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Acetyl coenzyme A acetyltransferase gene RKAcaT2 and application thereof

An acetyltransferase, acetyl coenzyme technology, applied in the directions of acyltransferase, transferase, application, etc., can solve the problems of complex process, low biological activity and high extraction cost

Active Publication Date: 2019-04-23
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with other methods, the production of carotenoids by chemical synthesis is the cheapest and most convenient method, but due to the low biological activity and certain toxic and side effects of pigment formation, it has been restricted in many countries and regions; the plant extraction method is due to The content of carotenoids in plants is low, the extraction cost is relatively high, and the process is complicated, etc., and the use is seldom; while the carotenoids extracted by biosynthesis are pure natural, safe and non-toxic to human body, so biosynthesis Extraction of carotenoids is currently the most promising extraction method

Method used

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  • Acetyl coenzyme A acetyltransferase gene RKAcaT2 and application thereof
  • Acetyl coenzyme A acetyltransferase gene RKAcaT2 and application thereof
  • Acetyl coenzyme A acetyltransferase gene RKAcaT2 and application thereof

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Experimental program
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Effect test

Embodiment 1

[0017] Embodiment 1: from Rhodosporidium yeast ( Rhodosporidium kratochvilovae ) Acetyl-CoA acetyltransferase gene isolated from YM25235 RKAcaT2 Nucleotide sequence

[0018] 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) was used for reverse transcription to synthesize cDNA, and 0.5 μL was used as a template for polymerase chain reaction. According to the RKAcaT2 sequence found in the transcriptome sequencing, specific primers RKAcaT2-F and RKAcaT2-R ( Primer 1 and primer 2), carry out PCR amplification on the PCR instrument (BIOER company) with the cDNA template obtained above, the primers, components and amplification conditions used in the reaction are as follows:

[0019] Primer 1: RKAcaT2-F: 5'-TCA...

Embodiment 2

[0025] Example 2: Construction of overexpression vector pRHRKAcaT2

[0026] Using the reverse-transcribed YM25235 cDNA as a template, RKAcaT2-F and RKAcaT2-R were used as primers to amplify the coding sequence of RKAcaT2, and the obtained RKAcaT2 fragment was about 1200bp in size. Noc I. Eco After RV two restriction endonucleases were digested, it was connected to the expression vector pRH2034 to obtain the recombinant plasmid pRHRKAcaT2 ( figure 2 ). The obtained recombinant plasmid was transformed into Escherichia coli DH5α for amplification, and then verified by colony PCR to extract the recombinant plasmid and use Noc I. Eco RⅤ carried out double enzyme digestion verification on pRHRKAcaT2. The results showed that the recombinant plasmid pRHRKAcaT2 produced two bands of about 1.2 kb and 10.7 kb after double enzyme digestion (the third lane in Figure 3), which were respectively related to RKAcaT2 The size of the fragment was the same as that of the pRH2034 vector ...

Embodiment 3

[0027] Example 3: RKAcaT2 Effect of Gene Overexpression on Carotenoid Synthesis in Rhodosporidium YM25235

[0028] 1. Agrobacterium-mediated transformation of Rhodosporidium YM25235

[0029] The recombinant plasmid pRHRKAcaT2 was transformed into Rhodosporidium YM25235 by the Agrobacterium-mediated method, and the transformants were selected with the YPD medium containing Hygromycin B (Hygromycin B) at a final concentration of 150 µg / mL, and then the Shanghai Sangon Bioengineering 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 as follows: Figure 4 shown.

[0030] 2, RKAcaT2 Analysis of Carotenoid Content in Gene Overexpressed Rhodosporidium YM25235

[0031] The overexpressed strain YM25235 / pRHRKAcaT2 containing pRHRKAcaT2 was cultured at 30°C for 144h to extract carotenoids, and the Rhodosporidium strain YM25235 / pRH2304 transformed into the empty pla...

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Abstract

The invention discloses an acetyl coenzyme A acetyltransferase gene RKAcaT2 and application thereof. The nucleotide sequence of the gene is shown in SEQ ID NO:1, the amino acid sequence encoded by thegene is shown in SEQ ID NO:2, the gene is a key enzyme gene synthesized by carotenoid in Rhodosporidium kratochvilovae YM25235 and has the functions of the acetyl coenzyme A acetyltransferase, and the carotenoid produced by the Rhodosporidium kratochvilovae YM25235 can be controlled; microorganisms are transformed through the genetic engineering means so as to increase the yield of carotenoid inthe microorganisms, and a foundation is laid for large-scale commercial production of the carotenoid.

Description

technical field [0001] The invention belongs to the fields of biotechnology and genetic engineering, and relates to an acetyl-CoA acetyltransferase gene RKAcaT2 , specifically related to the yeast---Rhodosporidium ( Rhodosporidium kratochvilovae ) Acetyl-CoA acetyltransferase gene cloned in YM25235 RKAcaT2 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 are lipophilic natural pigments synthesized by all photosynthetic organisms and some non-photosynthetic prokaryotes and fungi. They exist widely in nature and are generally yellow, orange-red, red or purple. Typical carotenoids are composed of 8 C 40 Terpenoids and their derivatives, the action of conjugated double bonds in their structure leads to the color difference of their various carotenoids. Some carotenoids have shorter side carbon cha...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/10
CPCC12N9/1029C12Y203/01009
Inventor 张琦张晓庆魏云林林连兵季秀玲
Owner KUNMING UNIV OF SCI & TECH
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