1. Definition of synthetic biology concepts
Metabolic engineering was defined in 1991 as ‘the directed improvement of production, formation, or cellular properties through the modification of specific biochemical reactions or the introduction of new ones with the use of recombinant DNA technology’ (Bailey, 1991). Since then, metabolic engineering has enabled spectacular advances in the production of a myriad of small compounds, including terpenoids, particularly in microbes. Now, demands have increased and biological functions that do not exist in nature are also desired. This can be achieved through synthetic biology, which can be defined as ‘the design and construction of new biological components, such as enzymes, genetic circuits, and cells, or the redesign of existing biological systems’ (Keasling, 2008). More elaborately, synthetic biology refers to the redesign of complex natural living systems in a rational and systematic way to simplified, predictable and controllable modules that can be modeled and manipulated to generate industrially scalable systems with a defined purpose. For many years, the term ‘synthetic biology’ was used to describe concepts that would be classified today as metabolic engineering. However, the definitions are not sharp-edged, and hence metabolic engineering might still be considered as the simplest form of synthetic biology (Channon et al., 2008).
Based on the approach used for synthetic biology, two main branches, commonly referred to as ‘top–down’ and ‘bottom–up’ synthetic biology, can be recognized. The top–down approach involves the introduction of exogenous genes into a host and the engineering of its native metabolic networks to reprogram cellular behavior by employing engineering and mathematical modeling toolkits. The bottom–up approach utilizes the biochemical toolkit for the de novo construction of synthetic genomes and unnatural components that behave in an analogous manner to their natural counterparts, and thereby allows the genesis of artificial living systems. The top–down approach of metabolic engineering for the production of useful products pertains to one of the most established concepts in the field of synthetic biology. Metabolic engineering combines transgene expression with the analysis of metabolic networks to optimize genetic and regulatory processes within cells for the production of a desired product. Metabolic engineering in a heterologous host may also involve the mathematical modeling of the host's native metabolic networks to calculate the yield of the desired product, the measurement of metabolic fluxes to pinpoint parts of the network that constrain production, genetic engineering of the host network to relieve these constraints and modeling of the modified network to calculate the product yield until an industrially applicable level is obtained (Koffas et al., 1999).
Contrary to cell-based synthetic biology, in which the cell's growth and survival objectives might interfere with the engineering objective, that is, the production of a desired compound, cell-free ‘in vitro synthetic biology’ provides a bottom–up platform, in which all available resources are concentrated on a user-defined objective, which could eventually result in improved production systems (Harris & Jewett, 2012). A cell-free environment is highly flexible and devoid of genetic regulation or transport barriers, facilitating substrate addition and product purification.
Alongside the engineering of organisms for enhanced production, synthetic biology also aims to create novel compounds with useful properties. One way to achieve this is by ‘combinatorial biosynthesis’, which allows the generation of new-to-nature compounds through the assembly of genes from different organisms, but catalyzing reactions in related pathways in a native or heterologous host, thereby establishing new enzyme–substrate combinations in vivo (Julsing et al., 2006). An alternative way to create novel compounds is by ‘directed evolution’ or ‘enzyme engineering’. The concept of directed enzyme evolution mimics the process of natural evolution and employs a set of methodologies to enhance or modify the function of a progenitor enzyme to accept an unnatural substrate or to catalyze a new biosynthetic reaction, thereby resulting in the formation of novel products (Dalby, 2011). Obviously, this concept can also be used in metabolic engineering for enhanced production by improving enzyme performance with its natural substrates.
2. Metabolic engineering and microbial biosynthesis of plant terpenoids
Compared with plant production systems, microorganisms are attractive alternatives as heterologous hosts because of their rapid doubling time, robustness under process conditions, ease of scalability, simplicity of product purification because of the absence of competing contaminants and cost-effectiveness resulting from the conversion of inexpensive feedstock to valuable compounds (Zhang et al., 2011). The choice of a suitable host (or ‘chassis’) is critical and should be based on multiple factors, including the chemical nature and complexity of the product to be synthesized, the genetic amenability of the host, the intrinsic availability of precursors for product biosynthesis, the codon usage bias of the host, the need for post-translational modifications and the feasibility to metabolically engineer the host to boost productivity (Keasling, 2010). Microbial synthesis of any plant natural product can be achieved by ‘precursor-mediated product synthesis’, in which an existing host pathway is altered to incorporate a heterologous pathway, or by ‘de novo synthesis’, in which new-to-host biosynthetic routes are imported, thereby avoiding feedback regulation (Chang & Keasling, 2006). After the establishment of heterologous synthesis, it is usually imperative to metabolically engineer the host to optimize the production yield and rate (Chemler & Koffas, 2008).
The colloquial hosts Escherichia coli and Saccharomyces cerevisiae have been employed for both precursor-mediated and de novo synthesis of mono-, di-, sesqui-, tri- and tetraterpenoids (Misawa, 2011), with artemisinic acid, the precursor of the antimalarial drug artemisinin, as the showcase for plant-derived terpenoids (Keasling, 2012). The prokaryotic E. coli has an inherent MEP pathway and the eukaryotic S. cerevisiae has the MVA pathway to produce IPP and its isomer DMAPP. Theoretically, terpenoid biosynthesis can be incorporated into these hosts by expressing the corresponding genes, but low yields may be obtained because of the limited intracellular IPP pool. The IPP and subsequent precursor levels have been supplemented by metabolic engineering of: (1) the MVA pathway in E. coli (Campos et al., 2001); (2) the MEP pathway and prenyltransferases in E. coli (Kajiwara et al., 1997); (3) the MVA pathway by a feedback regulation-deficient HMGR in S. cerevisiae (Ro et al., 2006); (4) the MVA pathway by decreasing downstream enzymes to accumulate precursors in S. cerevisiae (Paradise et al., 2008); (5) the global transcription factor regulating sterol biosynthesis in S. cerevisiae (Davies et al., 2005); and (6) protein scaffolds for the MVA pathway in S. cerevisiae (Dueber et al., 2009; Fig. 3).
Figure 3. Strategies employed to enhance the production of isopentenyl pyrophosphate (IPP) and terpenoids in Escherichia coli and Saccharomyces cerevisiae. (a) Expression of the S. cerevisiae mevalonate (MVA) pathway in E. coli. (b) Expression of rate-limiting 2-C-methyl-d-erythritol 4-phosphate (MEP) enzymes in E. coli. (c) Expression of a truncated form of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) in S. cerevisiae. ER, endoplasmic reticulum. (d) Downregulation of endogenous sterol biosynthesis to accumulate terpenoid precursors in S. cerevisiae. FPP, farnesyl pyrophosphate. (e) Expression of a mutant version (upc2-1) of the global transcription factor (UPC2) upregulates the expression of the native sterol biosynthesis genes in S. cerevisiae. (f) Protein scaffolding to prevent rate limitation in S. cerevisiae by the spatial organization of rate-limiting sterol biosynthetic enzymes in a modulated ratio. AACT, acetoacetyl-CoA thiolase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase.
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Alongside targeted engineering, global approaches have been applied to improve the terpenoid pathway flux in microbial hosts. A ‘chromosomal promoter engineering’ strategy was used to express some of the endogenous MEP genes from a strong bacteriophage T5 promoter in an E. coli strain harboring β-carotene biosynthetic genes, resulting in the enhanced production of β-carotene relative to the parental strain (Yuan et al., 2006). Similarly, a ‘global transcription machinery engineering’ on the rpoD gene encoding σ70, the primary sigma factor, resulted in increased lycopene production in E. coli (Alper & Stephanopoulos, 2007).
Once precursor synthesis has been optimized, another major hurdle to overcome is to achieve functional expression of the pathway genes downstream of the precursor, particularly CytP450s. Plant CytP450s are endoplasmic reticulum-localized enzymes with a prerequisite for a CytP450 reductase (CPR) partner for efficient functioning (Podust & Sherman, 2012). In this regard, S. cerevisiae, with its native CytP450s and CPR, has an advantage over E. coli for the expression of complex terpenoid pathways (Hamann & Møller, 2007). Nevertheless, plant CytP450s supplemented with a plant CPR have been successfully expressed in both E. coli and S. cerevisiae (Arsenault et al., 2008).
Saccharomyces cerevisiae has already been employed for the expression of triterpenoid saponin biosynthetic genes. Through its native ergosterol biosynthesis, S. cerevisiae produces oxidosqualene, the precursor of saponins. In engineered strains optimized to accumulate oxidosqualene, different OSCs and CytP450s have been expressed, mainly for their functional characterization (Augustin et al., 2011; Fig. 2). Engineering efforts have been limited to the production of β-amyrin only. Through a conventional pathway engineering approach, a final titer of 6 mg l−1 was demonstrated in an S. cerevisiae strain expressing a β-amyrin synthase (bAS) from Artemisia annua (Kirby et al., 2008). Subsequent to a genotype-to-phenotype linking study, a 500% improvement in β-amyrin production was achieved by overexpression of the native genes, ERG8, ERG9 and HFA1, in an S. cerevisiae strain expressing a Pisum sativum bAS, resulting in a final titer of 3.93 mg l−1 (Madsen et al., 2011). The β-amyrin levels produced by the parent strains in the above reports reflect the cyclization efficiency of the enzymes employed. Therefore, by employing a more efficient bAS (or any other saponin biosynthetic gene), followed by targeted and/or global engineering, it should be possible to further enhance β-amyrin (or triterpenoid) levels.
The β-amyrin-producing S. cerevisiae strains have been utilized as a tool for the in vivo expression and characterization of novel CytP450s. The co-expression of a CytP450 with a plant-derived CPR resulted in the generation of yeast strains producing different sapogenins. The expression of M. truncatula CYP716A12, together with the Lotus japonicus bAS and the L. japonicus CPR, resulted in the production of oleanolic acid in yeast (Fukushima et al., 2011; Fig. 2). β-Amyrin has also been modified to natural and rare triterpenoids by the combination of multiple CytP450s in yeast. The expression of M. truncatula CYP72A68v2 and CYP93E2 in the oleanolic acid-producing strain resulted in the production of gypsogenic acid and 4-epi-hederagenin, respectively (Fukushima et al., 2013). In addition to β-amyrin-producing strains, α-amyrin-, lupeol- and dammarenediol-producing yeasts have been employed for the functional characterization of CytP450s. The M. truncatula CYP716A12 also catalyzes the C-28 oxidations of α-amyrin to ursolic acid and lupeol to betulinic acid in yeast (Fukushima et al., 2011). Similarly, the C-6 and C-12 hydroxylations of dammarenediol by CYP716A53v2 and CYP716A47, respectively, have been demonstrated in yeast (Han et al., 2011, 2012).
To complement metabolic engineering, synthetic biology offers a plethora of tools through the generation of minimal hosts, standard biological parts, regulatory elements, vectors, assembly methods and in silico computer-aided design tools (Keasling, 2012). The first and main requirement for the production of any natural product is the availability of a robust host. Synthetic biology facilitates the generation of ‘minimal hosts’ that contain only the genes essential for their growth to synthesize macromolecules from simple and inexpensive feedstock. Minimal hosts of E. coli have been generated with c. 15% genome reduction by the deletion of non-essential genes (Pósfai et al., 2006). For S. cerevisiae, the synthetic yeast genome project Sc2.0 aims to design fully synthetic minimized hosts without transposable elements and telomeric sequences, with relocated tRNAs and with site-specific recombination sites incorporated into the genome. Two partially synthetic S. cerevisiae chromosomes with genome reductions of 15–20% have been generated and successfully reincorporated (Dymond et al., 2011). The Streptomyces avermitilis linear chromosome was reduced to 81.46% of the wild-type chromosome by stepwise deletion of a region of > 1.4 Mb, including genes coding for the synthesis of all endogenous secondary metabolites. The minimized strain was able to produce artemisin precursors on expression of a synthetic codon-optimized A. annua amorphadiene synthase gene (Komatsu et al., 2010). In addition, the feasibility of generating completely artificial synthetic hosts with a desired set of genes has been demonstrated by the cloning of a chemically synthesized and assembled Mycoplasma genitalium genome in S. cerevisiae (Gibson et al., 2008).
Most often metabolic engineering focuses on the maximization of the production of a final compound with less attention to the behavior of intermediates. Contrary to this, the bottom–up synthetic biology approach allows the deconvolution of metabolic pathways to independent parts that are optimized for host-specific expression, and are subsequently incorporated rationally to build production modules. The repositories of functional parts (promoters, ribosomal binding sites, protein domains, terminators, etc.), generated within synthetic biology initiatives, facilitate the assembly of metabolic pathways (Boyle & Silver, 2012). Two depositories with codon-optimized parts for pathway engineering in E. coli (The Registry of Standard Biological Parts, partsregistry.org/Main_Page) and terpenoid engineering in S. cerevisiae (Serber et al., 2012) have been described. Synthetic biology also promotes the variable expression of related biosynthetic genes to avoid metabolic bottlenecks. Robust synthetic promoter libraries with defined promoter strengths enable modular gene expression in bacteria and yeast (Hammer et al., 2006; Nevoigt et al., 2006). Tunable intergenic regions that generate mRNA secondary structures and RNase recognition sites have been employed for the differential stabilization of segments of mRNA encoding multiple enzymes in the form of operons (Pfleger et al., 2006). Synthetic protein scaffolds that are particularly efficient in overcoming rate-limiting steps have been generated to increase flux through metabolic pathways by tethering enzymes together (Dueber et al., 2009).
Natural product biosynthesis typically involves multigene pathways, thus implementing the necessity for the simultaneous expression of multiple genes in a microbial chassis. Both in vitro and in vivo methods facilitate multigene assembly in E. coli and S. cerevisiae (Ellis et al., 2011; Wang et al., 2012), some of which have been employed to assemble carotenoid biosynthetic pathways (Shao et al., 2009; Lemuth et al., 2011). In parallel, viral mechanisms, such as internal ribosome entry sites and 2A oligopeptide sequences, have been adapted for polycistronic gene expression (de Felipe, 2002). However, the latter tools have not yet been implemented for the expression of terpenoid pathway enzymes.
3. Combinatorial biosynthesis of plant terpenoids
Combinatorial biosynthesis-based reconstitution of pathways is a useful tool to generate known and novel natural products, which can be further modified by semi-synthesis. In its simplest form, combinatorial biosynthesis is the process of generating different, but structurally related, molecules through the assembly of genes from different organisms in a single host (Kirschning et al., 2007; Fig. 4a). Plants possess an immense potential for combinatorial biosynthesis (Pollier et al., 2011). However, apart from a pioneering study, in which the expression of a bacterial halogenase in C. roseus resulted in the generation of novel chlorinated TIAs (Runguphan et al., 2010), there have been no reports on a directed combinatorial biosynthesis approach for any other terpenoid or metabolite in plants to date. Nonetheless, the existing chemical diversity, together with our growing understanding of their biosynthesis, renders (tri)terpenoids appealing compounds for the combinatorial generation of novel analogs. For instance, the screening of a synthetic triterpenoid combinatorial library derived from betulinic and ursolic acid led to the identification of compounds with an enhanced anti-malarial activity relative to the parent compounds (Pathak et al., 2002).
Figure 4. Strategies to generate novel triterpenoid saponins. (a) Combinatorial biosynthesis in the model legume Medicago truncatula which produces 3-Glc-28-Glc-medicagenic acid endogenously. The overexpression of CYP88D6, a CytP450 from Glycyrrhiza uralensis roots that produces glycyrrhizin, in M. truncatula could lead to the formation of a combinatorial product together with the naturally occurring saponins. (b) Combinatorial biosynthesis of saponins in a sterol-reduced Saccharomyces cerevisiae strain by the heterologous expression of saponin biosynthetic genes from M. truncatula and G. uralensis. (c) The process of directed enzyme evolution involves mutagenesis and selection for desired enzyme properties. Here, the evolution of a multifunctional enzyme with an increased reaction specificity is depicted. Glc, glucose; GlcUA, glucuronic acid; IPP, isopentenyl pyrophosphate; MtbAS, M. truncatula β-amyrin synthase, MtCytP450s, M. truncatula cytochrome P450 monooxygenases; UGT, UDP-glucosyltransferase.
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Combinatorial biosynthesis of triterpenoid saponins holds great potential, as they exhibit a plethora of biological activities. Bardoxolone methyl, a semi-synthetic derivative of oleanolic acid, has been clinically evaluated for the treatment of chronic kidney disease. The synthesis of bardoxolone methyl occurs through chemical modifications of the three active portions of oleanolic acid that render the derivative biologically more potent than the starter molecule (Sporn et al., 2011). The enzymatic addition of extra functionalities to the triterpenoid backbone through combinatorial biosynthesis could increase the number of sites that can be accessed for further synthetic modifications (Pollier & Goossens, 2012).
A major drawback of the generation of novel molecules in planta lies in the complexity of plant metabolite extracts and the complications of purifying a compound of interest from a large pool of different molecules, including compounds with similar structures and physicochemical properties. Therefore, combinatorial biosynthesis of plant secondary metabolites has also been performed in microorganisms, which lack the production of compounds similar to the target compound (Fig. 4b). Novel carotenoid structures with an enhanced antioxidative activity have been generated in E. coli by the combinatorial expression of bacterial and plant genes (Sandmann, 2002). Recently, rare triterpenoids have been combinatorially produced in S. cerevisiae (Fukushima et al., 2013). A current obstacle to the wider utilization of combinatorial biosynthesis for plant-derived compounds is the limited availability of plant genes encoding the enzymes that catalyze the biosynthetic reactions. In the future, these bottlenecks may be solved by gene discovery in (medicinal) plants or, alternatively, by directed evolution of enzymes towards novel functions (Kwon et al., 2012).
4. Enzyme engineering or directed evolution of terpenoid biosynthetic enzymes
Small-molecule drugs, considered to be relevant as lead molecules, often have a high degree of chemical complexity with multiple functional groups and defined stereochemistry (Nannemann et al., 2011). In their natural source, these small molecules are most often synthesized by enzymes that have a high regio- and stereoselectivity, high catalysis rate and relaxed substrate specificity. Nonetheless, natural enzymes often cannot meet the requirements of industrial chemists in terms of substrate tolerance, efficiency, process tolerance and economic viability. Hence, enzymes have been engineered by directed evolution to improve one or more of their properties under defined conditions (Dalby, 2011; Fig. 4c). Directed enzyme evolution has progressed tremendously lately, and it is now feasible to engineer enzymes to accept unnatural substrates and to catalyze regio- and stereospecific reactions with an efficiency comparable with that of the natural enzymes (Goldsmith & Tawfik, 2012). The promiscuous nature of proteins gives them an inherent ability to generate novel or altered functions with a small number of amino acid substitutions (Aharoni et al., 2005), and computational methods, such as catalytic active site prediction (CLASP) and directed evolution using CLASP: an automated flow (Chakraborty et al., 2011; Chakraborty, 2012), utilize virtual screening for spatial, electrostatic and scaffold matching to identify target progenitor proteins. Enzymes catalyzing branch-point reactions in multi-branched pathways, in which a substrate is converted to multiple products, have a high evolvability. In addition, evolvable enzymes exhibit multiple mutational residues and are ‘locally specific’ as they recognize a common motif on structurally diverse substrates (Umeno et al., 2005).
Oxidosqualene, the immediate precursor of triterpenoid biosynthesis, is a versatile molecule that is cyclized into multiple products by different OSCs. Several of these OSCs are multifunctional in nature and generate multiple products in a single reaction (Phillips et al., 2006), highlighting the promiscuity, and thus evolvability, of the enzymes. Through directed evolution, the major cyclization product of a multifunctional OSC could be redefined to a specific or novel product. For other terpene synthases, this has already been successfully attempted. Following a site-saturation mutagenesis, the specificity of a carotenoid synthase was altered to generate unnatural C45 and C50 backbones in E. coli (Umeno & Arnold, 2004). The product specificity of a γ-humulene synthase from Abies grandis that cyclizes FPP to 52 different sesquiterpenoids was evolved by site-saturation mutagenesis to generate independent synthases, each producing one or a few products derived from a predominant reaction pathway (Yoshikuni et al., 2006).
This evolution approach could also be extended to downstream triterpenoid biosynthetic enzymes, in particular the CytP450s. Triterpenoid saponin backbones are made up of 30 carbons, c. 20 of which are accessible for CytP450-mediated modifications, as deduced from known saponins (Dinda et al., 2010). In addition, diverse functional groups are observed at the modifiable carbons, pointing to the existence of specific CytP450s that catalyze these specific reactions. For instance, the C-11 position of many triterpenoid backbones can be oxidized with an α- or β-hydroxy group, and a CytP450 that specifically catalyzes the α-hydroxylation has already been characterized (Seki et al., 2008). To date, only a few CytP450 families involved in triterpenoid modifications have been identified (Fig. 2). Through directed CytP450 evolution, it should be possible to: broaden their substrate acceptance to divergent backbones, target specific carbon positions, and specify the functional group to be added to the triterpenoid skeleton. Such approaches have been implemented on carotenoid desaturases that have been evolved by random mutagenesis to accept unnatural C35 carotenoid backbones in E. coli (Umeno & Arnold, 2003).
Protein engineering based on molecular evolution also serves as a tool to enhance enzyme efficiency or to abolish feedback regulation on enzymes. Through adaptive evolution, the unfavorable in vivo properties of truncated yeast HMGR were minimized for optimal functioning in E. coli, thereby also enhancing the final product yield by c. 1000-fold (Yoshikuni et al., 2008). Key to directed evolution studies is a profound understanding of sequence-to-structure-to-function relationships of a protein. Integrated databases of triterpenoid cyclases (TTCED; Racolta et al., 2012) and CytP450s (CYPED; Sirim et al., 2009) facilitate the identification of functionally relevant and selectivity-determining amino acid residues within members of a protein family by extensive sequence analysis. Therefore, the boosting of protein engineering efforts could enhance synthetic biology efforts in triterpenoid engineering in the future.
5. In vitro synthetic biology: an evolving tool
In vitro synthetic biology systems can comprise ‘synthetic enzymatic pathways’ (SEPs), in which purified enzymes are combined in an aqueous environment to convert a substrate to a product through a series of reactions. Alternatively, ‘crude extract cell-free’ (CECF) systems, in which cells are grown, harvested and lysed to obtain a crude extract, can be utilized for the conversion of a substrate to a product (Hodgman & Jewett, 2012; Fig. 5a). The choice between SEP and CECF is influenced by time, cost and the need for cellular reinforcement to support the desired network. A CECF approach, for instance, is more suited for a reaction requiring a constant supply of energy, such as protein synthesis (Carlson et al., 2012); however, unlike SEP, CECF reactions can exhibit undesirable activities because of the crude nature of the cellular extract.
Figure 5. In vitro synthetic biology platforms. (a) Synthetic enzymatic pathways in which purified enzymes are combined with reaction components in an aqueous environment to convert a substrate to a product through a series of reactions, and crude extract cell-free systems in which resources from the cell convert an exogenously provided substrate to a product. (b) In vitro compartmentalization using water-in-oil emulsions. The encapsulated water phase consists of a substrate coupled to a gene which is transcribed and translated in vitro to generate an enzyme that can convert the substrate to the product. (c–f) Metabolic channeling brings enzymes in close proximity with their substrate by (c) protein scaffolding, (d) tethering enzymes to a surface, (e) covalently linking related enzymes into aggregates and (f) foam dispersion techniques in which the enzymes are encapsulated using surfactants. a,b,c,d,e,f,g,h, native enzymes; b*,d*,g*, synthetically modified enzymes; B,C,D,E,F,G,H, intermediates; P, product; S, substrate.
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The multireaction nature of biochemical networks, low protein concentrations, enhanced substrate diffusion, low enzyme proximity and low reaction rates as a result of unbalanced enzyme activity still hamper the efficiency of cell-free synthetic biology. In vitro compartmentalization (IVC) is one way of achieving proximity of reaction components. In IVC, genes are coupled to a substrate and encapsulated in water-in-oil emulsions, together with transcription and translation machineries, to facilitate enzyme synthesis and consequent product formation (Fig. 5b). Novel enzymes have been uncovered by linking product formation to genes in a confined microenvironment through IVC (Rothe et al., 2006). IVC is also being employed as a screening approach for the directed evolution of enzymes (Arnold & Volkov, 1999; Forster & Church, 2007). In addition to enclosing reaction components in a defined environment through IVC, metabolic channeling has been employed as an alternative to reduce substrate diffusion lengths (Idan & Hess, 2013). Protein scaffolding (Fig. 5c), surface tethering of enzymes (Fig. 5d), covalently linked enzyme aggregates (Fig. 5e) and foam dispersion of enzymes with liposomes using surfactants (Fig. 5f) have been employed to facilitate the spatial organization of pathway components (Hodgman & Jewett, 2012).
Current applications of in vitro synthetic biology are limited to proteins, nucleic acids and small-molecule ligands. Nonetheless, these tools can undoubtedly be extended to natural product or (tri)terpenoid engineering in the future. For instance, IVC could be employed as a tool for the directed evolution of CytP450s. A potential hurdle is the membranous nature of CytP450s, which prevents their solubilization in the aqueous reaction environment, but which may be overcome by the utilization of nanodisc membranes (Denisov & Sligar, 2011). A great advantage of using in vitro synthetic biology in triterpenoid engineering is the simplicity and ease of catalysis of precise regio- and stereospecific reactions with a high efficiency in a relatively pure form, which may overcome the drawbacks of chemical synthesis, metabolic engineering and product purification.