Purification and optimization of sequential biosynthetic enzymes for co-localization onto a nanocarrier

Flavonoids are polyphenolic plant secondary metabolites with many purposes, including providing pigmentation in plants and anti-oxidant and anti-cancerous activities in humans. There are over 10,000 different flavonoids known, making them one of the largest groups of natural products. One major sub-class of flavonoids is the flavan-3-ols, which are known for their health benefits and the ability to form proanthocyanidins, or condensed tannins. Many of the intermediates in this pathway are unstable and have not been isolated in planta. Another natural product of interest is sclareol, a diterpene alcohol, which is important in the fragrance and flavor industry as a precursor to Ambrex, and ambergris analog that can be used as a fixative in high end perfumes. In this study we are aiming to create a synthetic complex using a polymer nanocarrier to co-localize two sequential enzymes in a pathway. The first system tried included the final two enzymes in flavan-3-ol biosynthesis, anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR). Each of these enzymes, along with the preceding enzyme, dihydroflavonol 4-reductase (DFR) has been recombinantly expressed in E. coli with a specific tag for binding to the nanocarrier. However, due to difficulty achieving ANS activity a backup system involved in sclareol synthesis was developed. Two enzymes, NgCPS and sSsSS, were used to convert geranylgeranyl-pyrophosphate (GGPP) to sclareol in a coupled assay. Each enzyme was purified with either a 6xHis tag or monomeric DM3 streptavidin tag for attachment to the nanocarrier. Assays to measure each enzymes activity have been designed, and our goal now is to test each enzymes activity before and after co-localization onto the nanocarrier

Probing Labdane-Related Diterpenoid Biosynthesis in the Fungal Genus Aspergillus

While terpenoid production is generally associated with plants, a variety of fungi contain operons predicted to lead to such biosynthesis. Notably, fungi contain a number of cyclases characteristic of labdane-related diterpenoid metabolism, which have not been much explored. These also are often found near cytochrome P450 (CYP) mono-oxygenases that presumably further decorate the ensuing diterpene, suggesting that these fungi might produce more elaborate diterpenoids. To probe the functional diversity of such biosynthetic capacity, an investigation of the phylogenetically diverse cyclases and associated CYPs from the fungal genus Aspergillus was undertaken, revealing their ability to produce isopimaradiene-derived diterpenoids. Intriguingly, labdane-related diterpenoid biosynthetic genes are largely found in plant-associated fungi, hinting that these natural products may play a role in such interactions. Accordingly, it is hypothesized here that isopimarane production may assist the plant-saprophytic lifestyle of Aspergillus fungi

Functional characterization of wheat ent-kaurene(-like) synthases indicates continuing evolution of labdane-related diterpenoid metabolism in the cereals

Wheat (Triticum aestivum) and rice (Oryza sativa) are two of the most agriculturally important cereal crop plants. Rice is known to produce numerous diterpenoid natural products that serve as phytoalexins and/or allelochemicals. Specifically, these are labdane-related diterpenoids, derived from a characteristic labdadienyl/copalyl diphosphate (CPP), whose biosynthetic relationship to gibberellin biosynthesis is evident from the relevant expanded and functionally diverse family of ent-kaurene synthase-like (KSL) genes found in rice (OsKSL). Here we report biochemical characterization of a similarly expansive family of KSL from wheat (the TaKSLs). In particular, beyond ent-kaurene synthases (KS), wheat also contains several biochemically diversified KSLs. These react either with the ent-CPP intermediate common to gibberellin biosynthesis or with the normal stereoisomer of CPP that also is found in wheat (as demonstrated by the accompanying description of wheat CPP synthases). Comparison with a barley (Hordeum vulgare) KS indicates conservation of monocot KS, with early and continued expansion and functional diversification of KSLs in at least the small grain cereals. In addition, some of the TaKSLs that utilize normal CPP also will react with syn-CPP, echoing previous findings with the OsKSL family, with such enzymatic promiscuity/plasticity providing insight into the continuing evolution of diterpenoid metabolism in the cereal crop plant family, as well as more generally, which is discussed here

Characterization of an Orphan Diterpenoid Biosynthetic Operon from Salinispora arenicola

While more commonly associated with plants than microbes, diterpenoid natural products have been reported to have profound effects in marine microbe–microbe interactions. Intriguingly, the genome of the marine bacterium Salinispora arenicola CNS-205 contains a putative diterpenoid biosynthetic operon, terp1. Here recombinant expression studies are reported, indicating that this three-gene operon leads to the production of isopimara-8,15-dien-19-ol (4). Although 4 is not observed in pure cultures of S. arenicola, it is plausible that the terp1 operon is only expressed under certain physiologically relevant conditions such as in the presence of other marine organisms

Purification and optimization of sequential biosynthetic enzymes for co-localization onto a nanocarrier

Flavonoids are polyphenolic plant secondary metabolites with many purposes, including providing pigmentation in plants and anti-oxidant and anti-cancerous activities in humans. There are over 10,000 different flavonoids known, making them one of the largest groups of natural products. One major sub-class of flavonoids is the flavan-3-ols, which are known for their health benefits and the ability to form proanthocyanidins, or condensed tannins. Many of the intermediates in this pathway are unstable and have not been isolated in planta. Another natural product of interest is sclareol, a diterpene alcohol, which is important in the fragrance and flavor industry as a precursor to Ambrex, and ambergris analog that can be used as a fixative in high end perfumes. In this study we are aiming to create a synthetic complex using a polymer nanocarrier to co-localize two sequential enzymes in a pathway. The first system tried included the final two enzymes in flavan-3-ol biosynthesis, anthocyanidin synthase (ANS) and anthocyanidin reductase (ANR). Each of these enzymes, along with the preceding enzyme, dihydroflavonol 4-reductase (DFR) has been recombinantly expressed in E. coli with a specific tag for binding to the nanocarrier. However, due to difficulty achieving ANS activity a backup system involved in sclareol synthesis was developed. Two enzymes, NgCPS and sSsSS, were used to convert geranylgeranyl-pyrophosphate (GGPP) to sclareol in a coupled assay. Each enzyme was purified with either a 6xHis tag or monomeric DM3 streptavidin tag for attachment to the nanocarrier. Assays to measure each enzymes activity have been designed, and our goal now is to test each enzymes activity before and after co-localization onto the nanocarrier.</p

Probing Labdane-Related Diterpenoid Biosynthesis in the Fungal Genus Aspergillus

While terpenoid production is generally associated with plants, a variety of fungi contain operons predicted to lead to such biosynthesis. Notably, fungi contain a number of cyclases characteristic of labdane-related diterpenoid metabolism, which have not been much explored. These also are often found near cytochrome P450 (CYP) mono-oxygenases that presumably further decorate the ensuing diterpene, suggesting that these fungi might produce more elaborate diterpenoids. To probe the functional diversity of such biosynthetic capacity, an investigation of the phylogenetically diverse cyclases and associated CYPs from the fungal genus Aspergillus was undertaken, revealing their ability to produce isopimaradiene-derived diterpenoids. Intriguingly, labdane-related diterpenoid biosynthetic genes are largely found in plant-associated fungi, hinting that these natural products may play a role in such interactions. Accordingly, it is hypothesized here that isopimarane production may assist the plant-saprophytic lifestyle of Aspergillus fungi.Reprinted (adapted) with permission from Probing Labdane-Related Diterpenoid Biosynthesis in the Fungal Genus Aspergillus. Meimei Xu, Matthew L. Hillwig, Mollie S. Tiernan, and Reuben J. Peters. Journal of Natural Products 2017 80 (2), 328-333. DOI: 10.1021/acs.jnatprod.6b00764. Copyright 2017 American Chemical Society.</p

Characterization of an Orphan Diterpenoid Biosynthetic Operon from Salinispora arenicola

While more commonly associated with plants than microbes, diterpenoid natural products have been reported to have profound effects in marine microbe–microbe interactions. Intriguingly, the genome of the marine bacterium Salinispora arenicola CNS-205 contains a putative diterpenoid biosynthetic operon, terp1. Here recombinant expression studies are reported, indicating that this three-gene operon leads to the production of isopimara-8,15-dien-19-ol (4). Although 4 is not observed in pure cultures of S. arenicola, it is plausible that the terp1 operon is only expressed under certain physiologically relevant conditions such as in the presence of other marine organisms.Reprinted (adapted) with permission from Journal of Natural Products 77 (2014): 2144, doi:10.1021/np500422d. Copyright 2014 American Chemical Society.</p

Domain loss has independently occurred multiple times in plant terpene synthase evolution

The extensive family of plant terpene synthases (TPSs) generally has a bi-domain structure, yet phylogenetic analyses consistently indicate that these evolved from larger diterpene synthases. In particular, that duplication of the diterpene synthase genes required for gibberellin phytohormone biosynthesis provided an early predecessor, whose loss of a ~220 amino acid “internal sequence element” (now recognized as the γ domain) gave rise to the precursor of modern mono- and sesqui-TPSs found in all higher plants. Intriguingly, TPSs are conserved by taxonomic relationships rather than function, demonstrating that such functional radiation has occurred both repeatedly and relatively recently, yet phylogenetic analyses assume that “internal/γ” domain loss represents a single evolutionary event. Here we provide evidence that such loss was not a singular event, but rather has occurred multiple times. Specifically, we provide an example of a bi-domain diterpene synthase, from Salvia miltiorrhiza, along with a sesquiterpene synthase from Triticum aestivum (wheat) that is not only closely related to diterpene synthases, but retains the ent-kaurene synthase activity relevant to the ancestral gibberellin metabolic function. Indeed, while the wheat sesquiterpene synthase clearly no longer contains the “internal/γ” domain, it is closely related to rice diterpene synthase genes that retain the ancestral tri-domain structure. Thus, these findings provide examples of key evolutionary intermediates underlying the bi-domain structure observed in the expansive plant TPS gene family, as well as indicating that “internal/γ” domain loss has independently occurred multiple times, highlighting the complex evolutionary history of this important enzymatic family.This is the peer reviewed version of the following article: Hillwig, M. L., Xu, M., Toyomasu, T., Tiernan, M. S., Wei, G., Cui, G., Huang, L. and Peters, R. J. (2011), Domain loss has independently occurred multiple times in plant terpene synthase evolution. The Plant Journal, 68: 1051–1060 , which has been published in final form at doi:10.1111/j.1365-313X.2011.04756.x . This article may be used for non-commercial purposes in accordance With Wiley Terms and Conditions for self-archiving.</p

Functional characterization of wheat ent-kaurene(-like) synthases indicates continuing evolution of labdane-related diterpenoid metabolism in the cereals

Wheat (Triticum aestivum) and rice (Oryza sativa) are two of the most agriculturally important cereal crop plants. Rice is known to produce numerous diterpenoid natural products that serve as phytoalexins and/or allelochemicals. Specifically, these are labdane-related diterpenoids, derived from a characteristic labdadienyl/copalyl diphosphate (CPP), whose biosynthetic relationship to gibberellin biosynthesis is evident from the relevant expanded and functionally diverse family of ent-kaurene synthase-like (KSL) genes found in rice (OsKSL). Here we report biochemical characterization of a similarly expansive family of KSL from wheat (the TaKSLs). In particular, beyond ent-kaurene synthases (KS), wheat also contains several biochemically diversified KSLs. These react either with the ent-CPP intermediate common to gibberellin biosynthesis or with the normal stereoisomer of CPP that also is found in wheat (as demonstrated by the accompanying description of wheat CPP synthases). Comparison with a barley (Hordeum vulgare) KS indicates conservation of monocot KS, with early and continued expansion and functional diversification of KSLs in at least the small grain cereals. In addition, some of the TaKSLs that utilize normal CPP also will react with syn-CPP, echoing previous findings with the OsKSL family, with such enzymatic promiscuity/plasticity providing insight into the continuing evolution of diterpenoid metabolism in the cereal crop plant family, as well as more generally, which is discussed here.This is the author’s version of a work that was accepted for publication in Phytochemistry. Changes resulting from the publishing process, such as peer review, editing, corrections, structural formatting, and other quality control mechanisms may not be reflected in this document. Changes may have been made to this work since it was submitted for publication. A definitive version was subsequently published in Phytochemistry, VOL 84, 2012, DOI: 10.1016/j.phytochem.2012.08.021.</p