ArticlePlant biotechnology journal2021
Developing a Nicotiana benthamiana transgenic platform for high-value diterpene production and candidate gene evaluation.
Article in Plant biotechnology journal, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 22 papers.
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22 citing papers in PubMed, 55 citations in OpenAlex.
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- Discovery of active mouse, plant and fungal cytochrome P450s in endogenous proteomes and upon expression in planta.Scientific reports · 2024Article
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- From qualitative to quantitative: the state of the art and challenges for plant synthetic biology.Quantitative biology (Beijing, China) · 2023Review
- An Engineered Heat-Inducible Expression System for the Production of Casbene inInternational journal of molecular sciences · 2023Article
- Chromosome-level Genome Assembly of Euphorbia peplus, a Model System for Plant Latex, Reveals that Relative Lack of Ty3 Transposons Contributed to Its Small Genome Size.Genome biology and evolution · 2023Article
- Engineering the Biosynthesis of Late-Stage Vinblastine Precursors Precondylocarpine Acetate, Catharanthine, Tabersonine inACS synthetic biology · 2023Article
- Identification of Gene Biomarkers for Tigilanol Tiglate Content inMolecules (Basel, Switzerland) · 2022Article
- Gene discovery and virus-induced gene silencing reveal branched pathways to major classes of bioactive diterpenoids inProceedings of the National Academy of Sciences of the United States of America · 2022Article
- Plant-based expression platforms to produce high-value metabolites and proteins.Frontiers in plant science · 2022Review
- CsAlaDC and CsTSI work coordinately to determine theanine biosynthesis in tea plants (Camellia sinensis L.) and confer high levels of theanine accumulation in a non-tea plant.Plant biotechnology journal · 2021Article
- Synthetic biology of plant natural products: From pathway elucidation to engineered biosynthesis in plant cells.Plant communications · 2021Review
- Developing a Nicotiana benthamiana transgenic platform for high-value diterpene production and candidate gene evaluation.Plant biotechnology journal · 2021Article
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Authors and funding
7 authors at 2 institutions in 1 country.
Funding
Abstract
To engineer Nicotiana benthamiana to produce novel diterpenoids, we first aimed to increase production of the diterpenoid precursor geranylgeranyl pyrophosphate (GGPP) by up-regulation of key genes of the non-mevalonate (MEP) pathway sourced from Arabidopsis thaliana. We used transient expression to evaluate combinations of the eight MEP pathway genes plus GGPP synthase and a Jatropha curcas casbene synthase (JcCAS) to identify an optimal combination for production of casbene from GGPP. AtDXS and AtHDR together with AtGGPPS and JcCAS gave a 410% increase in casbene production compared to transient expression of JcCAS alone. This combination was cloned into a single construct using the MoClo toolkit, and stably integrated into the N. benthamiana genome. We also created multigene constructs for stable transformation of two J. curcas cytochrome P450 genes, JcCYP726A20 and JcCYP71D495 that produce the more complex diterpenoid jolkinol C from casbene when expressed transiently with JcCAS in N. benthamiana. Stable transformation of JcCYP726A20, JcCYP71D495 and JcCAS did not produce any detectable jolkinol C until these genes were co-transformed with the optimal set of precursor-pathway genes. One such stable homozygous line was used to evaluate by transient expression the involvement of an 'alkenal reductase'-like family of four genes in the further conversion of jolkinol C, leading to the demonstration that one of these performs reduction of the 12,13-double bond in jolkinol C. This work highlights the need to optimize precursor supply for production of complex diterpenoids in stable transformants and the value of such lines for novel gene discovery.
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