Highlighting type A RRs as potential regulators of the dkHK1 multi-step phosphorelay pathway in Populus

In previous studies, we highlighted a multistep phosphorelay (MSP) system in poplars composed of two hybrid-type Histidine aspartate Kinases, dkHK1a and dkHK1b, which interact with three Histidine Phosphotransfer proteins, dkHPt2, 7, and 9, which in turn interact with six type B Response Regulators. These interactions correspond to the dkHK1a-b/dkHPts/dkRRBs MSP. This MSP is putatively involved in an osmosensing pathway, as dkHK1a-b are orthologous to the Arabidopsis osmosensor AHK1, and able to complement a mutant yeast deleted for its osmosensors. Since type A RRs have been characterized as negative regulators in cytokinin MSP signaling due to their interaction with HPt proteins, we decided in this study to characterize poplar type A RRs and their implication in the MSP. For a global view of this MSP, we isolated 10 poplar type A RR cDNAs, and determined their subcellular localization to check the in silico prediction experimentally. For most of them, the in planta subcellular localization was as predicted, except for three RRAs, for which this experimental approach gave a more precise localization. Interaction studies using yeast two-hybrid and in planta BiFC assays, together with transcript expression analysis in poplar organs led to eight dkRRAs being singled out as partners which could interfere the dkHK1a-b/dkHPts/dkRRBs MSP identified in previous studies. Consequently, the results obtained in this study now provide an exhaustive view of dkHK1a-b partners belonging to a poplar MSP

A microbial supply chain for production of the anti-cancer drug vinblastine

International audienceAbstract Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine 1 . As MIAs are difficult to chemically synthesize, the world’s supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus , which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale 2,3 . Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues

DNA Dosimetry Assessment for Sunscreen Genotoxic Photoprotection

Background: Due to the increase of solar ultraviolet radiation (UV) incidence over the last few decades, the use of sunscreen has been widely adopted for skin protection. However, considering the high efficiency of sunlight-induced DNA lesions, it is critical to improve upon the current approaches that are used to evaluate protection factors. An alternative approach to evaluate the photoprotection provided by sunscreens against daily UV radiation-induced DNA damage is provided by the systematic use of a DNA dosimeter. Methodology/Principal Findings: The Sun Protection Factor for DNA (DNA-SPF) is calculated by using specific DNA repair enzymes, and it is defined as the capacity for inhibiting the generation of cyclobutane pyrimidine dimers (CPD) and oxidised DNA bases compared with unprotected control samples. Five different commercial brands of sunscreen were initially evaluated, and further studies extended the analysis to include 17 other products representing various formulations and Sun Protection Factors (SPF). Overall, all of the commercial brands of SPF 30 sunscreens provided sufficient protection against simulated sunlight genotoxicity. In addition, this DNA biosensor was useful for rapidly screening the biological protection properties of the various sunscreen formulations. Conclusions/Significance: The application of the DNA dosimeter is demonstrated as an alternative, complementary, and reliable method for the quantification of sunscreen photoprotection at the level of DNA damage.Natura Inovacao e Tecnologia de Produtos LTDA (Sao Paulo, Brazil)Natura Inovacao e Tecnologia de Produtos LTDA (Sao Paulo, Brazil)FAPESP (Sao Paulo, Brazil)FAPESP (Sao Paulo, Brazil)CNPq (Brasilia, Brazil)CNPq (Brasilia, Brazil)Natura Inovacao e Tecnologia de Produtos LTDANatura Inovacao e Tecnologia de Produtos LTD

Antifungal Activity of Resveratrol Derivatives against Candida Species

trans-Resveratrol (1a) is a phytoalexin produced by plants in response to infections by pathogens. Its potential activity against clinically relevant opportunistic fungal pathogens has previously been poorly investigated. Evaluated herein are the candidacidal activities of oligomers (2a, 3–5) of 1a purified from Vitis vinifera grape canes and several analogues (1b–1j) of 1a obtained through semisynthesis using methylation and acetylation. Moreover, trans-ε-viniferin (2a), a dimer of 1a, was also subjected to methylation (2b) and acetylation (2c) under nonselective conditions. Neither the natural oligomers of 1a (2a, 3–5) nor the derivatives of 2a were active against Candida albicans SC5314. However, the dimethoxy resveratrol derivatives 1d and 1e exhibited antifungal activity against C. albicans with minimum inhibitory concentration (MIC) values of 29–37 μg/mL and against 11 other Candida species. Compound 1e inhibited the yeast-to-hyphae morphogenetic transition of C. albicans at 14 μg/mL

Larger yield of cyclobutane dimers than 8-oxo-7,8-dihydroguanine in the DNA of UVA-irradiated human skin cells

International audienc

Characterization of an autonomously replicating sequence inCandida guilliermondii

International audiencetCandida guilliermondii is an ascomycetous yeast widely studied due to its clinical importance, biotech-nological interest, and biological control potential. During a series of preliminary experiments aimingat optimizing the electroporation procedure of C. guilliermondii cells, we observed that the efficiencyof transformation of an ura5 recipient strain with the corresponding dominant marker URA5 was morethan a thousand fold higher as compared with the transformation of an ura3 strain with the URA3 wildtype allele. This result allowed the identification of an autonomously replicating sequence (ARS) withinan A/T rich region located upstream of the URA5 open reading frame (ORF). Interestingly, linear doublestrand DNAs (dsDNAs) containing this putative ARS are circularized and then autonomously replicatedin C. guilliermondii transformed cells. We demonstrated that the C. guilliermondii Lig4p ligase, involved inthe canonical non-homologous end-joining (NHEJ) pathway, was responsible for this phenomenon sincea lig4 mutant was unable to circularize and to autonomously maintain transforming dsDNAs containingthe putative ARS. Finally, a functional dissection of the C. guilliermondii A/T rich region located upstreamof the URA5 ORF revealed the presence of a 60 bp-length sequence essential and sufficient to confer ARSproperties to shuttle plasmid and linear dsDNAs

Antifungal Activity of Resveratrol Derivatives against Candida Species

International audiencetrans-Resveratrol (1a) is a phytoalexin produced by plants in response to infections by pathogens. Its potential activity against clinically relevant opportunistic fungal pathogens has previously been poorly investigated. Evaluated herein are the candidacidal activities of oligomers (2a, 3−5) of 1a purified from Vitis vinifera grape canes and several analogues (1b−1j) of 1a obtained through semisynthesis using methylation and acetylation. Moreover, trans-ε-viniferin (2a), a dimer of 1a, was also subjected to methylation (2b) and acetylation (2c) under nonselective conditions. Neither the natural oligomers of 1a (2a, 3−5) nor the derivatives of 2a were active against Candida albicans SC5314. However, the dimethoxy resveratrol derivatives 1d and 1e exhibited antifungal activity against C. albicans with minimum inhibitory concentration (MIC) values of 29−37 μg/mL and against 11 other Candida species. Compound 1e inhibited the yeast-to-hyphae morphogenetic transition of C. albicans at 14 μg/mL. trans Resveratrol (1a) is a natural stilbenoid produced by several plants in response to pathogenic infections. Although present in a restricted number of plant families, 1a forms the backbone of a wide range of stilbene biosynthetic products, leading to a spectrum of monomeric and oligomeric compounds with diverse biological activities.1,2 Stilbenes were first described for their fungicidal action against plant fungal diseases by Langcake and Pryce.3 Later, antimicrobial activities of stilbenes were reported against various phytopathogens, with 1a occurring as a major phytoalexin of the family Vitaceae.4,5 Resveratrol fully inhibits conidial germination of the gray mold agent Botrytis cinerea6 and reduces roughly 75% sporangial germination of the causal agent of grapevine downy mildew, Plasmopara viticola.7 Pterostilbene, the 3,5-dimethoxyresveratrol (1e), and trans ε-viniferin (2a) have been found to be 5- fold more active than 1a, indicating the potential of resveratrol derivatives as a source of effective antifungal agents.8,9 In the field of human health, several investigations have been conducted on 1a due to its multiple pharmacological activities including cardioprotective, antiaging, anticarcinogenic, antiinflammatory, estrogenic/antiestrogenic

Cycloheximide as a tool to investigate protein import in peroxisomes: a case study of the subcellular localization of isoprenoid biosynthetic enzymes.

International audienceCytosolic background fluorescence is often observed when native low-abundance peroxisomal proteins carrying a weak peroxisomal targeting sequence are expressed as fluorescent fusion protein using a strong constitutive promoter in transiently transformed plant cells. This cytosolic fluorescence usually comes from the strong expression of the low-abundance proteins exceeding the peroxisome import efficiency. This often results in a misinterpretation of the protein subcellular localization, as there is doubt as to whether proteins are dually targeted to the cytosol and peroxisome or are exclusively localized to peroxisomes. To circumvent this experimental difficulty, the protein peroxisome import study can be optimized by de novo protein synthesis inhibition in transiently transformed cells using the translation inhibitor cycloheximide. This approach was used here successfully for the study of the subcellular localization of distinct plant isoprenoid biosynthetic enzymes, allowing us to clearly demonstrate that 5-phosphomevalonate kinase, mevalonate 5-diphosphate decarboxylase and a short isoform of farnesyl diphosphate synthase from Catharanthus roseus are exclusively localized to peroxisomes