Probing the Synergistic Ratio of P450/CPR To Improve (+)-Nootkatone Production in Saccharomyces cerevisiae

Authors：Cha, Yaping; Li, Wen; Wu, Tao; You, Xia; Chen, Hefeng; Zhu, Chaoyi; Zhuo, Min; Chen, Bo^*; Li, Shuang^*

Source：Journal of Agricultural and Food Chemistry, 2022, 70(3): 815-825.

DOI：10.1021/acs.jafc.1c07035

• (+)-nootkatone
• (+)-valencene
• HPO/AtCPR
• high-throughput screening
• metabolic engineering

(+)-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.