An engineered strain MITXM-61 of Rhodococcus opacus PD630 capable of utilizing xylose was used to produce biofuel precursor-triacylglycerols (TAG) from mixed glucose-xylose substrates. Optimal nitrogen source and carbon source concentrations were investigated for cell growth and microbial lipid production by MITXM-61 in flask cultures. In a two-stage batch culture, the maximum lipid yield (0.152 g TAG per g consumed carbon source) was achieved by utilizing a mixture of glucose and xylose. The fed-batch culture featured intermittent feeding of refined xylose solution during the lipid accumulation stage provided 45 g L-1 of cell dry weight, 54% (g TAG per g cell dry weight) of lipid content, 0.179 g g(-1) (g TAG per g consumed carbon source) of lipid yield, and a 6.9 g L-1 day(-1) of lipid productivity. Fatty acids extracted from microbial lipids produced by MITXM-61 were predominately palmitic and oleic acids, which are the major components in TAG-derived biofuels. (C) 2015 Elsevier Ltd. All rights reserved.