Branched chain amino acid aminotransferase gene RkBat2 and application thereof

Abstract

The invention discloses a branched-chain amino acid aminotransferase gene RkBat2, the nucleotide sequence of the branched-chain amino acid aminotransferase gene RkBat2 is shown as SEQ ID NO: 1, the amino acid sequence coded by the gene is shown as SEQ ID NO: 2, the gene is obtained from rhodosporidium kratochvilovae YM25235, the gene is transformed into the rhodosporidium kratochvilovae YM25235, and the branched-chain amino acid aminotransferase gene RkBat2 is obtained after the gene is transformed into the rhodosporidium kratochvilovae YM25235. Experimental results show that overexpression of the gene RkBat2 can cause improvement of the synthesis level of carotenoid in the YM25235 strain; according to the invention, the microorganisms are modified by means of genetic engineering, so that the yield of carotenoid in the microorganisms is increased, and a foundation is laid for large-scale commercial production of carotenoid.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and genetic engineering, and specifically relates to a branched-chain amino acid aminotransferase gene RkBat2 and its role in promoting the growth of Rhodosporidium Rhodosporidium kratochvilovae ) Use in the production of carotenoids. Background Art

[0002] Carotenoids are a class of natural fat-soluble pigments that are widely found in higher plants, algae, fungi and bacteria. The presence of isoprenoid conjugated double bonds in the molecular structure of carotenoids gives them strong antioxidant activity. In terms of human health, carotenoids can reduce the risk of degenerative diseases such as cancer, cardiovascular disease, macular degeneration and cataracts, so carotenoids are widely used in the healthcare industry. In addition, carotenoids have a variety of colors, and they will show different colors as the number of conjugated double bonds, chain length and substitution groups in the molecule change. In the food and pharmaceutical industries, they are widely used as a natural pigment. In short, carotenoids play an important role in pharmaceuticals, chemicals, and food processing. The scrutiny and negative comments of modern consumers on synthetic food dyes have aroused people's strong interest in natural pigment substitutes. Compared with plant-extracted and chemically synthesized carotenoids, microbial synthesis and production of carotenoids have the advantages of low cost, strong natural activity and environmental friendliness. Generally speaking, carotenoids are synthesized in yeast mainly through the MVA pathway, using acetyl-CoA as the raw material, through a series of reactions such as condensation, dehydrogenation, and cyclization. β -carotene, γ -Carotene, rhodotorulaene, rhodotorula erythrin and astaxanthin.

[0003] The metabolism of branched-chain amino acids (BCAAs) has been intensively studied in mammals. Branched-chain amino acid metabolism is related to protein, glucose, and energy metabolism. BCAA aminotransferases (BCATs) catalyze the first step in BCAA metabolism, in which valine, leucine, and isoleucine are transaminated to α-ketoisovalerate (KIV), α-ketoisocaproate (KIC), and α-keto-β-methylvalerate (KMV), respectively. In eukaryotes, there are two isoforms of BCAT, mitochondrial BCAT (mBCAT) and cytosolic BCAT (cBCAT), which correspond to Bat1 and Bat2 in yeast, respectively. The first step in the metabolism of the three branched-chain amino acids is a reversible transamination and produces the corresponding branched-chain α-ketoacid (BCKA), which then undergoes oxidative decarboxylation to produce acyl-CoA derivatives with one carbon atom reduced, and finally undergoes steps similar to the fatty acid oxidation pathway to produce the corresponding end products. In Saccharomyces cerevisiaeBat1 and Bat2 It can play a role in both anabolic and catabolic pathways, acting as the final step in BCAA biosynthesis and the first step in degradation. Bat1 and Bat2 are homologous proteins with 77% primary sequence identity, but their localizations are significantly different. Bat1 Located in mitochondria, while those lacking MTS Bat2 It is present in the cytosol. Studies have reported that in Saccharomyces cerevisiae Bat1 and Bat2 With different functions, Bat1 The activity is mainly used for the biosynthesis of BCAA, while Bat2 Appears to play a role in catabolic pathways.

[0004] With the increasing demand for natural carotenoid products, the output and quality of natural carotenoids can no longer meet market demand. At present, the production methods of carotenoids mainly include plant extraction, chemical synthesis and biosynthesis. In the food industry, synthetic compounds have brought many negative effects, and the safety of synthetic food has received more and more attention from the society. Moreover, due to the low biological activity and certain toxic side effects of the generated pigments, it has been restricted for use by many countries and regions; plant extraction is less used due to the low content of carotenoids in plants, high extraction cost and complex process; while the carotenoids extracted by biosynthesis are pure natural, safe and non-toxic to the human body, so the extraction of carotenoids by biosynthesis is currently the most promising extraction method. At present, the microorganisms that can ferment and produce carotenoids mainly include fungi, bacteria and yeasts, among which the research on the production of carotenoids by yeast fermentation is the most common, and yeast is a confirmed feed microorganism in my country's feed industry. Therefore, it is a very feasible way to produce natural carotenoids by microbial fermentation using yeast as the production strain to meet people's needs. At present, there has been no report on the role of branched-chain amino acid aminotransferase in promoting the production of carotenoids by yeast. Summary of the invention

[0005] The present invention provides a branched-chain amino acid aminotransferase gene RkBat2 , which is derived from Rhodosporidium Rhodosporidium kratochvilovae ) YM25235, the nucleotide sequence of the gene is shown in SEQ ID NO:1 or is a fragment of the nucleotide sequence, or a nucleotide sequence complementary to SEQ ID NO:1, the gene sequence is 1149 bp (bases) long, and the amino acid sequence encoded by the gene is shown in SEQ ID NO:2.

[0006] The object of the present invention is achieved through the following technical solutions: 1. From red winter spore yeast ( Rhodosporidium kratochvilovae ) Amplification of branched-chain amino acid aminotransferase gene from YM25235 RkBat2 ; 2. Gene expression of branched-chain amino acid aminotransferase RkBat2 Connect to the expression vector pRH2034 to obtain a recombinant overexpression plasmid; 3. Recombinant overexpression plasmid pRH RkBat2 Transformed into Rhodosporidium sporogenes YM25235 for carotenoid production.

[0007] Advantages and technical effects of the present invention: The present invention is from red winter spore yeast ( Rhodosporidium kratochvilovae ) Branched-chain amino acid aminotransferase genes obtained from the total RNA gene of YM25235 RkBat2 Overexpression in red winter spore yeast YM25235 will increase the transcription level of this gene in the cell, indicating that the exogenous gene is transcribed in the bacteria and then translated into the corresponding protein, which makes the branched-chain amino acid aminotransferase in the yeast proliferate and causes the expression of enzymes related to carotenoid synthesis in the cell to increase. The results of this study help to clarify the mechanism of carotenoid production in red winter spore yeast YM25235, and help to improve the carotenoid content by genetic engineering. It has good application prospects and economic benefits for the industrial production of carotenoids and provides theoretical support for the production of carotenoids by biosynthesis. BRIEF DESCRIPTION OF THE DRAWINGS

[0008] Figure 1 The present invention is red winter spore yeast YM25235 RkBat2 Gene PCR amplification diagram; Figure 2 Recombinant plasmid pRH RkBat2 Plasmid map of Figure 3 Recombinant plasmid pRH RkBat2 PCR verification of transformation of red winter spore yeast YM25235; 1. DNA molecular scaler DL5000; 2. blank control group (no template added); 3. PCR product with YM25235 genome as template (containing introns); 4. RkBat2 5. Using YM25235 / pRH as template RkBat2 PCR products using the strain genome as template; Figure 4 For the overexpression strain YM25235 / pRH RkBat2 Compared with the total carotenoid content of the control strain YM25235 / pRH2034. DETAILED DESCRIPTION

[0009] The present invention is further described in detail below in conjunction with the accompanying drawings and examples, but the protection scope of the present invention is not limited to the contents described above. The reagents and methods used in the examples are conventional reagents and methods unless otherwise specified.

[0010] Example 1: From red winter spore yeast ( Rhodosporidium kratochvilovae ) Isolation and acquisition of branched-chain amino acid aminotransferase gene from YM25235 RkBat2 The total RNA of red winter spore yeast YM25235 was extracted using the UNlQ-10 column Trizol total RNA extraction kit (product number: SK1321) from Sangon Biotech (Shanghai) Co., Ltd., and then reverse transcribed and synthesized into cDNA using the TaKaRa kit PrimeScript® RT reagent Kit With gDNA Eraser (Perfect Real Time). 0.5 μL of cDNA was used as a template for polymerase chain reaction. According to the transcriptome sequencing results, RkBat2 Sequence and design specific primers RkBat2 -F and RkBat2 -R, PCR amplification was performed on a PCR instrument (BIOER) using the cDNA template obtained above. The primers, amplification system and amplification conditions used in the reaction were as follows: RkBat2 -F: 5'-ATCACTCACCATGGCG GATCCG ATGTCGATGCCTGTCGTTGCC-3' RkBat2 -R: 5'-CCGGTCGGCATCTAC GATATC CTACACCAGCACCGACCAGT-3′; ( GATCCG for BamH I Restriction site, GATATC for Eco RV restriction site); The PCR amplification system is as follows (50 μL):

[0011] The amplification conditions were: pre-denaturation at 94°C for 5 min, denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 2 min, 30 cycles, and final extension at 72°C for 10 min. After the reaction, 2 μL of the product was taken and analyzed by electrophoresis in a 1% agarose gel. The results are shown in the figure. Figure 1As shown, a fragment of about 1149 bp was amplified and recovered using an agarose gel DNA recovery kit (Beijing Solebow Technology Co., Ltd.), and the recovered fragment was ligated to pMD-18T (TaKaRa product), and the ligation product was transformed into competent Escherichia coli DH5α, cultured overnight on LB solid plates containing ampicillin (100µg / mL), and positive clones were verified by colony PCR. The positive clones were inoculated into LB liquid culture medium (containing 100µg / mL ampicillin) and cultured overnight, and the plasmid was extracted using a high-purity plasmid miniprep kit (spin column type) (Beijing Biotech Biotechnology Co., Ltd.), and sequenced (Kunming Shuoqing Biotechnology Co., Ltd.). The sequencing results showed that the size of the amplified fragment was 1149 bp, and the obtained fragment was named RkBat2 , the nucleotide sequence is shown in SEQ ID NO:1.

[0012] Example 2: Overexpression vector pRH RkBat2 Construction The reverse transcribed YM25235 cDNA was used as template. RkBat2 -F and RkBat2 -R as primer for amplification RkBat2 The coding sequence obtained RkBat2 The fragment size is about 1149 bp. RkBat2 Fragments and BamH I , Eco The vector pRH2034 digested by two restriction endonucleases RⅤ was connected to the expression vector pRH2034 to obtain the recombinant plasmid pRH RkBat2 ( Figure 2 ). The obtained recombinant plasmid was transferred into competent E. coli DH5α for amplification, and the recombinant plasmid was extracted after colony PCR verification; sequencing was performed using sequencing primers. The sequencing results showed that the sequence obtained by sequencing was completely consistent with the target sequence, without any base mutation or deletion.

[0013] Embodiment 3: RkBat2 Analysis of the relationship between genes and carotenoid biosynthesis in Rhodosporidium 1. Transformation of Rhodosporidium yeast YM25235 The recombinant plasmid pRH RKBAT2 Transformed into red winter spore yeast YM25235, the transformants were selected with YPD medium containing hygromycin B (final concentration of 150 μg / mL), and then the genomic DNA of the yeast transformants was extracted according to the steps in the DNA extraction kit instructions of Shanghai Sangon Biotechnology Co., Ltd., and then PCR verification was performed. The results are as follows Figure 3 shown.

[0014] 2. Detection of carotenoid content in red winter spore yeast pRH RkBat2 The overexpression strain was cultured at 28°C for 168 h, and carotenoids were extracted. The red winter spore yeast strain transformed with the empty plasmid pRH2034 was used as a control. The total carotenoid content (mg / g dry cell) was determined by UV-visible spectrophotometer at 445 nm. The results are shown in Figure 4 As shown; the results showed that the overexpression strain YM25235 / pRH RkBat2 The total carotenoid synthesis of the strain YM25235 / pRH was significantly increased compared with the control strain containing the empty plasmid pRH2034. The carotenoid synthesis of the control strain containing the empty plasmid pRH2034 was 6.02 mg / g, while the overexpression strain YM25235 / pRH RkBat2 The amount of carotenoid synthesis was 7.12 mg / g, that is, the overexpression strain YM25235 / pRH RkBat2 The amount of carotenoids synthesized was 1.2 times that of the control bacteria, proving that RkBat2 Genes can promote the synthesis of total carotenoids.

Claims

1. A branched-chain amino acid aminotransferase gene RkBat2 , whose nucleotide sequence is shown in SEQ ID NO:

1.

2. The branched-chain amino acid aminotransferase gene according to claim 1 RkBat2 In promoting red winter spore yeast ( Rhodosporidium kratochvilovae ) Application in the production of carotenoids.

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