利用基因组改组技术提高辅酶Q_10产量

Genome Shuffling of Rhodobacter Sphaeroides to Improve Coenzyme Q_10 Production

辅酶Q10(CoQ10)是生物细胞呼吸链中的重要递氢体,已广泛应用于心脏病、肝炎、帕金森症等多种疾病的治疗中。为了提高微生物法生产CoQ10的产量,本文利用基因组改组技术选育类球红细菌辅酶Q10高产菌株。根据CoQ10的合成途径及作用机理,确定了不同的抗性筛选标记物:罗红霉素、卡那霉素、对羟基苯甲酸、维生素K3和硫化钠。根据类球红细菌对标记物的耐受性确定了抗性筛选浓度。通过紫外线、紫外线/氯化锂、硫酸二乙酯、微波及钴605种诱变方式以及抗性培养基筛选获得了9株改良的突变株作为出发菌株。通过一轮基因组改组得到了几株高产菌株,其中PN13产CoQ10的量可达到2.39mg/g,是原菌的2.52倍。

Coenzyme Q10 （CoQ10） is an important electron transfer molecule in the respiratory chain and the indispensable coenzyme in the production of ATP, and it is widely used in therapeutic applications for several diseases such as heart diseases, hepatitis, Parkinson and so on. In order to improve the production of CoQ10, this paper studies the screening of the mutant Rhodobacter sphaeroides aiming for a higher CoQ10 production by genome shuffling. According to the biosynthetic pathway and its mechanism, various resistance markers are selected, including the roxithromycin, the kanamycin, the p-hydroxy benzcic acid, the vitamin K3 and sodium sulfide （Na2S）. Their concentrations are determined according to the tolerance experiments on Rhodobacter sphaeroides. The improved starting population including nine mutant strains is generated by different resistance markers and different mutagenesis ways, Such as the ultraviolet irradiation, the ultraviolet/lithium chloride, the diethyl sulfate, the microwave radiation and the 7 Co60. Several high CoQ10-producing colonies, including PN13, are selected from the first shuffled library. The CoQ10 content of the PNI3 reaches 2.39mg/g, 2.52 times of that of the wild-type strain.