A Modular Approach for Facile Biosynthesis of Labdane-Related Diterpenes

Cyr, Anthony R.; Wilderman, P. Ross; Determan, Mara K.; Peters, Reuben J.

doi:10.1021/ja071158n

Cited by 147 publications

(209 citation statements)

References 15 publications

(31 reference statements)

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“…This was originally attempted using the native genes, obtained from the KOME rice cDNA database (Kikuchi et al, 2003). Due to our previous successful expression of plant CYP in Escherichia coli (Swaminathan et al, 2009;Morrone et al, 2010a;Wang et al, 2011;Wu et al, 2011), this was carried out using our bacterial modular metabolic engineering system (Cyr et al, 2007). In particular, by coexpression with the requisite CYP reductase (CPR) and functional pairings of upstream CPS and KS(L) to produce all of the labdane-related diterpenes found in rice (see Supplemental Fig.…”

Section: Recombinant Expressionmentioning

confidence: 99%

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CYP701A8: A Rice ent-Kaurene Oxidase Paralog Diverted to More Specialized Diterpenoid Metabolism

et al. 2012

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All higher plants contain an ent-kaurene oxidase (KO), as such a cytochrome P450 (CYP) 701 family member is required for gibberellin (GA) phytohormone biosynthesis. While gene expansion and functional diversification of GA-biosynthesis-derived diterpene synthases into more specialized metabolism has been demonstrated, no functionally divergent KO/CYP701 homologs have been previously identified. Rice (Oryza sativa) contains five CYP701A subfamily members in its genome, despite the fact that only one (OsKO2/CYP701A6) is required for GA biosynthesis. Here we demonstrate that one of the other rice CYP701A subfamily members, OsKOL4/CYP701A8, does not catalyze the prototypical conversion of the ent-kaurene C4a-methyl to a carboxylic acid, but instead carries out hydroxylation at the nearby C3a position in a number of related diterpenes. In particular, under conditions where OsKO2 catalyzes the expected conversion of ent-kaurene to ent-kaurenoic acid required for GA biosynthesis, OsKOL4 instead efficiently reacts with ent-sandaracopimaradiene and ent-cassadiene to produce the corresponding C3a-hydroxylated diterpenoids. These compounds are expected intermediates in biosynthesis of the oryzalexin and phytocassane families of rice antifungal phytoalexins, respectively, and can be detected in rice plants under the appropriate conditions. Thus, it appears that OsKOL4 plays a role in the more specialized diterpenoid metabolism of rice, and our results provide evidence for divergence of a KO/CYP701 family member from GA biosynthesis. This further expands the range of enzymes recruited from the ancestral GA primary pathway to the more complex and specialized labdane-related diterpenoid metabolic network found in rice.

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Section: Recombinant Expressionmentioning

confidence: 99%

“…All CYP were expressed in the C41 overexpress strain of E. coli (Lucigen) using our previously described modular diterpene metabolic engineering system (Cyr et al, 2007). Briefly, these CYP were coexpressed with not only Figure 7.…”

Section: Recombinant Expressionmentioning

confidence: 99%

CYP701A8: A Rice ent-Kaurene Oxidase Paralog Diverted to More Specialized Diterpenoid Metabolism

et al. 2012

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“…8 The resulting recombinant bacterial strain produced ∼1 mg diterpenes/L culture, enabling isolation of sufficient amounts of the novel diterpene product of OsKSL4:T696I for structural determination by NMR, demonstrating that this predominant enzymatic product corresponds to aphidicol-15-ene (see Supporting Information). Notably, this appears to be the first report of an aphidicolene specific diterpene synthase.…”

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confidence: 99%

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

Morrone

Fulton

et al. 2008

J. Am. Chem. Soc.

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Terpene synthases often catalyze complex reactions involving an 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), 1 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. 2 Recent reports demonstrate that changes in a small number of amino acid residues can substantially alter specificity, although without generally increasing complexity of the catalyzed reaction and resulting product. 3 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 rearrangement reactions, resulting in the production of "simpler" diterpenes. 3e,g 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 synpimara-7,15-diene (1) to a rearranged tetracycle, aphidicol-15-ene (2). This result has implications for our understanding of the structure-function relationships underlying the reaction mechanism, engineering, and evolution of terpene synthases.Previously we found that substitution of a particular conserved Ile by Thr was sufficient to convert diverse ent-kaurene specific (>95%) synthases to the specific production of ent-pimara-8(14), 15-diene. 3e In addition, the abietadiene synthases involved in conifer resin acid production contain a nearby conserved Ala (Supporting Figure), substitution of which by Ser similarly leads to specific formation of pimaradienes rather than the original rearranged abeitane tricycles. 3g Accordingly, replacement of these particular aliphatic amino acids by residues containing a hydroxyl group can "short circuit" complex cyclization/rearrangement reactions. In particular, the mutant diterpene synthases presumably deprotonate the pimar-15-en-8-yl + intermediate formed by initial (tri)cyclization of their specific bicyclic copalyl/labdadienyl diphosphate (CPP) substrate. In both cases, the targeted residue can be modeled near the 8-yl carbocation position, with the shift in exact position presumably due to the differing stereochemistry of the relevant CPP substrate. Hence, a carbocation proximal aliphatic residue seems to be necessary, but possibly is not sufficient, for more complex reactions.There is a syn-pimara-7,15-diene synthase (OsKSL4) 4 involved in rice antifungal phytoalexin (momilactone) biosynthesis that contains a Thr in place of the aforementioned Ile found in entkaurene synthases, consistent with the identical C9 configuration of ent-and syn-CPP (3). The mechanistically important Ala identified in abietadiene synthases also is conserved in OsKSL4, as well as...

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“…We have developed a modular metabolic engineering system that enables facile transformation of Escherichia coli to engineer the production of any terpene for which the corresponding synthase or synthases are known (14). This has been further coupled to strategies for functional expression of plant CYPs in E. coli, to (re)create terpenoid biosynthetic pathways via a synthetic biology approach (15).…”

Section: Resultsmentioning

confidence: 99%

Probing the promiscuity of ent -kaurene oxidases via combinatorial biosynthesis

Mafu

Jia

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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The substrate specificity of enzymes from natural products' metabolism is a topic of considerable interest, with potential biotechnological use implicit in the discovery of promiscuous enzymes. However, such studies are often limited by the availability of substrates and authentic standards for identification of the resulting products. Here, a modular metabolic engineering system is used in a combinatorial biosynthetic approach toward alleviating this restriction. In particular, for studies of the multiply reactive cytochrome P450, ent-kaurene oxidase (KO), which is involved in production of the diterpenoid plant hormone gibberellin. Many, but not all, plants make a variety of related diterpenes, whose structural similarity to ent-kaurene makes them potential substrates for KO. Use of combinatorial biosynthesis enabled analysis of more than 20 such potential substrates, as well as structural characterization of 12 resulting unknown products, providing some insight into the underlying structure-function relationships. These results highlight the utility of this approach for investigating the substrate specificity of enzymes from complex natural products' biosynthesis.labdane-related diterpenoids | cytochrome P450 monooxygenases | natural products | enzyme specificity | gibberellins

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A Modular Approach for Facile Biosynthesis of Labdane-Related Diterpenes

Cited by 147 publications

References 15 publications

CYP701A8: A Rice ent-Kaurene Oxidase Paralog Diverted to More Specialized Diterpenoid Metabolism

CYP701A8: A Rice ent-Kaurene Oxidase Paralog Diverted to More Specialized Diterpenoid Metabolism

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

Probing the promiscuity of ent -kaurene oxidases via combinatorial biosynthesis

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