Genetic Engineering System And Metabolic Engineer Manipulation Of Coenzyme Q10 In Rhodobacter Sphaeroides

Coenzyme Q10 (CoQ10), an obligatory cofactor in the aerobic respiratory electron transfer or energy generation, is formed from the conjugation of a benzoquinone ring with a hydrophobic isoprenoid chain. CoQ10 is now used as a nutritional supplement because of its antioxidant properties and is beneficial in the treatment of several human diseases when administered orally。 It also protects the tissues and organs in the body by scavenging free radicals directly. Expanding its application in food, pharmaceutical and cosmetic industries, researchers and scientists pay great attention to improving its production. The companies from domestic and overseas closely concern on expanding commercial development. There are several methods of CoQ10 production. The most widely used technique is fermentation by microorganisms. Techniques of traditional breeding and fermentation optimization are in development bottlenect. In order to making industrial production of CoQ10, how to obtain a high-yield strain is the primary mission. A natural CoQ10 producer, Rhodobacter sphaeroides, was used as the only subject in the study. To improving its production of CoQ10 was the main part. This study has the following parts.Trying to manipulate Rhodobacter sphaeroides, the engineering system should be build. The broad host range plasmid pBBR1MCS-2 and its recombinant plasmid pBBRlMCS-2RFP were used. The density rate between the donor E.coli S17-1 and receptor of Rhodobacter sphaeroides was determined, compared the tolerant difference about K2TeO3 and nalidixic acid of the strains. All in all, an efficient and accurate conjugation and screening system of Rhodobacter sphaeroides was obtained.Six recombinant plasmid, pBBRlMCS2-tacGFP, pBBR1MCS2-119GFP, pBBR1MCS2-badGFP, PBBR1MCS2-cpa3GFP, pBBR1MCS2-cpa1GFP, pBBR1MCS2-wpa1GFP were constructed. Transfer to the host strain, Fluorospectrophotometer and FCM tested the fluorescence degree of every strain. The result indirect compared the intensity of six promoters. The order was tac,119, bad, cpa3, cpa1, wpa1.Three relatively strong promoters and one self-promoter, constructing the plasmid of pBBR1MCS2-tac-ddsa, pBBRlMCS2-cpa3-ddsa, pBBR1MCS2-119-ddsa, Gbbr1-ddsa were selected. Then transfer them to Rhodobacter sphaeroides, testing them with HPLC, the result showed that the host cell with Gbbrl-ddsa, the yield of C0Q10 reached 220.6 mg/L improving about 15.7%.Refer to the patent of DuPont company, one critical ORF of pBBR1MCS-2, known as rep, is involved in replication of plasmid. Temperature sensitive mutants was generated by random mutagenesis followed by screening to obtain mutants with temperature sensitive phenotype. One mutantions MpBBR1MCS-2#31 was selected as temperature sensitive plasmid.Directed evolution, is a rapid method to making gene mutation. Then adding appropriate screening agents, we can choose the ideal strain, which has the specific properties that we expect. Rhodobacter sphaeroides mutated by UV and ARTP, adding high concentration of analogues and respiratory inhibitors. Finally, production of CoQ10 in two mutational strains improving about 10.3%,12.5%, compared with the wide strain.