Abstract
(+)-Nootkatone is an expensive sesquiterpene substance found in grapefruit peels and the heartwood of yellow cedar. It can be used as a food additive, perfume, and insect repellent; therefore, its highly efficient production is greatly needed. However, the low catalytic efficiency of the membrane-anchored cytochrome P450/P450 reductase system (HPO/AtCPR) is the main challenge and limits the production of (+)-nootkatone. We developed an effective high-throughput screening system based on cell wall destruction to probe the optimal ratio of HPO/AtCPR, which achieved a twofold elevation in (+)-valencene oxidation in Saccharomyces cerevisiae. An engineered strain PK2RI-AtC/Hm6A was constructed to realize de novo (+)-nootkatone production by a series of metabolic engineering strategies. In biphasic fed-batch fermentation, maximum titers of 3.73 and 1.02 g/L for (+)-valencene and (+)-nootkatone, respectively, were achieved. The dramatically improved performance of the constructed S. cerevisiae provides an excellent approach for economical production of (+)-nootkatone from glucose.
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