Sustainable biosynthesis of squalene from waste cooking oil by the yeast Yarrowia lipolytica.

ABSTRACT

Squalene is a highly sought-after triterpene compound in growing demand, and its production offers a promising avenue for circular economy practices. In this study, we applied metabolic engineering principles to enhance squalene production in the nonconventional yeast Yarrowia lipolytica, using waste cooking oil as a substrate. By overexpressing key enzymes in the mevalonate pathway - specifically ERG9 encoding squalene synthase, ERG20 encoding farnesyl diphosphate synthase, and HMGR encoding hydroxy-methyl-glutaryl-CoA reductase - we achieved a yield of 779.9 mg/L of squalene. Further co-overexpression of DGA1, encoding diacylglycerol acyltransferase, and CAT2, encoding carnitine acetyltransferase, in combination with prior metabolic enhancements, boosted squalene production to 1381.4 mg/L in the engineered strain Po1g17. To enhance the supply of the precursor acetyl-CoA and inhibit downstream squalene conversion, we supplemented with 6 g/L pyruvic acid and 0.7 mg/L terbinafine, resulting in an overall squalene titer of 2594.1 mg/L. These advancements underscore the potential for sustainable, large-scale squalene production using Y. lipolytica cell factories, contributing to circular economy initiatives by valorizing waste materials.