Redox Biocatalysis Research Articles

Metabolic response of Pseudomonas putida during redox biocatalysis in the presence of a second octanol phase

A key limitation of whole-cell redox biocatalysis for the production of valuable, specifically functionalized products is substrate/product toxicity, which can potentially be overcome by using solvent-tolerant micro-organisms. To investigate the inter-relationship of solvent tolerance and energy-dependent biocatalysis, we established a model system for biocatalysis in the presence of toxic low logP(ow) solvents: recombinant solvent-tolerant Pseudomonas putida DOT-T1E catalyzing the stereospecific epoxidation of styrene in an aqueous/octanol two-liquid phase reaction medium. Using (13)C tracer based metabolic flux analysis, we investigated the central carbon and energy metabolism and quantified the NAD(P)H regeneration rate in the presence of toxic solvents and during redox biocatalysis, which both drastically increased the energy demands of solvent-tolerant P. putida. According to the driven by demand concept, the NAD(P)H regeneration rate was increased up to eightfold by two mechanisms: (a) an increase in glucose uptake rate without secretion of metabolic side products, and (b) reduced biomass formation. However, in the presence of octanol, only approximately 1% of the maximally observed NAD(P)H regeneration rate could be exploited for styrene epoxidation, of which the rate was more than threefold lower compared with operation with a non-toxic solvent. This points to a high energy and redox cofactor demand for cell maintenance, which limits redox biocatalysis in the presence of octanol. An estimated upper bound for the NAD(P)H regeneration rate available for biocatalysis suggests that cofactor availability does not limit redox biocatalysis under optimized conditions, for example, in the absence of toxic solvent, and illustrates the high metabolic capacity of solvent-tolerant P. putida. This study shows that solvent-tolerant P. putida have the remarkable ability to compensate for high energy demands by boosting their energy metabolism to levels up to an order of magnitude higher than those observed during unlimited growth.

Metabolic capacity estimation of Escherichia coli as a platform for redox biocatalysis: constraint‐based modeling and experimental verification

Whole-cell redox biocatalysis relies on redox cofactor regeneration by the microbial host. Here, we applied flux balance analysis based on the Escherichia coli metabolic network to estimate maximal NADH regeneration rates. With this optimization criterion, simulations showed exclusive use of the pentose phosphate pathway at high rates of glucose catabolism, a flux distribution usually not found in wild-type cells. In silico, genetic perturbations indicated a strong dependency of NADH yield and formation rate on the underlying metabolic network structure. The linear dependency of measured epoxidation activities of recombinant central carbon metabolism mutants on glucose uptake rates and the linear correlation between measured activities and simulated NADH regeneration rates imply intracellular NADH shortage. Quantitative comparison of computationally predicted NADH regeneration and experimental epoxidation rates indicated that the achievable biocatalytic activity is determined by metabolic and enzymatic limitations including non-optimal flux distributions, high maintenance energy demands, energy spilling, byproduct formation, and uncoupling. The results are discussed in the context of cellular optimization of biotransformation processes and may guide a priori design of microbial cells as redox biocatalysts.

NADH Availability Limits Asymmetric Biocatalytic Epoxidation in a Growing Recombinant Escherichia coli Strain

Styrene can efficiently be oxidized to (S)-styrene oxide by recombinant Escherichia coli expressing the styrene monooxygenase genes styAB from Pseudomonas sp. strain VLB120. Targeting microbial physiology during whole-cell redox biocatalysis, we investigated the interdependency of styrene epoxidation, growth, and carbon metabolism on the basis of mass balances obtained from continuous two-liquid-phase cultures. Full induction of styAB expression led to growth inhibition, which could be attenuated by reducing expression levels. Operation at subtoxic substrate and product concentrations and variation of the epoxidation rate via the styrene feed concentration allowed a detailed analysis of carbon metabolism and bioconversion kinetics. Fine-tuned styAB expression and increasing specific epoxidation rates resulted in decreasing biomass yields, increasing specific rates for glucose uptake and the tricarboxylic acid (TCA) cycle, and finally saturation of the TCA cycle and acetate formation. Interestingly, the biocatalysis-related NAD(P)H consumption was 3.2 to 3.7 times higher than expected from the epoxidation stoichiometry. Possible reasons include uncoupling of styrene epoxidation and NADH oxidation and increased maintenance requirements during redox biocatalysis. At epoxidation rates of above 21 micromol per min per g cells (dry weight), the absence of limitations by O(2) and styrene and stagnating NAD(P)H regeneration rates indicated that NADH availability limited styrene epoxidation. During glucose-limited growth, oxygenase catalysis might induce regulatory stress responses, which attenuate excessive glucose catabolism and thus limit NADH regeneration. Optimizing metabolic and/or regulatory networks for efficient redox biocatalysis instead of growth (yield) is likely to be the key for maintaining high oxygenase activities in recombinant E. coli.

Improvement the tolerance of baker's yeast to toxic substrate/product with cloud point system during the whole cell microbial transformation

The industrial process of a whole-cell microbial transformation is usually hindered by the toxicity or inhibition of a high substrate/product concentration. Especially in a biocatalytic redox process, the cofactor regeneration with the viable cells is a key of the whole cell microbial transformation. In present work, growing culture of Saccharomyces cerevisiae (baker's yeast) in a nonionic surfactant mediated cloud point system was carried out. Then the tolerances of baker's yeast cells against a high substrate/product concentration and a long term of biocatalytic reduction of acetophenone to produce 1-phenylethanol as a model were examined in the cloud point system. The long term of cofactor regeneration at a high substrate concentration in the cloud point system make the cloud point system potential for further development as a novel two-phase partitioning system for production of chiral compounds by microbial transformation.

Enzymatic Biofuel Cells for Implantable and Microscale Devices

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Enzymatic Biofuel Cells for Implantable and Microscale Devices

Introduction Technology for electrical power generation using enzyme catalysts, established four decades ago, has recently received increased attention associated with demand for micro-scale and implantable power supplies. The main challenges, namely the fragility of enzyme molecules, characteristic low current density, and poor fundamental understanding of redox biocatalysis, are currently being addressed from a variety of research perspectives, to take advantage of enzyme selectivity, low temperature and moderate pH activity, and manufacturability in small-scale devices. Such an effort benefits from four decades of multidisciplinary research in biosensors and related bioelectrochemical fields. This review paper summarizes the current state of enzymatic biofuel cell research in the context of foreseeable applications and assesses the future prospects of the technology. Emphasis is placed on device performance and engineering aspects, with a view toward practical portable power devices based on enzymatic biofuel cells. Research in biocatalytically modified electrodes, particularly for sensor applications, has provided a significant technological underpinning for current biofuel cell development. There exists significant overlap in technical requirements between sensors and biofuel cells, including chemical and mechanical stability, selectivity, and cost of materials. However, these two technologies diverge in the areas of current density, cell potential and stability. There exists extensive review literature in the area of biological fuel cells. Notably, Palmore and Whitesides summarized biological fuel cell concepts and performance up until about 1992. More recently, Katz and Willner discussed recent progress in novel electrode chemistries for both microbial and enzymatic fuel cells. We do not duplicate these valuable contributions, but instead focus on the strengths and weaknesses of state-of-art materials in the context of specific classes of applications, and point to areas where additional knowledge is currently needed to exploit biological fuel cells. With some exceptions, we focus on contributions made after 1992. Biofuel cells have traditionally been classified according to whether the catalytic enzymes were located inside or outside of living cells. If living cells are involved the system is considered to be microbial, and if not it is considered enzymatic. Although microbial fuel cells posses unique features unmatched by enzymatic cells, such as long-term stability and fuel efficiency, the power densities associated with such devices are typically much lower owing to resistance to mass transfer across cell membranes. Thus, microbial fuel cells are expected to find limited application in smallscale electronic devices. This review will focus on enzymatic biofuel cells. While such cells typically demonstrate reduced stability due to the limited lifetime of extracellular enzymes, and are typically unable to fully oxidize fuels, they allow for substantial concentration of catalysts and removal of mass transfer barriers and provide higher current and power densities, approaching the range of applicability to microand mini-scale electronics applications. Applications and Requirements The range of possible applications for biofuel cells may be broken down into three main subclasses: 1. Implantable power, such as micro-scale cells implanted in human or animal tissue, or larger cells implanted in blood vessels. 2. Power derived from ambient fuels or oxidants, mainly plant saps and juices, but extending to sewage and other waste streams. 3. Power derived from conventional fuels including hydrogen, methanol or higher alcohols. Classes 1 and 2 are closely related. The fuels available for implantable power, such as blood borne glucose or lactate, are ambient in the sense that they are present in a physiological environment in the absence of a fuel cell device. One major distinction between these two classes is that the ambient-fueled cell need not be implanted, and focuses on plantor waste-derived fuels, whereas the implantable cell focuses on animal-derived fuels and is present within the physiological system. Class 3 is unique in that this class competes with well-established conventional fuel cell technology. To a greater or lesser extent, all three classes share the fundamental technical requirements of high power density and high activity.

Biocatalytic Oxidation of Primary and Secondary Alcohols

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Electrochemical Regeneration of Oxidoreductases for Cell-free Biocatalytic Redox Reactions

Oxidoreductases represent a highly interesting and versatile class of biocatalysts for specific reduction, oxidation, and oxyfunctionalization reactions. Since oxidoreductases depend on cofactors and coenzymes to supply or withdraw redox equivalents released during the catalytic process, their application in cell-free environments requires external supply with these redox equivalents. Next to enzymatic approaches, a variety of non-enzymatic regeneration strategies have been developed. This review focuses on electrochemical methods for the in situ regeneration of nicotinamide cofactors as well as flavin- and heme-coenzymes, developed for synthetic application. The fields of electrochemical biosensors as well as biofuel cells are not discussed in detail. Electrochemical approaches bear much promise and in some cases are more efficient and more versatile than enzymatic regeneration approaches.

Biocatalytic Oxidation of Primary and Secondary Alcohols

AbstractDriven by the immaturity of many organic oxidation reactions and the necessity for ‘green’ chemical processes, biocatalytic redox processes are being investigated with increasing intensity in order to tap the full potential of the excellent chemo‐, regio‐ and enantioselectivity of enzymes. Despite their unmatched advantage in view of environmental aspects, the requirement of cofactors and the availability of redox enzymes able to tolerate high concentrations of organic (co)substrates sets limitations. However, during the past years, an increasing number of applications to the bio‐oxidation of primary and secondary alcohols with novel redox enzymes have been developed. This review gives an overview on the different methods and their potential and limits.

On the organic solvent and thermostability of the biocatalytic redox system of Rhodococcus ruber DSM 44541.

The sec-alcohol dehydrogenase activity of whole cells of Rhodococcus ruber DSM 44541 has been employed as an efficient biocatalytic redox system due to the use of acetone and 2-propanol at elevated concentrations for cofactor regeneration in the oxidation and reduction mode, respectively, and external addition of NADH/NAD(+) can be omitted. The operational half-life time of the redox system is 29 hours in 20% v/v acetone and 37 hours in 30% v/v 2-propanol. The Redox system allows the enantioselective oxidation of sec-alcohols and the asymmetric reduction of ketones to furnish (S)-configurated alcohols in high optical purity. The stability of the cells towards further organic solvents was investigated. In addition, the system displays thermostability of up to 60 degrees C and pH stability of up to pH 11. The system represents a simple to handle tool for environmentally benign redox reactions.

Modelling of the Baeyer-Villiger Monooxygenase Catalysed Synthesis of Optically Pure Lactones

While considerable progress has been made on the implementation of biocatalytic hydrolyses, there are still a number of engineering issues to address regarding the implementation of biocatalytic redox and carbon-carbon bond forming reactions. The use of process models linked to graphical tools (process maps) to describe the parameter boundaries for effective reactor operation have found particular use for these classes. Such process maps help to describe the properties of the biocatalyst and also the properties of the reactants and products which are frequently labile in the reactor environment. Data to form and verify such models can be rapidly collected at the microwell-scale. The models themselves can either be mechanistic or, as used here, based on experimental data. The output of such models can then be incorporated in graphical process maps and used as a predictive tool to carry out ‘what if’ studies. These may be, for example, on possible changes to the biocatalyst (via directed evolution) or the implementation of innovative process solutions (such as in-situ product removal). In this way, alternative process flow sheets and operating strategies can be evaluated more rapidly. In this paper, the utility of these numerical and graphical techniques using the Baeyer-Villiger oxidation of bicyclo[3.2.0]hept-2-en-6-one by a whole cell biocatalyst expressing cyclohexanone monooxygenase, is illustrated.

Artificial Electron Carriers for Preparative Biocatalytic Redox Reactions Forming Reversibly Carbon Hydrogen Bonds with Enzymes Present in Strict or Facultative Anaerobes

The application of various artificial electron mediators in combination with redox enzymes present in anaerobically grown cells is described. Examples include viologens of different redox potential, cobalt complexes, anthraquinones, phenoxazines, phenazines and others. The regeneration of the reduced or oxidised mediators by various methods is discussed, with hydrogen gas, carbon monoxide, formate or a cathode as electron donors, and dimethylsulphoxide, quinones, oxygen or an anode as electron acceptors. The enzymes used, usually in the form of resting cells or crude cell extracts of clostridia or Proteus species are mostly reversible. Examples for preparative reductions as well as for dehydrogenations are given.