The biosynthesis of thymol, carvacrol, and thymohydroquinone in Lamiaceae proceeds via cytochrome P450s and a short-chain dehydrogenase

Thymol and carvacrol are phenolic monoterpenes found in thyme, oregano, and several other species of the Lamiaceae. Long valued for their smell and taste, these substances also have antibacterial and anti-spasmolytic properties. They are also suggested to be precursors of thymohydroquinone and thymoquinone, monoterpenes with anti-inflammatory, antioxidant, and antitumor activities. Thymol and carvacrol biosynthesis has been proposed to proceed by the cyclization of geranyl diphosphate to γ-terpinene, followed by a series of oxidations via p-cymene. Here, we show that γ-terpinene is oxidized by cytochrome P450 monooxygenases (P450s) of the CYP71D subfamily to produce unstable cyclohexadienol intermediates, which are then dehydrogenated by a short-chain dehydrogenase/reductase (SDR) to the corresponding ketones. The subsequent formation of the aromatic compounds occurs via keto–enol tautomerisms. Combining these enzymes with γ-terpinene in in vitro assays or in vivo in Nicotiana benthamiana yielded thymol and carvacrol as products. In the absence of the SDRs, only p-cymene was formed by rearrangement of the cyclohexadienol intermediates. The nature of these unstable intermediates was inferred from reactions with the γ-terpinene isomer limonene and by analogy to reactions catalyzed by related enzymes. We also identified and characterized two P450s of the CYP76S and CYP736A subfamilies that catalyze the hydroxylation of thymol and carvacrol to thymohydroquinone when heterologously expressed in yeast and N. benthamiana. Our findings alter previous views of thymol and carvacrol formation, identify the enzymes involved in the biosynthesis of these phenolic monoterpenes and thymohydroquinone in the Lamiaceae, and provide targets for metabolic engineering of high-value terpenes in plants.

Comparative Phylogenomic Analysis of Cytochrome P450 Monooxygenases From Fusarium Species

Cytochrome P450s (P450s) are a unique multifamily class of enzymes that possess the capability to exhibit catalytic versatility in several biochemical reactions which entails metabolite biosynthesis, primary and secondary metabolism. Fusarium spp. is an important microorganism with many members known to produce secondary metabolites that cause plant diseases and mycotoxicoses in animals and humans. In this present study, from the initially screened 4,579 proteins, we elucidated the nature of abundance, evolutionary relationships, classification and cellular location of 320 cytochrome P450 from 17 phytopathogenic members of Fusarium species. The total CYPs protein sequences were phylogenetically grouped into seventeen (17) clades. Eighty-six (86) CYPs families and forty-eight (48) clans were identified. Twenty-seven (27) families were each found in only one species. The CYPs were found to be majorly localized in the endoplasmic reticulum. The non-ribosomal peptide synthetase-like (NRPS-like) gene cluster was the predominant secondary metabolic-related gene cluster across all the seventeen selected Fusarium species except in F. cucurbiticola and F. solani, where PolyKetide Synthase (PKS) was the most prevalent. The presence of numerous families and clans as observed in in this study shows the expansions of the CYPs families across Fusarium species, this CYPs family and clan expansion is often associated with the evolvement of several fungal traits that include their pathogenicity adaptation to survive on an extensive range of toxic substrates. Identification of P450 proteins in these pathogenic fungi provides fundamental information for further basic and applied biological research into the physiological and toxigenic roles of P450s in Fusarium species.

Discovering and harnessing oxidative enzymes for chemoenzymatic synthesis and diversification of anticancer camptothecin analogues

Semi-synthetic derivatives of camptothecin, a quinoline alkaloid found in the Camptotheca acuminata tree, are potent anticancer agents. Here we discovered two C. acuminata cytochrome P450 monooxygenases that catalyze regio-specific 10- and 11-oxidations of camptothecin, and demonstrated combinatorial chemoenzymatic C-H functionalizations of the camptothecin scaffold using the new enzymes to produce a suite of anticancer drugs, including topotecan (Hycamtin®) and irinotecan (Camptosar®). This work sheds new light into camptothecin metabolism, and represents greener approaches for accessing clinically relevant camptothecin derivatives.

Identification and characterization of a novel class of self-sufficient cytochrome P450 hydroxylase involved in cyclohexanecarboxylate degradation in Paraburkholderia terrae strain KU-64

ABSTRACT Cytochrome P450 monooxygenases play important roles in metabolism. Here, we report the identification and biochemical characterization of P450CHC, a novel self-sufficient cytochrome P450, from cyclohexanecarboxylate-degrading Paraburkholderia terrae KU-64. P450CHC was found to comprise a [2Fe-2S] ferredoxin domain, NAD(P)H-dependent FAD-containing reductase domain, FCD domain, and cytochrome P450 domain (in that order from the N terminus). Reverse transcription–polymerase chain reaction results indicated that the P450CHC-encoding chcA gene was inducible by cyclohexanecarboxylate. chcA overexpression in Escherichia coli and recombinant protein purification enabled functional characterization of P450CHC as a catalytically self-sufficient cytochrome P450 that hydroxylates cyclohexanecarboxylate. Kinetic analysis indicated that P450CHC largely preferred NADH (Km = 0.011 m m) over NADPH (Km = 0.21 m m). The Kd, Km, and kcat values for cyclohexanecarboxylate were 0.083 m m, 0.084 m m, and 15.9 s−1, respectively. The genetic and biochemical analyses indicated that the physiological role of P450CHC is initial hydroxylation in the cyclohexanecarboxylate degradation pathway.

Insect Resistance to Neonicotinoids - Current Status, Mechanism and Management Strategies

Pesticides are any substance used for controlling, preventing, destroying, repelling, or mitigating of pests. Neonicotinoids have been the most commonly used insecticide since the early 1990s, current market share of more than 25% of total global insecticide sales. Neonicotinoid insecticides are highly selective agonists of insect nicotinic acetylcholine receptors (nAChRs) that exhibit physicochemical properties, rendering them more useful over other classes of insecticides. This includes having a wide range of application techniques and efficacy in controlling sucking and biting insects. Although neonicotinoids are applied as foliar insecticides with possible direct exposure risks to honeybees, a large part of neonicotinoid use consists of seed coating or root drench application. There are three major detoxification enzymes involved in the development of resistance against insecticides viz., cytochrome P450 monooxygenases, carboxylesterases, and glutathione S-transferases. The repeatedly used use of compounds of the same active ingredients and application of excessive organophosphates (OPs) and pyrethroids in Bemisia tabaci. Resistance to insecticides resulting in loss of efficacy of many older insecticides has placed excessive pressure on novel products. One of the major limitations to resistance management is the occurrence of cross-resistance. This review briefly summarizes the current status of neonicotinoid resistance, the biochemical and mechanisms involved, and the implications for resistance management.

Conversion of viridicatic acid to crustosic acid by cytochrome P450 enzyme-catalysed hydroxylation and spontaneous cyclisation

Cytochrome P450 monooxygenases (P450s) are considered nature’s most versatile catalysts and play a crucial role in regio- and stereoselective oxidation reactions on a broad range of organic molecules. The oxyfunctionalisation of unactivated carbon-hydrogen (C-H) bonds, in particular, represents a key step in the biosynthesis of many natural products as it provides substrates with increased reactivity for tailoring reactions. In this study, we investigated the function of the P450 enzyme TraB in the terrestric acid biosynthetic pathway. We firstly deleted the gene coding for the DNA repair subunit protein Ku70 by using split marker-based deletion plasmids for convenient recycling of the selection marker to improve gene targeting in Penicillium crustosum. Hereby, we reduced ectopic DNA integration and facilitated genetic manipulation in P. crustosum. Afterward, gene deletion in the Δku70 mutant of the native producer P. crustosum and heterologous expression in Aspergillus nidulans with precursor feeding proved the involvement of TraB in the formation of crustosic acid by catalysing the essential hydroxylation reaction of viridicatic acid. Key points •Deletion of Ku70 by using split marker approach for selection marker recycling. •Functional identification of the cytochrome P450 enzyme TraB. •Fulfilling the reaction steps in the terrestric acid biosynthesis.

Evidence for Lignocellulose-Decomposing Enzymes in the Genome and Transcriptome of the Aquatic Hyphomycete Clavariopsis aquatica

Fungi are ecologically outstanding decomposers of lignocellulose. Fungal lignocellulose degradation is prominent in saprotrophic Ascomycota and Basidiomycota of the subkingdom Dikarya. Despite ascomycetes dominating the Dikarya inventory of aquatic environments, genome and transcriptome data relating to enzymes involved in lignocellulose decay remain limited to terrestrial representatives of these phyla. We sequenced the genome of an exclusively aquatic ascomycete (the aquatic hyphomycete Clavariopsis aquatica), documented the presence of genes for the modification of lignocellulose and its constituents, and compared differential gene expression between C. aquatica cultivated on lignocellulosic and sugar-rich substrates. We identified potential peroxidases, laccases, and cytochrome P450 monooxygenases, several of which were differentially expressed when experimentally grown on different substrates. Additionally, we found indications for the regulation of pathways for cellulose and hemicellulose degradation. Our results suggest that C. aquatica is able to modify lignin to some extent, detoxify aromatic lignin constituents, or both. Such characteristics would be expected to facilitate the use of carbohydrate components of lignocellulose as carbon and energy sources.

The Role of Cytochrome P450s in Insect Toxicology and Resistance

Insect cytochrome P450 monooxygenases (P450s) perform a variety of important physiological functions, but it is their role in the detoxification of xenobiotics, such as natural and synthetic insecticides, that is the topic of this review. Recent advances in insect genomics and postgenomic functional approaches have provided an unprecedented opportunity to understand the evolution of insect P450s and their role in insect toxicology. These approaches have also been harnessed to provide new insights into the genomic alterations that lead to insecticide resistance, the mechanisms by which P450s are regulated, and the functional determinants of P450-mediated insecticide resistance. In parallel, an emerging body of work on the role of P450s in defining the sensitivity of beneficial insects to insecticides has been developed. The knowledge gained from these studies has applications for the management of P450-mediated resistance in insect pests and can be leveraged to safeguard the health of important beneficial insects. Expected final online publication date for the Annual Review of Entomology, Volume 67 is January 2022. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Synthesis of (−)−deoxypodophyllotoxin and (−)−epipodophyllotoxin via a multi-enzyme cascade in E. coli

Background The aryltetralin lignan (−)−podophyllotoxin is a potent antiviral and anti-neoplastic compound that is mainly found in Podophyllum plant species. Over the years, the commercial demand for this compound rose notably because of the high clinical importance of its semi-synthetic chemotherapeutic derivatives etoposide and teniposide. To satisfy this demand, (−)−podophyllotoxin is conventionally isolated from the roots and rhizomes of Sinopodophyllum hexandrum, which can only grow in few regions and is now endangered by overexploitation and environmental damage. For these reasons, targeting the biosynthesis of (−)−podophyllotoxin precursors or analogues is fundamental for the development of novel, more sustainable supply routes. Results We recently established a four-step multi-enzyme cascade to convert (+)−pinoresinol into (−)−matairesinol in E. coli. Herein, a five-step multi-enzyme biotransformation of (−)−matairesinol to (−)−deoxypodophyllotoxin was proven effective with 98 % yield at a concentration of 78 mg/L. Furthermore, the extension of this cascade to a sixth step leading to (−)−epipodophyllotoxin was evaluated. To this end, seven enzymes were combined in the reconstituted pathway involving inter alia three plant cytochrome P450 monooxygenases, with two of them being functionally expressed in E. coli for the first time. Conclusions Both, (−)−deoxypodophyllotoxin and (−)−epipodophyllotoxin, are direct precursors to etoposide and teniposide. Thus, the reconstitution of biosynthetic reactions of Sinopodophyllum hexandrum as an effective multi-enzyme cascade in E. coli represents a solid step forward towards a more sustainable production of these essential pharmaceuticals.

The Basis of Tolerance Mechanism to Metsulfuron-Methyl in Roegneria kamoji (Triticeae: Poaceae)

Roegneria kamoji, a perennial monocot weed that belongs to the tribe Triticeae (family: Poaceae), is an emerging problematic weed in winter wheat (Triticum aestivum) fields in China. We have previously confirmed four R. kamoji populations tolerant to acetyl-CoA carboxylase (ACCase) inhibitors, and failed control of these populations by metsulfuron-methyl was observed. The objective of this study was to characterize the level of tolerance to metsulfuron-methyl, the basis of tolerance mechanism, and cross-tolerance to acetolactate synthase (ALS) inhibitors in R. kamoji. A whole-plant dose–response assay showed that plants of all R. kamoji populations (both from wheat fields and uncultivated areas) exhibited high tolerance to metsulfuron-methyl, based on their 100% survival at 6-fold recommended field dose (RFD) and ED50 values >6.84-fold RFD, no susceptible population was found. Gene sequencing indicated that no reported amino acid substitutions associated with resistance to ALS inhibitor were found in the ALS gene among the R. kamoji populations. Pretreatment with the known cytochrome P450 monooxygenases (CytP450) inhibitor malathion reduced the ED50 values of metsulfuron-methyl in two R. kamoji populations. These populations also exhibited cross-tolerance to RFD of mesosulfuron-methyl and bispyribac-sodium. The activities of glutathione-S-transferase (GST) and CytP450 could be induced by metsulfuron-methyl in R. kamoji, which is similar to the known tolerant crop wheat. This is the first report elucidating metsulfuron-methyl tolerance in R. kamoji. The reversal of tolerance by malathion and the GST and/or CytP450 enhanced herbicide metabolism suggests that non-target-site mechanisms confer tolerance to metsulfuron-methyl in R. kamoji.