Engineering Yarrowia lipolytica for high-level ergosterol production

Ergosterol is the key precursor of steroid drug synthesis. In this experiment, we systematically modified the synthesis of ergosterol. Firstly, we identified key rate-limiting enzymes through systematic screening of the post-squalene pathway. Combinatorial overexpression of IDI1 , tHMG1 , ERG4 , ERG5 , ERG27 , ERG1 and ERG11 achieved an ergosterol titer of 94.2 mg/L. Molecular dynamics guided mutagenesis of key substrate channel residues, particularly S372V Erg11, enhanced local flexibility and significantly increased ergosterol production. Introduction of the proton-donating mutations S372V-T305H- ERG11 established an artificial proton-dependent pathway, which, together with channel engineering, further increased the titer to 124 mg/L. Lipid droplet engineering and cellular compartmentalization strategies increased the titer to 148.3 mg/L. Ultimately, multi-copy integration of all ergosterol synthesis pathway genes increased the titer to 433.1 mg/L, and fed-batch fermentation in a 5-L bioreactor resulted in a final titer of 4.58 g/L. This study demonstrates a comprehensive hierarchical strategy for high-level sterol production.