Application of a model for gene local shuffling in ubiA mutation

Abstract:

Abstract: As a transhydrogen in the mitochondrial respiratory chain, Coenzyme Q (CoQ) has a critical role in the metabolism. 4-hydroxybenzoate polyprenyltransferase (UbiA), which coded by ubiA gene, is the rate-limiting enzyme in the CoQ biosynthesis progress. However, the relationship between structure and function remains unclear. [Objective] To set up a synthetic oligonucleotide-based shuffling model and apply in the random mutation of DNA sequence of ubiA gene which codes putative active site. [Methods] By using ubiA knockout mutant of E. coli. MC4100 as receptor, we set up a local Shuffling model by replacing target sequence with a random sequence fragment. Sequences of mutants and their function were analyzed. [Results] After local Shuffling and two cycle of screening, we gained seven mutants. Compared to the wild type, most of the mutant amino sequences showed obvious change, and had different change trend of CoQ production. Sequencing result showed that three aspartic acid sites may be highly related to UbiA catalyze activity. [Conclusion] The local Shuffling model we set up is feasible. By applying this model, we preliminary verified several location of key amino acid sites of UbiA.