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

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 downregulated 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.