The potential of metabolic engineering and synthetic biology is still being explored. For example, innovative work by the Keasling group has recently demonstrated that it is possible to metabolically engineer E. coli
to produce large quantities of artemisinic acid, a usable precursor for semisynthetic production of the antimalarial sesquiterpenoid natural product and medically relevant drug artemisinin (Chang et al. 2007
). This, and indeed most, published/reported work in the area of metabolic engineering has focused on the production of single or small groups of related, generally known compounds through manipulation of the corresponding specific biosynthetic pathway. However, this limits the utility of the developed system for investigation of novel metabolism. We wish to utilize metabolic engineering instead for biosynthetic pathway elucidation and enzymatic investigation via facile incorporation of putative biosynthetic enzymes and characterization of the resulting novel metabolites.
Our interest centers on the large superfamily of labdane-related diterpenoids, which is comprised of almost 7,000 known natural products. Many of these are exclusively found in plants, whose large and complex genomes preclude ready identification of the corresponding enzymatic genes by physical proximity or on the basis of homology (particularly in the case of multigenic families). To provide a platform for facile investigation of novel enzymatic activity, we have developed a modular system for metabolically engineering E. coli
to produce labdane-related diterpenes (Cyr et al. 2007
). This provides an elegant approach to elucidation of diterpenoid biosynthetic pathways and corresponding enzymatic investigation and one that is amendable, with slight adaptation, to the study of any type of terpenoid metabolism. However, in order to realize this potential, it must be possible to readily produce the multimilligram quantities of resulting metabolite(s) needed for structural analysis (i.e., by NMR).
Initial work on this system demonstrated the ability of E. coli
to produce desired diterpenes, albeit in small quantities of <100 µg/l of culture, presumably due to the limited endogenous isoprenoid precursor pools (Cyr et al. 2007
). Here, we have set out to shift carbon flux from E. coli
central metabolism for milligram production of diterpenes in liter scale culture growths. Two basic approaches were investigated, either supplementation of the endogenous MEP isoprenoid precursor pathway via overexpression of key enzymatic genes or addition of a heterologous MEV isoprenoid precursor pathway (this latter either in whole or, with supplementation, in part). The ability of these alternative approaches to improve diterpene production was assessed by analysis of their impact on abietadiene yield from cells coexpressing GGPP and abietadiene synthases.
Engineering MEP pathway enzymatic gene overexpression was found to increase abietadiene production. Interestingly, while only idi improved yield on its own, further improvement was evident upon overexpression of idi with both dxs and dxr, although these actually decreased yield on their own and when paired with each other or idi (Table ). As culture growth was unimpaired by any combination of MEP enzymes and only abietadiene diterpene product yield was affected, this suggests flux through the pathway may be most responsible for the varied outcome arising from different gene combinations. Further, these results suggest that the investigated MEP pathway enzymatic genes exert distributed metabolic control, specifically dxs and dxr. While the isomerization of DMAPP and IPP mediated by idi seems to be the initial rate-limiting step, further increases are dependent on overexpression of both dxs and dxr. Notably, the production of 1-deoxy-D-xylulose-5-phosphate (DXP) mediated by dxs is not unique to isoprenoid precursor production, which is initiated by the ensuing production of 2-C-methyl-D-erythitol-4-phosphate mediated by dxr. Thus, our results suggest that increased flux to DXP (i.e., from dxs overexpression) is shunted to alternative metabolic fates in the absence of increased dxr activity, although overexpression of dxr alone also is not sufficient on its own to compete with the alternative pathways.
Introduction of a heterologous MEV pathway, either in part (with the appropriate supplementation) or whole, increased abietadiene yield beyond that observed with engineering of the endogenous MEP pathway (Table ). These increases were particularly significant in bioreactor grown cultures, where yields >100 mg/L were observed, representing a >1,000-fold increase over that previously reported. However, this requires either costly mevalonate supplementation or the use of two plasmids for introduction of the whole MEP pathway, which limits the number of downstream steps that can be incorporated. In addition, obtaining truly significant increases further required use of a bioreactor. Nevertheless, baffled shake flask growth conditions with even the single plasmid associated with optimal MEP pathway engineering produces >10 mg/L of diterpene, providing a simple and economical means for obtaining the multimilligram quantities of novel metabolites necessary for their structural analysis.
Critically, the improvements reported here have been directly applied to the further incorporation of additional enzymatic genes (i.e., pairs of diterpene synthases rather than the bifunctional AgAS), carried on yet another plasmid, also with yields exceeding 100 mg/L in bioreactor fermentations. Thus, it should be possible to use the developed modular metabolic engineering system, with the yield improvements reported here, to investigate multistep biosynthetic pathways. We reiterate here that these include not only the labdane-related diterpenes that are the focus of our research, but other diterpenoid natural products and with slight modification (i.e., replacement of the GGPP synthase), other types of terpenoids as well.