A Model For Gene Local Shuffling And Its Application In UbiA Mutation

This study is a basic work for the construction of genetic engineering Escherichia coli which can overproduce CoQ 10.CoQ, a transhydrogen in the mitochondrial respiratory chain of organisms, has a great value of research in both application and academic sector. UbiA, which catalyzes the prenylation of 4-hydroxybenzoate in Escherichia coli, is the rate-limiting enzyme in the CoQ biosynthesis progress. In previous work, ubiA gene was disrupted on Escherichia coli MC4100 genome. Based on the character that Escherichia coli could not live under high temperature without UbiA, a screening model of active ubiA mutants has been set up and DNA shuffling condition of ubiA full sequence was established. In this work, a shuffling model of ubiA putative active site is set up and factors of the whole process of this model are optimized. On the other hand, reflux time of saponification is also been optimized. There is an obvious decrease of relative error of the CoQ analysis result. UbiA function and the yield of CoQ of shuffled ubiA genes are assayed.After two cycle of screening, nine active mutants which contain eight type of sequence are obtained. Two of them cause decrease of the yield of CoQ, three cause increase, others do not cause obvious change.