 30 cycles, and a final extension at 72°C for 10 minutes. Electrophoresis analysis was carried out in the agarose gel, the results are shown in Figure 1; the amplified fragment with a size of about 2000bp was named RKACS1 ; pRH2034 was double-digested with BamH Ⅰ and EcoR Ⅴ restriction enzymes ; The above two fragments were recovered with an agarose gel DNA recovery kit (Beijing Suo Laibao Technology Co., Ltd.), and the recovered two fragments were connected to obtain the recombinant plasmid pRHRKACS1 (Figure 2). The connection method was ClonExpress II One Step Cloning Kit kit, the connection system is as follows (20µL):

Exnase™ II 2µL fragment 79ng Linearized pRH2034 200ng 5×CEIIBuffer 4µL <![CDATA[ddH₂O]]> Add to 20µL

Gently blow and mix with a pipette, centrifuge briefly to collect the reaction solution to the bottom of the tube, then react at 37°C for 30min; cool down to 4°C or place on ice immediately.

Transfer the ligation product obtained above into Escherichia coli DH5α for amplification, culture on LB solid plate containing spectinomycin (100µg/mL) for 12~16h, pick the white colonies growing on the plate, and verify positive clones by colony PCR (As shown in Figure 3), put the verified positive clones into LB liquid medium (containing 100µg/mL spectinomycin) and culture for 12~16h, and use the high-purity plasmid miniprep kit (spin column type) ( Beijing Biotech Biotechnology Co., Ltd.) extracted the plasmid and sequenced (Kunming Shuoqing Biotechnology Co., Ltd.). The sequencing results showed that the size of the amplified fragment was 1977bp, which was consistent with the sequence of the transcriptome. The nucleotide sequence is as follows: SEQ ID NO :1 shown.

Example 2: Effect of RKACS1 Gene Overexpression on Carotenoid Synthesis in Rhodosporidium YM25235

1. Agrobacterium-mediated transformation of Rhodosporidium YM25235

The recombinant plasmid pRHRKACS1 was transformed into Rhodosporidium YM25235 by the Agrobacterium-mediated method, and the transformants were selected with the YPD medium containing the final concentration of Hygromycin B (HygromycinB) at a final concentration of 150 µg/mL. The steps in the instructions of the DNA extraction kit of Co., Ltd. extract the genomic DNA of the yeast transformant, and then perform PCR verification. The results are shown in Figure 4;

2. Analysis of carotenoid content in Rhodosporidium YM25235 overexpressed with RKACS1 gene

The overexpressed strain containing pRHRKACS1 was cultured at 28°C to extract carotenoids, and the wild-type Rhodosporidium YM25235 strain was used as a control, and the content of total carotenoids (mg /g dry thallus), the content is shown in Figure 5; as can be seen from the figure, the total carotenoid synthesis of the overexpressed strain YM25235/pRHRKACS1 was significantly higher than that of the wild-type Rhodosporidium YM25235 strain, and the wild-type Rhodosporidium YM25235 The carotenoid synthesis amount of the strain was 5.23±0.25mg/g, while the carotenoid synthesis amount of the overexpression strain YM25235/pRHRKACS1 was 9.57±0.57mg/g, that is, the carotenoid synthesis amount of the overexpression strain YM25235/pRHRKACS1 was the same as that of the control strain The results showed that the overexpression of the acetyl CoA synthetase gene RKACS1 can cause the increase of the total carotenoid content in the Rhodosporidium YM25235 strain, and the RKACS1 gene can promote the synthesis of the total carotenoid.

3. Analysis of oil content in Rhodosporidium YM25235 overexpressed with RKACS1 gene

The overexpressed strain containing pRHRKACS1 was cultured at 28°C to extract oil, and the wild-type Rhodosporidium YM25235 strain was used as a control to measure the oil content (% dry cells), and the content is shown in Figure 6; it can be seen from the figure , the lipid synthesis of the overexpression strain YM25235/pRHRKACS1 was significantly higher than that of the wild-type Rhodosporidium YM25235 strain, and the lipid synthesis of the wild-type Rhodosporidium YM25235 strain was 4.27±0.51%, while the lipid synthesis of the overexpression strain The amount of acetyl CoA synthase gene RKACS1 was 5.73±0.24%, that is, the amount of oil synthesis in the overexpressed strain YM25235/pRHRKACS1 was increased by 34.19% compared with the control strain; Increase, RKACS1 gene can promote oil synthesis;

In conclusion, the acetyl-CoA synthetase gene RKACS1 is associated with carotenoid and lipid synthesis in Rhodosporidium sp.

sequence listing

<110> Kunming University of Science and Technology

<120> Acetyl CoA synthetase gene RKACS1 and its application

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<170> SIPOSequenceListing 1.0

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<213> Rhodosporidium kratochvilovae YM25235 (Rhodosporidium kratochvilovaeYM25235)

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cccgaggacc gctacgcgtg catggcagac gtcggctgga tcaccggcca cacctacatc 960

gtttacggcc cgcttgcgaa cggcgtcacc accaccatct tcgagtcgac gccggtctac 1020

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acactggcgg agcctgccat catcgacgag atcaaggaga agttcgccaa ggcgtag 1977

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<213> Rhodosporidium kratochvilovae YM25235

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Gly Lys Pro Lys Gly Val Val His Ser Ser Ala Gly Tyr Leu Leu Gly

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Ala Phe Met Thr Leu Lys Tyr Val Phe Asp Val His Pro Glu Asp Arg

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Tyr Ala Cys Met Ala Asp Val Gly Trp Ile Thr Gly His Thr Tyr Ile

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Val Tyr Gly Pro Leu Ala Asn Gly Val Thr Thr Thr Ile Phe Glu Ser

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Thr Pro Val Tyr Pro Thr Pro Ser Arg Phe Trp Glu Thr Val Ala Lys

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His Lys Leu Thr Gln Phe Tyr Thr Ala Pro Thr Ala Ile Arg Leu Leu

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Claims (3)

1. An acetyl-CoA synthetase gene RKACS1 , the nucleotide sequence of which is shown in SEQ ID NO:1. 2. The application of the acetyl-CoA synthetase gene RKACS1 according to claim 1 in promoting carotenoid production by Rhodosporidium kratochvilovae . 3. The application of the acetyl-CoA synthetase gene RKACS1 according to claim 1 in promoting the oil production of Rhodosporidium kratochvilovae .

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