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Engineering redox balance through cofactor systems Xiulai Chen1,2,3, Shubo Li1,2,3, and Liming Liu1,2,3 1
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China 3 Laboratory of Food Microbial-Manufacturing Engineering, Jiangnan University, Wuxi 214122, China 2
Redox balance plays an important role in the production of enzymes, pharmaceuticals, and chemicals. To meet the demands of industrial production, it is desirable that microbes maintain a maximal carbon flux towards target metabolites with no fluctuations in redox. This requires functional cofactor systems that support dynamic homeostasis between different redox states or functional stability in a given redox state. Redox balance can be achieved by improving the self-balance of a cofactor system, regulating the substrate balance of a cofactor system, and engineering the synthetic balance of a cofactor system. This review summarizes how cofactor systems can be manipulated to improve redox balance in microbes. Cofactor systems in cellular physiology Cofactors provide redox carriers for biosynthetic and catabolic reactions, and act as important agents in transfer of energy for the cell [1]. In microorganisms, the NADH/ NAD+ and NADPH/NADP+ cofactor pairs are involved in 740 and 887 biochemical reactions and interact with 433 and 462 enzymes, respectively. They possess a series of physiological functions, including regulating energy metabolism [2], adjusting the intracellular redox state, controlling carbon flux, improving the function and activity of the mitochondrion [3], regulating the cell life cycle [4], and modulating microbial virulence [5]. It is clear that cofactors are essential to large numbers of biochemical reactions, and as expected, their manipulation has substantial effects on redox balance. Maintaining the cellular redox balance is a basic requirement for living cells to sustain metabolism [6]. The nucleotides, NAD(P)+ and NAD(P)H, as the most important redox carriers involved in metabolism, not only serve as electron acceptors in the breakdown of catabolic substrates but also provide the cell with the reducing power needed in energy-conserving redox reactions [7]. A balance in the rates of oxidation and reduction of these nucleotides is a prerequisite for both catabolism and anabolism [7]. Some examples have illustrated the requirement for redox balance and availability. An irreversible reaction catalyzed Corresponding author: Liu, L. ([email protected]). Keywords: cofactor systems; cofactor engineering; redox balance; synthetic biology. 0167-7799/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2014.04.003
by trans-enoyl-coenzyme A (CoA) reductase [8] is required to use accumulated NADH as a driving force for directing the flux to 1-butanol. To improve xylose fermentation, NADP+-dependent glyceraldehyde 3-phosphate dehydrogenase is used to increase the NADPH pool and create a better redox balance [9]. To gain a comprehensive understanding of physiology and cellular metabolism, the genome-scale metabolic models iJO1366 for Escherichia coli [10] and iMM904 for Saccharomyces cerevisiae [11] are used to search for NAD(P)H/NAD(P)+-related reactions. There are 221 reactions in iJO1366 and 223 reactions in iMM904, which are generally divided into three types: metabolic reactions, exchange reactions, and heterologous reactions. The heterologous reactions that are derived from synthetic pathways are different from the metabolic reactions that originate from metabolic pathways in models. Therefore, we assume the following structural design incorporating cofactor systems to manipulate redox balance (Figure 1): (i) self-balance of a cofactor system based on metabolic reactions; (ii) substrate balance of a cofactor system based on exchange reactions; (iii) synthetic balance of a cofactor system based on introduced reactions. According to these three systems, we show how certain strategies can be used to manipulate redox balance through microbial cofactor systems. Improving the self-balance of a cofactor system In microorganisms, sugars are broken down by respiration, leading to the reduction of NAD+ to NADH; in eukaryotic organisms, this occurs in separate cellular compartments. NADH is mainly formed by a rather limited set of cofactor reactions, for example, glycolysis, fatty acid oxidation, and the tricarboxylic acid (TCA) cycle. Therefore, the self-balance of a cofactor system is used to maintain the redox balance automatically through three routes in S. cerevisiae: overflow metabolism, respiratory chain, and mitochondrial redox shuttles. For example, glucose is exclusively broken down via acetate and glycerol fermentation, which is a redox-neutral process. Acetate overflow is believed to reoxidize NADH and balance the NAD(P)H/ NAD(P)+ ratio [12], and glycerol formation maintains the cytosolic redox balance and compensates for cellular reactions that produce NADH in S. cerevisiae [13]. In another example, the two sides of the mitochondria inner membrane couple NADH reoxidation to the respiratory chain [14]. Although mitochondrial NADH can be oxidized by Trends in Biotechnology xx (2014) 1–7
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Cofactor transion (A)
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Figure 1. Conceptual schematic representation of engineering of the redox balance. Schematic representation of strategies for engineering the redox balance, separated by different colored lines. Squares represent redox states. The yellow arrows, blue arrows, and green arrows in squares represent strategies to maintain redox balance. (A) Industrial microbes achieve redox balance by improving the self-balance of a cofactor system. State I represents the original redox state of the wild type strain, with the target to overcome redox imbalance, which is controlled by the original growth status. On encountering environmental perturbations (State II), redox state is altered, meaning that either metabolic flux redistribution or nonmetabolic property changes occur. (B) Industrial microbes achieve redox balance by regulating the substrate balance of a cofactor system. To meet the demand of the microbes for special physiology (such as chemical production), redox state is altered from State I (the original state) to State II (substrate perturbations) with redox balance achieved. (C) Industrial microbes achieve redox balance by engineering the synthetic balance of a cofactor system. When the original state (State I) is disturbed by synthetic imbalance, redox state is transmitted to transition state (State II) with redox imbalance. Further, redox state is enlarged by cofactor engineering via different biotechnologies and reshaped into State III (synthesis perturbations) with redox balance. When the perturbation is strong enough to drive cells to surpass State II space limits, redox state may fall into State III by transition, with the target functionality still stably maintained.
proton-translocating complex I or by an internal NADH dehydrogenase [15], cytosolic NADH can be oxidized in mitochondria either by an external NADH:ubiquinone oxidoreductase [16] or by a redox shuttle [17], such as the glycerol-3-phosphate shuttle. Similarly, the mitochondrial redox shuttles in S. cerevisiae, mainly containing the ethanol–acetaldehyde redox shuttle, the malate–oxaloacetate shuttle, the malate–aspartate shuttle, and the citrate–oxoglutarate shuttle, are important during respiratory growth, and are interconverted to balance NADH/NAD+ in both the cytosol and mitochondrial compartments [18]. Therefore, such a self-balance in cofactor systems can maintain functional stability and dynamic homeostasis in a given redox state automatically, so as to adapt to the changing environment. Regulating the substrate balance of a cofactor system The intracellular cofactor state can be improved by providing a variety of compounds that can serve as acceptors of electrons and precursors of NAD+, or by altering the environmental conditions of the culture. Such compounds can affect electron transfer by modifying NADH reoxidation, and thus achieve cofactor transition to improve the redox balance. Therefore, substrate balance of a cofactor system is used to achieve the optimal NADH/NAD+ and ATP/ADP ratios through regulating the redox state of a substrate, the co-substrates, and the environmental conditions. A good 2
example of regulation of the substrate redox state is anaerobic fermentation of glycerol. The utilization of glycerol as a substrate for the production of biochemicals and biofuels is one promising avenue for the coupling of processes within a biorefinery. The number of reducing equivalents produced during the conversion of glycerol into the metabolic intermediate pyruvate is twofold higher than during the metabolism of lignocellulosic sugars such as glucose or xylose [19]. These additional reducing equivalents provide glycerol with the natural advantage of higher theoretical product yields for reduced chemicals and fuels. The redox balance is maintained during anaerobic fermentation of glycerol in two ways: transferring of electrons to internally generate organic compounds [20] and shifting of electrons towards reduced products. These pathways provide channels for balancing redox and a means of maximizing the production of reduced chemicals and fuels [21]. Several studies have demonstrated that co-substrates that serve as external electron acceptors, such as ferricyanide, nitrate, organic acids, acetoin, aldehyde, and phenazine, can promote reoxidation of NADH, maintain an optimal NADH/NAD+ ratio and ATP level, and facilitate redox balance [22]. The intracellular redox balance is concerned not merely with the above-mentioned two aspects, but with the environmental conditions. When NADH is oxidized to NAD+, dissolved oxygen, as the electron acceptor in the oxidative phosphorylation, can
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be used to maintain redox balance and meet the total energy requirement for metabolism by influencing the activities of all enzymes that use or produce NAD+ and ATP [23]. In addition, the oxidoreduction potential can affect the synthesis or stability of certain enzymes such as the electron transport-related enzymes [24] and NAD+/ NADH-dependent enzymes [25], which in turn affects the ATP yield, NAD+/NADH ratio, and metabolic fluxes [26]. Thus, such a substrate balance in cofactor systems can often manipulate transitions between different redox states, so as to adapt to the fermentative condition. Engineering the synthetic balance of a cofactor system In a typical pathway, the status of redox is reflected by the cofactors NAD(P)H/NAD(P)+. Redox balance occurs when the production and consumption of cofactors are approximately equal. Imbalanced oxidoreduction potential can damage cells, waste energy, and/or carbon, and even lead to metabolic arrest. Fortunately, redox-imbalanced pathways can sometimes be rebalanced through six approaches (Figure 2): promoter engineering tuning cofactor-dependent
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Promoter engineering Microbial production of chemicals, involving the introduction of several genes encoding the enzymes of a metabolic pathway, is now an attractive alternative to chemical synthesis [27]. However, product titers and yields are often limited by redox imbalances, due to the utilization of cofactor-dependent enzymes in synthetic pathways [19]. To address this issue, several attempts at promoter engineering have shown great promise in helping generate the dynamic range necessary to enable fine-tuned gene expression for synthetic biology applications [28]. Several
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gene expression, genome-scale engineering reducing cofactor-dependent functional redundancy, protein engineering improving the specificity of cofactor-dependent enzymes, structural synthetic biotechnology enhancing cofactor transmission efficiency in synthetic pathways, systems metabolic engineering determining and optimizing the influence of cofactors at the systems level, and cofactor regeneration reconstructing metabolic pathways of cofactors.
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Figure 2. Redox balance can be maintained through (A) promoter engineering: the gene expression levels are regulated by engineering promoter strengths (green), properties (red), ribosome binding (blue), and intergenic regions (black); (B) genome-scale engineering: the top-down approach aims at further simplification of existing cells by removal of nonessential genes (green and gray); (C) structural synthetic biotechnology: structural synthetic biotechnology has fostered a variety of activities in cofactor-dependent metabolic pathways, aimed at designing and synthesizing DNA scaffolds, RNA scaffolds, and protein scaffolds; (D) systems metabolic engineering and protein engineering: systems metabolic engineering can be used to create new metabolic pathways, cellular regulatory circuits, and functions with the availability of necessary cofactors through the ‘omic’ techniques and computational techniques. During this process, protein engineering can be used to achieve redox balance by improving enzyme activity, changing substrate specificity, modifying cofactor specificity, constructing multi-enzyme complexes, and creating bioorthogonal redox systems; (E) cofactor regeneration: cofactor regeneration has provided an extra route to alter the intracellular cofactor pool and maintain the cellular redox balance by cytoplasmic H2O-forming NADH oxidase (NOX), mitochondrial alternative oxidase (AOX), phosphite dehydrogenase (PTDH), mitochondrial NADH kinase (POS5), mannitol dehydrogenase (MDH), and formate dehydrogenase (FDH).
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Review strategies have been developed to regulate gene expression, including engineering promoter strengths [29], properties [30], intergenic regions [31], and ribosome binding [32] (Figure 2A). Engineering promoter properties has been used to produce fatty acid ethyl ester (FAEE), an excellent diesel fuel replacement owing to its low water solubility and high energy density [30]. In this case, a dynamic sensor–regulator system is developed to improve FAEE biosynthetic pathway containing three modules: module A contains the E. coli native fatty acid synthase and a cytoplasmic thioesterase gene (tesA) and produces fatty acids; module B contains a pyruvate decarboxylase gene ( pdc) and an alcohol dehydrogenase gene (adhB) and produces ethanol; module C contains an acyl-CoA synthase gene ( fadD) and a wax-ester synthase gene (atfA) and produces FAEE as the end product. Controlling the strength of module B at a medium level and module C at high level results in a large increase in FAEE production (1.5 g/l). This result indicates that the medium expression level of module B improves the cofactor surplus (such as NADH) owing to the medium expression level of adhB and the metabolic imbalance as a result of the accumulation of cofactor. Another example, a fatty acid-producing strain has been developed by introducing a synthetic pathway that is re-cast into three modules: the upstream acetyl CoA formation module (GLY module, containing NAD(P)+-dependent reductions), the intermediary acetyl CoA activation module (ACA module), and the downstream fatty acid synthase module (FAS module, containing NAD(P)H-dependent oxidations) [33]. Combinatorial optimization of protein translation efficiency by customizing the ribosome binding sites (RBSs) of promoters for the GLY and FAS modules further improves fatty acid production. When the GLY module is expressed from a moderate-strength RBS, fatty acid production rises initially with the increased expression level of FAS module. This inconsistency could partially be attributed to the increased driving force for oxidation of NAD(P)H to NAD(P)+ and conversion of malonyl-acyl carrier proteins (ACPs) to the final product. These examples demonstrate that fine-tuned, cofactor-dependent gene expression for synthetic biology is greatly enhanced through promoter engineering, suggesting that a balance of carbon flux and redox to a certain degree can be optimally achieved. Genome-scale engineering One of the goals of synthetic biology is to synthesize a living cell chassis, which can be engineered with a set of useful properties predesigned for various tasks, ranging from gene therapy to biofuel production and biodegradation [34]. However, the traits of microorganisms in nature generally do not meet the expectations of the researcher, partially because cofactor metabolism is involved with cofactor-dependent genes. To deal with this problem, genome-scale engineering [35] has shown great potential in the creation of specialized cells [36]: top-down, aiming at further simplification of existing cells by removal of nonessential genes [37]; and bottom-up, attempting to create a cell by synthesizing its essential components [38] (Figure 2B). As an example of using the top-down approach, beginning with E. coli strain MGF-01 (3.62 Mbp 4
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genome) [39], E. coli DGF-298 (2.98 Mbp genome) generated with a deletion of 1429 genes, shows no auxotrophy, better growth fitness, and better cell yield in a rich medium [40]. Compared with the genome-scale metabolic model iAF1260, DGF-298 lacks 298 genes including 147 cofactor-dependent genes [41]. This result indicates that not all cofactors (such as NADH) are used during essential metabolism, and the deletion of nonessential cofactor-dependent genes can create a new redox balance and lower metabolic burden. Similarly, E. coli strain MDS42 [42] with genome reductions up to 14.3% (including cofactordependent genes), is re-engineered to increase the production of L-threonine [43]. When threonine dehydrogenase is deleted, L-threonine production exhibits an 89.2% increase in the strain MDS-203 compared to the wild type strain MG-103. The great potential of a designed cell harboring only the minimal genome lies in its simplicity [38], with no functional redundancy such as invalid redox metabolism. These achievements have opened a new avenue for metabolic engineering distinct from traditional genetic engineering and hold great promise in the construction of an ideal cell chassis for the efficient production of chemicals. Protein engineering Currently, advances in synthetic biology have enabled the production of many chemicals, including biofuels and drugs, from renewable resources [44]. However, synthetic pathways are assembled from biological components culled from nature, and they may not function optimally when simply put together in biological systems [45], partially owing to the influence of cofactor utilization. To overcome this issue, much of the effort in protein engineering to achieve redox balance has focused on: improving enzyme activity [46], changing substrate specificity [47,48], modifying cofactor specificity [49], constructing multi-enzyme complexes [50], and creating bioorthogonal redox systems [51] (Figure 2D). A good example of a natural chemical produced through modifying cofactor specificity is vitamin C [52]. In this case, the cofactor preference of Corynebacterium 2,5-diketo-Dgluconic acid reductase (2,5-DKG) was switched from NADPH to NADH. Banta and colleagues made a number of site-directed mutations at five sites in the cofactor binding pocket that interacts with the 20 -phosphate group of NADPH. Several beneficial mutations were identified and combined with other substitutions, resulting in a quadruple mutant, F22Y/K232G/R238H/A272G, which exhibits activity with NADH over two orders of magnitude higher than that of wild type 2,5-DKG. This method achieved good redox balance and pathway utilization of NADH, and may have greatly affected product yield and the formation of byproducts. In another example, a bioorthogonal redox system was created when the NAD+-dependent malic enzyme (ME), D-lactate dehydrogenase (DLDH), and malate dehydrogenase (MDH) were mutated to ME-L310R/Q401C, DLDHV152R, and MDH-L6R, respectively [51]. These mutations demonstrated excellent activity with nicotinamide flucytosine dinucleotide (NFCD) yet marginal activity with NAD+, which allows the insulation of artificially engineered pathways from cofactor metabolism. This opens the window to engineering bioorthogonal redox systems for a wide variety of applications in systems biology and synthetic biology.
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Review These examples show that the improvement of cofactor utilization reduces the influence of the cofactor in an artificial system, which is conducive to further engineering of a microbe with no consideration of cofactor imbalance. Structural synthetic biotechnology Structural synthetic biotechnology, which complements and integrates the methods and tools of structural biology and synthetic biology, could become an interdisciplinary field that provides a structure-based foundation for designing cellular metabolic and regulatory pathways [53]. It also provides a new way to localize and enhance cellular redox pathways and suggests additional opportunities for harnessing cofactor-mediated processes [1]. To date, this technology has fostered the engineering of a variety of cofactordependent metabolic pathways, aimed at designing and synthesizing protein scaffolds [54], RNA scaffolds [55], and DNA scaffolds [56] (Figure 2C). For example, butyrate, a short-chain fatty acid, is produced in recombinant E. coli through a series of pathway engineering maneuvers to enable stoichiometric redox balances of the butyrate pathway [57,58]. First, the native redox cofactor regeneration system is engineered by deleting alcohol dehydrogenase, lactate dehydrogenase, and fumarate reductase. Second, the synthetic butyrate pathway, which regenerates NAD+ from NADH using butyrate as the only final electron acceptor, is constructed using five genes from Clostridium acetobutylicum and Treponema denticola. Although it is possible to redesign the native redox cofactor regeneration system based on an understanding of its role in providing the driving force to achieve the production of butyrate, the pathway constructed from exogenous enzymes cannot efficiently convert carbon flux to butyrate. A synthetic scaffold protein that spatially co-localizes the three pathway enzymes, 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase, and transenoyl-CoA reductase, can improve butyrate production threefold, demonstrating that the scaffold can concentrate reactants to drive unfavorable reactions and protect enzymes or unstable intermediates from harmful cellular conditions or competing reactions [59]. These approaches have substantially expanded the metabolic tool kit into three dimensions and will most likely have an important role in a range of future pathway designs to reduce the influence of cofactor metabolism and, to a certain degree, achieve redox balance. Systems metabolic engineering Systems metabolic engineering, which incorporates the concepts and techniques of systems biology, synthetic biology, and evolutionary engineering at the systems level, emerges as a conceptual and technological framework to speed the creation of new metabolic enzymes and pathways or the modification of existing pathways for the optimal production of desired products [60]. It can thus be used to create new metabolic products and pathways and cellular regulatory circuits and functions with the availability of necessary cofactors as well as the balance of electronmediating organic cofactors, such as NADH and NADPH, through the ‘omic’ techniques [61], such as transcriptome, proteome, and metabolomics profiling, and computational
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techniques [60], such as chemical structure-based and knowledge-based methods (Figure 2D). For example, based on the comparison of transcriptome data sets from xylitolproducing and nonproducing conditions of the recombinant E. coli, the downregulated 56 genes are selected as candidate factors for suppression of the NADPH supply, and their defective mutants are tested for xylitol production [62]. Among the tested mutants, a yhbC-deficient strain shows improved xylitol production, due to the fact that yhbC, the second gene of the metY–rpsO operon, plays an important role in autoregulation of the operon when adapting to oxidative conditions caused by NADPH consumption. It also indicates that the deletion of the gene yhbC validated the function of the genes in supplying NADPH. Thus, the combination of transcriptome analysis and phenotype tests of single-gene-knockout mutants is a good approach to screen negative regulators for strictly controlled cofactor supply. Another representative example of systems metabolic engineering is 1,4-butanediol (BDO) production in E. coli [63]. By adopting biopathway prediction algorithms, Yim and colleagues elucidate multiple pathways for the biosynthesis of BDO from common metabolic intermediates. Because the conversion of 4-hydroxybutyrate to BDO requires two reduction steps catalyzed by dehydrogenases, the constraints-based, genome-scale metabolic model iJR904 and the OptKnock algorithm are used to design a strain in which production of BDO is the only way to balance redox and enable anaerobic growth. In this design, the oxidative TCA cycle is engineered to supply reducing equivalents under microaerobic cultivation conditions. First, the formation of the natural fermentation products ethanol, formate, lactate, and succinate is blocked, thereby forcing production of BDO to balance oxidation and reduction. In addition, a series of metabolic engineering strategies, such as overexpression of an NADH inhibitioninsensitive citrate synthase, replacement of an NADH inhibition-sensitive pyruvate dehydrogenase, and deletion of malate dehydrogenase, are adopted to direct the carbon flux into the TCA cycle in the presence of high intracellular NADH concentration. A systems-based metabolic engineering approach can efficiently design a host strain to take into account redox balance. These achievements demonstrate that systems biology approaches that combine modeling technologies with ‘omics’ data measurements can allow us to comprehend the cellular physiology more accurately, expand the understandable scope of engineering, and address metabolic bottlenecks (such as redox balance), and thus opens the window to efficiently engineer microorganisms with comprehensive consideration of cofactor systems. Cofactor regeneration Cofactor regeneration has provided another route for maintaining the cellular redox balance by altering the intracellular cofactor pool. Subsequently, cytoplasmic H2O-forming NADH oxidase (NOX) [64], mitochondrial alternative oxidase (AOX) [65], phosphite dehydrogenase (PTDH) [66], mitochondrial NADH kinase (POS5) [67], and formate dehydrogenase (FDH) [68], as indicators of the cofactor state of the cells, have been shown to directly affect 5
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Review the ratio of NADH/NAD+ or ATP/ADP (Figure 2E). Normally, L. lactis can convert most of the central intermediate pyruvate to lactate, a reaction catalyzed by LDH with the oxidation of NADH to NAD+ for maintaining a redox balance [69]. However, under aerobic conditions, the increased activity of NOX replaces the role of LDH in the regeneration of NAD+ and disturbs the original redox balance, resulting in the metabolic shift from homolactic to mixed-acid product formation, including lactate, acetate, and CO2 [70]. To finely tune lactate and diacetyl production, a constitutive promoter library bearing different strength noxE promoters is constructed to precisely regulate noxE expression and NADH/NAD+ ratio in L. lactis [64]. This approach indicates that the level of redox state is necessary for redirecting pyruvate flux to lactate via LDH. In another example, E. coli is engineered to produce mannitol from fructose by overexpression of MDH from Leuconostoc pseudomesenteroides and FDH from Mycobacterium vaccae N10 [71]. In this case, a recombinant redox cycle is established in which NADH produced from formate via FDH is used to drive the MDH-catalyzed reduction of fructose to mannitol, leading to a large increase in mannitol production. These observed re-routings of metabolism indicate that metabolic engineering strategies directed towards modulation of key cofactors may be a more direct way to manipulate metabolism and achieve redox balance. Concluding remarks Cofactor engineering has proved to be an interesting avenue for improving productivity, such as in the synthesis of medicines, biofuels, free fatty acids, and sugar alcohols, but traditional cofactor engineering has mainly focused on manipulating enzyme levels through the amplification, addition, or deletion of a particular pathway. Currently, emerging technologies, such as promoter engineering, genome-scale engineering, protein engineering, structural synthetic biotechnology, systems metabolic engineering, and cofactor regeneration, have dramatically expanded our capabilities to engineer cofactor systems, and these developments make rational design of a cofactor system more attractive, rapid, and powerful. Thus, cofactor engineering via different biotechnological approaches reshapes the whole-cell response to redox balance and optimizes dynamic control of the target metabolic flux. However, its development thus far is by no means complete and does not capture and regulate all of the cofactor metabolic characteristics. Therefore, the development of more accurate biotechnological approaches, preferably incorporating regulatory mechanisms, should be the first priority on the todo list of cofactor engineering. In addition, understanding of theinformation on the new class of cofactor transcription regulators (either metabolic enzymes or homologs) that use NADH/NAD+ and NADPH/NADP+ as cofactors [72], is currently lacking; future work will be required to elucidate their mechanisms. Acknowledgments This work was financially supported by the Major State Basic Research Development Program of China (2013CB733602); the National Natural Science Foundation of China (31270079); the Program for Young Talents in China; the Provincial Outstanding Youth Foundation of Jiangsu 6
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Province (BK2012002); and the Program for Innovative Research Team in University (IRT1249).
References 1 Wang, Y. et al. (2013) Cofactor engineering for advancing chemical biotechnology. Curr. Opin. Biotechnol. 24, 1–6 2 Liu, L. et al. (2006) Enhancement of pyruvate productivity in Torulopsis glabrata: increase of NAD+ availability. J. Biotechnol. 126, 173–185 3 La Piana, G. et al. (2005) Porin and cytochrome oxidase containing contact sites involved in the oxidation of cytosolic NADH. Arch. Biochem. Biophys. 436, 91–100 4 Lin, S.J. et al. (2002) Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration. Nature 418, 344–348 5 Domergue, R. et al. (2005) Nicotinic acid limitation regulates silencing of Candida adhesins during UTI. Science 308, 866–870 6 Heux, S. et al. (2006) Cofactor engineering in Saccharomyces cerevisiae: expression of a H2O-forming NADH oxidase and impact on redox metabolism. Metab. Eng. 8, 303–314 7 de Graef, M.R. et al. (1999) The steady-state internal redox state (NADH/NAD) reflects the external redox state and is correlated with catabolic adaptation in Escherichia coli. J. Bacteriol. 181, 2351–2357 8 Shen, C.R. et al. (2011) Driving forces enable high-titer anaerobic 1butanol synthesis in Escherichia coli. Appl. Environ. Microbiol. 77, 2905–2915 9 Bera, A.K. et al. (2011) A genetic overhaul of Saccharomyces cerevisiae 424A(LNH-ST) to improve xylose fermentation. J. Ind. Microbiol. Biotechnol. 38, 617–626 10 Orth, J.D. et al. (2011) A comprehensive genome-scale reconstruction of Escherichia coli metabolism–2011. Mol. Syst. Biol. 7, 535 11 Zomorrodi, A.R. and Maranas, C.D. (2010) Improving the iMM904 S. cerevisiae metabolic model using essentiality and synthetic lethality data. BMC Syst. Biol. 4, http://dx.doi.org/10.1186/1752-0509-4-178 12 Murray, D.B. et al. (2011) Redox regulation in respiring Saccharomyces cerevisiae. Biochim. Biophys. Acta 1810, 945–958 13 van Dijken, J.P. and Scheffers, W.A. (1986) Redox balances in the metabolism of sugars by yeasts. FEMS Microbiol. Lett. 32, 199–224 14 Overkamp, K.M. et al. (2000) In vivo analysis of the mechanisms for oxidation of cytosolic NADH by Saccharomyces cerevisiae mitochondria. J. Bacteriol. 182, 2823–2830 15 Palmer, J.M. (1979) The ‘‘uniqueness’’ of plant mitochondria. Biochem. Soc. Trans. 7, 246–252 16 von Jagow, G. and Klingenberg, M. (1970) Pathways of hydrogen in mitochondria of Saccharomyces carlsbergensis. Eur. J. Biochem. 12, 583–592 17 Kro¨mer, S. and Heldt, H.W. (1991) Respiration of pea leaf mitochondria and redox transfer between the mitochondrial and extramitochondrial compartment. Biochim. Biophys. Acta 1057, 42–50 18 Bakker, B.M. et al. (2001) Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae. FEMS Microbiol. Rev. 25, 15–37 19 Clomburg, J.M. and Gonzalez, R. (2013) Anaerobic fermentation of glycerol: a platform for renewable fuels and chemicals. Trends Biotechnol. 31, 20–28 20 Cashel, M. et al. (1996) The stringent response. In Escherichia coli and Salmonella typhimurium Cellular and Molecular Biology (Vol. 1) (Neidhardt, F.C., ed.), In pp. 1458–1496, ASM Press 21 Murarka, A. et al. (2008) Fermentative utilization of glycerol by Escherichia coli and its implications for the production of fuels and chemicals. Appl. Environ. Microbiol. 74, 1124–1135 22 Qin, Y. et al. (2009) Manipulation of NADH metabolism in industrial strains. Chinese J. Biotech. 25, 161–169 23 Diano, A. et al. (2006) Polyol synthesis in Aspergillus niger: influence of oxygen availability, carbon and nitrogen sources on the metabolism. Biotechnol. Bioeng. 94, 899–908 24 Elliott, S.J. et al. (2002) Detection and interpretation of redox potential optima in the catalytic activity of enzymes. Biochim. Biophys. Acta 1555, 54–59 25 Menzel, K. et al. (1998) Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture. IV. Enzymes and fluxes of pyruvate metabolism. Biotechnol. Bioeng. 60, 617–626
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Review 26 Riondet, C. et al. (2000) Extracellular oxidoreduction potential modifies carbon and electron flow in Escherichia coli. J. Bacteriol. 182, 620–626 27 Khosla, C. and Keasling, J.D. (2003) Metabolic engineering for drug discovery and development. Nat. Rev. Drug Discov. 2, 1019–1025 28 Blazeck, J. and Alper, H.S. (2013) Promoter engineering: recent advances in controlling transcription at the most fundamental level. Biotechnol. J. 8, 46–58 29 Na, D. et al. (2010) Construction and optimization of synthetic pathways in metabolic engineering. Curr. Opin. Microbiol. 13, 363–370 30 Zhang, F. et al. (2012) Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids. Nat. Biotechnol. 30, 354–359 31 Pfleger, B.F. et al. (2006) Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Nat. Biotechnol. 24, 1027–1032 32 Salis, H.M. et al. (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nat. Biotechnol. 27, 946– 950 33 Xu, P. et al. (2013) Modular optimization of multi-gene pathways for fatty acids production in E. coli. Nat. Commun. 4, 1409 34 Alper, H. and Stephanopoulos, G. (2009) Engineering for biofuels: exploiting innate microbial capacity or importing biosynthetic potential? Nat. Rev. Microbiol. 7, 715–723 35 Esvelt, K.M. and Wang, H.H. (2013) Genome-scale engineering for systems and synthetic biology. Mol. Syst. Biol. 9, http://dx.doi.org/ 10.1038/msb.2012.66 36 Juhas, M. et al. (2011) Essence of life: essential genes of minimal genomes. Trends Cell Biol. 21, 562–568 37 Moya, A. et al. (2009) Toward minimal bacterial cells: evolution vs. design. FEMS Microbiol. Rev. 33, 225–235 38 Jewett, M.C. and Forster, A.C. (2010) Update on designing and building minimal cells. Curr. Opin. Biotechnol. 21, 697–703 39 Mizoguchi, H. et al. (2007) Escherichia coli minimum genome factory. Biotechnol. Appl. Biochem. 46, 157–167 40 Hirokawa, Y. et al. (2013) Genetic manipulations restored the growth fitness of reduced-genome Escherichia coli. J. Biosci. Bioeng. 116, 52–58 41 Tang, B. et al. (2013) Genome minimization method based on metabolic network analysis and its application to Escherichia coli. Chinese J. Biotech. 29, 1173–1184 42 Posfai, G. et al. (2006) Emergent properties of reduced-genome Escherichia coli. Science 312, 1044–1046 43 Lee, J.H. et al. (2009) Metabolic engineering of a reduced-genome strain of Escherichia coli for L-threonine production. Microb. Cell Fact. 8, http://dx.doi.org/10.1186/1475-2859-8-2 44 Foo, J.L. et al. (2012) The imminent role of protein engineering in synthetic biology. Biotechnol. Adv. 30, 541–549 45 Li, Y. (2012) Beyond protein engineering: its applications in synthetic biology. Enz. Eng. 1, http://dx.doi.org/10.4172/eeg.1000e103 46 Paladini, D.H. et al. (2009) Induced fit and equilibrium dynamics for high catalytic efficiency in ferredoxin-NADP(H) reductases. Biochemistry 48, 5760–5768 47 Liu, X. et al. (2012) Structure-guided engineering of Lactococcus lactis alcohol dehydrogenase LlAdhA for improved conversion of isobutyraldehyde to isobutanol. J. Biotechnol. 164, 188–195 48 Zhang, K. et al. (2010) Expanding metabolism for total biosynthesis of the nonnatural amino acid L-homoalanine. Proc. Natl. Acad. Sci. U.S.A. 107, 6234–6239 49 Hoelsch, K. et al. (2013) Engineering of formate dehydrogenase: synergistic effect of mutations affecting cofactor specificity and chemical stability. Appl. Microbiol. Biotechnol. 97, 2473–2481 50 Zhou, Y.J. et al. (2012) Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production. J. Am. Chem. Soc. 134, 3234–3241
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51 Ji, D. et al. (2011) Creation of bioorthogonal redox systems depending on nicotinamide flucytosine dinucleotide. J. Am. Chem. Soc. 133, 20857–20862 52 Banta, S. et al. (2002) Optimizing an artificial metabolic pathway: engineering the cofactor specificity of Corynebacterium 2,5-diketo-Dgluconic acid reductase for use in vitamin C biosynthesis. Biochemistry 41, 6226–6236 53 Chen, Z. et al. (2010) Structural synthetic biotechnology: from molecular structure to predictable design for industrial strain development. Trends Biotechnol. 28, 534–542 54 Dueber, J.E. et al. (2009) Synthetic protein scaffolds provide modular control over metabolic flux. Nat. Biotechnol. 27, 753–759 55 Delebecque, C.J. et al. (2011) Organization of intracellular reactions with rationally designed RNA assemblies. Science 333, 470–474 56 Conrado, R.J. et al. (2012) DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency. Nucleic Acids Res. 40, 1879–1889 57 Baek, J.M. et al. (2013) Butyrate production in engineered Escherichia coli with synthetic scaffolds. Biotechnol. Bioeng. 110, 2790–2794 58 Lim, J.H. et al. (2013) Refactoring redox cofactor regeneration for highyield biocatalysis of glucose to butyric acid in Escherichia coli. Bioresour. Technol. 135, 568–573 59 Agapakis, C.M. et al. (2012) Natural strategies for the spatial optimization of metabolism in synthetic biology. Nat. Chem. Biol. 8, 527–535 60 Lee, J.W. et al. (2012) Systems metabolic engineering of microorganisms for natural and non-natural chemicals. Nat. Chem. Biol. 8, 536–546 61 Jang, Y.S. et al. (2012) Engineering of microorganisms for the production of biofuels and perspectives based on systems metabolic engineering approaches. Biotechnol. Adv. 30, 989–1000 62 Hibi, M. et al. (2007) Improvement of NADPH-dependent bioconversion by transcriptome-based molecular breeding. Appl. Environ. Microbiol. 73, 7657–7663 63 Yim, H. et al. (2011) Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol. Nat. Chem. Biol. 7, 445–452 64 Guo, T. et al. (2012) Fine tuning of the lactate and diacetyl production through promoter engineering in Lactococcus lactis. PLoS ONE 7, http://dx.doi.org/10.1371/journal.pone.0036296 65 Vemuri, G.N. et al. (2007) Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 104, 2402–2407 66 Zou, Y. et al. (2012) Crystal structures of phosphite dehydrogenase provide insights into nicotinamide cofactor regeneration. Biochemistry 51, 4263–4270 67 Lee, W.H. et al. (2013) Effects of NADH kinase on NADPH-dependent biotransformation processes in Escherichia coli. Appl. Microbiol. Biotechnol. 97, 1561–1569 68 Akinterinwa, O. et al. (2008) Metabolic engineering for bioproduction of sugar alcohols. Curr. Opin. Biotechnol. 19, 461–467 69 Neves, A.R. et al. (2005) Overview on sugar metabolism and its control in Lactococcus lactis – the input from in vivo NMR. FEMS Microbiol. Rev. 29, 531–554 70 Lopez de Felipe, F. et al. (1998) Cofactor engineering: a novel approach to metabolic engineering in Lactococcus lactis by controlled expression of NADH oxidase. J. Bacteriol. 180, 3804–3808 71 Kaup, B. et al. (2004) Metabolic engineering of Escherichia coli: construction of an efficient biocatalyst for D-mannitol formation in a whole-cell biotransformation. Appl. Microbiol. Biotechnol. 64, 333–339 72 Shi, Y. (2004) Metabolic enzymes and coenzymes in transcription – a direct link between metabolism and transcription? Trends Genet. 20, 445–452
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Review
Engineering redox balance through cofactor systems Xiulai Chen1,2,3, Shubo Li1,2,3, and Liming Liu1,2,3 1
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China Key Laboratory of Industrial Biotechnology, Ministry of Education, Jiangnan University, Wuxi 214122, China 3 Laboratory of Food Microbial-Manufacturing Engineering, Jiangnan University, Wuxi 214122, China 2
Redox balance plays an important role in the production of enzymes, pharmaceuticals, and chemicals. To meet the demands of industrial production, it is desirable that microbes maintain a maximal carbon flux towards target metabolites with no fluctuations in redox. This requires functional cofactor systems that support dynamic homeostasis between different redox states or functional stability in a given redox state. Redox balance can be achieved by improving the self-balance of a cofactor system, regulating the substrate balance of a cofactor system, and engineering the synthetic balance of a cofactor system. This review summarizes how cofactor systems can be manipulated to improve redox balance in microbes. Cofactor systems in cellular physiology Cofactors provide redox carriers for biosynthetic and catabolic reactions, and act as important agents in transfer of energy for the cell [1]. In microorganisms, the NADH/ NAD+ and NADPH/NADP+ cofactor pairs are involved in 740 and 887 biochemical reactions and interact with 433 and 462 enzymes, respectively. They possess a series of physiological functions, including regulating energy metabolism [2], adjusting the intracellular redox state, controlling carbon flux, improving the function and activity of the mitochondrion [3], regulating the cell life cycle [4], and modulating microbial virulence [5]. It is clear that cofactors are essential to large numbers of biochemical reactions, and as expected, their manipulation has substantial effects on redox balance. Maintaining the cellular redox balance is a basic requirement for living cells to sustain metabolism [6]. The nucleotides, NAD(P)+ and NAD(P)H, as the most important redox carriers involved in metabolism, not only serve as electron acceptors in the breakdown of catabolic substrates but also provide the cell with the reducing power needed in energy-conserving redox reactions [7]. A balance in the rates of oxidation and reduction of these nucleotides is a prerequisite for both catabolism and anabolism [7]. Some examples have illustrated the requirement for redox balance and availability. An irreversible reaction catalyzed Corresponding author: Liu, L. ([email protected]). Keywords: cofactor systems; cofactor engineering; redox balance; synthetic biology. 0167-7799/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tibtech.2014.04.003
by trans-enoyl-coenzyme A (CoA) reductase [8] is required to use accumulated NADH as a driving force for directing the flux to 1-butanol. To improve xylose fermentation, NADP+-dependent glyceraldehyde 3-phosphate dehydrogenase is used to increase the NADPH pool and create a better redox balance [9]. To gain a comprehensive understanding of physiology and cellular metabolism, the genome-scale metabolic models iJO1366 for Escherichia coli [10] and iMM904 for Saccharomyces cerevisiae [11] are used to search for NAD(P)H/NAD(P)+-related reactions. There are 221 reactions in iJO1366 and 223 reactions in iMM904, which are generally divided into three types: metabolic reactions, exchange reactions, and heterologous reactions. The heterologous reactions that are derived from synthetic pathways are different from the metabolic reactions that originate from metabolic pathways in models. Therefore, we assume the following structural design incorporating cofactor systems to manipulate redox balance (Figure 1): (i) self-balance of a cofactor system based on metabolic reactions; (ii) substrate balance of a cofactor system based on exchange reactions; (iii) synthetic balance of a cofactor system based on introduced reactions. According to these three systems, we show how certain strategies can be used to manipulate redox balance through microbial cofactor systems. Improving the self-balance of a cofactor system In microorganisms, sugars are broken down by respiration, leading to the reduction of NAD+ to NADH; in eukaryotic organisms, this occurs in separate cellular compartments. NADH is mainly formed by a rather limited set of cofactor reactions, for example, glycolysis, fatty acid oxidation, and the tricarboxylic acid (TCA) cycle. Therefore, the self-balance of a cofactor system is used to maintain the redox balance automatically through three routes in S. cerevisiae: overflow metabolism, respiratory chain, and mitochondrial redox shuttles. For example, glucose is exclusively broken down via acetate and glycerol fermentation, which is a redox-neutral process. Acetate overflow is believed to reoxidize NADH and balance the NAD(P)H/ NAD(P)+ ratio [12], and glycerol formation maintains the cytosolic redox balance and compensates for cellular reactions that produce NADH in S. cerevisiae [13]. In another example, the two sides of the mitochondria inner membrane couple NADH reoxidation to the respiratory chain [14]. Although mitochondrial NADH can be oxidized by Trends in Biotechnology xx (2014) 1–7
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Figure 1. Conceptual schematic representation of engineering of the redox balance. Schematic representation of strategies for engineering the redox balance, separated by different colored lines. Squares represent redox states. The yellow arrows, blue arrows, and green arrows in squares represent strategies to maintain redox balance. (A) Industrial microbes achieve redox balance by improving the self-balance of a cofactor system. State I represents the original redox state of the wild type strain, with the target to overcome redox imbalance, which is controlled by the original growth status. On encountering environmental perturbations (State II), redox state is altered, meaning that either metabolic flux redistribution or nonmetabolic property changes occur. (B) Industrial microbes achieve redox balance by regulating the substrate balance of a cofactor system. To meet the demand of the microbes for special physiology (such as chemical production), redox state is altered from State I (the original state) to State II (substrate perturbations) with redox balance achieved. (C) Industrial microbes achieve redox balance by engineering the synthetic balance of a cofactor system. When the original state (State I) is disturbed by synthetic imbalance, redox state is transmitted to transition state (State II) with redox imbalance. Further, redox state is enlarged by cofactor engineering via different biotechnologies and reshaped into State III (synthesis perturbations) with redox balance. When the perturbation is strong enough to drive cells to surpass State II space limits, redox state may fall into State III by transition, with the target functionality still stably maintained.
proton-translocating complex I or by an internal NADH dehydrogenase [15], cytosolic NADH can be oxidized in mitochondria either by an external NADH:ubiquinone oxidoreductase [16] or by a redox shuttle [17], such as the glycerol-3-phosphate shuttle. Similarly, the mitochondrial redox shuttles in S. cerevisiae, mainly containing the ethanol–acetaldehyde redox shuttle, the malate–oxaloacetate shuttle, the malate–aspartate shuttle, and the citrate–oxoglutarate shuttle, are important during respiratory growth, and are interconverted to balance NADH/NAD+ in both the cytosol and mitochondrial compartments [18]. Therefore, such a self-balance in cofactor systems can maintain functional stability and dynamic homeostasis in a given redox state automatically, so as to adapt to the changing environment. Regulating the substrate balance of a cofactor system The intracellular cofactor state can be improved by providing a variety of compounds that can serve as acceptors of electrons and precursors of NAD+, or by altering the environmental conditions of the culture. Such compounds can affect electron transfer by modifying NADH reoxidation, and thus achieve cofactor transition to improve the redox balance. Therefore, substrate balance of a cofactor system is used to achieve the optimal NADH/NAD+ and ATP/ADP ratios through regulating the redox state of a substrate, the co-substrates, and the environmental conditions. A good 2
example of regulation of the substrate redox state is anaerobic fermentation of glycerol. The utilization of glycerol as a substrate for the production of biochemicals and biofuels is one promising avenue for the coupling of processes within a biorefinery. The number of reducing equivalents produced during the conversion of glycerol into the metabolic intermediate pyruvate is twofold higher than during the metabolism of lignocellulosic sugars such as glucose or xylose [19]. These additional reducing equivalents provide glycerol with the natural advantage of higher theoretical product yields for reduced chemicals and fuels. The redox balance is maintained during anaerobic fermentation of glycerol in two ways: transferring of electrons to internally generate organic compounds [20] and shifting of electrons towards reduced products. These pathways provide channels for balancing redox and a means of maximizing the production of reduced chemicals and fuels [21]. Several studies have demonstrated that co-substrates that serve as external electron acceptors, such as ferricyanide, nitrate, organic acids, acetoin, aldehyde, and phenazine, can promote reoxidation of NADH, maintain an optimal NADH/NAD+ ratio and ATP level, and facilitate redox balance [22]. The intracellular redox balance is concerned not merely with the above-mentioned two aspects, but with the environmental conditions. When NADH is oxidized to NAD+, dissolved oxygen, as the electron acceptor in the oxidative phosphorylation, can
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be used to maintain redox balance and meet the total energy requirement for metabolism by influencing the activities of all enzymes that use or produce NAD+ and ATP [23]. In addition, the oxidoreduction potential can affect the synthesis or stability of certain enzymes such as the electron transport-related enzymes [24] and NAD+/ NADH-dependent enzymes [25], which in turn affects the ATP yield, NAD+/NADH ratio, and metabolic fluxes [26]. Thus, such a substrate balance in cofactor systems can often manipulate transitions between different redox states, so as to adapt to the fermentative condition. Engineering the synthetic balance of a cofactor system In a typical pathway, the status of redox is reflected by the cofactors NAD(P)H/NAD(P)+. Redox balance occurs when the production and consumption of cofactors are approximately equal. Imbalanced oxidoreduction potential can damage cells, waste energy, and/or carbon, and even lead to metabolic arrest. Fortunately, redox-imbalanced pathways can sometimes be rebalanced through six approaches (Figure 2): promoter engineering tuning cofactor-dependent
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Promoter engineering Microbial production of chemicals, involving the introduction of several genes encoding the enzymes of a metabolic pathway, is now an attractive alternative to chemical synthesis [27]. However, product titers and yields are often limited by redox imbalances, due to the utilization of cofactor-dependent enzymes in synthetic pathways [19]. To address this issue, several attempts at promoter engineering have shown great promise in helping generate the dynamic range necessary to enable fine-tuned gene expression for synthetic biology applications [28]. Several
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gene expression, genome-scale engineering reducing cofactor-dependent functional redundancy, protein engineering improving the specificity of cofactor-dependent enzymes, structural synthetic biotechnology enhancing cofactor transmission efficiency in synthetic pathways, systems metabolic engineering determining and optimizing the influence of cofactors at the systems level, and cofactor regeneration reconstructing metabolic pathways of cofactors.
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Figure 2. Redox balance can be maintained through (A) promoter engineering: the gene expression levels are regulated by engineering promoter strengths (green), properties (red), ribosome binding (blue), and intergenic regions (black); (B) genome-scale engineering: the top-down approach aims at further simplification of existing cells by removal of nonessential genes (green and gray); (C) structural synthetic biotechnology: structural synthetic biotechnology has fostered a variety of activities in cofactor-dependent metabolic pathways, aimed at designing and synthesizing DNA scaffolds, RNA scaffolds, and protein scaffolds; (D) systems metabolic engineering and protein engineering: systems metabolic engineering can be used to create new metabolic pathways, cellular regulatory circuits, and functions with the availability of necessary cofactors through the ‘omic’ techniques and computational techniques. During this process, protein engineering can be used to achieve redox balance by improving enzyme activity, changing substrate specificity, modifying cofactor specificity, constructing multi-enzyme complexes, and creating bioorthogonal redox systems; (E) cofactor regeneration: cofactor regeneration has provided an extra route to alter the intracellular cofactor pool and maintain the cellular redox balance by cytoplasmic H2O-forming NADH oxidase (NOX), mitochondrial alternative oxidase (AOX), phosphite dehydrogenase (PTDH), mitochondrial NADH kinase (POS5), mannitol dehydrogenase (MDH), and formate dehydrogenase (FDH).
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Review strategies have been developed to regulate gene expression, including engineering promoter strengths [29], properties [30], intergenic regions [31], and ribosome binding [32] (Figure 2A). Engineering promoter properties has been used to produce fatty acid ethyl ester (FAEE), an excellent diesel fuel replacement owing to its low water solubility and high energy density [30]. In this case, a dynamic sensor–regulator system is developed to improve FAEE biosynthetic pathway containing three modules: module A contains the E. coli native fatty acid synthase and a cytoplasmic thioesterase gene (tesA) and produces fatty acids; module B contains a pyruvate decarboxylase gene ( pdc) and an alcohol dehydrogenase gene (adhB) and produces ethanol; module C contains an acyl-CoA synthase gene ( fadD) and a wax-ester synthase gene (atfA) and produces FAEE as the end product. Controlling the strength of module B at a medium level and module C at high level results in a large increase in FAEE production (1.5 g/l). This result indicates that the medium expression level of module B improves the cofactor surplus (such as NADH) owing to the medium expression level of adhB and the metabolic imbalance as a result of the accumulation of cofactor. Another example, a fatty acid-producing strain has been developed by introducing a synthetic pathway that is re-cast into three modules: the upstream acetyl CoA formation module (GLY module, containing NAD(P)+-dependent reductions), the intermediary acetyl CoA activation module (ACA module), and the downstream fatty acid synthase module (FAS module, containing NAD(P)H-dependent oxidations) [33]. Combinatorial optimization of protein translation efficiency by customizing the ribosome binding sites (RBSs) of promoters for the GLY and FAS modules further improves fatty acid production. When the GLY module is expressed from a moderate-strength RBS, fatty acid production rises initially with the increased expression level of FAS module. This inconsistency could partially be attributed to the increased driving force for oxidation of NAD(P)H to NAD(P)+ and conversion of malonyl-acyl carrier proteins (ACPs) to the final product. These examples demonstrate that fine-tuned, cofactor-dependent gene expression for synthetic biology is greatly enhanced through promoter engineering, suggesting that a balance of carbon flux and redox to a certain degree can be optimally achieved. Genome-scale engineering One of the goals of synthetic biology is to synthesize a living cell chassis, which can be engineered with a set of useful properties predesigned for various tasks, ranging from gene therapy to biofuel production and biodegradation [34]. However, the traits of microorganisms in nature generally do not meet the expectations of the researcher, partially because cofactor metabolism is involved with cofactor-dependent genes. To deal with this problem, genome-scale engineering [35] has shown great potential in the creation of specialized cells [36]: top-down, aiming at further simplification of existing cells by removal of nonessential genes [37]; and bottom-up, attempting to create a cell by synthesizing its essential components [38] (Figure 2B). As an example of using the top-down approach, beginning with E. coli strain MGF-01 (3.62 Mbp 4
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genome) [39], E. coli DGF-298 (2.98 Mbp genome) generated with a deletion of 1429 genes, shows no auxotrophy, better growth fitness, and better cell yield in a rich medium [40]. Compared with the genome-scale metabolic model iAF1260, DGF-298 lacks 298 genes including 147 cofactor-dependent genes [41]. This result indicates that not all cofactors (such as NADH) are used during essential metabolism, and the deletion of nonessential cofactor-dependent genes can create a new redox balance and lower metabolic burden. Similarly, E. coli strain MDS42 [42] with genome reductions up to 14.3% (including cofactordependent genes), is re-engineered to increase the production of L-threonine [43]. When threonine dehydrogenase is deleted, L-threonine production exhibits an 89.2% increase in the strain MDS-203 compared to the wild type strain MG-103. The great potential of a designed cell harboring only the minimal genome lies in its simplicity [38], with no functional redundancy such as invalid redox metabolism. These achievements have opened a new avenue for metabolic engineering distinct from traditional genetic engineering and hold great promise in the construction of an ideal cell chassis for the efficient production of chemicals. Protein engineering Currently, advances in synthetic biology have enabled the production of many chemicals, including biofuels and drugs, from renewable resources [44]. However, synthetic pathways are assembled from biological components culled from nature, and they may not function optimally when simply put together in biological systems [45], partially owing to the influence of cofactor utilization. To overcome this issue, much of the effort in protein engineering to achieve redox balance has focused on: improving enzyme activity [46], changing substrate specificity [47,48], modifying cofactor specificity [49], constructing multi-enzyme complexes [50], and creating bioorthogonal redox systems [51] (Figure 2D). A good example of a natural chemical produced through modifying cofactor specificity is vitamin C [52]. In this case, the cofactor preference of Corynebacterium 2,5-diketo-Dgluconic acid reductase (2,5-DKG) was switched from NADPH to NADH. Banta and colleagues made a number of site-directed mutations at five sites in the cofactor binding pocket that interacts with the 20 -phosphate group of NADPH. Several beneficial mutations were identified and combined with other substitutions, resulting in a quadruple mutant, F22Y/K232G/R238H/A272G, which exhibits activity with NADH over two orders of magnitude higher than that of wild type 2,5-DKG. This method achieved good redox balance and pathway utilization of NADH, and may have greatly affected product yield and the formation of byproducts. In another example, a bioorthogonal redox system was created when the NAD+-dependent malic enzyme (ME), D-lactate dehydrogenase (DLDH), and malate dehydrogenase (MDH) were mutated to ME-L310R/Q401C, DLDHV152R, and MDH-L6R, respectively [51]. These mutations demonstrated excellent activity with nicotinamide flucytosine dinucleotide (NFCD) yet marginal activity with NAD+, which allows the insulation of artificially engineered pathways from cofactor metabolism. This opens the window to engineering bioorthogonal redox systems for a wide variety of applications in systems biology and synthetic biology.
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Review These examples show that the improvement of cofactor utilization reduces the influence of the cofactor in an artificial system, which is conducive to further engineering of a microbe with no consideration of cofactor imbalance. Structural synthetic biotechnology Structural synthetic biotechnology, which complements and integrates the methods and tools of structural biology and synthetic biology, could become an interdisciplinary field that provides a structure-based foundation for designing cellular metabolic and regulatory pathways [53]. It also provides a new way to localize and enhance cellular redox pathways and suggests additional opportunities for harnessing cofactor-mediated processes [1]. To date, this technology has fostered the engineering of a variety of cofactordependent metabolic pathways, aimed at designing and synthesizing protein scaffolds [54], RNA scaffolds [55], and DNA scaffolds [56] (Figure 2C). For example, butyrate, a short-chain fatty acid, is produced in recombinant E. coli through a series of pathway engineering maneuvers to enable stoichiometric redox balances of the butyrate pathway [57,58]. First, the native redox cofactor regeneration system is engineered by deleting alcohol dehydrogenase, lactate dehydrogenase, and fumarate reductase. Second, the synthetic butyrate pathway, which regenerates NAD+ from NADH using butyrate as the only final electron acceptor, is constructed using five genes from Clostridium acetobutylicum and Treponema denticola. Although it is possible to redesign the native redox cofactor regeneration system based on an understanding of its role in providing the driving force to achieve the production of butyrate, the pathway constructed from exogenous enzymes cannot efficiently convert carbon flux to butyrate. A synthetic scaffold protein that spatially co-localizes the three pathway enzymes, 3-hydroxybutyryl-CoA dehydrogenase, 3-hydroxybutyryl-CoA dehydratase, and transenoyl-CoA reductase, can improve butyrate production threefold, demonstrating that the scaffold can concentrate reactants to drive unfavorable reactions and protect enzymes or unstable intermediates from harmful cellular conditions or competing reactions [59]. These approaches have substantially expanded the metabolic tool kit into three dimensions and will most likely have an important role in a range of future pathway designs to reduce the influence of cofactor metabolism and, to a certain degree, achieve redox balance. Systems metabolic engineering Systems metabolic engineering, which incorporates the concepts and techniques of systems biology, synthetic biology, and evolutionary engineering at the systems level, emerges as a conceptual and technological framework to speed the creation of new metabolic enzymes and pathways or the modification of existing pathways for the optimal production of desired products [60]. It can thus be used to create new metabolic products and pathways and cellular regulatory circuits and functions with the availability of necessary cofactors as well as the balance of electronmediating organic cofactors, such as NADH and NADPH, through the ‘omic’ techniques [61], such as transcriptome, proteome, and metabolomics profiling, and computational
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techniques [60], such as chemical structure-based and knowledge-based methods (Figure 2D). For example, based on the comparison of transcriptome data sets from xylitolproducing and nonproducing conditions of the recombinant E. coli, the downregulated 56 genes are selected as candidate factors for suppression of the NADPH supply, and their defective mutants are tested for xylitol production [62]. Among the tested mutants, a yhbC-deficient strain shows improved xylitol production, due to the fact that yhbC, the second gene of the metY–rpsO operon, plays an important role in autoregulation of the operon when adapting to oxidative conditions caused by NADPH consumption. It also indicates that the deletion of the gene yhbC validated the function of the genes in supplying NADPH. Thus, the combination of transcriptome analysis and phenotype tests of single-gene-knockout mutants is a good approach to screen negative regulators for strictly controlled cofactor supply. Another representative example of systems metabolic engineering is 1,4-butanediol (BDO) production in E. coli [63]. By adopting biopathway prediction algorithms, Yim and colleagues elucidate multiple pathways for the biosynthesis of BDO from common metabolic intermediates. Because the conversion of 4-hydroxybutyrate to BDO requires two reduction steps catalyzed by dehydrogenases, the constraints-based, genome-scale metabolic model iJR904 and the OptKnock algorithm are used to design a strain in which production of BDO is the only way to balance redox and enable anaerobic growth. In this design, the oxidative TCA cycle is engineered to supply reducing equivalents under microaerobic cultivation conditions. First, the formation of the natural fermentation products ethanol, formate, lactate, and succinate is blocked, thereby forcing production of BDO to balance oxidation and reduction. In addition, a series of metabolic engineering strategies, such as overexpression of an NADH inhibitioninsensitive citrate synthase, replacement of an NADH inhibition-sensitive pyruvate dehydrogenase, and deletion of malate dehydrogenase, are adopted to direct the carbon flux into the TCA cycle in the presence of high intracellular NADH concentration. A systems-based metabolic engineering approach can efficiently design a host strain to take into account redox balance. These achievements demonstrate that systems biology approaches that combine modeling technologies with ‘omics’ data measurements can allow us to comprehend the cellular physiology more accurately, expand the understandable scope of engineering, and address metabolic bottlenecks (such as redox balance), and thus opens the window to efficiently engineer microorganisms with comprehensive consideration of cofactor systems. Cofactor regeneration Cofactor regeneration has provided another route for maintaining the cellular redox balance by altering the intracellular cofactor pool. Subsequently, cytoplasmic H2O-forming NADH oxidase (NOX) [64], mitochondrial alternative oxidase (AOX) [65], phosphite dehydrogenase (PTDH) [66], mitochondrial NADH kinase (POS5) [67], and formate dehydrogenase (FDH) [68], as indicators of the cofactor state of the cells, have been shown to directly affect 5
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Review the ratio of NADH/NAD+ or ATP/ADP (Figure 2E). Normally, L. lactis can convert most of the central intermediate pyruvate to lactate, a reaction catalyzed by LDH with the oxidation of NADH to NAD+ for maintaining a redox balance [69]. However, under aerobic conditions, the increased activity of NOX replaces the role of LDH in the regeneration of NAD+ and disturbs the original redox balance, resulting in the metabolic shift from homolactic to mixed-acid product formation, including lactate, acetate, and CO2 [70]. To finely tune lactate and diacetyl production, a constitutive promoter library bearing different strength noxE promoters is constructed to precisely regulate noxE expression and NADH/NAD+ ratio in L. lactis [64]. This approach indicates that the level of redox state is necessary for redirecting pyruvate flux to lactate via LDH. In another example, E. coli is engineered to produce mannitol from fructose by overexpression of MDH from Leuconostoc pseudomesenteroides and FDH from Mycobacterium vaccae N10 [71]. In this case, a recombinant redox cycle is established in which NADH produced from formate via FDH is used to drive the MDH-catalyzed reduction of fructose to mannitol, leading to a large increase in mannitol production. These observed re-routings of metabolism indicate that metabolic engineering strategies directed towards modulation of key cofactors may be a more direct way to manipulate metabolism and achieve redox balance. Concluding remarks Cofactor engineering has proved to be an interesting avenue for improving productivity, such as in the synthesis of medicines, biofuels, free fatty acids, and sugar alcohols, but traditional cofactor engineering has mainly focused on manipulating enzyme levels through the amplification, addition, or deletion of a particular pathway. Currently, emerging technologies, such as promoter engineering, genome-scale engineering, protein engineering, structural synthetic biotechnology, systems metabolic engineering, and cofactor regeneration, have dramatically expanded our capabilities to engineer cofactor systems, and these developments make rational design of a cofactor system more attractive, rapid, and powerful. Thus, cofactor engineering via different biotechnological approaches reshapes the whole-cell response to redox balance and optimizes dynamic control of the target metabolic flux. However, its development thus far is by no means complete and does not capture and regulate all of the cofactor metabolic characteristics. Therefore, the development of more accurate biotechnological approaches, preferably incorporating regulatory mechanisms, should be the first priority on the todo list of cofactor engineering. In addition, understanding of theinformation on the new class of cofactor transcription regulators (either metabolic enzymes or homologs) that use NADH/NAD+ and NADPH/NADP+ as cofactors [72], is currently lacking; future work will be required to elucidate their mechanisms. Acknowledgments This work was financially supported by the Major State Basic Research Development Program of China (2013CB733602); the National Natural Science Foundation of China (31270079); the Program for Young Talents in China; the Provincial Outstanding Youth Foundation of Jiangsu 6
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Province (BK2012002); and the Program for Innovative Research Team in University (IRT1249).
References 1 Wang, Y. et al. (2013) Cofactor engineering for advancing chemical biotechnology. Curr. Opin. Biotechnol. 24, 1–6 2 Liu, L. et al. (2006) Enhancement of pyruvate productivity in Torulopsis glabrata: increase of NAD+ availability. J. Biotechnol. 126, 173–185 3 La Piana, G. et al. (2005) Porin and cytochrome oxidase containing contact sites involved in the oxidation of cytosolic NADH. Arch. Biochem. Biophys. 436, 91–100 4 Lin, S.J. et al. (2002) Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration. Nature 418, 344–348 5 Domergue, R. et al. (2005) Nicotinic acid limitation regulates silencing of Candida adhesins during UTI. Science 308, 866–870 6 Heux, S. et al. (2006) Cofactor engineering in Saccharomyces cerevisiae: expression of a H2O-forming NADH oxidase and impact on redox metabolism. Metab. Eng. 8, 303–314 7 de Graef, M.R. et al. (1999) The steady-state internal redox state (NADH/NAD) reflects the external redox state and is correlated with catabolic adaptation in Escherichia coli. J. Bacteriol. 181, 2351–2357 8 Shen, C.R. et al. (2011) Driving forces enable high-titer anaerobic 1butanol synthesis in Escherichia coli. Appl. Environ. Microbiol. 77, 2905–2915 9 Bera, A.K. et al. (2011) A genetic overhaul of Saccharomyces cerevisiae 424A(LNH-ST) to improve xylose fermentation. J. Ind. Microbiol. Biotechnol. 38, 617–626 10 Orth, J.D. et al. (2011) A comprehensive genome-scale reconstruction of Escherichia coli metabolism–2011. Mol. Syst. Biol. 7, 535 11 Zomorrodi, A.R. and Maranas, C.D. (2010) Improving the iMM904 S. cerevisiae metabolic model using essentiality and synthetic lethality data. BMC Syst. Biol. 4, http://dx.doi.org/10.1186/1752-0509-4-178 12 Murray, D.B. et al. (2011) Redox regulation in respiring Saccharomyces cerevisiae. Biochim. Biophys. Acta 1810, 945–958 13 van Dijken, J.P. and Scheffers, W.A. (1986) Redox balances in the metabolism of sugars by yeasts. FEMS Microbiol. Lett. 32, 199–224 14 Overkamp, K.M. et al. (2000) In vivo analysis of the mechanisms for oxidation of cytosolic NADH by Saccharomyces cerevisiae mitochondria. J. Bacteriol. 182, 2823–2830 15 Palmer, J.M. (1979) The ‘‘uniqueness’’ of plant mitochondria. Biochem. Soc. Trans. 7, 246–252 16 von Jagow, G. and Klingenberg, M. (1970) Pathways of hydrogen in mitochondria of Saccharomyces carlsbergensis. Eur. J. Biochem. 12, 583–592 17 Kro¨mer, S. and Heldt, H.W. (1991) Respiration of pea leaf mitochondria and redox transfer between the mitochondrial and extramitochondrial compartment. Biochim. Biophys. Acta 1057, 42–50 18 Bakker, B.M. et al. (2001) Stoichiometry and compartmentation of NADH metabolism in Saccharomyces cerevisiae. FEMS Microbiol. Rev. 25, 15–37 19 Clomburg, J.M. and Gonzalez, R. (2013) Anaerobic fermentation of glycerol: a platform for renewable fuels and chemicals. Trends Biotechnol. 31, 20–28 20 Cashel, M. et al. (1996) The stringent response. In Escherichia coli and Salmonella typhimurium Cellular and Molecular Biology (Vol. 1) (Neidhardt, F.C., ed.), In pp. 1458–1496, ASM Press 21 Murarka, A. et al. (2008) Fermentative utilization of glycerol by Escherichia coli and its implications for the production of fuels and chemicals. Appl. Environ. Microbiol. 74, 1124–1135 22 Qin, Y. et al. (2009) Manipulation of NADH metabolism in industrial strains. Chinese J. Biotech. 25, 161–169 23 Diano, A. et al. (2006) Polyol synthesis in Aspergillus niger: influence of oxygen availability, carbon and nitrogen sources on the metabolism. Biotechnol. Bioeng. 94, 899–908 24 Elliott, S.J. et al. (2002) Detection and interpretation of redox potential optima in the catalytic activity of enzymes. Biochim. Biophys. Acta 1555, 54–59 25 Menzel, K. et al. (1998) Kinetic, dynamic, and pathway studies of glycerol metabolism by Klebsiella pneumoniae in anaerobic continuous culture. IV. Enzymes and fluxes of pyruvate metabolism. Biotechnol. Bioeng. 60, 617–626
TIBTEC-1159; No. of Pages 7
Review 26 Riondet, C. et al. (2000) Extracellular oxidoreduction potential modifies carbon and electron flow in Escherichia coli. J. Bacteriol. 182, 620–626 27 Khosla, C. and Keasling, J.D. (2003) Metabolic engineering for drug discovery and development. Nat. Rev. Drug Discov. 2, 1019–1025 28 Blazeck, J. and Alper, H.S. (2013) Promoter engineering: recent advances in controlling transcription at the most fundamental level. Biotechnol. J. 8, 46–58 29 Na, D. et al. (2010) Construction and optimization of synthetic pathways in metabolic engineering. Curr. Opin. Microbiol. 13, 363–370 30 Zhang, F. et al. (2012) Design of a dynamic sensor-regulator system for production of chemicals and fuels derived from fatty acids. Nat. Biotechnol. 30, 354–359 31 Pfleger, B.F. et al. (2006) Combinatorial engineering of intergenic regions in operons tunes expression of multiple genes. Nat. Biotechnol. 24, 1027–1032 32 Salis, H.M. et al. (2009) Automated design of synthetic ribosome binding sites to control protein expression. Nat. Biotechnol. 27, 946– 950 33 Xu, P. et al. (2013) Modular optimization of multi-gene pathways for fatty acids production in E. coli. Nat. Commun. 4, 1409 34 Alper, H. and Stephanopoulos, G. (2009) Engineering for biofuels: exploiting innate microbial capacity or importing biosynthetic potential? Nat. Rev. Microbiol. 7, 715–723 35 Esvelt, K.M. and Wang, H.H. (2013) Genome-scale engineering for systems and synthetic biology. Mol. Syst. Biol. 9, http://dx.doi.org/ 10.1038/msb.2012.66 36 Juhas, M. et al. (2011) Essence of life: essential genes of minimal genomes. Trends Cell Biol. 21, 562–568 37 Moya, A. et al. (2009) Toward minimal bacterial cells: evolution vs. design. FEMS Microbiol. Rev. 33, 225–235 38 Jewett, M.C. and Forster, A.C. (2010) Update on designing and building minimal cells. Curr. Opin. Biotechnol. 21, 697–703 39 Mizoguchi, H. et al. (2007) Escherichia coli minimum genome factory. Biotechnol. Appl. Biochem. 46, 157–167 40 Hirokawa, Y. et al. (2013) Genetic manipulations restored the growth fitness of reduced-genome Escherichia coli. J. Biosci. Bioeng. 116, 52–58 41 Tang, B. et al. (2013) Genome minimization method based on metabolic network analysis and its application to Escherichia coli. Chinese J. Biotech. 29, 1173–1184 42 Posfai, G. et al. (2006) Emergent properties of reduced-genome Escherichia coli. Science 312, 1044–1046 43 Lee, J.H. et al. (2009) Metabolic engineering of a reduced-genome strain of Escherichia coli for L-threonine production. Microb. Cell Fact. 8, http://dx.doi.org/10.1186/1475-2859-8-2 44 Foo, J.L. et al. (2012) The imminent role of protein engineering in synthetic biology. Biotechnol. Adv. 30, 541–549 45 Li, Y. (2012) Beyond protein engineering: its applications in synthetic biology. Enz. Eng. 1, http://dx.doi.org/10.4172/eeg.1000e103 46 Paladini, D.H. et al. (2009) Induced fit and equilibrium dynamics for high catalytic efficiency in ferredoxin-NADP(H) reductases. Biochemistry 48, 5760–5768 47 Liu, X. et al. (2012) Structure-guided engineering of Lactococcus lactis alcohol dehydrogenase LlAdhA for improved conversion of isobutyraldehyde to isobutanol. J. Biotechnol. 164, 188–195 48 Zhang, K. et al. (2010) Expanding metabolism for total biosynthesis of the nonnatural amino acid L-homoalanine. Proc. Natl. Acad. Sci. U.S.A. 107, 6234–6239 49 Hoelsch, K. et al. (2013) Engineering of formate dehydrogenase: synergistic effect of mutations affecting cofactor specificity and chemical stability. Appl. Microbiol. Biotechnol. 97, 2473–2481 50 Zhou, Y.J. et al. (2012) Modular pathway engineering of diterpenoid synthases and the mevalonic acid pathway for miltiradiene production. J. Am. Chem. Soc. 134, 3234–3241
Trends in Biotechnology xxx xxxx, Vol. xxx, No. x
51 Ji, D. et al. (2011) Creation of bioorthogonal redox systems depending on nicotinamide flucytosine dinucleotide. J. Am. Chem. Soc. 133, 20857–20862 52 Banta, S. et al. (2002) Optimizing an artificial metabolic pathway: engineering the cofactor specificity of Corynebacterium 2,5-diketo-Dgluconic acid reductase for use in vitamin C biosynthesis. Biochemistry 41, 6226–6236 53 Chen, Z. et al. (2010) Structural synthetic biotechnology: from molecular structure to predictable design for industrial strain development. Trends Biotechnol. 28, 534–542 54 Dueber, J.E. et al. (2009) Synthetic protein scaffolds provide modular control over metabolic flux. Nat. Biotechnol. 27, 753–759 55 Delebecque, C.J. et al. (2011) Organization of intracellular reactions with rationally designed RNA assemblies. Science 333, 470–474 56 Conrado, R.J. et al. (2012) DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency. Nucleic Acids Res. 40, 1879–1889 57 Baek, J.M. et al. (2013) Butyrate production in engineered Escherichia coli with synthetic scaffolds. Biotechnol. Bioeng. 110, 2790–2794 58 Lim, J.H. et al. (2013) Refactoring redox cofactor regeneration for highyield biocatalysis of glucose to butyric acid in Escherichia coli. Bioresour. Technol. 135, 568–573 59 Agapakis, C.M. et al. (2012) Natural strategies for the spatial optimization of metabolism in synthetic biology. Nat. Chem. Biol. 8, 527–535 60 Lee, J.W. et al. (2012) Systems metabolic engineering of microorganisms for natural and non-natural chemicals. Nat. Chem. Biol. 8, 536–546 61 Jang, Y.S. et al. (2012) Engineering of microorganisms for the production of biofuels and perspectives based on systems metabolic engineering approaches. Biotechnol. Adv. 30, 989–1000 62 Hibi, M. et al. (2007) Improvement of NADPH-dependent bioconversion by transcriptome-based molecular breeding. Appl. Environ. Microbiol. 73, 7657–7663 63 Yim, H. et al. (2011) Metabolic engineering of Escherichia coli for direct production of 1,4-butanediol. Nat. Chem. Biol. 7, 445–452 64 Guo, T. et al. (2012) Fine tuning of the lactate and diacetyl production through promoter engineering in Lactococcus lactis. PLoS ONE 7, http://dx.doi.org/10.1371/journal.pone.0036296 65 Vemuri, G.N. et al. (2007) Increasing NADH oxidation reduces overflow metabolism in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A. 104, 2402–2407 66 Zou, Y. et al. (2012) Crystal structures of phosphite dehydrogenase provide insights into nicotinamide cofactor regeneration. Biochemistry 51, 4263–4270 67 Lee, W.H. et al. (2013) Effects of NADH kinase on NADPH-dependent biotransformation processes in Escherichia coli. Appl. Microbiol. Biotechnol. 97, 1561–1569 68 Akinterinwa, O. et al. (2008) Metabolic engineering for bioproduction of sugar alcohols. Curr. Opin. Biotechnol. 19, 461–467 69 Neves, A.R. et al. (2005) Overview on sugar metabolism and its control in Lactococcus lactis – the input from in vivo NMR. FEMS Microbiol. Rev. 29, 531–554 70 Lopez de Felipe, F. et al. (1998) Cofactor engineering: a novel approach to metabolic engineering in Lactococcus lactis by controlled expression of NADH oxidase. J. Bacteriol. 180, 3804–3808 71 Kaup, B. et al. (2004) Metabolic engineering of Escherichia coli: construction of an efficient biocatalyst for D-mannitol formation in a whole-cell biotransformation. Appl. Microbiol. Biotechnol. 64, 333–339 72 Shi, Y. (2004) Metabolic enzymes and coenzymes in transcription – a direct link between metabolism and transcription? Trends Genet. 20, 445–452
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