Engineering biosynthetic pathways in heterologous microbial host organisms offers an elegant approach to pathway elucidation via the incorporation of putative biosynthetic enzymes and characterization of resulting novel metabolites. Our previous work in Escherichia coli demonstrated the feasibility of a facile modular approach to engineering the production of labdane-related diterpene (20 carbon) natural products. However, yield was limited (<0.1 mg/L), presumably due to reliance on endogenous production of the isoprenoid precursors dimethylallyl diphosphate and isopentenyl diphosphate. Here, we report incorporation of either a heterologous mevalonate pathway (MEV) or enhancement of the endogenous methyl erythritol phosphate pathway (MEP) with our modular metabolic engineering system. With MEP pathway enhancement, it was found that pyruvate supplementation of rich media and simultaneous overexpression of three genes (idi, dxs, and dxr) resulted in the greatest increase in diterpene yield, indicating distributed metabolic control within this pathway. Incorporation of a heterologous MEV pathway in bioreactor grown cultures resulted in significantly higher yields than MEP pathway enhancement. We have established suitable growth conditions for diterpene production levels ranging from 10 to >100 mg/L of E. coli culture. These amounts are sufficient for nuclear magnetic resonance analyses, enabling characterization of enzymatic products and hence, pathway elucidation. Furthermore, these results represent an up to >1,000-fold improvement in diterpene production from our facile, modular platform, with MEP pathway enhancement offering a cost effective alternative with reasonable yield. Finally, we reiterate here that this modular approach is expandable and should be easily adaptable to the production of any terpenoid natural product.Electronic supplementary materialThe online version of this article (doi:10.1007/s00253-009-2219-x) contains supplementary material, which is available to authorized users.

Rice (Oryza sativa) produces momilactone diterpenoids as both phytoalexins and allelochemicals. Accordingly, the committed step in biosynthesis of these natural products is catalyzed by the class I terpene synthase that converts syn-copalyl diphosphate to the corresponding polycyclic hydrocarbon intermediate syn-pimara-7,15-diene. Here, a functional genomics approach was utilized to identify a syn-copalyl diphosphate specific 9b-pimara-7,15-diene synthase (OsDTS2). To our knowledge, this is the first identified terpene synthase with this particular substrate stereoselectivity and, by comparison with the previously described and closely related ent-copalyl diphosphate specific cassa-12,15-diene synthase (OsDTC1), provides a model system for investigating the enzymatic determinants underlying the observed difference in substrate specificity. Further, OsDTS2 mRNA in leaves is up-regulated by conditions that stimulate phytoalexin biosynthesis but is constitutively expressed in roots, where momilactones are constantly synthesized as allelochemicals. Therefore, transcription of OsDTS2 seems to be an important regulatory point for controlling production of these defensive compounds. Finally, the gene identified here as OsDTS2 has previously been mapped at 14.3 cM on chromosome 4. The class II terpene synthase producing syn-copalyl diphosphate from the universal diterpenoid precursor geranylgeranyl diphosphate was also mapped to this same region. These genes catalyze sequential cyclization steps in momilactone biosynthesis and seem to have been evolutionarily coupled by physical linkage and resulting cosegregation. Further, the observed correlation between physical proximity and common metabolic function indicates that other such class I and class II terpene synthase gene clusters may similarly catalyze consecutive reactions in shared biosynthetic pathways.Plants produce a vast and diverse array of low-molecular weight organic compounds, the overwhelming majority of which are secondary metabolites with nonessential, yet important, functions such as defense . For example, phytoalexins are produced in response to microbial infections and exhibit antimicrobial activity (VanEtten et al., 1994), while allelochemicals are secreted to the rhizosphere and suppress the growth of neighboring plants (Bais et al., 2004). Often found serving in such roles are terpenoids, which are particularly abundant in plant metabolism and form the largest class of natural products, exhibiting wide diversity in chemical structure and biological function . Much of the structural variation within this class arises from the diverse carbon backbones formed by terpene synthases (cyclases). These divalent metal dependent enzymes carry out complex electrophilic cyclizations/ rearrangements to create these diverse skeletal structures from relatively simple acyclic precursors (Davis and Croteau, 2000). Notably, production of a specific backbone structure either dictates, or at least severely restricts, the metabolic fate of that particular molecule. Thus,...

There have been few insights into the biochemical origins of natural product biosynthesis from primary metabolism. Of particular interest are terpene synthases, which often mediate the committed step in particular biosynthetic pathways so that alteration of their product outcome is a key step in the derivation of novel natural products. These enzymes also catalyze complex reactions of significant mechanistic interest. Following an evolutionary lead from two recently diverged, functionally distinct diterpene synthase orthologs from different subspecies of rice, we have identified a single residue that can switch product outcome. Specifically, the mutation of a conserved isoleucine to threonine that acts to convert not only the originally targeted isokaurene synthase into a specific pimaradiene synthase but also has a much broader effect, which includes conversion of the ent-kaurene synthases found in all higher plants for gibberellin phytohormone biosynthesis to the production of pimaradiene. This surprisingly facile switch for diterpene synthase catalytic specificity indicates the ease with which primary (gibberellin) metabolism can be subverted to secondary biosynthesis and may underlie the widespread occurrence of pimaradiene-derived natural products. In addition, because this isoleucine is required for the mechanistically more complex cyclization to tetracyclic kaurene, whereas substitution with threonine ''short-circuits'' this mechanism to produce the ''simpler'' tricyclic pimaradiene, our results have some implications regarding the means by which terpene synthases specify product outcome.enzyme specificity ͉ natural product biosynthesis ͉ terpene synthase ͉ biochemical evolution T he evolution of secondary metabolism presumably originates via changes in the catalytic specificity of enzymes recruited from primary metabolism. Although a recent report demonstrates that small numbers of changes in a sesquiterpene synthase can dramatically shift product outcome (i.e., plasticity), the target enzyme was already involved in secondary metabolism, and the parent wild-type enzyme was quite promiscuous (i.e., produced many different products) (1). Thus, how readily the typically specific enzymes involved in primary metabolism can be subverted into secondary metabolism remains a matter of conjecture.Terpene synthases carry out complex electrophilic cyclization/ rearrangement reactions, creating diverse hydrocarbon skeletal structures from simpler isoprenoid precursors, which often represents the committed step in particular biosynthetic pathways. Hence, altering the function of these enzymes is expected to represent a key step in the evolution of secondary metabolism. In addition, terpene synthases have attracted a great deal of interest because of their complex reaction mechanisms and wide variety of resulting products. Of particular interest is how these enzymes specify product outcome (2). Recent reports have demonstrated that specificity can be dramatically shifted by changes in a small number of amino acid resid...

Rice (Oryza sativa) produces ent-copalyl diphosphate for both gibberellin (GA) phytohormone and defensive phytoalexin biosynthesis, raising the question of how this initial biosynthetic step is carried out for these distinct metabolic processes. Here, a functional genomics approach has been utilized to identify two disparate ent-copalyl diphosphate synthases from rice (OsCPS1 ent and OsCPS2 ent ). Notably, it was very recently demonstrated that only one of these (OsCPS1 ent ) normally operates in GA biosynthesis as mutations in this gene result in severely impaired growth. Evidence is presented here strongly indicating that the other (OsCPS2 ent ) is involved in related secondary metabolism producing defensive phytochemicals. In particular, under appropriate conditions, OsCPS2 ent mRNA is specifically induced in leaves prior to production of the corresponding phytoalexins. Thus, transcriptional control of OsCPS2 ent seems to be an important means of regulating defensive phytochemical biosynthesis. Finally, OsCPS1 ent is significantly more similar to the likewise GA-specific gene An1/ZmCPS1 ent in maize (Zea mays) than its class II terpene synthase paralogs involved in rice secondary metabolism. Hence, we speculate that this crossspecies conservation by biosynthetic process reflects derivation of related secondary metabolism from the GA primary biosynthetic pathway prior to the early divergence between the separate lineages within the cereal/grass family (Poaceae) resulting in modern rice and maize.Plants produce a vast and diverse array of low-M r organic compounds. A small number of these are primary metabolites, which are common to all plant species as they are directly required for growth and development. The remaining, overwhelming majority of these natural products are considered secondary metabolites, although many have important ecological roles, particularly in plant defense (Dixon, 2001). Terpenoids, which comprise the largest class of natural products, are particularly abundant in plants and can be found in both primary and secondary metabolism . Notably, a substantial fraction of the known terpenoids can be classified as labdanerelated diterpenoids (20 carbon). These are defined here as minimally containing the bicyclic hydrocarbon structure found in the labdane class of diterpenoids, although this core structure can be further cyclized and/or rearranged, as in the related/derived structural classes (e.g. kauranes, abietanes, and [iso] pimaranes). Significantly, this includes the GA growth hormones as primary metabolites. Nevertheless, the vast majority of the .5,000 labdane-related diterpenoids are secondary metabolites.Biosynthesis of labdane-related diterpenoids is initiated by class II terpene synthases, which catalyze formation of the characteristic bicyclic backbone in producing specific stereoisomers of labdadienyl/ copalyl diphosphate (CPP) from the universal diterpenoid precursor (E,E,E)-geranylgeranyl diphosphate (GGPP). This protonation-initiated cyclization is fundamentally different than ...