A Preliminary Study On The Production Of Coenzyme Q10 Through Fermentation Process By Photosynthetic Bacteria

Background: Photosynthetic bacteria(PSB), with high protein content, good amounts of CoQ10 and vitamins, can be used as an animal feed additive, characterized enhancing the livability, immunity and growth rate of livestock. In the meanwhile, extensive attention has been attracted on exploitation and utilization of CoQ10 as its excellent therapeutic effects and health protection. As a natural health product or medicine, it has been widely used to improve the immunity of human or animal. However, the content of CoQ10 in wild strains of PSB is limited after all. Through construction of recombinant strain producing CoQ10 and optimization of the culture medium and fermentation conditions of a selected strain, the content of CoQ10 in PSB will be Increased markedly. It can not only remarkably strengthen the effect of PSB feed additive, but also lay foundation for the exploitation of CoQ10.It was confirmed that the key enzyme of CoQ10 synthesis is p-hydroxybenzoate polyprenyltransferase, encoded by gene ubiA in E. coli and lacks specificity to the aggregation length of its substrate (polyisoprenyl pyrophosphate). If gene ubiA can be introduced into PSB and is high expression in PSB, a recombinant strain producing CoQ10 could be obtained.Consequently, the productive cost will decrease sharply.Object: The culture medium and culture conditions of PSB will be optimized and gene ubiA will be cloned for the construction of recombinant strain producing and foundation of the fermentative technology in large scale.Methods: The productive strain was screened according to the content ofCoQio which was extracted by saponification method from the biomass of PSB. Uniform design was employed to optimize the culture conditions and the component of culture medium including carbon source, nitrogen source, phosphor source, growth factors, inorganic salts and precusor as well. The experimental data was analyzed by the uniform design software package. Designing a specific primer pair based on the sequence of the gene ubiA in E.coli MC4100, a DNA fragment was amplified from the genomic DNA of E.coli MC4100 by PCR and then was cloned into pUCm-T Vector. The cloned fragment was sequenced.Results: (1) R.capsulatus MT1131 was determined as the productive strain in this study. (2) A rapid mensuration of the content of CoQio in microbial cells has been set up.(3) For maximal CoQio production by R.capsulatus MT1131, an optimal culture medium in the presence of 3.13 g/1 yeast extract, 0.8 g/1 (NH4)2SO4, 0.64 g/1 Mg2+, 45.2 mg/1 Fe2+, 18 mg/1 Mn2*, 16 mg/1 Co2+ was required. When the strain grew in the optimal culture under conditions of initial pH 7.0 , 30癈, 2000Lx for 4 days, the content of CoQio in R.capsulatus MT1131 cells rise from 15.213mg/l to 20.365mg/l and increased by 33.87% compared with data before. (4) The gene ubiA was amplified from the genomic DNA of E.coli MC4100 by PCR. The DNA sequence of the amplified fragment is identical with the published sequence of gene ubiA in GenBank with BLAST, apart from that the 1,007th base was mutated from T to C.Conclusions: A rapid mensuration of the content of CoQio in microbial cells has been set up in this study. The culture medium and culture conditions had been optimized successfully through uniform design method for maximal CoQio produced by R.capsulatus MT1131. The gene ubiA was successfully cloned in this study. It lays a foundation for construction of genetic engineering bacteria producing CoQio.