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.
State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, 250100, People's Republic of China.
Junming
J
Wang
State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, 250100, People's Republic of China.
Dongfang
D
Gao
State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, 250100, People's Republic of China.
Xiaoli
X
Yu
State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, 250100, People's Republic of China.
Wen
W
Li
State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan, 250100, People's Republic of China. qiqingsheng@sdu.edu.cn.
Qingsheng
Q
Qi
D001222:Aspartate Kinase / Q000235:genetics* / Q000378:metabolism*; D001412:Bacillus subtilis / Q000201:enzymology* / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005820:Genetic Testing / Q000379:methods; D008239:Lysine / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics* / Q000378:metabolism*; D016963:Thermus thermophilus / Q000201:enzymology* / Q000235:genetics
D011993:Recombinant Fusion Proteins; D001222:Aspartate Kinase; D008239:Lysine
Aspartate kinase; Escherichia coli; Evolution; L-lysine; Riboswitch