caa a22 4500 605499624 CHVBK 20210128100552.0 cr unu---uuuuu 210128e20151001xx s 000 0 eng 10.1007/s00253-015-6615-0 doi (NATIONALLICENCE)springer-10.1007/s00253-015-6615-0 Evolution of a chimeric aspartate kinase for L-lysine production using a synthetic RNA device [Elektronische Daten] [Junming Wang, Dongfang Gao, Xiaoli Yu, Wen Li, Qingsheng Qi] 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 674mg/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. Springer-Verlag Berlin Heidelberg, 2015 Riboswitch nationallicence L-lysine nationallicence Evolution nationallicence Aspartate kinase nationallicence Escherichia coli nationallicence Wang Junming State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, 250100, Jinan, People's Republic of China aut Gao Dongfang State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, 250100, Jinan, People's Republic of China aut Yu Xiaoli State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, 250100, Jinan, People's Republic of China aut Li Wen State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, 250100, Jinan, People's Republic of China aut Qi Qingsheng State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, 250100, Jinan, People's Republic of China aut Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/20(2015-10-01), 8527-8536 0175-7598 99:20<8527 2015 99 253 https://doi.org/10.1007/s00253-015-6615-0 text/html Onlinezugriff via DOI BK010053 XK010053 XK010000 Metadata rights reserved Springer special CC-BY-NC licence nationallicence 1 research-article jats NATIONALLICENCE NATIONALLICENCE NL-springer NATIONALLICENCE 856 40 https://doi.org/10.1007/s00253-015-6615-0 text/html Onlinezugriff via DOI NATIONALLICENCE 700 1- Wang Junming State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, 250100, Jinan, People's Republic of China aut NATIONALLICENCE 700 1- Gao Dongfang State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, 250100, Jinan, People's Republic of China aut NATIONALLICENCE 700 1- Yu Xiaoli State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, 250100, Jinan, People's Republic of China aut NATIONALLICENCE 700 1- Li Wen State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, 250100, Jinan, People's Republic of China aut NATIONALLICENCE 700 1- Qi Qingsheng State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, 250100, Jinan, People's Republic of China aut NATIONALLICENCE 773 0- Applied Microbiology and Biotechnology Springer Berlin Heidelberg 99/20(2015-10-01), 8527-8536 0175-7598 99:20<8527 2015 99 253