Construction and optimization of Saccharomyces cerevisiae for synthesizing forskolin

• Published in: Applied microbiology and biotechnology 2022-03, Vol.106 (5-6), p.1933-1944
• Main Authors: Ju, Haiyan, Zhang, Chuanbo, He, Shifan, Nan, Weihua, Lu, Wenyu
• Format: Article
• Language: English
• Subjects: Accumulation; Adaptation; Analysis; Anticancer properties; Antihypertensives; Batch culture; Biomedical and Life Sciences; Bioreactors; Biosynthesis; Biotechnological Products and Process Engineering; Biotechnology; Brewer's yeast; Cell culture; Cytochrome P450; Cytochromes P450; Diterpenes; Endoplasmic reticulum; Fermentation; Flasks; Forskolin; Fungi; Gene regulation; Identification and classification; Life Sciences; Metabolic engineering; Metabolism; Metabolites; Microbial Genetics and Genomics; Microbiology; Optimization; Saccharomyces cerevisiae; Terpenes; Yeast
• ISSN: 0175-7598; 1432-0614
• DOI: 10.1007/s00253-022-11819-z

Forskolin, one of the primary active metabolites of labdane-type diterpenoids, exhibits significant medicinal value, such as anticancer, antiasthmatic, and antihypertensive activities. In this study, we constructed a Saccharomyces cerevisiae cell factory that efficiently produced forskolin. First, a chassis strain that can accumulate 145.8 mg/L 13R-manoyl oxide (13R-MO), the critical precursor of forskolin, was constructed. Then, forskolin was produced by integrating CfCYP76AH15 , CfCYP76AH11 , CfCYP76AH16 , ATR1 , and CfACT1-8 into the 13R-MO chassis with a titer of 76.25 μg/L. We confirmed that cytochrome P450 enzymes (P450s) are the rate-limiting step by detecting intermediate metabolite accumulation. Forskolin production reached 759.42 μg/L by optimizing the adaptations between Cf CYP76AHs, t66 Cf CPR, and t30 Aa CYB5. Moreover, multiple metabolic engineering strategies, including regulation of the target genes’ copy numbers, amplification of the endoplasmic reticulum (ER) area, and cofactor metabolism enhancement, were implemented to enhance the metabolic flow to forskolin from 13R-MO, resulting in a final forskolin yield of 21.47 mg/L in shake flasks and 79.33 mg/L in a 5 L bioreactor. These promising results provide guidance for the synthesis of other natural terpenoids in S. cerevisiae , especially for those containing multiple P450s in their synthetic pathways. Key points • The forskolin biosynthesis pathway was optimized from the perspective of system metabolism for the first time in S. cerevisiae . • The adaptation and optimization of CYP76AHs, t66CfCPR, and t30AaCYB5 promote forskolin accumulation, which can provide a reference for diterpenoids containing complex pathways, especially multiple P450s pathways. • The forskolin titer of 79.33 mg/L is the highest production currently reported and was achieved by fed-batch fermentation in a 5 L bioreactor.