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HMG-CoA synthetase gene RKHMGCS and application thereof

A synthase, gene technology, applied in applications, genetic engineering, plant genetic improvement, etc., can solve the problems of lack of strategy, cost-effectiveness, yield and isolation and extraction limitations

Active Publication Date: 2019-05-21
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although many strategies have been used to promote the synthesis of carotenoids in microorganisms, due to the limitations of cost-effectiveness, yield and separation and extraction, the production of various carotenoids by traditional microbial fermentation technology is not suitable for industrial-scale fermentation production. There is still no corresponding strategy

Method used

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  • HMG-CoA synthetase gene RKHMGCS and application thereof
  • HMG-CoA synthetase gene RKHMGCS and application thereof
  • HMG-CoA synthetase gene RKHMGCS and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0018] Embodiment 1: from Rhodosporidium yeast ( Rhodosporidium kratochvilovae ) Isolation of key enzymes for carotenoid synthesis from YM25235 RKHMGCS Nucleotide sequence

[0019] The total RNA of Rhodosporidium YM25235 was extracted using the UNlQ-10 Column Trizol Total RNA Extraction Kit (product number: SK1321) of Sangon Bioengineering (Shanghai) Co., Ltd., and then the PrimeScript ® RT reagent of the TaKaRa company kit was used to extract the total RNA. Kit With gDNA Eraser (Perfect Real Time) for reverse transcription to synthesize cDNA, take 0.5 μL as a template for polymerase chain reaction, according to the results found in transcriptome sequencing RKHMGCS sequence, design specific primers RKHMGCS -F and RKHMGCS -R (primer 1 and primer 2), use the cDNA template obtained above to carry out PCR amplification on a PCR instrument (BIOER company), the primers, components and amplification conditions used in the reaction are as follows:

[0020] Primer 1: RKHMGCS -F:5...

Embodiment 2

[0026] Example 2: HMG-CoA Synthetase Gene RKHMGCS Construction of overexpression vector pRHRKHMGCS

[0027] Using the reverse transcribed YM25235 cDNA as a template, use RKHMGCS -F and RKHMGCS -R as a primer for amplification RKHMGCS The coding sequence obtained RKHMGCS The fragment size is about 1449bp, the amplified RKHMGCS Fragment by Nco I. Eco After cutting with two restriction endonucleases, RV was connected to the expression vector pRH2034 to obtain the recombinant plasmid pRHRKHMGCS ( figure 2 ). The obtained recombinant plasmid was transferred into Escherichia coli DH5α for amplification, and then verified by colony PCR to extract the recombinant plasmid and use Nco I. Eco RⅤ carried out double digestion verification on pRHRKHMGCS; the results showed that the recombinant plasmid pRHRKHMGCS produced two bands of about 1.5 kb and 10.0 kb after double digestion ( image 3 Lane 3), these two bands were compared with RKHMGCS The size of the fragment and the...

Embodiment 3

[0028] Example 3: RKHMGCS Effect of Gene Overexpression on Carotenoid Synthesis in Rhodosporidium YM25235

[0029] 1. Agrobacterium-mediated transformation of Rhodosporidium YM25235

[0030] The recombinant plasmid pRHRKHMGCS was transformed into Rhodosporidium YM25235 by the Agrobacterium-mediated method, and the transformants were selected with YPD medium containing Hygromycin B (HygromycinB) at a final concentration of 150 µg / mL, and then the Shanghai Sangon Bioengineering Co., Ltd. Co., Ltd. DNA Extraction Kit manual extracts the genomic DNA of the yeast transformant, and then performs PCR verification. The results are shown in Figure 4 .

[0031] 2, RKHMGCS Analysis of Carotenoid Content in Gene Overexpressed Rhodosporidium YM25235

[0032] The overexpressed strain containing pRHRKHMGCS was cultured at 15°C, carotenoids were extracted, and the content of β-carotene was determined. The Rhodosporidium strain transformed into the empty plasmid pRH2034 was used as a cont...

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Abstract

The invention discloses an HMG-CoA synthetase gene RKHMGCS, the nucleotide sequence is shown as the SEQ ID NO:1, and the amino acid sequence of the gene coding is shown as the SEQ ID NO:2; the gene isthe key enzyme gene for synthesis of the carotenoid in the Rhodosporidium kratochvilovae YM 25235, the gene has the HMG-CoA synthetase function, and the Rhodosporidium kratochvilovae YM 25235 can becontrolled to produce the carotenoid; the microorganism is modified by the means of genetic engineering, the yield of the carotenoid in the microorganism body is improved, and a foundation is laid forlarge-scale commercialized production of the carotenoid.

Description

technical field [0001] The invention belongs to the fields of biotechnology and genetic engineering, and relates to a gene for HMG-CoA synthetase RKHMGCS , specifically related to the yeast--Rhodosporidium ( Rhodosporidium kratochvilovae ) HMG-CoA synthetase gene cloned in YM25235 RKHMGCS And the gene is directly connected with different vectors and transferred into yeast cells to increase the expression level of this gene and finally promote the synthesis of carotenoids. Background technique [0002] Carotenoids (carotenoids) are a class of colored substances that widely exist in nature, generally appearing yellow, orange-red or red; there are many kinds of carotenoids in nature, and more than 600 carotenoids have been discovered with clear structures; Most known carotenoids consist of 8 isoprenoids; most are tetraterpenes with 40 carbon atoms, but there are also carotenoids with less than 40 carbon atoms, such as β-apo- carrot aldehyde. [0003] Carotenoids are the ge...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/81C12P23/00C12R1/645
Inventor 张琦陈波魏云林季秀玲
Owner KUNMING UNIV OF SCI & TECH
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