초록
<P><B>Abstract</B></P> <P>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 <I>Saccharomyces cerevisiae</I> 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 <I>ERG9</I> with the <I>MET3</I> 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/OD<SUB>600</SUB>. Here, we describe a systematic synthetic biology method to produce 13R-MO from a simple carbon source using <I>Saccharomyces cerevisiae</I>. This not only enabled a relatively high level of 13R-MO production but also provided an efficient platform for the production of other diterpenoids.</P> <P><B>Highlights</B></P> <P> <UL> <LI> Heterologous biosynthesis of 13R-manoyl oxide in <I>Saccharomyces cerevisiae</I> was achieved. </LI> <LI> Production titer was stepwise increased by optimizing entire MVA pathway. </LI> <LI> Positive and negative fusion of genes to explore the influence on production of 13R-manoyl oxide. </LI> <LI> Three feed strategies were investigated to enhance titer of 13R-manoyl oxide. </LI> </UL> </P> <P><B>Graphical abstract</B></P> <P>[DISPLAY OMISSION]</P>