Probing the synergistic ratio of HPO/AtCPR to improve (+)-nootkatone production in Saccharomyces cerevisiae

(+)-Nootkatone is one type of expensive sesquiterpene aroma found in grapefruit peel and heartwood of yellow cedar. It could be used as food additives, perfume, insect repellent and therefor its highly efficient production is greatly requested. However, the low catalysis efficiency of membrane-anchored cytochrome P450/P450 reductase system (HPO/AtCPR) was the main challenge and limited the production of (+)-nootkatone. We developed an effective high-throughput screening system based on the cell-wall destruction to probe the optimal ratio of HPO/AtCPR, which achieved a 2-fold elevation in (+)-valencene oxidation in S. cerevisiae . In combination with the introduction of valencene synthase from Callitropsis nootkatensis and alcohol dehydrogenase form Pichia pastoris , an engineered strain was constructed to realize de novo (+)-nootkatone production. Then a series of engineering strategies, including knockout of the transcription factor (ROX1), down-regulating the squalene synthase enzyme (EGR9), overexpression of a truncated version of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (tHMG1) and type III membrane protein (ICE2), were applied to generate PK2RI-AtC/H m6 A. In bi-phasic fed-batch fermentation, a maximum yield of 3.73 and 1.02 g/L for valencene and (+)-nootkatone were achieved. The dramatically improved performance of the constructed S. cerevisiae provides an excellent approach for economical production of (+)-nootkatone from glucose.