Engineering metabolism and product formation in Corynebacterium glutamicum by coordinated gene overexpression

Single gene overexpression in product pathways such as lysine synthesis has often been employed in metabolic engineering efforts aiming at pathway flux amplification and metabolite overproduction. This approach is limited due to metabolic flux imbalances that often lead to unpredictable physiological responses and suboptimal metabolite productivity. This deficiency can be overcome by the coordinated overexpression of more than one flux controlling genes in a production pathway selected by considering their individual contributions on the cell physiology This concept is demonstrated by the simultaneous overexpression of pyruvate carboxylase and aspartate kinase, two key enzymes in central carbon metabolism and the lysine production pathway in Corynebacterium glutamicum. Contrary to expectations based on the importance of each of these two genes in lysine production, the monocistronic overexpression of either gene results in marginal changes in the overall lysine productivity due to either reduced cell growth or reduced lysine specific productivity. In contrast, the simultaneous amplification of the activities of the two enzymes yielded more than 250% increase of the lysine specific productivity in lactate minimal medium without affecting the growth rate or final cell density of the culture. These results demonstrate that significant flux amplification in complex pathways involving central carbon metabolism is possible through coordinated overexpression of more than one gene in the pathway. This can be achieved either by external, gene expression inducing, controls or controls responding to the physiological cellular state.     

Metabolite damage and repair in metabolic engineering design

The necessarily sharp focus of metabolic engineering and metabolic synthetic biology on pathways and their fluxes has tended to divert attention from the damaging enzymatic and chemical side-reactions that pathway metabolites can undergo. Although historically overlooked and underappreciated, such metabolite damage reactions are now known to occur throughout metabolism and to generate (formerly enigmatic) peaks detected in metabolomics datasets. It is also now known that metabolite damage is often countered by dedicated repair enzymes that undo or prevent it. Metabolite damage and repair are highly relevant to engineered pathway design: metabolite damage reactions can reduce flux rates and product yields, and repair enzymes can provide robust, host-independent solutions. Herein, after introducing the core principles of metabolite damage and repair, we use case histories to document how damage and repair processes affect efficient operation of engineered pathways – particularly those that are heterologous, non-natural, or cell-free. We then review how metabolite damage reactions can be predicted, how repair reactions can be prospected, and how metabolite damage and repair can be built into genome-scale metabolic models. Lastly, we propose a versatile ‘plug and play’ set of well-characterized metabolite repair enzymes to solve metabolite damage problems known or likely to occur in metabolic engineering and synthetic biology projects.     

Enhanced lycopene productivity by manipulation of carbon flow to isopentenyl diphosphate in Escherichia coli

Lycopene is a useful phytochemical that holds great commercial value. In our study the lycopene production pathway in E. coli originating from the precursor isopentenyl diphosphate (IPP) of the non-mevalonate pathway was reconstructed. This engineered strain of E. coli accumulated lycopene intracellularly under aerobic conditions. As a next step, the production of lycopene was enhanced through metabolic engineering methodologies. Various competing pathways at the pyruvate and acetyl-CoA nodes were inactivated to divert more carbon flux to IPP and subsequently to lycopene. It was found that the ackA-pta, nuo mutant produced a higher amount of lycopene compared to the parent strain. To further enhance lycopene production, a novel mevalonate pathway, in addition to the already existing non-mevalonate pathway, was engineered. This pathway utilizes acetyl-CoA as precursor, condensing it to form acetoacetyl-CoA and subsequently leading to formation of IPP. Upon the introduction of this new pathway, lycopene production increased by over 2-fold compared to the ackA-pta, nuo mutant strain.     

Disruption of a Global Regulatory Gene to Enhance Central Carbon Flux into Phenylalanine Biosynthesis in Escherichia coli

Genetic engineering of microbes for commercial metabolite production traditionally has sought to alter the levels and/or intrinsic activities of key enzymes in relevant biosynthetic pathway(s). Microorganisms exploit similar strategies for flux control, but also coordinate flux through sets of related pathways by using global regulatory circuits. We have engineered a global regulatory system of Escherichia coli, Csr (carbon storage regulator), to increase precursor for aromatic amino acid biosynthesis. Disruption of csrA increases gluconeogenesis, decreases glycolysis, and thus elevates phosphoenolpyruvate, a limiting precursor of aromatics. A strain in which the aromatic (shikimate) pathway had been optimized produced twofold more phenylalanine when csrA was disrupted. Overexpression of tktA (transketolase) to increase the other precursor, erythrose-4-phosphate, yielded ∼1.4-fold enhancement, while both changes were additive. These effects of csrA were not mediated by increasing the regulatory enzymes of phenylalanine biosynthesis. This study introduces the concept of “global metabolic engineering” for second-generation strain improvement. Received: 25 October 2000 / Accepted: 8 December 2000     

Diversion of flux toward sesquiterpene production in Saccharomyces cerevisiae by fusion of host and heterologous enzymes

The ability to transfer metabolic pathways from the natural producer organisms to the well-characterized cell factory Saccharomyces cerevisiae is well documented. However, as many secondary metabolites are produced by collaborating enzymes assembled in complexes, metabolite production in yeast may be limited by the inability of the heterologous enzymes to collaborate with the native yeast enzymes. This may cause loss of intermediates by diffusion or degradation or due to conversion of the intermediate through competitive pathways. To bypass this problem, we have pursued a strategy in which key enzymes in the pathway are expressed as a physical fusion. As a model system, we have constructed several fusion protein variants in which farnesyl diphosphate synthase (FPPS) of yeast has been coupled to patchoulol synthase (PTS) of plant origin (Pogostemon cablin). Expression of the fusion proteins in S. cerevisiae increased the production of patchoulol, the main sesquiterpene produced by PTS, up to 2-fold. Moreover, we have demonstrated that the fusion strategy can be used in combination with traditional metabolic engineering to further increase the production of patchoulol. This simple test case of synthetic biology demonstrates that engineering the spatial organization of metabolic enzymes around a branch point has great potential for diverting flux toward a desired product.     

Genetic engineering of carotenoid formation in tomato fruit and the potential application of systems and synthetic biology approaches

The health benefits conferred by numerous carotenoids have led to attempts to elevate their levels in foodstuffs. Tomato fruit and its products contain the potent antioxidant lycopene and are the predominant source of lycopene in the human diet. In addition, tomato products are an important source of provitamin A (β-carotene). The presence of other health promoting phytochemicals such as tocopherols and flavonoids in tomato has led to tomato and its products being termed a functional food. Over the past decade genetic/metabolic engineering of carotenoid biosynthesis and accumulation has resulted in the generation of transgenic varieties containing high lycopene and β-carotene contents. In achieving this important goal many fundamental lessons have been learnt. Most notably is the observation that the endogenous carotenoid pathways in higher plants appear to resist engineered changes. Typically, this resistance manifests itself through intrinsic regulatory mechanisms that are “silent” until manipulation of the pathway is initiated. These mechanisms may include feedback inhibition, forward feed, metabolite channelling, and counteractive metabolic and cellular perturbations. In the present article we will review progress made in the genetic engineering of carotenoids in tomato fruit, highlighting the limiting regulatory mechanisms that have been observed experimentally. The predictability and efficiency of the present engineering strategies will be questioned and the potential of more Systems and Synthetic Biology approaches to the enhancement of carotenoids will be assessed.     

Precursor balancing for metabolic engineering of lycopene production in Escherichia coli

One issue that must be addressed in the rational design of metabolic pathways is the elimination of potential bottlenecks in the upstream pathways. We have reconstructed the isoprenoid pathway to overproduce the carotenoid lycopene in Escherichia coli. Here we show that the distribution between pyruvate and glyceraldehyde 3-phosphate (G3P), the originating precursors of the isoprenoid pathway, is a major factor that can limit isoprenoid production yields in E. coli. In particular, alterations in the central metabolism that redirect flux from pyruvate back to G3P enhance lycopene production, while alterations that channel carbon flux away from the G3P pool have the opposite effect. These results suggest that G3P may be limiting in the biosynthesis of lycopene, and modifications that achieve a more equitable distribution between the two precursors are able to increase the lycopene yield in metabolically engineered E. coli.     

Dynamic metabolic control: towards precision engineering of metabolism

Advances in metabolic engineering have led to the synthesis of a wide variety of valuable chemicals in microorganisms. The key to commercializing these processes is the improvement of titer, productivity, yield, and robustness. Traditional approaches to enhancing production use the “push–pull-block” strategy that modulates enzyme expression under static control. However, strains are often optimized for specific laboratory set-up and are sensitive to environmental fluctuations. Exposure to sub-optimal growth conditions during large-scale fermentation often reduces their production capacity. Moreover, static control of engineered pathways may imbalance cofactors or cause the accumulation of toxic intermediates, which imposes burden on the host and results in decreased production. To overcome these problems, the last decade has witnessed the emergence of a new technology that uses synthetic regulation to control heterologous pathways dynamically, in ways akin to regulatory networks found in nature. Here, we review natural metabolic control strategies and recent developments in how they inspire the engineering of dynamically regulated pathways. We further discuss the challenges of designing and engineering dynamic control and highlight how model-based design can provide a powerful formalism to engineer dynamic control circuits, which together with the tools of synthetic biology, can work to enhance microbial production.     

Synergy between methylerythritol phosphate pathway and mevalonate pathway for isoprene production in Escherichia coli

Isoprene, a key building block of synthetic rubber, is currently produced entirely from petrochemical sources. In this work, we engineered both the methylerythritol phosphate (MEP) pathway and the mevalonate (MVA) pathway for isoprene production in E. coli. The synergy between the MEP pathway and the MVA pathway was demonstrated by the production experiment, in which overexpression of both pathways improved the isoprene yield about 20-fold and 3-fold, respectively, compared to overexpression of the MEP pathway or the MVA pathway alone. The ¹³C metabolic flux analysis revealed that simultaneous utilization of the two pathways resulted in a 4.8-fold increase in the MEP pathway flux and a 1.5-fold increase in the MVA pathway flux. The synergy of the dual pathway was further verified by quantifying intracellular flux responses of the MEP pathway and the MVA pathway to fosmidomycin treatment and mevalonate supplementation. Our results strongly suggest that coupling of the complementary reducing equivalent demand and ATP requirement plays an important role in the synergy of the dual pathway. Fed-batch cultivation of the engineered strain overexpressing the dual pathway resulted in production of 24.0 g/L isoprene with a yield of 0.267 g/g of glucose. The synergy of the MEP pathway and the MVA pathway also successfully increased the lycopene productivity in E. coli, which demonstrates that it can be used to improve the production of a broad range of terpenoids in microorganisms.     

Metabolic control analysis of Aspergillus niger L-arabinose catabolism

A mathematical model of the L-arabinose/D-xylose catabolic pathway of Aspergillus niger was constructed based on the kinetic properties of the enzymes. For this purpose L-arabinose reductase, L-arabitol dehydrogenase and D-xylose reductase were purified using dye-affinity chromatography, and their kinetic properties were characterized. For the other enzymes of the pathway the kinetic data were available from the literature. The metabolic model was used to analyze flux and metabolite concentration control of the L-arabinose catabolic pathway. The model demonstrated that flux control does not reside at the enzyme following the intermediate with the highest concentration, L-arabitol, but is distributed over the first three steps in the pathway, preceding and following L-arabitol. Flux control appeared to be strongly dependent on the intracellular L-arabinose concentration. At 5 mM intracellular L-arabinose, a level that resulted in realistic intermediate concentrations in the model, flux control coefficients for L-arabinose reductase, L-arabitol dehydrogenase and L-xylulose reductase were 0.68, 0.17 and 0.14, respectively. The analysis can be used as a guide to identify targets for metabolic engineering aiming at either flux or metabolite level optimization of the L-arabinose catabolic pathway of A. niger. Faster L-arabinose utilization may enhance utilization of readily available organic waste containing hemicelluloses to be converted into industrially interesting metabolites or valuable enzymes or proteins.     

Microbial engineering strategies to improve cell viability for biochemical production

Efficient production of biochemicals using engineered microbes as whole-cell biocatalysts requires robust cell viability. Robust viability leads to high productivity and improved bioprocesses by allowing repeated cell recycling. However, cell viability is negatively affected by a plethora of stresses, namely chemical toxicity and metabolic imbalances, primarily resulting from bio-synthesis pathways. Chemical toxicity is caused by substrates, intermediates, products, and/or by-products, and these compounds often interfere with important metabolic processes and damage cellular infrastructures such as cell membrane, leading to poor cell viability. Further, stresses on engineered cells are accentuated by metabolic imbalances, which are generated by heavy metabolic resource consumption due to enzyme overexpression, redistribution of metabolic fluxes, and impaired intracellular redox state by co-factor imbalance. To address these challenges, herein, we discuss a range of key microbial engineering strategies, substantiated by recent advances, to improve cell viability for commercially sustainable production of biochemicals from renewable resources.     

Increasing the flux in metabolic pathways: A metabolic control analysis perspective

The problems of engineering increased flux in metabolic pathways are analyzed in terms of the understanding provided by metabolic control analysis. Over-expression of a single enzyme is unlikely to be effective unless it is known to have a high flux control coefficient, which can be used as an approximate predictive tool. This is likely to rule out enzymes subject to feedback inhibition, because it transfers control downstream from the inhibited enzyme to the enzymes utilizing the feedback metabolite. Although abolishing feedback inhibition can restore flux control to an enzyme, it is also likely to cause large increases in the concentrations of metabolic intermediates. Simultaneous and coordinated over-expression of most of the enzymes in a pathway can, in principle, produce substantial flux increases without changes in metabolite levels, though technically it may be difficult to achieve. It is, however, closer to the method used by cells to change flux levels, where coordinated changes in the level of activity of pathway enzymes are the norm. Another option is to increase the demand for the pathway product, perhaps by increasing its rate of excretion or removal. Copyright 1998 John Wiley & Sons, Inc.     

Matrix method for determining steps most rate-limiting to metabolic fluxes in biotechnological processes

The metabolic control theory developed by Kacser, Burns, Heinrich, and Rapoport is briefly outlined, extended, and transformed so as optimally to address some biotechnological questions. The extensions include (i) a new theorem that relates the control of metabolite concentrations by enzyme activities to flux ratios at branches in metabolic pathways; (ii) a new theorem that does the same for the control of the distribution of the flux over two branches; (iii) a method that expresses these controls into properties (the so-called elasticity coefficients) of the enzymes in the pathway; and (iv) a theorem that relates the effects of changes in metabolite concentrations on reaction rates to the effects of changes in enzyme properties on the same rates. Matrix equations relating the flux control and concentration control coefficients to the elasticity coefficients of enzymes in simple linear and branched pathways incorporating feedback are given, together with their general solutions and a numerical example. These equations allow one to develop rigorous criteria by which to decide the optimal strategy for the improvement of a microbial process. We show how this could be used in deciding which property of which enzyme should be changed in order to obtain the maximal concentration of a metabolite or the maximal metabolic flux.     

Engineering carotenoid production in mammalian cells for nutritionally enhanced cell-cultured foods

Metabolic engineering of mammalian cells has to-date focused primarily on biopharmaceutical protein production or the manipulation of native metabolic processes towards therapeutic aims. However, significant potential exists for expanding these techniques to diverse applications by looking across the taxonomic tree to bioactive metabolites not synthesized in animals. Namely, cross-taxa metabolic engineering of mammalian cells could offer value in applications ranging fromfood and nutrition to regenerative medicine and gene therapy. Towards the former, recent advances in meat production through cell culture suggest the potential to produce meat with fine cellular control, where tuning composition through cross-taxa metabolic engineering could enhance nutrition and food-functionality. Here we demonstrate this possibility by engineering primary bovine and immortalized murine muscle cells with prokaryotic enzymes to endogenously produce the antioxidant carotenoids phytoene, lycopene and β-carotene. These phytonutrients offer general nutritive value and protective effects against diseases associated with red and processed meat consumption, and so offer a promising proof-of-concept for nutritional engineering in cultured meat. We demonstrate the phenotypic integrity of engineered cells, the ability to tune carotenoid yields, and the antioxidant functionality of these compounds in vitro towards both nutrition and food-quality objectives. Our results demonstrate the potential for tailoring the nutritional profile of cultured meats. They further lay a foundation for heterologous metabolic engineering of mammalian cells for applications outside of the clinical realm.     

Channeling in native microbial pathways: Implications and challenges for metabolic engineering

Intracellular enzymes can be organized into a variety of assemblies, shuttling intermediates from one active site to the next. Eukaryotic compartmentalization within mitochondria and peroxisomes and substrate tunneling within multi-enzyme complexes have been well recognized. Intriguingly, the central pathways in prokaryotes may also form extensive channels, including the heavily branched glycolysis pathway. In vivo channeling through cascade enzymes is difficult to directly measure, but can be inferred from in vitro tests, reaction thermodynamics, transport/reaction modeling, analysis of molecular diffusion and protein interactions, or steady state/dynamic isotopic labeling. Channeling presents challenges but also opportunities for metabolic engineering applications. It rigidifies fluxes in native pathways by trapping or excluding metabolites for bioconversions, causing substrate catabolite repressions or inferior efficiencies in engineered pathways. Channeling is an overlooked regulatory mechanism used to control flux responses under environmental/genetic perturbations. The heterogeneous distribution of intracellular enzymes also confounds kinetic modeling and multiple-omics analyses. Understanding the scope and mechanisms of channeling in central pathways may improve our interpretation of robust fluxomic topology throughout metabolic networks and lead to better design and engineering of heterologous pathways.     

Metabolic regulation analysis of wild-type and arcA mutant Escherichia coli under nitrate conditions using different levels of omics data

It is of practical interest to investigate the effect of nitrates on bacterial metabolic regulation of both fermentation and energy generation, as compared to aerobic and anaerobic growth without nitrates. Although gene level regulation has previously been studied for nitrate assimilation, it is important to understand this metabolic regulation in terms of global regulators. In the present study, therefore, we measured gene expression using DNA microarrays, intracellular metabolite concentrations using CE-TOFMS, and metabolic fluxes using the (13)C-labeling technique for wild-type E. coli and the ΔarcA (a global regulatory gene for anoxic response control, ArcA) mutant to compare the metabolic state under nitrate conditions to that under aerobic and anaerobic conditions without nitrates in continuous culture conditions at a dilution rate of 0.2 h(-1). In wild-type, although the measured metabolite concentrations changed very little among the three culture conditions, the TCA cycle and the pentose phosphate pathway fluxes were significantly different under each condition. These results suggested that the ATP production rate was 29% higher under nitrate conditions than that under anaerobic conditions, whereas the ATP production rate was 10% lower than that under aerobic conditions. The flux changes in the TCA cycle were caused by changes in control at the gene expression level. In ΔarcA mutant, the TCA cycle flux was significantly increased (4.4 times higher than that of the wild type) under nitrate conditions. Similarly, the intracellular ATP/ADP ratio increased approximately two-fold compared to that of the wild-type strain.