Engineering Yarrowia lipolytica to produce biodiesel from raw starch

Background: In the last year, the worldwide concern about the abuse of fossil fuels and the seeking for alternatives sources to produce energy have found microbial oils has potential candidates for diesel substitutes. Yarrowia lipolytica has emerged as a paradigm organism for the production of bio-lipids in white biotechnology. It accumulates high amounts of lipids from glucose as sole carbon sources. Nonetheless, to lower the cost of microbial oil production and rival plant-based fuels, the use of raw and waste materials as fermentation substrate is required. Starch is one of the most abundant carbohydrates in nature and it is constituted by glucose monomers. Y. lipolytica lacks the capacity to breakdown this polymer and thus expensive enzymatic and/or physical pre-treatments are needed. Results: In this work, we express heterologous alpha-amylase and glucoamylase enzymes in Y. lipolytica. The modified strains were able to produce and secrete high amounts of active form of both proteins in the culture media. These strains were able to grow on starch as sole carbon source and produce certain amount of lipids. Thereafter, we expressed both enzymes in an engineered strain able to overaccumulate lipids. This strain was able to produce up to 21 % of DCW as fatty acids from soluble starch, 5.7 times more than the modified strain in the wild-type background. Media optimization to increase the C/N ratio to 90 increased total lipid content up to 27 % of DCW. We also tested these strains in industrial raw starch as a proof of concept of the feasibility of the consolidated bioprocess. Lipid production from raw starch was further enhanced by the expression of a second copy of each enzyme. Finally, we determined in silico that the properties of a biodiesel produced by this strain from raw starch would fit the established standards. Conclusions: In this work, we performed a strain engineering approach to obtain a consolidated bioprocess to directly produce biolipids from raw starch. Additionally, we proved that lipid production from starch can be enhanced by both metabolic engineering and culture condition optimization, setting up the basis for further studies.