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Rapid Screening of Strains Improved Coenzyme Q10 Production by Genome Shuffling

Author: QiaoZhiXin
Tutor: ZhangJinGang；YuQun
School: PLA Military Academy of Medical Sciences
Course: Immunology
Keywords: Coenzyme Q10 high-throughput screening mutagenesis genome shuffling
CLC: Q93
Type: Master's thesis
Year: 2009
Downloads: 170
Quote: 2
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Abstract


Coenzyme Q10(CoQ10) is a prenylated benzoquinone lipid, and it is also named ubiquinone. CoQ10 is an important electron transfer molecule in the respiratory chain, and the necessary coenzyme of the production of ATP. CoQ10 is supposed to be an important endogenous lipid-soluble antioxidant and extensively present in the cell of animals, plants and bacteria. CoQ10 has been widely used in the treatment of various diseases such as heart diseases, cancer, hepatitis, Parkinson and so on.The production of CoQ10 by microbial fermentation has become the most promising production method and to obtain a good strain with high level of CoQ10 is the key to the production. Alter the expression of one or a few key enzymes couldn’t really significantly improve the content of stains by gene engineering methods since the biosynthetic pathway of CoQ10 is too complicated. The traditional mutation breeding is still the important ways for improving the content of CoQ10.Genome shuffling is the extension of molecular directed evolution at the level of genome, extending the shuffling from a single gene to the whole genome. Through the recursive fusion of forward mutants, screen the the strains with phenotype significant improvement. Most cellular phenotypes are affected by many genes can be improved quickly and effectively through Genome shuffling method. Genome shuffling can rapidly optimize metabolic pathways and has been applied to biosynthesis of many metabolites in metabolic engineering.In this paper, we apply the genome shuffling combined the traditional mutation to improve the content of CoQ10 by fusioning the forward mutants. Determine the resistance marker according to the biosynthetic pathway and physiological function of CoQ10.Combined the resistance marker and variety of mutagenesis methods to establish a library of forward mutants. To obtain the high-yielding mutants fusioning variety of forwand mutants through rounds of genome shuffling. This paper is divided into four parts: The culture conditions study of Rhodobacter.Sphaeoides; establish the high-throughput screening platform; the research of mutation breeding; the study of the protoplast preparing and fusion. The main result of research are as following:1. Establish the high-throughput screening platformAccording to the quinone ring and polyisoprene characteristics of CoQ10 chemical structure, combined with the physiological function of CoQ10, establish the high-throughput screening platform. Based on the platform, achieve the rapid and efficient screening. It’s possible to extract and detect almost hundreds samples one tine by the alkaline lysis method and the improved craven test method.2. Determine the parameter of variety of mutagenesis methods As the growth curve show, R.S begin at logarithmic phase in 5h. R.S OD reach the peak at 28h and begin the the stable growth period. The content of CoQ10 reach the maximum at 36h. Determine the mutagenesis parameter of UV radiation, DES,MW radiation and Co60γradiation. Based on the high-throughput screening platform, obtain the forward mutants. Establish the forward mutants library.3. Determine the parameter of resistance markers and proper protoplast preparing and regenerationDetermine the parameter of Roxithromycin, Kanamycin, PHB, Vitamin K3, Na2S, Roxithromycin 7-10mg/L; Kanamycin 9-10 mg/L; PHB 0.1%-0.15%; Vitamin K 10-15mg/L; Na2S 10-15mg/ml. Determine the parameter of proper protoplast preparing and regeneration: enzymatic time 1h, enzymatic temperature 37℃; enzymatic concentration 1mg/ml. Among the forward mutants obtain through protoplast fusion, the CoQ10 content of PN13 achieved 2.39mg/g, producing 2.52 times as content of R.S.

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