A branched-chain amino acid aminotransferase gene RkBat2 and use thereof

Abstract

The application discloses a branched-chain amino acid aminotransferase gene RkBat2 , wherein the nucleotide sequence is shown as SEQ ID NO:1, the amino acid sequence coded by the gene is shown as SEQ ID NO:2, and the gene is obtained from Rhodosporidium diobovatum YM25235, and the gene is transformed into the Rhodosporidium diobovatum YM25235 through transformation. Rhodosporidium kratochvilovae Experimental results show that overexpression of the gene RkBat2 can cause the improvement of the carotenoid synthesis level in the YM25235 strain; the application improves the carotenoid yield in microorganisms through genetic engineering, and lays a foundation for large-scale commercial production of carotenoids.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology and genetic engineering, and particularly relates to a branched-chain amino acid aminotransferase gene RkBat2 and its use in promoting the production of carotenoids by Rhodotorula diobovata Rhodosporidium kratochvilovae . BACKGROUND

[0002] Carotenoids are a class of natural liposoluble pigments, which are widely present in higher plants, algae, fungi and bacteria; the presence of isoprenoid conjugated double bonds in the molecular structure of carotenoids makes them have strong antioxidant activity. In terms of human health, carotenoids can reduce the risk of cancer, cardiovascular disease, macular degeneration and cataract and other degenerative diseases, so carotenoids have a wide application in the health care industry. In addition, carotenoids have rich colors, and will present different colors with the change of the number of conjugated double bonds, chain length and substituent groups in the molecule, and are widely used as a natural pigment in the food and pharmaceutical industries. In summary, carotenoids play an important role in pharmaceuticals, chemical industry, food processing, and modern consumers' scrutiny and negative evaluation of synthetic food dyes have aroused strong interest in natural pigment substitutes. Compared with plant extraction and chemical synthesis of carotenoids, microbial synthesis of carotenoids has the advantages of low cost, strong natural activity and environmental friendliness. Generally speaking, in yeast, carotenoids are mainly synthesized through the MVA pathway, using acetyl-CoA as the raw material, through condensation, dehydrogenation, cyclization and a series of reactions to form β - carotenes, gamma - lutein, astaxanthin and phytoene.

[0003] The metabolism of branched-chain amino acids (BCAAs) has been well studied in mammals. Branched-chain amino acid metabolism is related to protein, glucose and energy metabolism. BCAA transaminase (BCAT) catalyzes the first step of BCAA catalytic 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 subtypes of BCAT, mitochondrial BCAT (mBCAT) and cytosolic BCAT (cBCAT), which correspond to Bat1 and Bat2 in yeast, respectively. The first step of the metabolism of the three branched-chain amino acids is a reversible transamination and produces the corresponding branched-chain α-keto acid (BCKA), which is then oxidatively decarboxylated to generate an acyl-CoA derivative with one less carbon atom, and finally through steps similar to the fatty acid oxidation pathway, the corresponding end product is generated. In Saccharomyces cerevisiae,Bat1 and Bat2 It can play a role in both anabolism and catabolism, serving as the final step in BCAA biosynthesis and the first step in its degradation. Although Bat1 and Bat2 They are homologous proteins with 77% primary sequence identity, but their localizations are significantly different. One possesses a mitochondrial targeting signal (MTS). Bat1 Located in mitochondria, but lacking MTS Bat2 It exists in cytosol. Studies have reported its presence in *Saccharomyces cerevisiae*. Bat1 and Bat2 They have different functions. Bat1 Its activity is mainly used in the biosynthesis of BCAA, while Bat2 It appears to play a role in the catabolic pathway.

[0004] With the increasing demand for natural carotenoid products, the quantity and quality of natural carotenoids can no longer meet market demand. Currently, the main production methods for carotenoids include plant extraction, chemical synthesis, and biosynthesis. In the food industry, synthetic compounds have brought many negative impacts, and the safety of synthetic foods is receiving increasing attention. Furthermore, due to their low biological activity and the toxic side effects of the pigments produced, their use has been restricted in many countries and regions. Plant extraction is also less common due to the low carotenoid content in plants, high extraction costs, and complex processes. Biosynthetic carotenoids are purely natural and safe for humans, making it the most promising extraction method. Currently, the microorganisms capable of fermenting to produce carotenoids mainly include fungi, bacteria, and yeasts. Research on yeast fermentation for carotenoid production is the most common, and yeast is a recognized feed microorganism in my country's feed industry. Therefore, using yeast as the production strain for microbial fermentation to produce natural carotenoids to meet consumer demand is a very feasible approach. Currently, there are no reports on branched-chain amino acid aminotransferases promoting carotenoid production by yeast. Summary of the Invention

[0005] This invention provides a branched-chain amino acid aminotransferase gene. RkBat2 This gene was derived from *Rhodotorula rubrum* (…). Rhodosporidium kratochvilovae The gene was isolated from YM25235. The nucleotide sequence of the gene is shown in SEQ ID NO:1 or is a fragment of the nucleotide sequence, or is 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 objective of this invention is achieved through the following technical solution:

[0007] 1. From Rhodotorula rubrum ( Rhodosporidium kratochvilovae Branched-chain amino acid aminotransferase gene was amplified from YM25235. RkBat2 ;

[0008] 2. Branched-chain amino acid aminotransferase gene RkBat2 The recombinant overexpression plasmid was obtained by ligating it into the expression vector pRH2034;

[0009] 3. The recombinant overexpression plasmid pRH RkBat2 The yeast was converted into Rhodotorula rubrum YM25235 for the production of carotenoids.

[0010] Advantages and technical effects of the present invention:

[0011] This invention is derived from *Rhodotorula rubrum* (… Rhodosporidium kratochvilovae Branched-chain amino acid aminotransferase gene obtained from total RNA of YM25235 RkBat2 Overexpression of this gene in *Rhodotorula rubrum* YM25235 leads to an increase in the intracellular transcription level of the gene, indicating that the exogenous gene is transcribed within the cell and subsequently translated into the corresponding protein. This results in the proliferation of branched-chain aminotransferases in the yeast, leading to an increase in the expression of enzymes related to carotenoid synthesis. The findings of this study contribute to elucidating the mechanism of carotenoid production in *Rhodotorula rubrum* YM25235, and facilitate the modification of the yeast using genetic engineering to increase carotenoid content. This provides promising application prospects and economic benefits for the industrial production of carotenoids, and offers theoretical support for the biosynthetic production of carotenoids. Attached Figure Description

[0012] Figure 1 The red syringomyelia YM25235 of the present invention RkBat2 Gene PCR amplification diagram;

[0013] Figure 2 For recombinant plasmid pRH RkBat2 plasmid map;

[0014] Figure 3 pRH is a recombinant plasmid RkBat2 PCR validation of transformation of *Rhodotorula rubrum* YM25235; including: 1. DNA molecular scalar DL5000; 2. Blank control group (no template added); 3. PCR product using the YM25235 genome as template (containing introns); 4. PCR product using plasmid pRH. RkBat2 PCR products using YM25235 / pRH as template, 5. RkBat2 PCR products using the bacterial genome as a template;

[0015] RkBat2 For overexpression strain YM25235 / pRH Figure 4 Total carotenoid content compared to control strain YM25235 / pRH2034. Detailed Implementation

[0016] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments. However, the scope of protection of the present invention is not limited to the contents described. Unless otherwise specified, the reagents and methods used in the embodiments are all conventional reagents and conventional methods.

[0017] Example 1: From Rhodotorula rubrum ( RkBat2 Branched-chain amino acid aminotransferase gene isolated from YM25235 Rhodosporidium kratochvilovae

[0018] Total RNA was extracted from *Rhodotorula rubrum* YM25235 using the UNlQ-10 column-based Trizol total RNA extraction kit (product number: SK1321) from Sangon Biotech (Shanghai) Co., Ltd. Then, cDNA was synthesized via reverse transcription using the TaKaRa PrimeScript® RT reagent Kit With gDNA Eraser (Perfect Real Time). 0.5 μL of cDNA was used as a template for polymerase chain reaction (PCR), and the RNA was extracted based on findings from transcriptome sequencing. RkBat2 Sequence, design specific primers RkBat2 -F and RkBat2 -R, using the cDNA template obtained above, PCR amplification was performed on a PCR instrument (BIOER). The primers, amplification system, and amplification conditions used are as follows:

[0019] RkBat2 -F:5'-ATCACTCACCATGGCG RkBat2 ATGTCGATGCCTGTCGTTGCC-3'

[0020] GATCCG -R:5'-CCGGTCGGCATCTAC RkBat2 CTACACCAGCACCGACCAGT-3' ;

[0021] ( GATATC for GATCCG Enzyme cleavage site, BamH I for GATATC RV restriction site);

[0022] The PCR amplification system is as follows (50 μL):

[0023]

[0024] The amplification conditions were as follows: pre-denaturation at 94℃ for 5 min, followed by denaturation at 94℃ for 30 s, annealing at 60℃ for 30 s, and extension at 72℃ for 2 min, for 30 cycles. A final extension at 72℃ for 10 min was performed. After the reaction, 2 μL of the product was collected and analyzed by electrophoresis on a 1% agarose gel. The results are as follows. Eco As shown, a fragment of approximately 1149 bp was amplified and recovered using an agarose gel DNA recovery kit (Beijing Solarbio Science & Technology Co., Ltd.). The recovered fragment was ligated into pMD-18T (TaKaRa product), and the ligation product was transformed into competent *E. coli* DH5α cells. The cells were cultured overnight on LB agar plates containing ampicillin (100 µg / mL), and positive clones were verified by colony PCR. The verified positive clones were inoculated into LB liquid medium (containing 100 µg / mL ampicillin) and cultured overnight. Plasmids were extracted using a high-purity plasmid mini-preparation kit (centrifuge column type) (Beijing Biotech Biotechnology Co., Ltd.), and sequenced (Kunming Shuoqing Biotechnology Co., Ltd.). The sequencing results showed that the amplified fragment was 1149 bp in size. The obtained fragment was named... Figure 1 The nucleotide sequence is shown in SEQ ID NO:1.

[0025] Example 2: Overexpression vector pRH RkBat2 Construction

[0026] Using reverse-transcribed YM25235 cDNA as a template, RkBat2 -F and RkBat2 -R is used as a primer for amplification. RkBat2 The encoded sequence obtained RkBat2 The fragment size is approximately 1149 bp, and the amplified fragment will be obtained... RkBat2 Fragments and being RkBat2 I , BamH The vector pRH2034, digested with two restriction endonucleases (R and V), was ligated into the expression vector pRH2034 to obtain the recombinant plasmid pRH. Eco ( RkBat2 The obtained recombinant plasmid was transformed into competent E. coli DH5α for amplification. After verification by colony PCR, the recombinant plasmid was extracted and sequenced using sequencing primers. The sequencing results showed that the sequence obtained was completely identical to the target sequence, with no base mutations or deletions.

[0027] Example 3: Figure 2 Analysis of the relationship between genes and carotenoid synthesis in Rhodotorula rubrum

[0028] 1. Transformation of Rhodotorula rubrum YM25235

[0029] The recombinant plasmid pRH RKBAT2 was transformed into red yeast YM25235 by Agrobacterium-mediated method, and the transformants were screened on YPD medium containing hygromycin B (final concentration 150 µg / mL). Then, the genomic DNA of the yeast transformants was extracted according to the steps in the DNA extraction kit instruction of Shanghai Generay Biotech Co., Ltd., and PCR verification was performed, and the results are shown in Figure 3 .

[0030] 2. Detection of carotenoid content in red yeast

[0031] The overexpression strain containing pRH RkBat2 was cultured at 28°C for 168 h, and then carotenoids were extracted. The red yeast strain into which the empty plasmid pRH2034 was transferred was used as a control. The content of total carotenoids (mg / g dry mycelium) was determined at 445 nm by ultraviolet-visible spectrophotometry, and the results are shown in Figure 4 . The results show that the total carotenoid synthesis amount of the overexpression strain YM25235 / pRH RkBat2 is obviously improved compared with the control strain containing the empty plasmid pRH2034. The carotenoid synthesis amount of the control strain containing the empty plasmid pRH2034 is 6.02 mg / g, while the carotenoid synthesis amount of the overexpression strain YM25235 / pRH RkBat2 is 7.12 mg / g, i.e., the carotenoid synthesis amount of the overexpression strain YM25235 / pRH RkBat2 is 1.2 times that of the control strain, proving that the genes can promote the synthesis of total carotenoids. RkBat2 ​

Claims

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

1. RkBat2 2. The branched-chain amino acid aminotransferase gene according to claim 1, wherein the nucleotide sequence is shown as SEQ ID NO:

2.

2. The branched-chain amino acid aminotransferase gene of claim 1 RkBat2 application in promoting the production of carotenoids by Rhodotorula Rhodosporidium kratochvilovae ).

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