Engineering Saccharomyces cerevisiae for production of 2(or 5)-ethyl-5(or 2)-methyl-4-hydroxy-3(2H)-furanone by screening and remolding of enone oxidoreductase

HEMF（4-Hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone）is widely used as a food flavoring agent due to its unique fragrance and is in great demand. The purpose of this study is to combine enzyme remodeling and metabolic engineering methods for constructing a HEMF yield yeast strain. First, five enone oxidoreductase were selected with high sequence similarity from NCBI database and an enone oxidoreductase derived from Naumovozyma dairenensis CBS 421, named NDEO, produced higher HEMF level compared to other source of enzymes. Then, the mutant NDEOT183W, K290W was obtained by semi-rational protein engineering, which showed 75.2% increase in HEMF productivity in vitro reaction compared with NDEO. Finally, the NDEOT183W, K290W was integrated into the strain optimized for precursor supply; combined with the optimization of fermentation medium and fermentation conditions, the HEMF yield of the strain BM4 reached 103.60 mg L-1. Our study not only improved HEMF production in yeast, but also provided an effective method to improve activities of enone oxidoreductase. This work has great potential in the industrial synthesis of furanone.