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1.  Optimization of recombinant expression enables discovery of novel cytochrome P450 activity in rice diterpenoid biosynthesis 
Applied microbiology and biotechnology  2015;99(18):7549-7558.
The oxygenation reactions catalyzed by cytochromes P450 (CYPs) play critical roles in plant natural products biosynthesis. At the same time, CYPs are one of most challenging enzymes to functionally characterize due to the difficulty of recombinantly expressing these membrane-associated monooxygenases. In the course of investigating rice diterpenoid biosynthesis we have developed a synthetic biology approach for functional expression of relevant CYPs in Escherichia coli. In certain cases activity was observed for only one of two closely related paralogs although it seems clear that related reactions are required for production of the known diterpenoids. Here we report that optimization of the recombinant expression system enabled characterization of not only these previously recalcitrant CYPs, but also discovery of additional activity relevant to rice diterpenoid biosynthesis. Of particular interest, CYP701A8 was found to catalyze 3β-hydroxylation of syn-pimaradiene, which is presumably relevant to momilactone biosynthesis, while CYP71Z6 & 7 were found to catalyze multiple reactions, with CYP71Z6 catalyzing the production of 2α,3α-dihydroxy-ent-isokaurene via 2α-hydroxy- ent-isokaurene, and CYP71Z7 catalyzing the production of 3α-hydroxy-ent-cassadien-2- one via 2α-hydroxy-ent-cassadiene and ent-cassadien-2-one, which may be relevant to oryzadione and phytocassane biosynthesis, respectively.
PMCID: PMC4546517  PMID: 25758958
metabolic engineering; natural products; labdane-related diterpenoids; phytoalexin; antibiotic; evolution
2.  Characterization of an Orphan Diterpenoid Biosynthetic Operon from Salinispora arenicola 
Journal of Natural Products  2014;77(9):2144-2147.
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.
PMCID: PMC4176389  PMID: 25203741
3.  Biochemical characterization of the castor bean ent-kaurene synthase(-like) family supports quantum chemical view of diterpene cyclization 
Phytochemistry  2014;103:13-21.
It has become apparent that plants have extensively diversified their arsenal of labdane-related diterpenoids (LRDs), in part via gene duplication and neo-functionalization of the ancestral ent-kaurene synthase (KS) required for gibberellin metabolism. For example, castor bean (Ricinus communis) was previously shown to produce an interesting set of biosynthetically related diterpenes, specifically ent-sandracopimaradiene, ent-beyerene, and ent-trachylobane, in addition to ent-kaurene, using four separate diterpene synthases, albeit these remain unidentified. Notably, despite mechanistic similarity of the underlying reaction to that catalyzed by KSs, ent-beyerene and ent-trachylobane synthases have not yet been identified. Given our interest in LRD biosynthesis, and the recent availability of the castor bean genome sequence, we applied a synthetic biology approach to biochemically characterize the four KS(-like) enzymes [KS(L)s] found in Ricinus communis [i.e., the RcKS(L)s]. In particular, using bacteria engineered to produce the relevant ent-copalyl diphosphate precursor and synthetic genes based on the predicted RcKS(L)s, although this ultimately required correction of a “splicing” error in one of the predicted genes, highlighting the dependence of such a synthetic biology approach on accurate gene sequences. Nevertheless, we can assign each of the four RcKS(L)s to one of the previously observed diterpene synthase activities, providing access to functionally novel enzymes. Intriguingly, the product distribution of the RcKS(L)s seems to support the distinct diterpene synthase reaction mechanism proposed by quantum chemical calculations, rather than the classically proposed pathway.
PMCID: PMC4062354  PMID: 24810014
natural products biosynthesis; diterpenoids; terpene synthases
4.  Non-Linear Association between Exposure to Ambient Temperature and Children’s Hand-Foot-and-Mouth Disease in Beijing, China 
PLoS ONE  2015;10(5):e0126171.
Hand, foot and mouth disease (HFMD) was listed as a notifiable communicable disease in 2008 and is an emerging public health problem in China, especially for children. However, few data are available on the risk assessment of the potential reasons for HFMD in Beijing. This study examined the association of temperature with the incidence of children’s HFMD in Beijing at the daily scale for the first time.
A newly developed case-crossover design with a distributed lag nonlinear model (DLNM) was used to assess the delayed and cumulative associations of daily temperature with gender- and age-specific HFMD in Beijing, China, during 2010–2012. Relative humidity, day of the week, public holiday, season and long-term trends were controlled in the model.
Among the total of 113,475 cases, the ratio between males and females was 1.52:1. HFMD was more prevalent in May-July. The temperature-HFMD relationships were non-linear in most age groups except for children aged 6–15 years, with a peak at 25.0~27.5°C. The high-temperature risks were greater, appeared earlier and lasted longer than the low-temperature risks. The relative risks for female children and those aged 6–15 years were higher than those among other groups.
Rising temperatures increased the incidence of children’s HFMD, with the largest association at 25.0~27.5°C. Females and children aged 6–15 years were more vulnerable to changes in temperature with regard to the transmission of HFMD than males and other age groups, respectively. Further studies are warranted to confirm these findings in other populations.
PMCID: PMC4444089  PMID: 26010147
5.  Spatiotemporal analysis of particulate air pollution and ischemic heart disease mortality in Beijing, China 
Environmental Health  2014;13:109.
Few studies have used spatially resolved ambient particulate matter with an aerodynamic diameter of <10 μm (PM10) to examine the impact of PM10 on ischemic heart disease (IHD) mortality in China. The aim of our study is to evaluate the short-term effects of PM10 concentrations on IHD mortality by means of spatiotemporal analysis approach.
We collected daily data on air pollution, weather conditions and IHD mortality in Beijing, China during 2008 and 2009. Ordinary kriging (OK) was used to interpolate daily PM10 concentrations at the centroid of 287 township-level areas based on 27 monitoring sites covering the whole city. A generalized additive mixed model was used to estimate quantitatively the impact of spatially resolved PM10 on the IHD mortality. The co-effects of the seasons, gender and age were studied in a stratified analysis. Generalized additive model was used to evaluate the effects of averaged PM10 concentration as well.
The averaged spatially resolved PM10 concentration at 287 township-level areas was 120.3 ± 78.1 μg/m3. Ambient PM10 concentration was associated with IHD mortality in spatiotemporal analysis and the strongest effects were identified for the 2-day average. A 10 μg/m3 increase in PM10 was associated with an increase of 0.33% (95% confidence intervals: 0.13%, 0.52%) in daily IHD mortality. The effect estimates using spatially resolved PM10 were larger than that using averaged PM10. The seasonal stratification analysis showed that PM10 had the statistically stronger effects on IHD mortality in summer than that in the other seasons. Males and older people demonstrated the larger response to PM10 exposure.
Our results suggest that short-term exposure to particulate air pollution is associated with increased IHD mortality. Spatial variation should be considered for assessing the impacts of particulate air pollution on mortality.
Electronic supplementary material
The online version of this article (doi:10.1186/1476-069X-13-109) contains supplementary material, which is available to authorized users.
PMCID: PMC4293109  PMID: 25495440
Spatiotemporal analysis; Ischemic heart disease; Particulate matter; Ordinary kriging; Generalized additive mixed model
6.  Functional characterization of wheat ent-kaurene(-like) synthases indicates continuing evolution of labdane-related diterpenoid metabolism in the cereals 
Phytochemistry  2012;84C:47-55.
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.
PMCID: PMC3483413  PMID: 23009879
ent-kaurene synthase; phytoalexin; phytoanticipin; allelochemicals; natural products biosynthesis; plant defense
7.  Effect of Isotopically Sensitive Branching on Product Distribution for Pentalenene Synthase – Support for a Mechanism Predicted by Quantum Chemistry 
Journal of the American Chemical Society  2012;134(28):11369-11371.
Mechanistic proposals for the carbocation cas cade reaction leading to the tricyclic sesquiterpene pentalenene are assessed in light of the results of isotopically sensitive branching experiments with the H309A mutant of pentalenene synthase. These experimental results support a mechanism for pentalenene formation involving a 7-protoilludyl cation intermediate that was first predicted using quantum chemical calculations.
PMCID: PMC3402234  PMID: 22738258
terpene; kinetic isotope effect; quantum chemical calculations; carbocation; reaction mechanism; natural product biosynthesis
8.  Genetic evidence for natural product mediated plant–plant allelopathy in rice 
The New Phytologist  2011;193(3):570-575.
A role for specific natural products in directly mediating antagonistic plant–plant interactions –that is, allelopathy –has been controversial. If proven, such phenomena would hold considerable promise for agronomic improvement of staple food crops such as rice (Oryza sativa).However, while substantiated by the presence of phytotoxic compounds at potentially relevant levels, demonstrating a direct role for specific natural products in allelopathy has been difficult due to the chemical complexity of root and plant litter exudates. This complexity can be bypassed via selective genetic manipulation to ablate production of putative allelopathic compounds, but such an approach previously has not been applied.The rice diterpenoid momilactones provide an example of natural products for which correlative biochemical evidence has been obtained for a role in allelopathy. Here, we apply reverse genetics, using knock-outs of the relevant diterpene synthases (OsCPS4 and OsKSL4), to demonstrate that rice momilactones are involved in allelopathy, including suppressing growth of the widespread rice paddy weed, barnyard grass (Echinochloa crus-galli).Thus, our results not only provide novel genetic evidence for natural product mediated allelopathy, but also furnish a molecular target for breeding and metabolic engineering of this important crop plant.
PMCID: PMC3257406  PMID: 22150231
allelopathy; biosynthetic gene cluster; diterpenoids; momilactones; rice (Oryza sativa); weed suppression
9.  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.
PMCID: PMC3237789  PMID: 21999670
10.  Functional characterization and evolution of the isotuberculosinol operon in Mycobacterium tuberculosis and related Mycobacteria 
Terpenoid metabolites are important to the cellular function, structural integrity, and pathogenesis of the human-specific pathogen Mycobacterium tuberculosis (Mtb). Genetic and biochemical investigations have indicated a role for the diterpenoid isotuberculosinol (isoTb) early in the infection process. There are only two genes (Rv3377c and Rv3378c) required for production of isoTb, yet these are found in what appears to be a five-gene terpenoid/isoprenoid biosynthetic operon. Of the three remaining genes (Rv3379c, Rv3382c, and Rv3383c), previous work has indicated that Rv3379c is an inactive pseudo-gene. Here we demonstrate that Rv3382c and Rv3383c encode biochemically redundant machinery for isoprenoid metabolism, encoding a functional 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (LytB) for isoprenoid precursor production and a geranylgeranyl diphosphate (GGPP) synthase, respectively, for which the Mtb genome contains other functional isozymes (Rv1110 and Rv0562, respectively). These results complete the characterization of the isoTb biosynthetic operon, as well as further elucidating isoprenoid metabolism in Mtb. In addition, we have investigated the evolutionary origin of this operon, revealing Mtb-specific conservation of the diterpene synthase genes responsible for isoTb biosynthesis, which supports our previously advanced hypothesis that isoTb acts as a human-specific pathogenic metabolite and is consistent with the human host specificity of Mtb. Intriguingly, our results revealed that many mycobacteria contain orthologs for both Rv3383c and Rv0562, suggesting a potentially important role for these functionally redundant GGPP synthases in the evolution of terpenoid/isoprenoid metabolism in the mycobacteria.
PMCID: PMC3470408  PMID: 23091471
isoprenoid biosynthesis; molecular evolution; virulence; terpenoids
12.  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.
Electronic supplementary material
The online version of this article (doi:10.1007/s00253-009-2219-x) contains supplementary material, which is available to authorized users.
PMCID: PMC2811251  PMID: 19777230
Terpenoid; Natural products biosynthesis; Metabolic engineering; Isoprenoid
13.  Characterization and Inhibition of a Class II Diterpene Cyclase from Mycobacterium tuberculosis 
The Journal of Biological Chemistry  2009;284(35):23574-23579.
Mycobacterium tuberculosis remains a widespread and devastating human pathogen, whose ability to infiltrate macrophage host cells from the human immune system is an active area of investigation. We have recently reported the discovery of a novel diterpene from M. tuberculosis, edaxadiene, whose ability to arrest phagosomal maturation in isolation presumably contributes to this critical process in M. tuberculosis infections. (Mann, F. M., Xu, M., Chen, X., Fulton, D. B., Russell, D. G., and Peters, R. J. (2009) J. Am. Chem. Soc., in press). Here, we present characterization of the class II diterpene cyclase that catalyzes the committed step in edaxadiene biosynthesis, i.e. the previously identified halimadienyl-diphosphate synthase (HPS; EC Intriguingly, our kinetic analysis suggests a potential biochemical regulatory mechanism that triggers edaxadiene production upon phagosomal engulfment. Furthermore, we report characterization of potential HPS inhibitors: specifically, two related transition state analogs (15-aza-14,15-dihydrogeranylgeranyl diphosphate (7a) and 15-aza-14,15-dihydrogeranylgeranyl thiolodiphosphate (7b)) that exhibit very tight binding. Although arguably not suitable for clinical use, these nevertheless provide a basis for pharmaceutical design against this intriguing biosynthetic pathway. Finally, we provide evidence indicating that this pathway exists only in M. tuberculosis and is not functional in the closely related Mycobacterium bovis because of an inactivating frameshift in the HPS-encoding gene. Thus, we hypothesize that the inability to produce edaxadiene may be a contributing factor in the decreased infectivity and/or virulence of M. bovis relative to M. tuberculosis in humans.
PMCID: PMC2749132  PMID: 19574210
14.  Edaxadiene: A New Bioactive Diterpene from Mycobacterium tuberculosis 
Journal of the American Chemical Society  2009;131(48):17526-17527.
Mycobacterium tuberculosis remains a widespread and devastating human pathogen. Presented here is the characterization of an atypical class I diterpene cyclase from M. tuberculosis that catalyzes an unusual cyclization reaction in converting the known M. tuberculosis metabolite halimadienyl diphosphate to a further cyclized novel diterpene, which we have termed edaxadiene, as it directly inhibits maturation of the phagosomal compartment in which the bacterium is taken up during infection.
PMCID: PMC2787244  PMID: 19583202

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