Glycerol generated during renewable fuel production processes is potentially an attractive substrate for the production of value-added materials by fermentation. An engineered strain MITXM-61 of the oleaginous bacterium Rhodococcus opacus produces large amounts of intracellular triacylglycerols (TAGs) for lipid-based biofuels on high concentrations of glucose and xylose. However, on glycerol medium, MITXM-61 does not produce TAGs and grows poorly. The aim of the present work was to construct a TAG-producing R. opacus strain capable of high-cell-density cultivation at high glycerol concentrations.