The use of mixed sugars containing glucose and xylose in lignocellulosic biomass is desirable for the microbial production of chemicals and fuels. We investigated the effect of individual or simultaneous overexpression of glycolytic genes on n-lactate production from a mixture of glucose and xylose by a recombinant xylose-assimilating Corynebacterium glutamicum strain. The individual overexpression of genes encoding phosphofructokinase (PFK) and triosephosphate isomerase (WI) increased D-lactate production rate by 71% and 34%, respectively, with corresponding increases (2.4- and 1.8-fold) in the glucose consumption; however, the amount of xylose consumed not altered. D-Lactate yield was also increased by 5.5%, but only in the strain overexpressing the gene encoding PFK. In the parent strain and the strains overexpressing the genes encoding PFK or WI, a reduction in D-lactate production occurred at approximately 900 mM after 32 h. However, the strain that simultaneously overexpressed the genes encoding PFK and TPI continued to produce n-lactate after 32 h, with the eventual production of 1326 mM after production for 80 h in mineral salts medium. Our findings contribute to the cost-effective, large-scale production of p-lactate from mixed sugars. (C) 2018, The Society for Biotechnology, Japan. All rights reserved.