High-level diterpene production by transient expression in Nicotiana benthamiana.
Identifieur interne : 000465 ( Main/Exploration ); précédent : 000464; suivant : 000466High-level diterpene production by transient expression in Nicotiana benthamiana.
Auteurs : Kathleen Brückner ; Alain Tissier [Allemagne]Source :
- Plant methods [ 1746-4811 ] ; 2013.
Abstract
BACKGROUND
Characterization of plant terpene synthases is typically done by production of recombinant enzymes in Escherichia coli. This is often difficult due to solubility and codon usage issues. Furthermore, plant terpene synthases which are targeted to the plastids, such as diterpene synthases, have to be shortened in a more or less empirical approach to improve expression. We report here an optimized Agrobacterium-mediated transient expression assay in Nicotiana benthamiana for plant diterpene synthase expression and product analysis.
RESULTS
Agrobacterium-mediated transient expression of plant diterpene synthases in N. benthamiana led to the accumulation of diterpenes within 3 days of infiltration and with a maximum at 5 days. Over 50% of the products were exported onto the leaf surface, thus considerably facilitating the analysis by reducing the complexity of the extracts. The robustness of the method was tested by expressing three different plant enzymes, cembratrien-ol synthase from Nicotiana sylvestris, casbene synthase from Ricinus communis and levopimaradiene synthase from Gingko biloba. Furthermore, co-expression of a 1-deoxy-D-xylulose-5-phosphate synthase from tomato and a geranylgeranyl diphosphate synthase from tobacco led to a 3.5-fold increase in the amount of cembratrien-ol produced, with maximum yields reaching 2500 ng/cm2.
CONCLUSION
With this optimized method for diterpene synthase expression and product analysis, a single infiltrated leaf of N. benthamiana would be sufficient to produce quantities required for the structure elucidation of unknown diterpenes. The method will also be of general use for gene function discovery, pathway reconstitution and metabolic engineering of diterpenoid biosynthesis in plants.
DOI: 10.1186/1746-4811-9-46
PubMed: 24330621
PubMed Central: PMC3878842
Affiliations:
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Alain.Tissier@ipb-halle.de.</nlm:affiliation><country xml:lang="fr">Allemagne</country><wicri:regionArea>Department of Cell and Metabolic Biology, Leibniz Institute of Plant Biochemistry, Weinberg 3, 06120 Halle-Saale</wicri:regionArea><wicri:noRegion>06120 Halle-Saale</wicri:noRegion><wicri:noRegion>06120 Halle-Saale</wicri:noRegion><wicri:noRegion>06120 Halle-Saale</wicri:noRegion></affiliation></author></analytic><series><title level="j">Plant methods</title><idno type="ISSN">1746-4811</idno><imprint><date when="2013" type="published">2013</date></imprint></series></biblStruct></sourceDesc></fileDesc><profileDesc><textClass></textClass></profileDesc></teiHeader><front><div type="abstract" xml:lang="en"><p><b>BACKGROUND</b></p><p>Characterization of plant terpene synthases is typically done by production of recombinant enzymes in Escherichia coli. This is often difficult due to solubility and codon usage issues. Furthermore, plant terpene synthases which are targeted to the plastids, such as diterpene synthases, have to be shortened in a more or less empirical approach to improve expression. We report here an optimized Agrobacterium-mediated transient expression assay in Nicotiana benthamiana for plant diterpene synthase expression and product analysis.</p></div><div type="abstract" xml:lang="en"><p><b>RESULTS</b></p><p>Agrobacterium-mediated transient expression of plant diterpene synthases in N. benthamiana led to the accumulation of diterpenes within 3 days of infiltration and with a maximum at 5 days. Over 50% of the products were exported onto the leaf surface, thus considerably facilitating the analysis by reducing the complexity of the extracts. The robustness of the method was tested by expressing three different plant enzymes, cembratrien-ol synthase from Nicotiana sylvestris, casbene synthase from Ricinus communis and levopimaradiene synthase from Gingko biloba. Furthermore, co-expression of a 1-deoxy-D-xylulose-5-phosphate synthase from tomato and a geranylgeranyl diphosphate synthase from tobacco led to a 3.5-fold increase in the amount of cembratrien-ol produced, with maximum yields reaching 2500 ng/cm2.</p></div><div type="abstract" xml:lang="en"><p><b>CONCLUSION</b></p><p>With this optimized method for diterpene synthase expression and product analysis, a single infiltrated leaf of N. benthamiana would be sufficient to produce quantities required for the structure elucidation of unknown diterpenes. The method will also be of general use for gene function discovery, pathway reconstitution and metabolic engineering of diterpenoid biosynthesis in plants.</p></div></front></TEI><pubmed><MedlineCitation Status="PubMed-not-MEDLINE" Owner="NLM"><PMID Version="1">24330621</PMID><DateCompleted><Year>2013</Year><Month>12</Month><Day>23</Day></DateCompleted><DateRevised><Year>2020</Year><Month>09</Month><Day>29</Day></DateRevised><Article PubModel="Electronic"><Journal><ISSN IssnType="Print">1746-4811</ISSN><JournalIssue CitedMedium="Print"><Volume>9</Volume><Issue>1</Issue><PubDate><Year>2013</Year><Month>Dec</Month><Day>12</Day></PubDate></JournalIssue><Title>Plant methods</Title><ISOAbbreviation>Plant Methods</ISOAbbreviation></Journal><ArticleTitle>High-level diterpene production by transient expression in Nicotiana benthamiana.</ArticleTitle><Pagination><MedlinePgn>46</MedlinePgn></Pagination><ELocationID EIdType="doi" ValidYN="Y">10.1186/1746-4811-9-46</ELocationID><Abstract><AbstractText Label="BACKGROUND" NlmCategory="BACKGROUND">Characterization of plant terpene synthases is typically done by production of recombinant enzymes in Escherichia coli. This is often difficult due to solubility and codon usage issues. Furthermore, plant terpene synthases which are targeted to the plastids, such as diterpene synthases, have to be shortened in a more or less empirical approach to improve expression. We report here an optimized Agrobacterium-mediated transient expression assay in Nicotiana benthamiana for plant diterpene synthase expression and product analysis.</AbstractText><AbstractText Label="RESULTS" NlmCategory="RESULTS">Agrobacterium-mediated transient expression of plant diterpene synthases in N. benthamiana led to the accumulation of diterpenes within 3 days of infiltration and with a maximum at 5 days. Over 50% of the products were exported onto the leaf surface, thus considerably facilitating the analysis by reducing the complexity of the extracts. The robustness of the method was tested by expressing three different plant enzymes, cembratrien-ol synthase from Nicotiana sylvestris, casbene synthase from Ricinus communis and levopimaradiene synthase from Gingko biloba. Furthermore, co-expression of a 1-deoxy-D-xylulose-5-phosphate synthase from tomato and a geranylgeranyl diphosphate synthase from tobacco led to a 3.5-fold increase in the amount of cembratrien-ol produced, with maximum yields reaching 2500 ng/cm2.</AbstractText><AbstractText Label="CONCLUSION" NlmCategory="CONCLUSIONS">With this optimized method for diterpene synthase expression and product analysis, a single infiltrated leaf of N. benthamiana would be sufficient to produce quantities required for the structure elucidation of unknown diterpenes. 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