A common strategy of metabolic engineering is to increase the endogenous supply of precursor metabolites 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 highlevel levopimaradiene production in Escherichia coli. To increase levopimaradiene synthesis, we amplified the flux toward isopentenyl diphosphate and dimethylallyl diphosphate precursors and reprogrammed the rate-limiting downstream pathway by generating combinatorial mutations in geranylgeranyl diphosphate synthase and levopimaradiene synthase. The mutant library contained pathway variants that not only increased diterpenoid production but also tuned the selectivity toward levopimaradiene. The most productive pathway, combining precursor flux amplification and mutant synthases, conferred approximately 2,600-fold increase in levopimaradiene levels. A maximum titer of approximately 700 mg∕L was subsequently obtained by cultivation in a benchscale bioreactor. 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.GGPP synthase | levopimaradiene synthase | metabolic engineering | Escherichia coli | molecular reprogramming M etabolic engineering is the enabling technology for the manipulation of organisms to synthesize high-value compounds of both natural and heterologous origin (1-4). In the case of heterologous production, well-characterized microorganisms are used as production hosts because targeted optimization can be performed using widely available genetic tools and synthetic biology frameworks (5, 6). One important application of engineered microbial systems is geared toward the synthesis of terpenoid natural products (7-9). Terpenoids represent one of the largest classes of secondary metabolites that includes pharmaceuticals, cosmetics, and potential biofuels candidates (8,(10)(11)(12). Metabolic engineering approaches to produce terpenoids in microbial systems such as Escherichia coli and yeast have commonly focused on increasing the precursor flux into the heterologous terpenoid pathway by rerouting endogenous isoprenoid metabolism (13-15). These engineering strategies have relied heavily on changing the enzyme concentrations in the product pathway.Many properties of a metabolic pathway, however, are not limited solely by the enzyme concentration, as is particularly true for the terpenoid pathway. In nature, terpenoid biosynthesis is regulated at multiple metabolic branch points to create large structural and functional diversity (16-18). In the major metabolic branch point in terpenoid biosynthesis, the prenyl transferases and terpenoid synthases catalyze the formation of a wide range of structurally diverse acyclic and cyclic terp...

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