Combining metabolic and protein engineering of a terpenoid biosynthetic pathway for overproduction and selectivity control

Abstract

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 high-level 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 bench-scale 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.

Fig. 1.

(A) Engineering levopimaradiene synthesis in E. coli. A plant-derived pathway was constructed by introducing T. chinensis ggpps and G. biloba lps codon-optimized genes. To amplify the endogenous precursor pools of GGPPS substrates (IPP and DMAPP), copy numbers of rate-limiting steps (dxs, idi, ispD, ispF) in the MEP pathway were amplified by additional episomal expression. (B) General reaction mechanism of “LPS-type” enzymes. Levopimaradiene, the major product of G. biloba LPS is the gateway precursor of ginkgolides. Coproducts of LPS include abietadiene, neoabiatadiene, and sandaracopimaradiene that stem from the different deprotonation patterns throughout intermediates in the reaction cascade.

Fig. 2.

Summary of LPS mutations and the impact with respect to product distribution and productivity of the engineered pathway. Trace amount (TA), not detected (ND). Numbers indicate percentage of each isomer (1-levopimaradiene; 2-abietadiene; 3-sandaracopimaradiene).

Fig. 3.

Characteristics (productivity and product distribution) of the preengineered strains expressing wild-type ggpps and lps saturation mutagenesis library of: (A) Met593, (B) Tyr700, (C) Ala620, and (D) Tyr700 using lps M593I. In the product distribution charts, gray bars, orange bars, white bars represent proportion of levopimaradiene, abietadiene, and sandaracopimaradiene in the product mixture, respectively. Two toned (white and light gray) bars represent nil production. WT represents the preengineered strain expressing wild-type ggpps and lps.

Fig. 4.

Generation of GGPPS library based on stochastic mutation. (A) Creation of a facile high-throughput screening assay by fusing a lycopene pathway (crtB and crtI) with ggpps libraries. Mutant ggpps genes that conferred improved lycopene production (red colonies) were isolated. These variants were then coexpressed with lps carrying M593I/Y700F mutations for diterpenoid production assay. (B) Production phenotype of the preengineered E. coli strains coexpressing selected ggpps variants and lps M593I/Y700F. WT represents the strain expressing the wild-type ggpps and lps M593I/Y700F.

Fig. 5.

Cultivation of the E. coli strain overexpressing the MEP pathway and the “reprogrammed” plant-derived pathway constituting GGPPS S239C/G295D and LPS M593I/Y700F mutants. (A) Diterpenoid production curves. Total diterpenoid, levopimaradiene, abietadiene, and sandaracopimaradiene are in circles, squares, triangles, and crosses, respectively. (B) Feed, fermentative by-product, and biomass curves. Glycerol, acetate, and cell density are in triangles, diamonds, and circles, respectively. Inverse triangle denotes the time point where 3 g of glycerol was added every 8 h.