Developing a <i>Nicotiana benthamiana</i> transgenic platform for high?value diterpene production and candidate gene evaluation

To engineer Nicotiana benthamiana to produce novel diterpenoids, we first aimed increase production of the diterpenoid precursor geranylgeranyl pyrophosphate (GGPP) by up-regulation key genes non-mevalonate (MEP) pathway sourced from Arabidopsis thaliana. We used transient expression evaluate combinations eight MEP plus GGPP synthase and a Jatropha curcas casbene (JcCAS) identify an optimal combination for GGPP. AtDXS AtHDR together with AtGGPPS JcCAS gave 410% in compared alone. This was cloned into single construct using MoClo toolkit, stably integrated N. genome. also created multigene constructs stable transformation two J. cytochrome P450 genes, JcCYP726A20 JcCYP71D495 that more complex jolkinol C when expressed transiently benthamiana. Stable JcCYP726A20, did not any detectable until these were co-transformed set precursor-pathway genes. One such homozygous line involvement ‘alkenal reductase’-like family four further conversion C, leading demonstration one performs reduction 12,13-double bond C. work highlights need optimize supply diterpenoids transformants value lines gene discovery.