The use of biodiesel-derived glycerol as a carbon source for single cell oil (SCO) production is a biorefinery engineering strategy that aims to valorize the by-product waste and make the microbial lipid production process more cost-effective. This work aimed to improve the capacity of Yarrowia lipolytica to produce large amounts of lipids and replace genetic engineering by a metabolic approach based on the stimulation of the rate-limiting enzymes and reducing the activation energy thereby increasing the rate of lipids synthesis. The effects of biotin and leucine addition on the lipid content of Y. lipolytica have been investigated. The lipid content of Y. lipolytica was strongly influenced by the addition of biotin. In fact, an increase in biotin concentration from 25 μg/L to 200 μg/L practically increased the lipid concentration up to 15 g/L. Besides, to channel metabolic flux into lipid biosynthesis, the addition of citric acid as lipid precursor led to an increase in total catabolism activation and lipid accumulation to reach around 63% (w/w). The biochemical approach can be a useful target for improving the efficiency of lipid-producing yeast strain rather than genetic engineering.