Strain Selection Of Rhodobacter Sphaeroides In Coenzyme Q₁₀ Production And Optimization Of Fermentation Conditions

Coenzyme Q10 (also known as ubiquinone,abbreviated as CoQ10) is a 1,4-benzoquinone,and it is a component of the electron transport chain and participates in aerobic cellular respiration. This oil-soluble substance is supposed to be an important antioxidant and generally present in the cells of animals, plants and bacterias.CoQ10 has been increasingly used in the treatment of various diseases such as heart disease, acute and chronic hepatitis, Parkinson's disease and so on, and gradually applied to the field of health care products.The microbial fermentation has become the main method of the CoQ10 production and the premise is to obtain a good strain with high level of CoQ10 production. The optimization of fermentation conditions can improve the production of CoQ10,so it is widely used in industrial production.Because the biosynthesis pathway of CoQ10 is too complicated,it is difficult to significantly increase the productive capacity of the fermentation strain by changing one or a few key genes.The traditional physical and chemical mutation breeding techniques is still an important means of breeding in CoQ10 production.In this paper, we use Co60 gamma rays and UV for mutation breeding and select the high yield strains by the high-throughput screening platform. We adjust the fermentation medium with different carbon and nitrogen ratio,choose different precursors in accordance with CoQ10 biosynthesis and metabolic pathways,determine the optimal fermentation precursors ratio by Orthogonal test and validate the optimized conditions using the fermentor. This paper is divided into three parts: The mutation breeding of Rhodobacter.Sphaeoides; The optimization of fermentation conditions in CoQ10 production by Rhodobacter.Sphaeoides;The pilot-scale fermentation tests. The main results of research are as following:We obtain 6 breeding mutant strains using the Co60 gamma rays and UV,select for the ratio of carbon and nitrogen in the fermentation medium and the optimal combination of fermentation precursors and verify that the conditions is effective using the fermentor.1. The mutation breeding of Rhodobacter.Sphaeoides According to the literature,the strength of Co60 irradiation is determined 5Gy. The death rate is more than 95%, consistent with mutagenesis requirements; the strains selected by the high-throughput screening platform were used to UV mutagenesis, and 6 breeding mutant strains were eventually selected out.2. The optimization of fermentation conditions in CoQ10 production by Rhodobacter.SphaeoidesWe adjust the ratio of carbon source in the fermentation medium to improve the coenzyme Q10 production. The precursors conditions was optimized by using the orthogonal experiments and was tested by using fermentor, The optimum condition for the CoQ10 production is:Vitamin B1(VB1) 10 mg/L;sodium acetate 100 mg/L; Hydroxybenzoic acid 20 mg/L; peanut oil 0.01%.The production of CoQ10 was improved from 13.296 mg/L to 31.824 mg/L.3. The pilot-scale fermentation tests.Verify the experimental optimizing results in the shaking flasks by the fermentor, determine the dynamic consumption of carbon sources by detecting the total sugar in the fermentor at different time points and examine the optimum precursors conditions. The results showed that:adding the filtering precursor 24h post-fermentation can improve the CoQ10 production from 84.252 mg/L to129.972 mg/L,while adding the sterilized one can not effectively improve the CoQ10 production, but it can greatly reduce the period of coenzyme Q10 fermentation.The CoQ10 production by strain CU20-9 decreases in the low dissolved oxygen conditions.