Carnosic acid and carnosol, two supra-antioxidant of rosemary (Rosmarinus officinalis) : roles, mecanisms, physiology and applications

L’acide carnosique (CA) et le carnosol (CARN), deux diterpènes spécifiques des Lamiacées, sont en abondance dans le romarin. Le CA extrait de cette plante est largement utilisé dans l’industrie pour ses propriétés antioxydantes portées par le groupe catéchol. Malgré beaucoup d’applications, les rôles et les modes d'action de ces composés in planta n’ont reçu que peu d’attention. Des analyses par HPLC-UV et imagerie d’autoluminescence révèlent que le CA et le CARN protègent les lipides contre des oxydations in vitro par les ERO. Lors de la préservation des lipides, des analyses de MS indiquent que le CA est oxydé en une variété de dérivés alors que le CARN résiste. L’utilisation d’une sonde de spin et de la spectroscopie RPE montre que le CA est un piégeur chimique des ERO. L’action inhibitrice du CARN sur des oxydations de lipides induites in vitro ou in vivo indique que le CARN interfère avec le processus de peroxydation lipidique. Des études in vivo de deux variétés de romarin contrastées en CA exposées à un stress photooxydant montrent que le CA protège les lipides in planta. Une étude des variations de CA et de CARN en fonctions des facteurs abiotiques met en avant qu’une forte intensité lumineuse et des fluctuations de températures favorisent la réponse antioxydante du CA qui s’oxyde en CARN. Des analyses de RT-qPCR montrent que les facteurs abiotiques ne stimulent pas la voie de biosynthèse du CA. En condition de stress oxydant, le CA du romarin préserve les membranes par piégeage des ERO produisant des dérivés d’oxydation, dont le CARN, qui protègent aussi les membranes en bloquant la réaction en chaîne de la peroxydation lipidique.Carnosic acid (CA) and carnosol (CARN), two diterpenes specific of the Lamiaceae, are highly abundant in rosemary species. CA extracted from rosemary is used by industries for its antioxidative features, endowed by it catechol group. Despite numerous applications, the role and the mode of action of CA in planta has received little attention. Analyses, using HPLC-UV and luminescence imaging revealed that CA and CARN protect lipids from in vitro oxidation by ROS. Upon ROS oxidation of lipids, MS analyses indicated that CA was oxidized into various derivatives while CARN resisted. Using spin probes and EPR detection, we confirmed that CA, rather than CARN, is a ROS quencher. The inhibitory effect of CARN on lipid peroxidation induced in vitro or in vivo indicated that CARN interferes with lipid peroxidation. In vivo studies of two rosemary varieties contrasted in their CA content exposed to photooxidative stress showed that CA protects lipids in planta. A study of CA and CARN variations in response to abiotic factors showed that high light and temperature fluctuations lead to CA oxidation into CARN. RT-qPCR analyses revealed that abiotic factors do not stimulate CA biosynthesis genes. Under oxidative stress condition, rosemary CA preserves biological membranes by ROS scavenging, hence producing a set of oxidative derivatives, including CARN, which protect biological membranes by blocking the lipid peroxidation chain reaction

Carnosic Acid and Carnosol, Two Major Antioxidants of Rosemary, Act through Different Mechanisms

International audienceCarnosic acid, a phenolic diterpene specific to the Lamiaceae family, is highly abundant in rosemary (Rosmarinus officinalis). Despite numerous industrial and medicinal/pharmaceutical applications of its antioxidative features, this compound in planta and its antioxidant mechanism have received little attention, except a few studies of rosemary plants under natural conditions. In vitro analyses, using high-performance liquid chromatography-ultraviolet and luminescence imaging, revealed that carnosic acid and its major oxidized derivative, carnosol, protect lipids from oxidation. Both compounds preserved linolenic acid and monogalactosyldiacylglycerol from singlet oxygen and from hydroxyl radical. When applied exogenously, they were both able to protect thylakoid membranes prepared from Arabidopsis (Arabidopsis thaliana) leaves against lipid peroxidation. Different levels of carnosic acid and carnosol in two contrasting rosemary varieties correlated with tolerance to lipid peroxidation. Upon reactive oxygen species (ROS) oxidation of lipids, carnosic acid was consumed and oxidized into various derivatives, including into carnosol, while carnosol resisted, suggesting that carnosic acid is a chemical quencher of ROS. The antioxidative function of carnosol relies on another mechanism, occurring directly in the lipid oxidation process. Under oxidative conditions that did not involve ROS generation, carnosol inhibited lipid peroxidation, contrary to carnosic acid. Using spin probes and electron paramagnetic resonance detection, we confirmed that carnosic acid, rather than carnosol, is a ROS quencher. Various oxidized derivatives of carnosic acid were detected in rosemary leaves in low light, indicating chronic oxidation of this compound, and accumulated in plants exposed to stress conditions, in parallel with a loss of carnosic acid, confirming that chemical quenching of ROS by carnosic acid takes place in planta

An HMM approach expands the landscape of sesquiterpene cyclases across the kingdom Fungi.

Sesquiterpene cyclases (STC) catalyse the cyclization of the C15 molecule farnesyl diphosphate into a vast variety of mono- or polycyclic hydrocarbons and, for a few enzymes, oxygenated structures, with diverse stereogenic centres. The huge diversity in sesquiterpene skeleton structures in nature is primarily the result of the type of cyclization driven by the STC. Despite the phenomenal impact of fungal sesquiterpenes on the ecology of fungi and their potentials for applications, the fungal sesquiterpenome is largely untapped. The identification of fungal STC is generally based on protein sequence similarity with characterized enzymes. This approach has improved our knowledge on STC in a few fungal species, but it has limited success for the discovery of distant sequences. Besides, the tools based on secondary metabolite biosynthesis gene clusters have shown poor performance for terpene cyclases. Here, we used four sets of sequences of fungal STC that catalyse four types of cyclization, and specific amino acid motives to identify phylogenetically related sequences in the genomes of basidiomycetes fungi from the order Polyporales. We validated that four STC genes newly identified from the genome sequence of Leiotrametes menziesii, each classified in a different phylogenetic clade, catalysed a predicted cyclization of farnesyl diphosphate. We built HMM models and searched STC genes in 656 fungal genomes genomes. We identified 5605 STC genes, which were classified in one of the four clades and had a predicted cyclization mechanism. We noticed that the HMM models were more accurate for the prediction of the type of cyclization catalysed by basidiomycete STC than for ascomycete STC

An HMM approach expands the landscape of sesquiterpene cyclases across the kingdom Fungi

International audienceSesquiterpene cyclases (STC) catalyse the cyclization of the C15 molecule farnesyl diphosphate into a vast variety of mono- or polycyclic hydrocarbons and, for a few enzymes, oxygenated structures, with diverse stereogenic centres. The huge diversity in sesquiterpene skeleton structures in nature is primarily the result of the type of cyclization driven by the STC. Despite the phenomenal impact of fungal sesquiterpenes on the ecology of fungi and their potentials for applications, the fungal sesquiterpenome is largely untapped. The identification of fungal STC is generally based on protein sequence similarity with characterized enzymes. This approach has improved our knowledge on STC in a few fungal species, but it has limited success for the discovery of distant sequences. Besides, the tools based on secondary metabolite biosynthesis gene clusters have shown poor performance for terpene cyclases. Here, we used four sets of sequences of fungal STC that catalyse four types of cyclization, and specific amino acid motives to identify phylogenetically related sequences in the genomes of basidiomycetes fungi from the order Polyporales. We validated that four STC genes newly identified from the genome sequence of Leiotrametes menziesii , each classified in a different phylogenetic clade, catalysed a predicted cyclization of farnesyl diphosphate. We built HMM models and searched STC genes in 656 fungal genomes genomes. We identified 5605 STC genes, which were classified in one of the four clades and had a predicted cyclization mechanism. We noticed that the HMM models were more accurate for the prediction of the type of cyclization catalysed by basidiomycete STC than for ascomycete STC

In vitro Applications of the Terpene Mini‐Path 2.0

International audienceIn 2019 four groups reported independently the development of a simplified enzymatic access to the diphosphates (IPP and DMAPP) of isopentenol and dimethylallyl alcohol (IOH and DMAOH). The former are the two universal precursors of all terpenes. We report here on an improved version of what we call the terpene mini-path as well as its use in enzymatic cascades in combination with various transferases. The goal of this study is to demonstrate the in vitro utility of the TMP in, i) synthesizing various natural terpenes, ii) revealing the product selectivity of an unknown terpene synthase, or iii) generating unnatural cyclobutylated terpenes