Production of sesquiterpenoid zerumbone from metabolic engineered Saccharomyces cerevisiae

Zerumbone, the predominant sesquiterpenoid component of Zingiber zerumbet, exhibits diverse pharmacological properties. In this study, de novo production of zerumbone was achieved in a metabolically engineered yeast cell factory by introducing α-humulene synthase (ZSS1), α-humulene 8-hydroxylase (CYP71BA1) and zerumbone synthase variant (ZSD1^S114A) from Z. zerumbet, together with AtCPR1 from Arabidopsis thaliana into the yeast strain. Multistep metabolic engineering strategies were applied, including the over-expression of the mevalonate (MVA) pathway rate-limiting enzymes tHMG1 and ERG20, regulation of ERG9 by an inducible promoter and competitive pathway deletion to redirect metabolic flux toward the desired product. In the engineered strain, α-humulene production increased by 18-fold, to 92 mg/L compared to that in the original strain. Five cytochrome P450 reductases (CPRs) from different sources were selected for CYP71BA1 adaptability tests, and AtCPR1 from A. thaliana was found to be the optimal, producing 113.16 μg/L of 8-hydroxy-α-humulene. Multicopy integration of CYP71BA1, AtCPR1, ZSS1 and ICE2 (type III membrane protein) genes resulting in strain LW14 increased the production of 8-hydroxy-α-humulene by 134-fold to 15.2 mg/L. Expressing ZSD1^S114A in the ura3 site of strain LW14 resulted in the production of 7 mg/L zerumbone. Multicopy integration of ZSD1^S114A increased the production of zerumbone to 20.6 mg/L. The high zerumbone-producing strain was used for batch and fed-batch fermentation in a 5-L bioreactor and zerumbone degradation by yeast was observed; the production of zerumbone finally reached 40 mg/L by fed-batch fermentation in a 5-L bioreactor.