Knockout mutation to enhance sophorolipid production in Starmerella bombicola based on computational structural analysis of fatty acid oxidation enzymes

Sophorolipids (SLs), produced by Starmerella bombicola, consist of a hydrophilic sophorose linked to a hydrophobic ω/ω-1 hydroxyl fatty acid. Fatty acid oxidation competes with the synthesis of SL, particularly under conditions of low content of glucose or high accumulation of fatty acid. While many studies have focused on modifying key enzyme genes within the synthesis pathway of SL, research to identify key genes outside of this pathway, such as those involved in the β- and ω-oxidation of fatty acids, remains limited. This study aimed to prevent ω hydroxyl fatty acids from entering β- and ω-oxidation pathways, thus directing them toward SL synthesis. Key enzymes, SbMFE-2, SbADH1, and SbADH2, were selected for investigation. Gene knockout mutants revealed that the ΔSbADH1 mutant significantly increased SL yield when linoleic acid and oleic acid (C18) was used as the lipid source. No significant differences were observed with lauric acid (C12) as the lipid source. Additionally, computational structural analyses using AlphaFold 3, docking, and molecular dynamics simulations provided insights into the enzyme functions and their substrate preferences, offering a mechanistic explanation for the enhanced SL production in the ΔSbADH1 mutant.