Engineering Terpenoid Biosynthetic Pathway for Overproduction and Selectivity Control

A common strategy of metabolic engineering is to increase the endogenous supply of precursor metabolites in order to improve pathway productivity. The ability to further enhance heterologous production of a desired compound may be limited by the inherent capacity of the imported pathway to accommodate high precursor supply. Here, we present engineered diterpenoid biosynthesis as a case where insufficient downstream pathway capacity limits high level levopimaradiene production in Escherichia coli. To increase levopimaradiene synthesis, we amplified the flux towards isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) precursors and reprogrammed the rate-limiting downstream pathway by generating combinatorial mutations in geranylgeranyl diphosphate synthase (GGPPS) and levopimaradiene synthase (LPS). The mutant library contained pathway variants that not only increased diterpenoid production but also tuned the selectivity towards levopimaradiene. The most productive pathway, combining precursor flux amplification and mutant synthases, conferred >2,600-fold increase in levopimaradiene levels, corresponding to a titer of ~700 mg/L in bench-scale fermentation. The present study highlights the importance of engineering proteins along with pathways as a key strategy in achieving microbial biosynthesis and overproduction of pharmaceutical and chemical products.