Abstract
SummaryTo 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.
Highlights
Plant diterpenes containing a gem-dimethylcyclopropane subunit, mostly found in species of the Euphorbiaceaea, are of much interest across various industrial sectors including pharmaceuticals due to their bioactivity and structural complexity (Duran-Pen~a et al, 2014)
We propose that 6,9-dihydroxy-5-ketocasbene is the product of incomplete oxidation by JcCYP71D495, resulting in a hydroxyl rather than a keto-group at C-9. 6,9-Dihydroxy-5-ketocasbene is unable to participate in the same spontaneous aldol reaction forming jolkinol C as 6-hydroxy-5,9-diketocasbene and we propose that this causes it to accumulate in the A-CP transformants
In various combinations, of the entire suite of MEP pathway genes from A. thaliana allowed us to define an optimal combination of three genes, DXS, hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR) and GGPPS, for production of casbene in N. benthamiana
Summary
Plant diterpenes containing a gem-dimethylcyclopropane subunit, mostly found in species of the Euphorbiaceaea, are of much interest across various industrial sectors including pharmaceuticals due to their bioactivity and structural complexity (Duran-Pen~a et al, 2014). Ingenol mebutate from Euphorbia peplus and tigilanol tiglate from Fontainea picrosperma are examples of casbene-derived gemdimethylcyclopropane diterpenes that exemplify the fact that even when these compounds are developed as products, the supply chain remains challenging for industry. Tigilanol tiglate is an experimental drug already approved for treating dog tumours mast cells (Ridder et al, 2020) but obtained solely from the extraction from seeds of Fontainea picrosperma, a sub-canopy tree from a restricted area of Queensland rainforest (Lamont et al, 2016). Developing new sustainable production platforms for high value diterpenoids would improve the supply chains of existing diterpene-derived drugs and provide the confidence needed to exploit the huge potential that this class of compounds has to offer
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