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Mevalonate kinase gene RKMK and application thereof

A kind of mevalonate kinase, gene technology, applied in genetic engineering, biological field

Active Publication Date: 2022-03-01
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] There is no report on the role of mevalonate kinase gene in promoting microbial production of carotenoids

Method used

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  • Mevalonate kinase gene RKMK and application thereof
  • Mevalonate kinase gene RKMK and application thereof
  • Mevalonate kinase gene RKMK and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1: Isolation of mevalonate kinase gene from Rhodosporidium YM25235 RKMK And the construction of overexpression vector pRHRKMK

[0020] 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 followed by Vazyme’s kit (product number: R212-02) According to the operating instructions in HiScript II 1st Strand cDNA Synthesis Kit (+gDNA wiper), carry out reverse transcription to synthesize cDNA, take 1 μL of cDNA as a template for polymerase chain reaction, according to the results found in transcriptome sequencing RKMK Sequence, design specific primers RKMK-F and RKMK-R (primer 1 and primer 2), use the cDNA template obtained above, use primers RKMK-F and RKMK-R, in a PCR machine (Beijing Liuyi Biotechnology Co., Ltd.) The primers, amplification system and amplification conditions used in the reaction are as follows:

[0021] Prime...

Embodiment 2

[0030] Example 2: RKMK Analysis of Carotenoid Content in Rhodosporidium YM25235 Overexpressed

[0031] 1. Transform Rhodosporidium YM25235

[0032] Pick the single clone of the DH5α strain successfully transferred to the correct recombinant vector pRHRKMK and insert it into LB liquid medium (containing 100 μg / mL spectinomycin) for overnight culture, extract the plasmid (OMEGA Plasmid Mini Kit I, OMEGA, USA), and measure the concentration , stored at -20°C for later use; pick a single colony of Rhodosporidium YM25235 and inoculate it in 5mL of YPD liquid medium, and culture it overnight at 30°C and 200rpm with shaking; Connect to 50mL of YPD liquid medium at 30°C and shake at 200rpm and culture until the OD of the bacterial solution 600 Centrifuge the culture at 4°C, 4500 rpm for 5 min to collect the bacteria; use the previously prepared citric acid buffer (30mM citric acid, 83mM sodium citrate, 600mM mannitol, NaOH to adjust the pH value to 5.4) to wash the bacteria Centrif...

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Abstract

The invention discloses a mevalonate kinase gene RKMK and application thereof, the nucleotide sequence of the mevalonate kinase gene RKMK 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 separated from Rhodosporidium kratochvilovae YM25235, the gene is connected with a carrier and is transferred into a Rhodosporidium cell, and an experimental result shows that overexpression of the RKMK gene can promote synthesis of carotenoid by the Rhodosporidium kratochvilovae; 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 commercialized production of carotenoid.

Description

technical field [0001] The invention belongs to the field of biotechnology and genetic engineering technology, and relates to a mevalonate kinase gene RKMK and its role in improving Rhodosporidium ( Rhodosporidium kratochvilovae ) application in carotenoid production. Background technique [0002] Mevalonate kinase (MVK) is the first of three consecutive ATP-dependent enzymes in the mevalonate (MVA) pathway and is one of the rate-limiting enzymes that control the entire metabolic pathway. Mevalonate kinase The N-terminus of the protein contains a conserved region rich in Gly / Ser, which is involved in the binding of ATP (adenosine triphosphate). Mevalonate kinase is responsible for transferring a phosphate group on the ATP-γ position to the hydroxyl group at the 5th position of mevalonate to form mevalonate-5-phosphate and release ADP. Mevalonate kinases from different biological sources are usually monomers or dimers composed of the same subunits, with a molecular weight...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/81C12N1/19C12P23/00C12R1/645
CPCC12N9/1205C12N15/815C12P23/00C12Y207/01036
Inventor 张琦郭彩娜魏云林
Owner KUNMING UNIV OF SCI & TECH
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