Nicotiana benthamiana as a Production Platform for Artemisinin Precursors

Background Production of pharmaceuticals in plants provides an alternative for chemical synthesis, fermentation or natural sources. Nicotiana benthamiana is deployed at commercial scale for production of therapeutic proteins. Here the potential of this plant is explored for rapid production of precursors of artemisinin, a sesquiterpenoid compound that is used for malaria treatment. Methodology/Principal Findings Biosynthetic genes leading to artemisinic acid, a precursor of artemisinin, were combined and expressed in N. benthamiana by agro-infiltration. The first committed precursor of artemisinin, amorpha-4,11-diene, was produced upon infiltration of a construct containing amorpha-4,11-diene synthase, accompanied by 3-hydroxy-3-methylglutaryl-CoA reductase and farnesyl diphosphate synthase. Amorpha-4,11-diene was detected both in extracts and in the headspace of the N. benthamiana leaves. When the amorphadiene oxidase CYP71AV1 was co-infiltrated with the amorphadiene-synthesizing construct, the amorpha-4,11-diene levels strongly decreased, suggesting it was oxidized. Surprisingly, no anticipated oxidation products, such as artemisinic acid, were detected upon GC-MS analysis. However, analysis of leaf extracts with a non-targeted metabolomics approach, using LC-QTOF-MS, revealed the presence of another compound, which was identified as artemisinic acid-12-ß-diglucoside. This compound accumulated to 39.5 mg.kg-1 fwt. Apparently the product of the heterologous pathway that was introduced, artemisinic acid, is further metabolized efficiently by glycosyl transferases that are endogenous to N. benthamiana. Conclusion/Significance This work shows that agroinfiltration of N. bentamiana can be used as a model to study the production of sesquiterpenoid pharmaceutical compounds. The interaction between the ectopically introduced pathway and the endogenous metabolism of the plant is discussed

Metatranscriptomics Reveals the Diversity of Genes Expressed by Eukaryotes in Forest Soils

Eukaryotic organisms play essential roles in the biology and fertility of soils. For example the micro and mesofauna contribute to the fragmentation and homogenization of plant organic matter, while its hydrolysis is primarily performed by the fungi. To get a global picture of the activities carried out by soil eukaryotes we sequenced 2×10,000 cDNAs synthesized from polyadenylated mRNA directly extracted from soils sampled in beech (Fagus sylvatica) and spruce (Picea abies) forests. Taxonomic affiliation of both cDNAs and 18S rRNA sequences showed a dominance of sequences from fungi (up to 60%) and metazoans while protists represented less than 12% of the 18S rRNA sequences. Sixty percent of cDNA sequences from beech forest soil and 52% from spruce forest soil had no homologs in the GenBank/EMBL/DDJB protein database. A Gene Ontology term was attributed to 39% and 31.5% of the spruce and beech soil sequences respectively. Altogether 2076 sequences were putative homologs to different enzyme classes participating to 129 KEGG pathways among which several were implicated in the utilisation of soil nutrients such as nitrogen (ammonium, amino acids, oligopeptides), sugars, phosphates and sulfate. Specific annotation of plant cell wall degrading enzymes identified enzymes active on major polymers (cellulose, hemicelluloses, pectin, lignin) and glycoside hydrolases represented 0.5% (beech soil)–0.8% (spruce soil) of the cDNAs. Other sequences coding enzymes active on organic matter (extracellular proteases, lipases, a phytase, P450 monooxygenases) were identified, thus underlining the biotechnological potential of eukaryotic metatranscriptomes. The phylogenetic affiliation of 12 full-length carbohydrate active enzymes showed that most of them were distantly related to sequences from known fungi. For example, a putative GH45 endocellulase was closely associated to molluscan sequences, while a GH7 cellobiohydrolase was closest to crustacean sequences, thus suggesting a potentially significant contribution of non-fungal eukaryotes in the actual hydrolysis of soil organic matter

Aspergillus as a host for heterologous protein production: the problem of proteases.

Homologous and heterologous protein production by filamentous fungi is often limited by the expression of proteases at high levels. By eliminating specific protease activities, protein production in Aspergillus niger can be improved considerably. Both classical mutagenesis and gene disruption techniques have yielded strains with reduced protease expression. Combinations of these mutations and disruptions result in a further reduction of protease activity. The coupling of efficient promoters to target genes allows their expression under conditions that repress the expression of several proteases, which further improves product yields. The strategies used have led to the development of a set of tester strains from which the appropriate genetic background for production can be selected

Aspergillus as a host for heterologous protein production: the problem of proteases.

Homologous and heterologous protein production by filamentous fungi is often limited by the expression of proteases at high levels. By eliminating specific protease activities, protein production in Aspergillus niger can be improved considerably. Both classical mutagenesis and gene disruption techniques have yielded strains with reduced protease expression. Combinations of these mutations and disruptions result in a further reduction of protease activity. The coupling of efficient promoters to target genes allows their expression under conditions that repress the expression of several proteases, which further improves product yields. The strategies used have led to the development of a set of tester strains from which the appropriate genetic background for production can be selected

erformance of the COX1 gene as a marker for the study of metabolically active Pezizomycotina and Agaricomycetes fungal communities from the analysis of soil RNA

International audienceIn temperate forest soils, filamentous ectomycorrhizal and saprotrophic fungi affiliated to the Agaricomycetes and Pezizomycotina contribute to key biological processes. The diversity of soil fungal communities is usually estimated by studying molecular markers such as nuclear ribosomal gene regions amplified from soil-extracted DNA. However, this approach only reveals the presence of the corresponding genomic DNA in the soil sample and may not reflect the diversity of the metabolically active species. To circumvent this problem, we investigated the performance of the mitochondrial cytochrome c oxidase 1 (COX1)-encoding gene as a fungal molecular marker for environmental RNA-based studies. We designed PCR primers to specifically amplify Agaricomycetes and Pezizomycotina COX1 partial sequences and amplified them from both soil DNA and reverse-transcribed soil RNA. As a control, we also amplified the nuclear internal transcribed spacer ribosomal region from soil DNA. Fungal COX1 sequences were readily amplified from soil-extracted nucleic acids and were not significantly contaminated by nontarget sequences. We show that the relative abundance of fungal taxonomic groups differed between the different sequence data sets, with for example ascomycete COX1 sequences being more abundant among sequences amplified from soil DNA than from soil cDNAs

Tansley review Hebeloma cylindrosporum -a model species to study ectomycorrhizal symbiosis from gene to ecosystem

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