Developing a Nicotiana benthamiana transgenic platform for high‐value diterpene production and candidate gene evaluation

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.

Engineering Production of a Novel Diterpene Synthase Precursor in Nicotiana benthamiana

“…Arabidopsis thaliana cDNAs of 1-DEOXY-D-XYLULOSE-5-PHOSPHATE SYNTHASE ( DXS , AT4G15560.1), 4-HYDROXY-3-METHYLBUT-2-ENYL DIPHOSPHATE REDUCTASE ( HDR , AT4G34350.1, Phillips et al, 2008 ), and GERANYLGERANYL PYROPHOSPHATE SYNTHASE 11 ( GGPPS11 , AT4G36810.1, Beck et al, 2013 ) coding for plastidial enzymes referenced in TAIR (The Arabidopsis Information Resource) and cDNA of CASBENE SYNTHASE ( CAS ) from Jatropha curcas ( King et al, 2014 ) were cloned into pEAQ-HT ( Sainsbury et al, 2009 ) vector as described in Forestier et al (2021) . A codon optimized cDNA of NaGLS based on the sequence accession number XM_019410085 was synthesized by gBlock IDT with extensions allowing to clone it directly with In-Fusion® in AgeI/StuI linearized pEAQ-HT vector.…”

“…A codon optimized cDNA of NaGLS based on the sequence accession number XM_019410085 was synthesized by gBlock IDT with extensions allowing to clone it directly with In-Fusion® in AgeI/StuI linearized pEAQ-HT vector. Transient expression in wild-type N. benthamiana was performed as described in Forestier et al (2021) .…”

“…Many of the gem-dimethylcyclopropyl class of bioactive casbene-derived diterpenoids from plants have been shown to have pharmacological activities. Low abundance in their natural host ( Hohmann et al, 2000 ; Johnson et al, 2008 ) along with their structural complexity ( Jorgensen et al, 2013 ; Kawamura et al, 2016 ; Hashimoto et al, 2017 ) has led to efforts to engineer alternative microbial ( Hill et al, 1996 ; Callari et al, 2018 ; Wong et al, 2018 ), algal ( Mehrshahi et al, 2020 ), or plant-based production platforms ( Forestier et al, 2021 ). The majority of diterpenes in plants are biosynthesized in chloroplasts ( Rohmer et al, 1993 ; McGarvey and Croteau, 1995 ; Lichtenthaler et al, 1997 ) from the five-carbon building blocks, isopentyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP), provided by the MEP pathway and combined together in a three-to-one ratio to form the C 20 precursor, GGPP ( Gershenzon and Croteau, 1993 ).…”

“…We recently developed a Nicotiana benthamiana platform optimized for production of casbene and derivatives by engineering flux through the MEP pathway ( Forestier et al, 2021 ). We demonstrated how this platform could be used for production of the lathyrane jolkinol C by introduction of functionally characterized P450 oxidases ( Forestier et al, 2021 ).…”

“…We recently developed a Nicotiana benthamiana platform optimized for production of casbene and derivatives by engineering flux through the MEP pathway ( Forestier et al, 2021 ). We demonstrated how this platform could be used for production of the lathyrane jolkinol C by introduction of functionally characterized P450 oxidases ( Forestier et al, 2021 ). The elucidation of the biosynthetic steps from casbene to the tigliane ( Kulkosky et al, 2001 ; Kissin and Szallasi, 2011 ; De Ridder et al, 2021 ), ingenane ( Siller et al, 2010 ) and jatrophane ( Corea et al, 2009 ; Hadi et al, 2013 ) classes of diterpenoids has also been investigated but remains to be resolved.…”