Heterologous expression of the isopimaric acid pathway in Nicotiana benthamiana and the effect of N-terminal modifications of the involved cytochrome P450 enzyme

Abstract

BackgroundPlant terpenoids are known for their diversity, stereochemical complexity, and their commercial interest as pharmaceuticals, food additives, and cosmetics. Developing biotechnology approaches for the production of these compounds in heterologous hosts can increase their market availability, reduce their cost, and provide sustainable production platforms. In this context, we aimed at producing the antimicrobial diterpenoid isopimaric acid from Sitka spruce. Isopimaric acid is synthesized using geranylgeranyl diphosphate as a precursor molecule that is cyclized by a diterpene synthase in the chloroplast and subsequently oxidized by a cytochrome P450, CYP720B4.ResultsWe transiently expressed the isopimaric acid pathway in Nicotiana benthamiana leaves and enhanced its productivity by the expression of two rate-limiting steps in the pathway (providing the general precursor of diterpenes). This co-expression resulted in 3-fold increase in the accumulation of both isopimaradiene and isopimaric acid detected using GC-MS and LC-MS methodology. We also showed that modifying or deleting the transmembrane helix of CYP720B4 does not alter the enzyme activity and led to successful accumulation of isopimaric acid in the infiltrated leaves. Furthermore, we demonstrated that a modified membrane anchor is a prerequisite for a functional CYP720B4 enzyme when the chloroplast targeting peptide is added. We report the accumulation of 45–55 μg/g plant dry weight of isopimaric acid four days after the infiltration with the modified enzymes.ConclusionsIt is possible to localize a diterpenoid pathway from spruce fully within the chloroplast of N. benthamiana and a few modifications of the N-terminal sequences of the CYP720B4 can facilitate the expression of plant P450s in the plastids. The coupling of terpene biosynthesis closer to photosynthesis paves the way for light-driven biosynthesis of valuable terpenoids.

Photosynthetic fuel for heterologous enzymes: the role of electron carrier proteins

“…Ferredoxin was already suggested 20 years ago to enable coupling of cytochrome P450 activity to photosynthesis (Lacour and Ohkawa 1999), but the first demonstrations of this idea were only recently realized in vitro (Jensen et al 2011) and in vivo (Nielsen et al 2013;Gnanasekaran et al 2015Gnanasekaran et al , 2016Wlodarczyk et al 2016). Although work on redirecting photosynthetic reducing equivalents remains mostly proof of concept, light-driven P450 activity has yielded reasonable titers.…”

“…Expression of the biosynthetic pathway consisting of two P450s (CYP79A1 and CYP71E1) and a glucosyltransferase (UGT85B1) yielded 1-2 mg g DW −1 and 3-5 mg L −1 , respectively, of the cyanogenic glucoside dhurrin in N. tabacum chloroplast or in Synechocystis PCC 6803 (Wlodarczyk et al 2016). In another example, 40 µg g DW −1 of the antimicrobial diterpenoid isopimaric acid resulted from transiently expressing CYP720B4 from Sitka spruce in tobacco (Gnanasekaran et al 2015). While the physiological consequences of coupling cytochrome P450 enzymes to photosynthetic reducing power are not yet fully explored, introduction of the mammalian CYP1A1 into Synechococcus elongatus PCC 7002 caused a 31% increase in photosynthetic electron transfer rates (Berepiki et al 2016), which suggests that the photosynthetic machinery has the capacity to tolerate increased sink activity.…”

Transient Expression of Human Cytochrome P450s 2D6 and 3A4 in Nicotiana benthamiana Provides a Possibility for Rapid Substrate Testing and Production of Novel Compounds

“…In spite of the multiple examples of favorable effect of chloroplast targeting on heterologous protein accumulation, it was noticed that the optimal subcellular localization might be selected rather empirically, because of individual hardly predictable protein peculiarities . Few recent reports on targeting heterologous CYP450s into chloroplasts, on the one hand, validated their capability to use photosynthetically generated electrons for oxidation reactions; on the other hand, indicated drastic dependence of their physiological activity in chloroplasts on molecular structure of transmembrane domain . In the latter work, truncation of the membrane anchor with simultaneous introduction of point mutations into the remaining N‐terminal part of CYP720B4 from Picea sitchensis was considered to enable its activity in chloroplasts although strongly reduced transport efficiency.…”

Compartmentalized metabolic engineering for biochemical and biofuel production

“…These localization tags are roughly 60 amino acids in length and are rich in hydroxylated, hydrophobic and basic residues. Localization of membrane proteins to the chloroplast requires additional signal sequences for proper membrane attachment . While not as extensively used for the production of secondary metabolites, chloroplast localization has also been characterized for non‐plant systems such as diatoms .…”

“…These localization tags are roughly 60 amino acids in length and are rich in hydroxylated, hydrophobic and basic residues. Localization of membrane proteins to the chloroplast requires additional signal sequences for proper membrane attachment [28].…”