Forestier, Edith Catherine Felicie, Czechowski, Tomasz, Cording, Amy et al. (4 more authors) (2021) Developing a Nicotiana benthamiana transgenic platform for high value diterpene production and candidate gene evaluation. Plant biotechnology journal. pp. 1614-1623. ISSN 1467-7644
Abstract
To engineer Nicotiana benthamiana to produce novel diterpenoids, we first aimed to increase production of the diterpenoid precursor geranylgeranyl pyrophosphate (GGPP) by upregulation 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 optimise precursor supply for production of complex diterpenoids in stable transformants and the value of such lines for novel gene discovery.
Metadata
Item Type: | Article |
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Authors/Creators: |
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Copyright, Publisher and Additional Information: | © 2021 The Authors. |
Keywords: | plant diterpenes,Euphorbiaceae,Jatropha curcas,casbene,jolkinol,lathyranes,double bond reductase,Nicotiana benthamiana,stable transformations |
Dates: |
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Institution: | The University of York |
Academic Units: | The University of York > Faculty of Sciences (York) > Biology (York) > Centre for Novel Agricultural Products (CNAP) (York) The University of York > Faculty of Sciences (York) > Biology (York) |
Funding Information: | Funder Grant number BBSRC (BIOTECHNOLOGY AND BIOLOGICAL SCIENCES RESEARCH COUNCIL) BB/M018210/1 |
Depositing User: | Pure (York) |
Date Deposited: | 10 Mar 2021 11:10 |
Last Modified: | 29 Oct 2024 01:04 |
Published Version: | https://doi.org/10.1111/pbi.13574 |
Status: | Published |
Refereed: | Yes |
Identification Number: | 10.1111/pbi.13574 |
Open Archives Initiative ID (OAI ID): | oai:eprints.whiterose.ac.uk:172029 |