Metabolic Engineering of <i>Saccharomyces cerevisiae</i> for High Bioproduction of (+)-Nootkatone

(+)-Nootkatone, a high-value sesquiterpenoid compound, has found great applications in food, agriculture, and pharmaceutical fields. The biosynthesis of (+)-nootkatone is considered as a green and sustainable synthetic method but is mainly challenged by the significant accumulation of the precursor (+)-valencene during the fermentation process. In the present study, the genes CnVS, HPOV480A/V482A, AtCPR1, and ZSD1 were integrated into the genome of strain SQ1 and the original promoter of ERG9 was replaced by PHXT1 for constructing the (+)-nootkatone biosynthetic pathway and inhibiting the competitive ergosterol synthesis pathway, respectively. Subsequently, the flexible linkers and promoters of ERG20 and CnVS were screened and optimized to reduce the shuttling distance of the substrate between the enzymes. Further, ERG20-(GSG)2-CnVS, HPOV480A/V482A, AtCPR1, and ZSD1 were overexpressed, resulting in a titer of 244.1 mg/L (+)-nootkatone. The signal peptide of HPOV480A/V482A was rationally engineered to improve the titer of (+)-nootkatone to 466.1 mg/L, while the titer of valencene was reduced to 29.3 mg/L. Molecular dynamics simulations showed more stable binding between HPOV480A/V482A and the substrate after modifying the signal peptide and thus enhanced enzyme activity. Finally, the maximum (+)-nootkatone titer of 6.5 g/L was obtained in a 5 L bioreactor. This is the highest (+)-nootkatone titer reported to date. Thus, the present study provides a solid foundation for the future large-scale production of (+)-nootkatone.