Opportunities and challenges for the sustainable production of structurally complex diterpenoids in recombinant microbial systems

Abstract

With over 50.000 identified compounds terpenes are the largest and most structurally diverse group of natural products. They are ubiquitous in bacteria, plants, animals and fungi, conducting several biological functions such as cell wall components or defense mechanisms. Industrial applications entail among others pharmaceuticals, food additives, vitamins, fragrances, fuels and fuel additives. Central building blocks of all terpenes are the isoprenoid compounds isopentenyl diphosphate and dimethylallyl diphosphate. Bacteria like Escherichia coli harbor a native metabolic pathway for these isoprenoids that is quite amenable for genetic engineering. Together with recombinant terpene biosynthesis modules, they are very suitable hosts for heterologous production of high value terpenes. Yet, in contrast to the number of extracted and characterized terpenes, little is known about the specific biosynthetic enzymes that are involved especially in the formation of highly functionalized compounds. Novel approaches discussed in this review include metabolic engineering as well as site-directed mutagenesis to expand the natural terpene landscape. Focusing mainly on the validation of successful integration of engineered biosynthetic pathways into optimized terpene producing Escherichia coli, this review shall give an insight in recent progresses regarding manipulation of mostly diterpene synthases.

Keywords: enzyme engineering; heterologous production in E. coli; metabolic pathway optimization; modular biosynthesis; plant diterpenes.

Scheme 1

Isoprenoid biosynthetic pathways and examples for their engineering in heterologous production systems. a) Formation of central isoprenoid metabolites isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) occurs via two distinct natural pathways. Designations MEV and MEP derive from significant intermediates: MEV = mevalonate-dependent and MEP= methylerythritol phosphate-dependent. b) Subsequent condensation of IPP and DMAPP by isoprenyl diphosphate synthases provides specific terpene synthases with their linear substrates. Terpenes are classified according to the carbon atom number in their basic scaffold, beginning with hemiterpenes (C5) and continuing in multiples of five. c) A possible strategy for MEP-pathway optimization for the improved production of the diterpene taxadiene reported by Ajikumar et al. [28]; targeted elements of the biosynthetic pathways and their expression manipulations are given. d) Selection of overexpression targets for the production of ent-kaurene reported by Kong et al. [29]; HMGR = hydroxymethylglutaryl(HMG)-CoA-reductase; dxs = 1-deoxy-D-xylulose-5-phosphate synthase; dxr = 1-deoxy-D-xylulose-5-phosphate reductoisomerase; ispD = 2-C-methyl-D-erythritol(ME)-4-phosphate cytidylyltransferase; ispE = 4-(cyt-5’-diphospho)-ME kinase; ispF = ME-2,4-cyclodiphosphate synthase; ispG = hydroxylmethylbutenyl(HMB)-4-diphosphate synthase; ispH = HMB-4-diphosphate reductase; ispA = farnesyl diphosphate synthase from Escherichia coli; idi = IPP isomerase; GPPS = geranyl diphosphate synthase; FPPS = farnesyl diphosphate synthase, GGPPS = geranylgeranyl diphosphate synthase; GGPPS^RS = GGPPS from Rhodobacter sphaeroides; KS^SR = ent-kaurene synthase from Stevia rebaudiana, CPPS^SR = ent-copalyl diphosphate synthase from Stevia rebaudiana, Trc = Trc promoter; T7 = T7 promoter.

Scheme 2

Mutational engineering of different classes of terpene synthases. Left side: The natural product of wild-type cyclooctat-9-en-7-ol-synthase (CotB2) is a tricyclic diterpene whereas mutations in positions 107 and 288 yield in monocyclic cembrene A and bicyclic 3,7,18-dolabellatriene [57]. Changing the main product specificity of taxadiene synthase from Taxus brevifolia (TXS) without significant loss in synthase activity was realized in bicyclic verticillia-3,7,12-triene production through mutation of valin584. Another mutation (W753H) resulted in 100% product specificity for cembrene A but TXS activity was reduced by half in comparison to the wild-type [51]. Right side: Methyl group shifts in Class II peregrinol diphosphate synthase from Marrubium vulgare (MvCPS1) were drastically rearranged by introduction of two mutations leading to a previously undescribed halimadane type diphosphate [58], a possible new precursor for valuable halimadane diterpenes with antimicrobial or anti-allergic potential.

Figure 1

Implementation of a microbial cell factory. 1: Selection of enzymes from different species. P450 and related reductase enzymes (indicated with blue and red knots respectively) derive almost exclusively from plants; terpene synthases and other enzymes that are involved in precursor formation (indicated with green knot) can be obtained from various organisms (indicated through symbolic bacteria, leaf (representing plants in general) and fungi). 2: Eukaryotic enzymes have to be engineered for functional and soluble expression in prokaryotic hosts like E. coli, Removal of the N-terminal and thereby the cell-wall-localization domain (indicated through scissors) is a standard procedure in engineering plant enzymes; 3: Further engineering steps are not mandatory but often entail site-directed mutagenesis (indicated through wrench) of TPS (green) for product modulation or introduction of a linker-coding sequence for co-expression of P450 monooxygenase and reductase (blue and red); 4: Heterologous expression in E. coli (depicted in orange). Construction of synthetic operons and screening for highest yield of plasmid systems generally precedes genomic integration; 5: Isoprenoid precursor supply: precursor flux has to be balanced carefully to avoid metabolic overload and accumulation of unwanted byproducts; 6: Downstream terpenoid biosynthesis using heterologous enzymes; 7: Upscaling of terpene production in fermentation systems using different carbon sources (left) for optimally engineered E. coli strains is a potential future source for valuable diterpenes like miltiradiene (a), sclareol (b) or taxadiene (c).