Stepwise increase in the production of 13R-manoyl oxide through metabolic engineering of Saccharomyces cerevisiae

Abstract

Forskolin, a labdane diterpenoid, possesses a wide range of pharmacological activities. (13R)-manoyl oxide (13R-MO) is the precursor of forskolin. As forskolin is a structurally complex, highly oxidized compound, chemical synthesis is tedious and difficult. Herein, we present a biosynthesis method involving metabolic engineering of Saccharomyces cerevisiae to produce 13R-MO at an initial titer of 2.3 mg/l. We further optimized the entire MVA pathway, which increased the FPP supply pool, but resulted in a sharp decrease in the 13R-MO production due to low metabolic flux toward geran ylgeranyl pyrophosphate (GGPP). To mitigate this, we down-regulated the competing pathway by replacing the original promoter of the squalene synthase gene ERG9 with the MET3 promoter, significantly improving the 13R-MO production to 45.2 mg/l. Finally, three feed strategies were investigated in fed-batch fermentation; the glucose feeding strategy enabled the engineered yeast to produce 167.1 ± 5.2 mg/l 13R-MO, equivalent to 2.6 ± 0.08 mg/l/OD600. Here, we describe a systematic synthetic biology method to produce 13R-MO from a simple carbon source using Saccharomyces cerevisiae. This not only enabled a relatively high level of 13R-MO production but also provided an efficient platform for the production of other diterpenoids.