title: Characterization of aspartate kinase and homoserine dehydrogenase from Corynebacterium glutamicum IWJ001 and systematic investigation of L-isoleucine biosynthesis.
abstract: Previously we have characterized a threonine dehydratase mutant TD(F383V) (encoded by ilvA1) and an acetohydroxy acid synthase mutant AHAS(P176S, D426E, L575W) (encoded by ilvBN1) in Corynebacterium glutamicum IWJ001, one of the best L-isoleucine producing strains. Here, we further characterized an aspartate kinase mutant AK(A279T) (encoded by lysC1) and a homoserine dehydrogenase mutant HD(G378S) (encoded by hom1) in IWJ001, and analyzed the consequences of all these mutant enzymes on amino acids production in the wild type background. In vitro enzyme tests confirmed that AK(A279T) is completely resistant to feed-back inhibition by L-threonine and L-lysine, and that HD(G378S) is partially resistant to L-threonine with the half maximal inhibitory concentration between 12 and 14 mM. In C. glutamicum ATCC13869, expressing lysC1 alone led to exclusive L-lysine accumulation, co-expressing hom1 and thrB1 with lysC1 shifted partial carbon flux from L-lysine (decreased by 50.1 %) to L-threonine (4.85 g/L) with minor L-isoleucine and no L-homoserine accumulation, further co-expressing ilvA1 completely depleted L-threonine and strongly shifted carbon flux from L-lysine (decreased by 83.0 %) to L-isoleucine (3.53 g/L). The results demonstrated the strongly feed-back resistant TD(F383V) might be the main driving force for L-isoleucine over-synthesis in this case, and the partially feed-back resistant HD(G378S) might prevent the accumulation of toxic intermediates. Information exploited from such mutation-bred production strain would be useful for metabolic engineering.
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China.|Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, China.
authors.forename: Xunyan
authors.initials: X
authors.lastname: Dong
authors.affiliation: Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, China.
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi, 214122, China.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, China.
authors.forename: Jianxun
authors.initials: J
authors.lastname: Zhao
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi, 214122, China. xiaoyuanwang@hotmail.com.|Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, China. xiaoyuanwang@hotmail.com.
authors.forename: Xiaoyuan
authors.initials: X
authors.lastname: Wang
mesh_terms: D000596:Amino Acids / Q000378:metabolism; D001222:Aspartate Kinase / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D048230:Corynebacterium glutamicum / Q000201:enzymology* / Q000235:genetics; D003470:Culture Media / Q000737:chemistry; D053938:DNA Fragmentation; D005285:Fermentation; D006715:Homoserine Dehydrogenase / Q000378:metabolism*; D007218:Industrial Microbiology; D007532:Isoleucine / Q000096:biosynthesis*; D008239:Lysine / Q000378:metabolism; D060847:Metabolic Engineering; D013912:Threonine / Q000096:biosynthesis
chemical_list: D000596:Amino Acids; D001426:Bacterial Proteins; D003470:Culture Media; D007532:Isoleucine; D013912:Threonine; D006715:Homoserine Dehydrogenase; D001222:Aspartate Kinase; D008239:Lysine
keywords: Aspartate kinase; Corynebacterium glutamicum; Feed-back inhibition; Homoserine dehydrogenase; L-Isoleucine biosynthesis; Metabolic engineering