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The authors discuss occurrence of heterotrophic microbes, specifically bacteria, yeast, and molds, in biodiesel production is well discussed. Besides, these oleaginous microorganisms are reported to accumulate triacylglycerols (TAGs) that contain long-chain fatty acids. Most of the evidence suggests that TAGs accumulated are suitable for biodiesel production. Lipid accumulation (de novo or ex novo) required a set of enzymes and expression of various genes that are under complex regulatory system. The authors take a step in this direction and provides a summary of the actual knowledge on heterotrophic oleaginous species, their fatty acids, TAGs, and sterol biosynthesis with a biochemical and genetic approaches to better understand their regulation. Likewise, the impact of abiotic factors that can contribute to higher lipid accumulation is summarized. This review also highlights various issues concerning genetic engineering that may contribute to an advanced path forward for microbial feedstock-based biodiesel.