Multienzyme Pathways Research Articles

Microtiter Plate Immobilization Screening for Prototyping Heterogeneous Enzyme Cascades.

Immobilization is a key enabling technology in applied biocatalysis that facilitates the separation, recovery, and reuse of heterogeneous biocatalysts. However, finding a consensus immobilization protocol for several enzymes forming a multi-enzyme system is extremely difficult and relies on a combinatorial trial-and-error approach. Herein, we describe a protocol in which 17 different carriers functionalized with different reactive groups are tested in a 96-well microtiter plate to screen up to 21 immobilization protocols for up to 18 enzymes. This screening includes an activity and stability assay to select the optimal immobilization chemistry to achieve the most active and stable heterogeneous biocatalysts. The information retrieved from the screening can be rationalized using a Python-based application CapiPy. Finally, through scoring the screening results, we find the consensus immobilization protocol to assemble an immobilized four-enzyme system to transform vinyl acetate into (S)-3-hydroxybutyric acid. This methodology opens a path to speed up the prototyping of immobilized multi-enzyme pathways for chemical manufacturing.

Bacterial microcompartments as a next-generation metabolic engineering tool: utilizing nature's solution for confining challenging catabolic pathways.

Advancements in synthetic biology have facilitated the incorporation of heterologous metabolic pathways into various bacterial chassis, leading to the synthesis of targeted bioproducts. However, total output from heterologous production pathways can suffer from low flux, enzyme promiscuity, formation of toxic intermediates, or intermediate loss to competing reactions, which ultimately hinder their full potential. The self-assembling, easy-to-modify, protein-based bacterial microcompartments (BMCs) offer a sophisticated way to overcome these obstacles by acting as an autonomous catalytic module decoupled from the cell's regulatory and metabolic networks. More than a decade of fundamental research on various types of BMCs, particularly structural studies of shells and their self-assembly, the recruitment of enzymes to BMC shell scaffolds, and the involvement of ancillary proteins such as transporters, regulators, and activating enzymes in the integration of BMCs into the cell's metabolism, has significantly moved the field forward. These advances have enabled bioengineers to design synthetic multi-enzyme BMCs to promote ethanol or hydrogen production, increase cellular polyphosphate levels, and convert glycerol to propanediol or formate to pyruvate. These pioneering efforts demonstrate the enormous potential of synthetic BMCs to encapsulate non-native multi-enzyme biochemical pathways for the synthesis of high-value products.

Reconstruction of the Native Biosynthetic System of Carotenoids in E. coli─Biosynthesis of a Series of Carotenoids Specific to Paprika Fruit

Capsanthin, capsorubin, cucurbitaxanthin A, and capsanthin 3,6-epoxide, a series of carotenoids specific to the red fruit of paprika (Capsicum annuum), were produced in pathway-engineered Escherichia coli cells. These cells functionally expressed multiple genes for eight carotenogenic enzymes, two of which, paprika capsanthin/capsorubin synthase (CaCCS) and zeaxanthin epoxidase (CaZEP), were designed to be located adjacently. The biosynthesis of these carotenoids, except for capsanthin, was the first successful attempt in E. coli. In a previous study, the levels of capsanthin synthesized were low despite the high expression of the CaCCS gene, which may have been due to the dual activity of CaCCS as a lycopene β-cyclase and CCS. An enhanced interaction between CaCCS and CaZEP that supplies antheraxanthin and violaxanthin, substrates for CaCCS, was considered to be crucial for an efficient reaction. To achieve this, we adapted S·tag and S-protein binding. The S·tag Thrombin Purification Kit (Novagen) is merchandized for in vitro affinity purification, and S·tag-fused proteins in the E. coli lysate are specifically trapped by S-proteins fixed on the agarose carrier. Furthermore, S-proteins have been reported to oligomerize via C-terminal swapping. In the present study, CaCCS and CaZEP were individually fused to the S·tag and designed to interact on oligomerized S-protein scaffolds in E. coli, which led to the biosynthesis of not only capsanthin and capsorubin but also cucurbitaxanthin A and capsanthin 3,6-epoxide. The latter reaction by CaCCS was assigned for the first time. This approach reinforces the scaffold's importance for multienzyme pathways when native biosynthetic systems are reconstructed in microorganisms.

Spatially Segregated MOF Bioreactor Enables Versatile Modular Glycoenzyme Assembly for Hierarchical Glycan Library Construction.

The multienzyme cascade has received growing attention to obtain structurally defined glycans in vitro. However, due to poor enzyme stability and low compatibility between glycoenzymes, artificially designed multienzyme pathways to access glycans are often inefficient. Herein, based on the strategy "Modular-Enzymes Assembly by Spatial Segregation" (MASS), we developed a universal immobilization platform to assemble multiple glycoenzymes in compartmentalized MOF particles, inside and outside, significantly reducing the undesired interference and cross-inhibitions. By changing the enzyme modules, a series of glycosyl donor, disaccharides, oligosaccharides, and polysaccharides bearing cofactor regeneration were efficiently prepared. This bioreactor was further successfully applied to the reaction system with high substrate concentration to demonstrate its industrial potential. This robust multienzyme immobilization platform should serve to promote the enzymatic synthesis of more complex glycans.

Modulation of Enzyme Cascade Activity by Local Substrate Enrichment and Exclusion on DNA Nanostructures.

Substrate confinement and channeling play a critical role in multienzyme pathways and are considered to impact the catalytic efficiency and specificity of biomimetic and artificial nanoreactors. Here we reported a modulation of a multienzyme system with the cascade activity impacted by the surface affinity binding to substrate molecules. A DNA origami modified with aptamers was used to bind and enrich ATP molecules in the local area of immobilized enzymes, thereby enhancing the activity of an enzyme cascade by more than 2-fold. Alternatively, DNA nanostructure modified with blocked aptamers does not bind with ATP, thereby reducing the activity of the enzyme cascade. The Michaelis-Menten kinetics showed decreased apparent KM values (∼3-fold lower) for enzyme nanostructures modified with aptamers, suggesting the higher effective substrate concentration near enzymes due to the local enrichment of substrates. Conversely, increased apparent KM values (∼2-fold higher) were observed for enzyme nanostructures modified with blocked aptamers, possibly due to the exclusion of substrates approaching the surface. The similar concept of this modified surface-substrate interaction should be applicable to other multienzyme systems immobilized on nanostructures, which could be useful in the development of biomimetic nanoreactors.

A sustainability strategy: Convert methanol into valuable chemicals via multi-enzyme cascades

“Turning waste into treasure,” one of nature’s most attractive abilities, inspires scientists to develop advanced synthetic strategies for various applications. In a recent article from Chem Catalysis , Tan and co-workers demonstrate such a “turning” strategy, whereby they design three multi-enzyme cascade pathways to convert methanol into valuable C 2 /C 4 compounds. “Turning waste into treasure,” one of nature’s most attractive abilities, inspires scientists to develop advanced synthetic strategies for various applications. In a recent article from Chem Catalysis , Tan and co-workers demonstrate such a “turning” strategy, whereby they design three multi-enzyme cascade pathways to convert methanol into valuable C 2 /C 4 compounds.

Three multi-enzyme cascade pathways for conversion of C1 to C2/C4 compounds

A feasible and promising carbon reduction strategy is to convert CO 2 into C1 compounds through renewable energy-driven first and then use a multi-enzyme cascade to obtain compounds with higher carbon numbers and market value. In this study, three multi-enzyme cascade pathways for preparing important C2/C4 compounds (ethylene glycol [EG], glycolic acid [GA], and d -erythrose) from methanol were proposed. All pathways use glycolaldehyde as the key intermediate, which can be obtained from methanol through glycolaldehyde synthase and alcohol oxidase cascade. Then, the target compounds can be acquired by adding different enzymes. As a result, 0.90 g/L EG, 0.33 g/L GA, and 53.65 mg/L d -erythrose were produced from methanol. Compared with the titers of EG (6.6 mM) and GA (1.2 g/L) synthesis routes from formaldehyde reported in the literature, the titers of this study (27.58 mM EG and 1.59 g/L GA from formaldehyde) were significantly improved. • Glycolic acid and d -erythrose pathways from CH 3 OH were proposed for the first time • DERA were found to have the function of condensing glycolaldehyde to d -erythrose • GOX were used to synthesize ethylene glycol from glycolaldehyde • ALDH were used to oxidize glycolaldehyde to glycolic acid Under the background of CO 2 is one of the main gases leading to the greenhouse effect and the inexpensive and widespread feedstock. Coupling the renewable energy-driven CO 2 capture technology with multi-enzyme cascade catalytic conversion of C1 compounds to multi-carbon compounds is a promising method for CO 2 utilization. In this case, we successfully constructed three multi-enzyme cascade pathways to synthesize important C2/C4 compounds (ethylene glycol, glycolic acid, and d -erythrose) from C1 compounds. The pathways proposed herein could contribute to realizing the recycling of carbon resources and solving the problems of plastic pollution and energy shortage. They could also provide a new impetus for the design of chemo-bio routes for the efficient conversion of CO 2 into compounds with higher carbon numbers and market value. Junhui et al. successfully constructed three multi-enzyme pathways for synthesizing C2/C4 compounds (ethylene glycol, glycolic acid, and d -erythrose) from C1 compounds. Glycolic acid or d -erythrose pathways were proposed for the first time. Three new kinds of enzymes were found to catalyze intermediate glycolaldehyde to synthesize such C2/C4 compounds.

High-Yield Synthesis of Enantiopure 1,2-Amino Alcohols from l-Phenylalanine via Linear and Divergent Enzymatic Cascades.

Enantiomerically pure 1,2-amino alcohols are important compounds due to their biological activities and wide applications in chemical synthesis. In this work, we present two multienzyme pathways for the conversion of l-phenylalanine into either 2-phenylglycinol or phenylethanolamine in the enantiomerically pure form. Both pathways start with the two-pot sequential four-step conversion of l-phenylalanine into styrene via subsequent deamination, decarboxylation, enantioselective epoxidation, and enantioselective hydrolysis. For instance, after optimization, the multienzyme process could convert 507 mg of l-phenylalanine into (R)-1-phenyl-1,2-diol in an overall isolated yield of 75% and >99% ee. The opposite enantiomer, (S)-1-phenyl-1,2-diol, was also obtained in a 70% yield and 98–99% ee following the same approach. At this stage, two divergent routes were developed to convert the chiral diols into either 2-phenylglycinol or phenylethanolamine. The former route consisted of a one-pot concurrent interconnected two-step cascade in which the diol intermediate was oxidized to 2-hydroxy-acetophenone by an alcohol dehydrogenase and then aminated by a transaminase to give enantiomerically pure 2-phenylglycinol. Notably, the addition of an alanine dehydrogenase enabled the connection of the two steps and made the overall process redox-self-sufficient. Thus, (S)-phenylglycinol was isolated in an 81% yield and >99.4% ee starting from ca. 100 mg of the diol intermediate. The second route consisted of a one-pot concurrent two-step cascade in which the oxidative and reductive steps were not interconnected. In this case, the diol intermediate was oxidized to either (S)- or (R)-2-hydroxy-2-phenylacetaldehyde by an alcohol oxidase and then aminated by an amine dehydrogenase to give the enantiomerically pure phenylethanolamine. The addition of a formate dehydrogenase and sodium formate was required to provide the reducing equivalents for the reductive amination step. Thus, (R)-phenylethanolamine was isolated in a 92% yield and >99.9% ee starting from ca. 100 mg of the diol intermediate. In summary, l-phenylalanine was converted into enantiomerically pure 2-phenylglycinol and phenylethanolamine in overall yields of 61% and 69%, respectively. This work exemplifies how linear and divergent enzyme cascades can enable the synthesis of high-value chiral molecules such as amino alcohols from a renewable material such as l-phenylalanine with high atom economy and improved sustainability.

Molecular Dynamics of Rare-Event Electrostatic Channeling

Cascade reactions are potential green reactions since all the reaction transformations take place in a single pot without purification steps, but their reaction efficiency is limited by the transport efficiency of intermediates between adjacent steps [1]. Understanding the mechanism of multi-enzyme metabolic pathways is of great importance for the design of practical cascades for chemical conversion. Molecular dynamics simulations of cascades can elucidate and quantify physicochemical effects on intermediate transport, but is challenged by processes whose time scales exceed the typical picosecond to nanosecond range. Here, we show how infrequent metadynamics (InMetaD) [2], umbrella sampling (US) and kinetic Monte Carlo (KMC) can be combined to elucidate rare-event transport processes. We build on a previously studied glucose-6-phosphate (G6P) intermediate within a bridged supramolecular structure comprising the enzymes hexokinase (HK) and glucose-6-dehydrogenase (G6PDH) [3].When the previous polylysine bridge is replaced by polyarginine, electrostatic interactions between negatively-charged G6P and the positively charged bridge are enhanced, but diffusive transport of G6P on the bridge surface, via a hopping mechanism, is slowed to time scales exceeding 10 ns. InMetaD is an advanced sampling technique that boosts a system out of energy minima by biasing the predefined collective variables and estimates unbiased kinetics by reweighting the biased time. We investigated the hopping mechanism of G6P on arginine peptides as a rare event using InMetaD, and computed the hopping time to be 125.5 ± 2.7 ns at 310 K and hopping activation energy to be 25.1 ± 3.7 kJ/mol at the ionic strength of 0 mM.We conducted US simulations to capture the configurational change in the desorption process and calculate the desorption energy. By considering the energy barriers calculated above, the KMC simulations were performed to study the transfer efficiency of G6P. The results show that even at a high ionic strength of 120 mM, arginine peptides are still able to transfer G6P with the lag time 5 seconds, much smaller than lysine peptides with the lag time of 59 seconds reported before [4]. Our work indicates that arginine peptides are a better bridge candidate for electrostatic channeling in terms of decreased lag time compared with lysine peptides. This can help in the future design of efficient artificial cascade reactions by using electrostatic channeling. References ： K. C. Nicolaou, D. J. Edmonds, and P. G. Bulger, Angew. Chemie - Int. Ed., 45, 7134–7186 (2006). doi:10.1002/anie.200601872R. Casasnovas, V. Limongelli, P. Tiwary, P. Carloni, and M. Parrinello, J. Am. Chem. Soc., 139, 4780–4788 (2017). doi:10.1021/jacs.6b12950Y. Liu, I. Matanovic, D. P. Hickey, S. D. Minteer, P. Atanassov, and S. Calabrese Barton, ACS Catal., 8, 7719–7726 (2018). doi:10.1021/acscatal.8b01041Y. Liu, D. P. Hickey, J. Guo, E. Earl, S. Abdellaoui, R. D. Milton, M. S. Sigman, S. D. Minteer, and S. Calabrese Barton, ACS Catal., 7, 2486–2493 (2017). doi:10.1021/acscatal.6b03440 Figure 1

Tunable expression rate control of a growth-decoupled T7 expression system by l-arabinose only

BackgroundPrecise regulation of gene expression is of utmost importance for the production of complex membrane proteins (MP), enzymes or other proteins toxic to the host cell. In this article we show that genes under control of a normally Isopropyl β-d-1-thiogalactopyranoside (IPTG)-inducible PT7-lacO promoter can be induced solely with l-arabinose in a newly constructed Escherichia coli expression host BL21-AI<gp2>, a strain based on the recently published approach of bacteriophage inspired growth-decoupled recombinant protein production.ResultsHere, we show that BL21-AI<gp2> is able to precisely regulate protein production rates on a cellular level in an l-arabinose concentration-dependent manner and simultaneously allows for reallocation of metabolic resources due to l-arabinose induced growth decoupling by the phage derived inhibitor peptide Gp2. We have successfully characterized the system under relevant fed-batch like conditions in microscale cultivation (800 µL) and generated data proofing a relevant increase in specific yields for 6 different Escherichia coli derived MP-GFP fusion proteins by using online-GFP signals, FACS analysis, SDS-PAGE and western blotting.ConclusionsIn all cases tested, BL21-AI<gp2> outperformed the parental strain BL21-AI, operated in growth-associated production mode. Specific MP-GFP fusion proteins yields have been improved up to 2.7-fold. Therefore, this approach allows for fine tuning of MP production or expression of multi-enzyme pathways where e.g. particular stoichiometries have to be met to optimize product flux.

Engineering of Phytosterol-Producing Yeast Platforms for Functional Reconstitution of Downstream Biosynthetic Pathways.

As essential structural molecules for plant plasma membranes, phytosterols are key intermediates for the synthesis of many downstream specialized metabolites of pharmaceutical or agricultural significance, such as brassinosteroids and withanolides. Saccharomyces cerevisiae has been widely used as an alternative producer for plant secondary metabolites. Establishment of heterologous sterol pathways in yeast, however, has been challenging due to either low efficiency or structural diversity, likely a result of crosstalk between the heterologous phytosterol and the endogenous ergosterol biosynthesis. For example, in this study, we engineered campesterol production in yeast using plant enzymes; although we were able to enhance the titer of campesterol to ∼40 mg/L by upregulating the mevalonate pathway, no conversion to downstream products was detected upon the introduction of downstream plant enzymes. Further investigations uncovered two interesting observations about sterol engineering in yeast. First, many heterologous sterols tend to be efficiently and intensively esterified in yeast, which drastically impedes the function of downstream enzymes. Second, yeast can overcome the growth deficiency caused by altered sterol metabolism through repeated culture. By employing metabolic engineering, strain evolution, fermentation engineering, and pathway reconstitution, we were able to reconstruct the multienzyme pathways for the synthesis of a set of phytosterols: campesterol (∼7 mg/L), β-sitosterol (∼2 mg/L), 22-hydroxycampesterol (∼1 mg/L), and 22-hydroxycampest-4-en-3-one (∼4 mg/L). This work identified and addressed some of the technical bottlenecks in phytosterol-derived pathway reconstitution in the baker's yeast and opens up opportunities for efficient bioproduction and metabolic pathway elucidation of this group of phytochemicals.

In vitro multi-enzyme molecular machines - a review

In vitro multi-enzyme molecular machines that follow the designed multi-enzyme pathways, require the rational optimization and adaptation of several purified or partially purified enzyme components, in order to convert certain substrates into target compounds in vitro in an efficient manner. This type of molecular machine is component-based and modularized, so that its design, assembly, and regulation processes are highly flexible. Recently, the advantages of in vitro multi-enzyme molecular machines on the precise control of reaction process and the enhancement of product yield have suggested their great application potential in biomanufacturing. Studies on in vitro multi-enzyme molecular machines have become an important branch of synthetic biology, and are gaining increasing attentions. This article systematically reviews the enzyme component-/module-based construction strategy of in vitro multi-enzyme molecular machines, as well as the research progress on the improvement of compatibility among enzyme components/modules. The current challenges and future prospects of in vitro multi-enzyme molecular machines are also discussed.

A designed bacterial microcompartment shell with tunable composition and precision cargo loading

Microbes often augment their metabolism by conditionally constructing proteinaceous organelles, known as bacterial microcompartments (BMCs), that encapsulate enzymes to degrade organic compounds or assimilate CO2. BMCs self-assemble and are spatially delimited by a semi-permeable shell made up of hexameric, trimeric, and pentameric shell proteins. Bioengineers aim to recapitulate the organization and efficiency of these complex biological architectures by redesigning the shell to incorporate non-native enzymes from biotechnologically relevant pathways. To meet this challenge, a diverse set of synthetic biology tools are required, including methods to manipulate the properties of the shell as well as target and organize cargo encapsulation. We designed and determined the crystal structure of a synthetic shell protein building block with an inverted sidedness of its N- and C-terminal residues relative to its natural counterpart; the inversion targets genetically fused protein cargo to the lumen of the shell. Moreover, the titer of fluorescent protein cargo encapsulated using this strategy is controllable using an inducible tetracycline promoter. These results expand the available set of building blocks for precision engineering of BMC-based nanoreactors and are compatible with orthogonal methods which will facilitate the installation and organization of multi-enzyme pathways.

Assembly of TALE-based DNA scaffold for the enhancement of exogenous multi-enzymatic pathway

Synthetic scaffold systems, which exhibit enzyme clustering effect, have been considered as an important parallel approach for metabolic flux control and pathway enhancement. Here, we described an improved DNA-based scaffold system for synthetic tri-enzymatic pathway in Escherichia coli. With plasmid DNA serving as scaffold and exogenous enzymes fused with rationally designed transcription activator-like effectors (TALEs), our approach successfully clustered three TALE-fused enzymes and significantly increased the production of a mevalonate-producing tri-enzymatic pathway with the optimized scaffold structure and plasmid copy number. These results further suggested the scalability and robustness of the TALE-based scaffold system, and we can assume that it can be used on numerous multi-enzyme metabolic pathways due to its programmable features.

Cell-Free Protein Synthesis and Its Perspectives for Assembling Cells from the Bottom-Up.

The underlying idea of synthetic biology is that biological reactions/modules/systems can be precisely engineered and controlled toward desired products. Numerous efforts in the past decades in deciphering the complexity of biological systems in vivo have led to a variety of tools for synthetic biology, especially based on recombinant DNA. However, one generic limitation of all living systems is that the vast majority of energy input is dedicated to maintain the system as a whole, rather than the small part of interest. Cell-free synthetic biology is aiming at exactly this fundamental limitation, providing the next level of flexibility for engineering and designing biological systems in vitro. New technology has continuously inspired cell-free biology and extended its applications, including gene circuits, spatiotemporally controlled pathways, coactivated catalysts systems, and rationally designed multienzyme pathways, in particular, minimal cell construction. In the context of this special issue, discussing work being carried out in the "MaxSynBio" consortium, the advances in characterizing stochasticity and dynamics of cell-free protein synthesis within cell-sized compartments, as well as the molecular crowding effect, are discussed. The organization of spatial heterogeneity is the key prerequisite for achieving hierarchy and stepwise assembly of minimal cells from the bottom-up.

Analysis of Interdependent Kinetic Controls of Fatty Acid Synthases.

Biocatalytic systems (e.g., multienzyme pathways or complexes) enable the conversion of simple sugars into complex products under ambient conditions and, thus, represent promising platforms for the synthesis of renewable fuels and chemicals. Unfortunately, to date, many of these systems have proven difficult to engineer without a detailed understanding of the kinetic relationships that regulate the concerted action of their constituent enzymes. This study develops a mechanistic kinetic model of the fatty acid synthase (FAS) of Escherichia coli and uses that model to determine how different FAS components work together to control the production of free fatty acids-precursors to a wide range of oleochemicals. Perturbational analyses indicate that the modification or overexpression of a single FAS component can depress fatty acid production (a commonly observed phenomenon) by sequestering the proteins with which it interacts and/or by depleting common substrate pools. Compositional studies, in turn, suggest that simple changes in the ratios of FAS components can alter the average length of fatty acids but show that specialized enzymes (i.e., highly specific ketoacyl synthases or thioesterases) are required for narrow product profiles. Intriguingly, a sensitivity analysis indicates that two components primarily influence-and, thus, enable fine control over-total production, but suggests that the enzymes that regulate product profile are more broadly influential. Findings thus reveal the general importance of kinetic considerations in efforts to engineer fatty acid biosynthesis and provide strategies-and a kinetic model-for incorporating those considerations into FAS designs.

Spatial Regulation of Biomolecular Interactions with a Switchable Trident-Shaped DNA Nanoactuator.

DNA nanostructures with controllable motions and functions have been used as flexible scaffolds to precisely and spatially organize molecular reactions at the nanoscale. The construction of dynamic DNA nanostructures with site-specifically incorporated functional elements is a critical step toward building nanomachines. Artificial self-assembled DNA nanostructures have also been developed to mimic key biological processes like various small biomolecule- and protein-based functional biochemistry pathways. Here, we report a self-assembled dynamic trident-shaped DNA (TS DNA) nanoactuator, in which biomolecules can be tethered to the three "arms" of the TS DNA nanoactuator. The TS DNA nanoactuator is implemented as the mechanical scaffold for the reconfiguration of fluorescent/quenching molecules and the assembly of gold nanoparticles, which exhibit controlled spatial separation. Furthermore, two enzymes (glucose oxidase and horseradish peroxidase) are attached to the two outer arms of the TS DNA nanoactuator, which show an enhanced cascade reaction efficiency compared to free enzymes. The efficiency of the two-enzyme cascade reaction can be spatially regulated by switching the TS DNA nanoactuator between opened, semiopened, and closed states through adding the "thermodynamic drivers" (fuels or antifuels). This is the first report to precisely modulate the relative position of coupled enzyme with multiple states and only based on one dynamic DNA scaffold. The present TS DNA nanoactuator with multistage conformational transition functionality could be applied as a potential platform to precisely and dynamically control the multienzyme pathways and would broaden the scope of DNA nanostructures in single-molecule biology applications.

Strategies for microbial synthesis of high-value phytochemicals.

Phytochemicals are of great pharmaceutical and agricultural importance, but often exhibit low abundance in nature. Recent demonstrations of industrial-scale production of phytochemicals in yeast have shown that microbial production of these high-value chemicals is a promising alternative to sourcing these molecules from native plant hosts. However, a number of challenges remain in the broader application of this approach, including the limited knowledge of plant secondary metabolism and the inefficient reconstitution of plant metabolic pathways in microbial hosts. In this Review, we discuss recent strategies to achieve microbial biosynthesis of complex phytochemicals, including strategies to: (1) reconstruct plant biosynthetic pathways that have not been fully elucidated by mining enzymes from native and non-native hosts or by enzyme engineering; (2) enhance plant enzyme activity, specifically cytochrome P450 activity, by improving efficiency, selectivity, expression or electron transfer; and (3) enhance overall reaction efficiency of multi-enzyme pathways by dynamic control, compartmentalization or optimization with the host's metabolism. We also highlight remaining challenges to - and future opportunities of - this approach.

Biosynthetic Pathway and Metabolic Engineering of Plant Dihydrochalcones.

Dihydrochalcones are plant natural products containing the phenylpropanoid backbone and derived from the plant-specific phenylpropanoid pathway. Dihydrochalcone compounds are important in plant growth and response to stresses and, thus, can have large impacts on agricultural activity. In recent years, these compounds have also received increased attention from the biomedical community for their potential as anticancer treatments and other benefits for human health. However, they are typically produced at relatively low levels in plants. Therefore, an attractive alternative is to express the plant biosynthetic pathway genes in microbial hosts and to engineer the metabolic pathway/host to improve the production of these metabolites. In the present review, we discuss in detail the functions of genes and enzymes involved in the biosynthetic pathway of the dihydrochalcones and the recent strategies and achievements used in the reconstruction of multi-enzyme pathways in microorganisms in efforts to be able to attain higher amounts of desired dihydrochalcones.

Network design and analysis for multi-enzyme biocatalysis

BackgroundAs more and more biological reaction data become available, the full exploration of the enzymatic potential for the synthesis of valuable products opens up exciting new opportunities but is becoming increasingly complex. The manual design of multi-step biosynthesis routes involving enzymes from different organisms is very challenging. To harness the full enzymatic potential, we developed a computational tool for the directed design of biosynthetic production pathways for multi-step catalysis with in vitro enzyme cascades, cell hydrolysates and permeabilized cells.ResultsWe present a method which encompasses the reconstruction of a genome-scale pan-organism metabolic network, path-finding and the ranking of the resulting pathway candidates for proposing suitable synthesis pathways. The network is based on reaction and reaction pair data from the Kyoto Encyclopedia of Genes and Genomes (KEGG) and the thermodynamics calculator eQuilibrator. The pan-organism network is especially useful for finding the most suitable pathway to a target metabolite from a thermodynamic or economic standpoint. However, our method can be used with any network reconstruction, e.g. for a specific organism. We implemented a path-finding algorithm based on a mixed-integer linear program (MILP) which takes into account both topology and stoichiometry of the underlying network. Unlike other methods we do not specify a single starting metabolite, but our algorithm searches for pathways starting from arbitrary start metabolites to a target product of interest. Using a set of biochemical ranking criteria including pathway length, thermodynamics and other biological characteristics such as number of heterologous enzymes or cofactor requirement, it is possible to obtain well-designed meaningful pathway alternatives. In addition, a thermodynamic profile, the overall reactant balance and potential side reactions as well as an SBML file for visualization are generated for each pathway alternative.ConclusionWe present an in silico tool for the design of multi-enzyme biosynthetic production pathways starting from a pan-organism network. The method is highly customizable and each module can be adapted to the focus of the project at hand. This method is directly applicable for (i) in vitro enzyme cascades, (ii) cell hydrolysates and (iii) permeabilized cells.