Pyruvate kinase (PYK) is an important enzyme in the intermediary metabolism and has attracted much attention as a target for metabolic engineering of Corynebacterium glutamicum. Genome sequencing revealed that the 308 residue of PYK was mutated from methionine in model strain C. glutamicum ATCC14067 to isoleucine in L-serine-producing strain C. glutamicum SYPS-062. Consequently, a significantly lower PYK activity (77%) was noted in C. glutamicum SYPS-062, when compared with that in C glutamicum ATCC14067. To confirm the role of this point mutation, pyk in both C. glutamicum SYPS-062 and C. glutamicum SYPS-062-33a Delta SSAA was reversely mutated to restore the PYK enzyme activity, which led to a 33.1% and 28.8% decrease in L-serine titer, respectively. This is the first report to show that the (Met-308 -> Ile) mutation site of pyk is closely associated with its activity and apparently affected L-serine production. Furthermore, pyk was deleted in strain C glutamicum SYPS-062-33a Delta SSAA, and the resulting strain did not show alteration in growth rate and presented a 12% increase in L-serine production. (C) 2017 Elsevier Ltd. All rights reserved.