Evolution of a chimeric aspartate kinase for L-lysine production using a synthetic RNA device

Aspartate kinase (AK) is a key enzyme involved in catalyzing the first step of the aspartate-derived amino acid biosynthesis, including L-lysine and L-threonine, which is regulated by the end-metabolites through feedback inhibition. In order to accumulate the end-metabolites in the host, the feedback inhibition of AK has to be released. In this study, a chimeric aspartate kinase, which is composed of the N-terminal catalytic region from Bacillus subtilis AKII and the C-terminal region from Thermus thermophilus , was evolved through random mutagenesis and then screened using a high-throughput synthetic RNA device which comprises of an L-lysine-sensing riboswitch and a selection module. Of three evolved aspartate kinases, the best mutant BT3 showed 160 % increased in vitro activity compared to the wild-type enzyme. Recombinant Escherichia coli harboring BT3 produced 674 mg/L L-lysine in batch cultivation, similar to that produced by the strain harboring the typical commercial widely used feedback resistant aspartate kinase AKC fbr from E. coli . The results suggested that this strategy can be extended for screening of other key enzymes involved in lysine biosynthesis pathways.