Research Projects

Every year, food industries and households generate approximately 1200 kilotons of waste oil in India, causing severe environmental pollution due to improper disposal. These significant low-value carbon resources must be turned into high-value profitable products. Therefore, various technological improvements are required to add value to waste oil to increase sustainability and reduce pollution. Though many chemical industries are making secondary products to mitigate waste oils, it is high time to develop modern technologies to ease waste oil management. In the proposed objectives, we intend to implement synthetic biology strategies to convert waste oil into natural triterpenes using a non-conventional and oleaginous yeast, Yarrowia lipolytica (Yl), as a microbial platform. Our metabolic engineering strategies involve an effective waste oil uptake mechanism (Fatty acid-pull strategy) and its maximum utilization by peroxisomes followed by lipid pathway engineering for expanded endoplasmic reticulum biogenesis (Fatty Acid-Push strategy), leading to elevated mevalonate-ergosterol (MEV-ERG) pathway. Peroxisomes produce large acetyl-CoA flux through beta-oxidation, a primary precursor of the MEV-ERG pathway, and expanded endoplasmic reticulum provides larger surface area for maximal activity of the MEV-ERG pathway. This pathway provides significant amounts of intermediate metabolites, such as squalene and 2,3 oxido-squalene, for triterpene biosynthesis. The final strategy introduces plant triterpene biosynthetic genes to produce a triterpene compound, Betulinic Acid (Hereafter BA). BA is a natural pentacyclic triterpene with high medicinal properties and significant demand from the pharmaceutical sector. The proposed strategy enables low-value carbon resources (i.e., waste oil) to be converted into high-value BA. The technologies generated from this project proposal will enable a ‘waste to wealth’ approach by converting waste oil into high-value triterpenoids. The work will not only be a viable solution to control environmental problems caused by improper disposal of waste oil but will also uncover new knowledge about the fatty acid transport mechanism in Yl, and its exploitation for metabolic engineering strategies. The proposed strategies involve the construction of four genetic circuits which drive 1) high fatty acid mobilization from waste oil into the cytosol, followed by 2) maximal uptake of high fatty acid flux from the cytosol to peroxisomes, where fatty acids are oxidized to produce high acetyl-CoAs flux through enhanced beta-oxidation pathway; 3) simultaneously blocking neutral lipid pathway and diverting excess fatty acid to endoplasmic reticulum proliferation and phospholipid signalling for elevated endogenous MEV-ERG pathway activity; 4) overexpressing betulinic acid biosynthetic genes in engineered Yarrowia lipolytica platform strain. Finally, the engineered strain will be optimized for high BA production.