A Modular Approach for Facile Biosynthesis of Labdane-Related Diterpenes

Labdane-related diterpenoids are a large group of over 5,000 natural products whose biosynthesis typically proceeds through a labdadienyl/copalyl diphosphate (CPP) intermediate to a further cyclized and/or rearranged hydrocarbon diterpene en route to more elaborated compounds. Here we report a modular approach for facile biosynthesis of labdane-related diterpenes wherein base pGGxC vectors capable of introducing bacterial production of any one of the three common stereoisomers of CPP can be co-introduced with diterpene synthases that convert these CPP intermediates to specific diterpene hydrocarbon skeletal structures. The utility of this approach is demonstrated by individually engineering E. coli to produce any one of eight different diterpene skeletal structures, which collectively serve as precursors to literally thousands of distinct natural products

Increasing Complexity of a Diterpene Synthase Reaction with a Single Residue Switch

Terpene synthases often catalyze complex reactions involving intricate series of carbocation intermediates. The resulting, generally cyclical, structures provide initial hydrocarbon frameworks that underlie the astonishing structural diversity of the enormous class of terpenoid natural products (\u3e50,000 known), and these enzymes often mediate the committed step in their particular biosynthetic pathway. Accordingly, how terpene synthases specify product outcome has drawn a great deal of attention. In previous work, we have shown that mutational introduction of a hydroxyl group at specific positions within diterpene synthase active sites can short circuit complex cyclization and/or rearrangement reactions, resulting in the production of simpler \u27 diterpenes. Here we demonstrate that the converse change, substitution of an Ile for Thr at the relevant position in a native pimaradiene synthase, leads to a dramatic increase in reaction complexity. Product outcome is shifted from the tricyclic pimaradiene to a rearranged tetracycle, aphidicol-15-ene. Thus, the nature of the residue at this position acts as a true switch for product outcome. In addition, the ability of aliphatic residue substitution to enable a more complex reaction emphasizes the importance of substrate conformation imposed by a largely inert active site. Furthermore, the profound plasticity of diterpene synthases exemplified by this single residue switch for product outcome is consistent with the screening/diversity-oriented hypothesis of natural products metabolism

Increasing diterpene yield with a modular metabolic engineering system in E. coli: comparison of MEV and MEP isoprenoid precursor pathway engineering

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

A Modular Approach for Facile Biosynthesis of Labdane-Related Diterpenes

Labdane-related diterpenoids are a large group of over 5,000 natural products whose biosynthesis typically proceeds through a labdadienyl/copalyl diphosphate (CPP) intermediate to a further cyclized and/or rearranged hydrocarbon diterpene en route to more elaborated compounds. Here we report a modular approach for facile biosynthesis of labdane-related diterpenes wherein base pGGxC vectors capable of introducing bacterial production of any one of the three common stereoisomers of CPP can be co-introduced with diterpene synthases that convert these CPP intermediates to specific diterpene hydrocarbon skeletal structures. The utility of this approach is demonstrated by individually engineering E. coli to produce any one of eight different diterpene skeletal structures, which collectively serve as precursors to literally thousands of distinct natural products.Reprinted with permission from Journal of the American Chemical Society 129 (2007): 6684, doi:10.1021/ja071158n. Copyright 2007 American Chemical Society.</p

A Single Residue Switch for Mg2+-dependent Inhibition Characterizes Plant Class II Diterpene Cyclases from Primary and Secondary Metabolism

Class II diterpene cyclases mediate the acid-initiated cycloisomerization reaction that serves as the committed step in biosynthesis of the large class of labdane-related diterpenoid natural products, which includes the important gibberellin plant hormones. Intriguingly, these enzymes are differentially susceptible to inhibition by their Mg2+ cofactor, with those involved in gibberellin biosynthesis being more sensitive to such inhibition than those devoted to secondary metabolism, which presumably limits flux toward the potent gibberellin phytohormones. Such inhibition has been suggested to arise from intrasteric Mg2+ binding to the DXDD motif that cooperatively acts as the catalytic acid, whose affinity must then be modulated in some fashion. While further investigating class II diterpene cyclase catalysis, we discovered a conserved basic residue that seems to act as a counter ion to the DXDD motif, enhancing the ability of aspartic acid to carry out the requisite energetically difficult protonation of a carbon-carbon double bond and also affecting inhibitory Mg2+binding. Notably, this residue is conserved as a histidine in enzymes involved in gibberellin biosynthesis and as an arginine in those dedicated to secondary metabolism. Interchanging the identity of these residues is sufficient to switch the sensitivity of the parent enzyme to inhibition by Mg2+. These striking findings indicate that this is a single residue switch for Mg2+ inhibition, which not only supports the importance of this biochemical regulatory mechanism in limiting gibberellin biosynthesis, but the importance of its release, presumably to enable higher flux, into secondary metabolism.This research was originally published in Journal of Biological Chemistry. Mann FM, Prisic S, Davenport EK, Determan MK, Coates RM, Peters RJ. A single residue switch for Mg2+-dependent inhibition characterizes plant class II diterpene cyclases from primary and secondary metabolism. Journal of Biological Chemistry. 2010 Jul 2;285:20558-63. © the American Society for Biochemistry and Molecular Biology.</p

Increasing Complexity of a Diterpene Synthase Reaction with a Single Residue Switch

Terpene synthases often catalyze complex reactions involving intricate series of carbocation intermediates. The resulting, generally cyclical, structures provide initial hydrocarbon frameworks that underlie the astonishing structural diversity of the enormous class of terpenoid natural products (>50,000 known), and these enzymes often mediate the committed step in their particular biosynthetic pathway. Accordingly, how terpene synthases specify product outcome has drawn a great deal of attention. In previous work, we have shown that mutational introduction of a hydroxyl group at specific positions within diterpene synthase active sites can "short circuit" complex cyclization and/or rearrangement reactions, resulting in the production of "simpler"' diterpenes. Here we demonstrate that the converse change, substitution of an Ile for Thr at the relevant position in a native pimaradiene synthase, leads to a dramatic increase in reaction complexity. Product outcome is shifted from the tricyclic pimaradiene to a rearranged tetracycle, aphidicol-15-ene. Thus, the nature of the residue at this position acts as a true switch for product outcome. In addition, the ability of aliphatic residue substitution to enable a more complex reaction emphasizes the importance of substrate conformation imposed by a largely inert active site. Furthermore, the profound plasticity of diterpene synthases exemplified by this single residue switch for product outcome is consistent with the screening/diversity-oriented hypothesis of natural products metabolism.Reprinted with permission from Journal of the American Chemical Society 130 (2008): 5400, doi:10.1021/ja710524w. Copyright 2008 American Chemical Society</p

Increasing diterpene yield with a modular metabolic engineering system in E. coli: comparison of MEV and MEP isoprenoid precursor pathway engineering

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 (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.This is an article published as Morrone, D., Lowry, L., Determan, M.K. et al. Appl Microbiol Biotechnol (2010) 85: 1893. doi:10.1007/s00253-009-2219-x. Posted with permission.</p