Increased Product Formation Research Articles

Coupling of the catalytic reactions from formate dehydrogenase and hydrogenase in solution: Insights from computations and in situ IR spectroscopy

Sophisticated enzymatic systems have evolved in nature to efficiently couple distinct biochemical reactions in form of cascades. As such they serve as reference models to understand the indirect interactions of catalytic centers. Herein, we studied, in solution, the coupling of the reactions from membrane‐bound [NiFe] hydrogenase (MBH) from Cupriavidus necator (reversible H2 splitting into H+ and e‐) and the molybdenum‐dependent formate dehydrogenase from Rhodobacter capsulatus (reversible formate to CO2 interconversion). To follow their interplay via the characteristic absorptions from the MBH's active site or the respective substrate and product bands of FDH, we utilized in situ IR spectrocopy and GC(‐MS), in the absence or presence of soluble redox mediators. Coarse grained molecular dynamics (cgMD) computations revealed the lack of productive enzyme complexes for direct electron transfer (ET). Thus, the observed minor amounts of H2 or formate were produced from transient interactions between the two enzymes. On the contrary, the significantly increased product formation in the presence of methylene viologen can be related to the putative multiple interaction sites of the redox mediator with FDH identified by cgMD. Our study represents a proof‐of‐concept approach that can be used in future to develop novel coupled biocatalytic systems by identifying potential ET pathways.

Developing hybrid systems to address oxygen uncoupling in multi-component Rieske oxygenases

Rieske non-heme iron oxygenases (ROs) are redox enzymes essential for microbial biodegradation and natural product synthesis. These enzymes utilize molecular oxygen for oxygenation reactions, making them very useful biocatalysts due to their broad reaction scope and high selectivities. The mechanism of oxygen activation in ROs involves electron transfers between redox centers of associated protein components, forming an electron transfer chain (ETC). Although the ETC is essential for electron replenishment, it carries the risk of reactive oxygen species (ROS) formation due to electron loss during oxygen activation. Our previous study linked ROS formation to O2 uncoupling in the flavin-dependent reductase of the three-component cumene dioxygenase (CDO). In the present study, we extend this finding by investigating the effects of ROS formation on the multi-component CDO system in a cell-free environment. In particular, we focus on the effects of hydrogen peroxide (H2O2) formation in the presence of a NADH cofactor regeneration system on the catalytic efficiency of CDO in vitro. Based on this, we propose the implementation of hybrid systems with alternative (non-native) redox partners for CDO, which are highly advantageous in terms of reduced H2O2 formation and increased product formation. The hybrid system consisting of the RO-reductase from phthalate dioxygenase (PDR) and CDO proved to be the most promising for the oxyfunctionalization of indene, showing a 4-fold increase in product formation (20 mM) over 24 h (TTN of 1515) at a 3-fold increase in production rate.

Experimentally implemented dynamic optogenetic optimization of ATPase expression using knowledge-based and Gaussian-process-supported models

Optogenetic modulation of adenosine triphosphatase (ATPase) expression represents a novel approach to maximize bioprocess efficiency by leveraging enforced adenosine triphosphate (ATP) turnover. In this study, we experimentally implement a model-based open-loop optimization scheme for optogenetic modulation of the expression of ATPase. Increasing the intracellular concentration of ATPase, and thus the level of ATP turnover, in bioprocesses with product synthesis coupled with ATP generation, can lead to increased substrate uptrake and product formation. Previous simulation studies formulated optimal control problems using dynamic constraint-based models to find optimal light inputs in fermentations with optogenetically mediated ATPase expression. However, using these models poses challenges due to resulting bilevel optimizations and complex parameterization. Here, we outline a simplified unsegregated and quasi-unstructured kinetic modeling approach that reduces the number of dynamic states and leads to single-level optimizations. The models can be augmented with Gaussian processes to compensate for model uncertainties. We implement optimal control constrained by knowledge-based and hybrid models for optogenetic ATPase expression in Escherichia coli with lactate as the main product. To do so, we genetically engineer E. coli to obtain optogenetic expression of ATPase using the CcaS/CcaR system. This represents the first experimental implementation of model-based optimization of ATPase expression in bioprocesses.

Biotransamination with racemic amines as amine donors: kill three birds with one stone through a dual-enzyme cascade

A novel dual-enzyme cascade process utilizing carbonyl reductase and ω-transaminase enzyme was developed to remove by-products via the asymmetric reduction and increase product formation, making it ideal for both industrial and academic applications.

Intensification of bioprocesses with filamentous microorganisms

Abstract Many industrial biotechnological processes use filamentous microorganisms to produce platform chemicals, proteins, enzymes and natural products. Product formation is directly linked to their cellular morphology ranging from dispersed mycelia over loose clumps to compact pellets. Therefore, the adjustment and control of the filamentous cellular morphology pose major challenges for bioprocess engineering. Depending on the filamentous strain and desired product, optimal morphological shapes for achieving high product concentrations vary. However, there are currently no overarching strain- or product-related correlations to improve process understanding of filamentous production systems. The present book chapter summarizes the extensive work conducted in recent years in the field of improving product formation and thus intensifying biotechnological processes with filamentous microorganisms. The goal is to provide prospective scientists with an extensive overview of this scientifically diverse, highly interesting field of study. In the course of this, multiple examples and ideas shall facilitate the combination of their acquired expertise with promising areas of future research. Therefore, this overview describes the interdependence between filamentous cellular morphology and product formation. Moreover, the currently most frequently used experimental techniques for morphological structure elucidation will be discussed in detail. Developed strategies of morphology engineering to increase product formation by tailoring and controlling cellular morphology and thus to intensify processes with filamentous microorganisms will be comprehensively presented and discussed.

H2O2-Driven Hydroxylation of Steroids Catalyzed by Cytochrome P450 CYP105D18: Exploration of the Substrate Access Channel.

CYP105D18 supports H2O2 as an oxygen surrogate for catalysis well and shows high H2O2 resistance capacity. We report the hydroxylation of different steroids using H2O2 as a cosubstrate. Testosterone was regiospecifically hydroxylated to 2β-hydroxytestosterone. Based on the experimental data and molecular docking, we predicted that hydroxylation of methyl testosterone and nandrolone would occur at position 2 in the A-ring, while hydroxylation of androstenedione and adrenosterone was predicted to occur in the B-ring. Further, structure-guided rational design of the substrate access channel was performed with the mutagenesis of residues S63, R82, and F184. Among the mutants, S63A showed a marked decrease in product formation, while F184A showed a significant increase in product formation in testosterone, nandrolone, methyl testosterone, androstenedione, and adrenosterone. The catalytic efficiency (kcat/Km) toward testosterone was increased 1.36-fold in the F184A mutant over that in the wild-type enzyme. These findings might facilitate the potential use of CYP105D18 and further engineering to establish the basis of biotechnological applications. IMPORTANCE The structural modification of steroids is a challenging chemical reaction. Modifying the core ring and the side chain improves the biological activity of steroids. In particular, bacterial cytochrome P450s are used as promiscuous enzymes for the activation of nonreactive carbons of steroids. In the present work, we reported the H2O2-mediated hydroxylation of steroids by CYP105D18, which also overcomes the use of expensive cofactors. Further, exploring the substrate access channel and modifying the bulky amino acid F184A increase substrate conversion while modifying the substrate recognizing amino acid S63 markedly decreases product formation. Exploring the substrate access channel and the rational design of CYP105D18 can improve the substrate conversion, which facilitates the engineering of P450s for industrial application.

A tunable metabolic valve for precise growth control and increased product formation in Pseudomonas putida

Metabolic engineering of microorganisms aims to design strains capable of producing valuable compounds under relevant industrial conditions and in an economically competitive manner. From this perspective, and beyond the need for a catalyst, biomass is essentially a cost-intensive, abundant by-product of a microbial conversion. Yet, few broadly applicable strategies focus on the optimal balance between product and biomass formation. Here, we present a genetic control module that can be used to precisely modulate growth of the industrial bacterial chassis Pseudomonas putida KT2440. The strategy is based on the controllable expression of the key metabolic enzyme complex pyruvate dehydrogenase (PDH) which functions as a metabolic valve. By tuning the PDH activity, we accurately controlled biomass formation, resulting in six distinct growth rates with parallel overproduction of excess pyruvate. We deployed this strategy to identify optimal growth patterns that improved the production yield of 2-ketoisovalerate and lycopene by 2.5- and 1.38-fold, respectively. This ability to dynamically steer fluxes to balance growth and production substantially enhances the potential of this remarkable microbial chassis for a wide range of industrial applications.

Operando XAS Study of Pt-Doped CeO2 for the Nonoxidative Conversion of Methane

The methane to olefins, aromatics, and hydrogen (MTOAH) process via Pt/CeO2 catalysts poses an attractive route to improve yield and stability for the direct catalytic conversion of methane. In this study, two sets of samples, one composed of PtOx single sites on ceria and the other with additional Pt agglomerates, were prepared. Both sets of samples showed enhanced catalytic activity for the direct conversion of methane exceeding the performance of pure ceria. Pulsed reaction studies unraveled three reaction stages: reduction of the ceria support during activation, an induction phase with increasing product formation, and finally, stable running of the catalytic reactions. The reduction of ceria was confirmed by X-ray absorption spectroscopy (XAS) after conducting the MTOAH reaction. Operando X-ray absorption spectroscopy at challenging reaction temperatures of up to 975 °C in combination with theoretical simulations further evidenced an increased Pt–Ce interaction upon reaction with CH4. Analysis of the extended X-ray absorption fine structure (EXAFS) spectra proved decoration and encapsulation of the Pt particles by the CeO2/Ce2O3 support or a partial Ce–Pt alloy formation due to the strong metal–support interaction that developed under reaction conditions. Moreover, methyl radicals were detected as reaction intermediates indicating a reaction pathway through the gas-phase coupling of methyl radicals. The results indicate that apart from single-atom Pt sites reported in the literature, the observed Pt–Ce interface may have eased the activation of CH4 by forming methyl radicals and suppressed coke formation, significantly improving the catalytic performance of the ceria-based catalysts in general.

Impact of glgA1, glgA2 or glgC overexpression on growth and glycogen production in Synechocystis sp. PCC 6803

Low production rates are still one limiting factor for the industrial climate-neutral production of biovaluable compounds in cyanobacteria. Next to optimized cultivation conditions, new production strategies are required. Hence, the use of established molecular tools could lead to increased product yields in the cyanobacterial model organism Synechocystis sp. PCC6803. Its main storage compound glycogen was chosen to be increased by the use of these tools. In this study, the three genes glgC, glgA1 and glgA2, which are part of the glycogen synthesis pathway, were combined with the Pcpc560 promoter and the neutral cloning site NSC1. The complete genome integration, protein formation, biomass production and glycogen accumulation were determined to select the most productive transformants. The overexpression of glgA2 did not increase the biomass or glycogen production in short-term trials compared to the other two genes but caused transformants death in long-term trials. The transformants glgA1_11 and glgC_2 showed significantly increased biomass (1.6-fold - 1.7-fold) and glycogen production (3.5-fold - 4-fold) compared to the wild type after 96 h making them a promising energy source for further applications. Those could include for example a two-stage production process, with first energy production (glycogen) and second increased product formation (e.g. ethanol).

Exploiting RNA thermometer-driven molecular bioprocess control as a concept for heterologous rhamnolipid production

A key challenge to advance the efficiency of bioprocesses is the uncoupling of biomass from product formation, as biomass represents a by-product that is in most cases difficult to recycle efficiently. Using the example of rhamnolipid biosurfactants, a temperature-sensitive heterologous production system under translation control of a fourU RNA thermometer from Salmonella was established to allow separating phases of preferred growth from product formation. Rhamnolipids as bulk chemicals represent a model system for future processes of industrial biotechnology and are therefore tied to the efficiency requirements in competition with the chemical industry. Experimental data confirms function of the RNA thermometer and suggests a major effect of temperature on specific rhamnolipid production rates with an increase of the average production rate by a factor of 11 between 25 and 38 °C, while the major part of this increase is attributable to the regulatory effect of the RNA thermometer rather than an unspecific overall increase in bacterial metabolism. The production capacity of the developed temperature sensitive-system was evaluated in a simple batch process driven by a temperature switch. Product formation was evaluated by efficiency parameters and yields, confirming increased product formation rates and product-per-biomass yields compared to a high titer heterologous rhamnolipid production process from literature.

Understanding D-xylonic acid accumulation: a cornerstone for better metabolic engineering approaches.

The xylose oxidative pathway (XOP) has been engineered in microorganisms for the production of a wide range of industrially relevant compounds. However, the performance of metabolically engineered XOP-utilizing microorganisms is typically hindered by D-xylonic acid accumulation. It acidifies the media and perturbs cell growth due to toxicity, thus curtailing enzymatic activity and target product formation. Fortunately, from the growing portfolio of genetic tools, several strategies that can be adapted for the generation of efficient microbial cell factories have been implemented to address D-xylonic acid accumulation. This review centers its discussion on the causes of D-xylonic acid accumulation and how to address it through different engineering and synthetic biology techniques with emphasis given on bacterial strains. In the first part of this review, the ability of certain microorganisms to produce and tolerate D-xylonic acid is also tackled as an important aspect in developing efficient microbial cell factories. Overall, this review could shed some insights and clarity to those working on XOP in bacteria and its engineering for the development of industrially applicable product-specialist strains. KEY POINTS: D-Xylonic acid accumulation is attributed to the overexpression of xylose dehydrogenase concomitant with basal or inefficient expression of enzymes involved in D-xylonic acid assimilation. Redox imbalance and insufficient cofactors contribute to D-xylonic acid accumulation. Overcoming D-xylonic acid accumulation can increase product formation among engineered strains. Engineering strategies involving enzyme engineering, evolutionary engineering, coutilization of different sugar substrates, and synergy of different pathways could potentially address D-xylonic acid accumulation.

Redirection of the central metabolism of Klebsiella pneumoniae towards dihydroxyacetone production

BackgroundKlebsiella pneumoniae is a bacterium that can be used as producer for numerous chemicals. Glycerol can be catabolised by K. pneumoniae and dihydroxyacetone is an intermediate of this catabolism pathway. Here dihydroxyacetone and glycerol were produced from glucose by this bacterium based a redirected glycerol catabolism pathway.ResultstpiA, encoding triosephosphate isomerase, was knocked out to block the further catabolism of dihydroxyacetone phosphate in the glycolysis. After overexpression of a Corynebacterium glutamicum dihydroxyacetone phosphate dephosphorylase (hdpA), the engineered strain produced remarkable levels of dihydroxyacetone (7.0 g/L) and glycerol (2.5 g/L) from glucose. Further increase in product formation were obtained by knocking out gapA encoding an iosenzyme of glyceraldehyde 3-phosphate dehydrogenase. There are two dihydroxyacetone kinases in K. pneumoniae. They were both disrupted to prevent an inefficient reaction cycle between dihydroxyacetone phosphate and dihydroxyacetone, and the resulting strains had a distinct improvement in dihydroxyacetone and glycerol production. pH 6.0 and low air supplement were identified as the optimal conditions for dihydroxyacetone and glycerol production by K, pneumoniae ΔtpiA-ΔDHAK-hdpA. In fed batch fermentation 23.9 g/L of dihydroxyacetone and 10.8 g/L of glycerol were produced after 91 h of cultivation, with the total conversion ratio of 0.97 mol/mol glucose.ConclusionsThis study provides a novel and highly efficient way of dihydroxyacetone and glycerol production from glucose.

On the influence of carbon nanoparticles as additives in the electrosynthesis of bromoarenes

Kinetic studies on an electrochemical bromination reaction are presented. In the presence of different carbon nanoparticles bromide salts were used as safe bromine sources and the interplay of graphene derivates with different electrode materials was investigated. Boron-doped graphene in combination with graphite electrodes was found to be an efficient promotor for the regioselective bromination of arenes. Graphite electrodes not only provided the best results in combination with Boron-doped graphene but also proved to be stable under the reaction conditions as shown by SEM and EDX measurements while other electrodes displayed some fouling. Among the beneficial effects of the addition of Boron-doped graphene is the increased product formation constant and the extension of the applied substrate scope, while still providing a safe and user-friendly bromination protocol.

Challenges of influencing cellular morphology by morphology engineering techniques and mechanical induced stress on filamentous pellet systems-A critical review.

Filamentous microorganisms are main producers of organic acids, enzymes, and pharmaceutical agents such as antibiotics and other active pharmaceutical ingredients. With their complex cell morphology, ranging from dispersed mycelia to dense pellets, the cultivation is challenging. In recent years, various techniques for tailor‐made cell morphologies of filamentous microorganisms have been developed to increase product formation and have been summarised under the term morphology engineering. These techniques, namely microparticle‐enhanced cultivation, macroparticle‐enhanced cultivation, and alteration of the osmolality of the culture medium by addition of inorganic salts, the salt‐enhanced cultivation, are presented and discussed in this review. These techniques have already proven to be useful and now await further proof‐of‐concept. Furthermore, the mechanical behaviour of individual pellets is of special interest for a general understanding of pellet mechanics and the productivity of biotechnological processes with filamentous microorganisms. Correlating them with substrate uptake and finally with productivity would be a breakthrough not to be underestimated for the comprehensive characterisation of filamentous systems. So far, this research field is under‐represented. First results on filamentous pellet mechanics are discussed and important future aspects, which the filamentous expert community should deal with, will be presented and critically discussed.

Thermodynamic Contribution of Water in Cryptophane Host-Guest Binding Reaction.

A detailed examination of binding thermodynamics is undertaken for the interaction between rubidium ion and a water-soluble cryptophane molecule using isothermal titration calorimetry. The equilibrium-binding quotient for this host-guest pair decreases with increasing product formation. When analyzed with a thermodynamic framework that considers water explicitly in the governing equation, the shift in equilibrium is ascribed to an unfavorable change in the free energy of solvation upon formation of the inclusion complex. A van't Hoff analysis of the binding data, as well as an observation of aggregation between inclusion complexes, suggests that charge-charge interactions between rubidium ion and the phenolate groups of the cryptophane host provide the driving force for association in water that overcomes a large and unfavorable change in solvent enthalpy.

C3PE: counter-current continuous phase extraction for improved precision of in-droplet chemical reactions

To improve tools for controlling and optimizing miniaturized chemistry, a novel oil extraction architecture, designated as the Counter-Current Continuous Phase Extraction (C3PE) module, was developed to enable precise control over reaction incubation in water-in-oil droplet microfluidic reactors. Using a symmetric pillar array coupled to adjustable oil flows prevented sample loss and droplet breakup, even at high final volume fractions, and cross-flow added novel stabilization of oil extraction against instability in control pressures. By integrating this dynamic functionality, C3PE enabled rational selection of the oil extraction magnitude across a range of achievable final droplet volume fractions (up to 85%) when processing droplets at 40–200 Hz. Further, this versatile device handled many droplet sizes (70–500 pL demonstrated here). Next, this approach to controlling droplet volume fraction enabled incubation time monitoring and optimization when coupled to a K-channel direct injection feature to label selected droplets. In profiling system characteristics like volume fraction, channel geometry, and continuous phase viscosity, this technique provided a powerful tool to control, measure, and improve incubation performance. Finally, applying C3PE principles to an in-droplet β-galactosidase enzyme reaction (useful in immunoassay systems) increased product formation while significantly decreasing variance in product yield among droplets relative to a non-extracted comparison. We envision that this method will inform future design and implementation of high precision in-droplet chemistry while being of general interest in the study of emulsion fluid dynamics.

KINETIC STUDIES OF NEISSERIA MENINGITIDIS SEROGROUP W CAPSULE POLYMERASE USING BIOLUMINESCENCE‐BASED ASSAYS

Neisseria meningitidis is a Gram‐negative bacterium that causes meningitis. Our overall goal is increased understanding of the N. meningitidis serogroup W capsule polymerase. This enzyme creates the capsular polysaccharide found in this serogroup. During the reaction, the enzyme transfers galactose and sialic acid from two nucleotide donor sugars (UDP‐Galactose and CMP‐Sialic Acid) to an acceptor. This capsule polymerase can be used as a new tool for glycoconjugate vaccine development. In this work, we describe our efforts to determine kinetic parameters of the enzyme which are currently unknown. We have optimized commercially available bioluminescence‐based assays to investigate activity of the enzyme (CMP‐Glo and UDP‐Glo by Promega). Reactions were performed to determine Km and Vmax values in a 10‐minute reaction. Km and Vmax values of 629.2 μg/mL and 0.8965 μM/min were determined for the acceptor (hydrolyzed serogroup W polysaccharide). To simplify our studies, we have recently moved to use of a homogeneous acceptor, a sialic acid trimer (DP3) and focused strictly on the UDP‐Glo assay. In our studies with this acceptor, we confirm an increase in product formation with increasing enzyme amounts (0–1250 ng enzyme). Km and Vmax values were determined for UDP‐Galactose (44.61 μM and 0.009457 μM/min) and DP3 acceptor (2984.2 μM and 0.01099 μM/min). In future work, we will perform kinetic studies with a modified acceptor (DP3‐Galactose) to determine Km and Vmax for this acceptor and CMP Sialic acid.Support or Funding InformationNIH 5SC2GM125517‐02NIH UL1GM118973

Flux Balance Analysis for Media Optimization and Genetic Targets to Improve Heterologous Siderophore Production.

SummarySiderophores are small molecule metal chelators secreted in sparse quantities by their native microbial hosts but can be engineered for enhanced production from heterologous hosts like Escherichia coli. These molecules have been proved to be capable of binding heavy metals of commercial and/or environmental interest. In this work, we incorporated, as needed, the appropriate pathways required to produce several siderophores (anguibactin, vibriobactin, bacillibactin, pyoverdine, and enterobactin) into the base E. coli K-12 MG1655 metabolic network model to computationally predict, via flux balance analysis methodologies, gene knockout targets, gene over-expression targets, and media modifications capable of improving siderophore reaction flux. E. coli metabolism proved supportive for the underlying production mechanisms of various siderophores. Within such a framework, the gene deletion and over-expression targets identified, coupled with complementary insights from medium optimization predictions, portend experimental implementation to both enable and improve heterologous siderophore production. Successful production of siderophores would then spur novel metal-binding applications.

Beneficial mutations for carotenoid production identified from laboratory-evolved Saccharomyces cerevisiae.

Adaptive laboratory evolution (ALE) is a powerful tool used to increase strain fitness in the presence of environmental stressors. If production and strain fitness can be coupled, ALE can be used to increase product formation. In earlier work, carotenoids hyperproducing mutants were obtained using an ALE strategy. Here, de novo mutations were identified in hyperproducers, and reconstructed mutants were explored to determine the exact impact of each mutation on production and tolerance. A single mutation in YMRCTy1-3 conferred increased carotenoid production, and when combined with other beneficial mutations led to further increased β-carotene production. Findings also suggest that the ALE strategy selected for mutations that confer increased carotenoid production as primary phenotype. Raman spectroscopy analysis and total lipid quantification revealed positive correlation between increased lipid content and increased β-carotene production. Finally, we demonstrated that the best combinations of mutations identified for β-carotene production were also beneficial for production of lycopene.

A 96-multiplex capillary electrophoresis screening platform for product based evolution of P450 BM3

The main challenge that prevents a broader application of directed enzyme evolution is the lack of high-throughput screening systems with universal product analytics. Most directed evolution campaigns employ screening systems based on colorimetric or fluorogenic surrogate substrates or universal quantification methods such as nuclear magnetic resonance spectroscopy or mass spectrometry, which have not been advanced to achieve a high-throughput. Capillary electrophoresis with a universal UV-based product detection is a promising analytical tool to quantify product formation. Usage of a multiplex system allows the simultaneous measurement with 96 capillaries. A 96-multiplexed capillary electrophoresis (MP-CE) enables a throughput that is comparable to traditional direct evolution campaigns employing 96-well microtiter plates. Here, we report for the first time the usage of a MP-CE system for directed P450 BM3 evolution towards increased product formation (oxidation of alpha-isophorone to 4-hydroxy-isophorone; highest reached total turnover number after evolution campaign: 7120 mol4-OH molP450−1). The MP-CE platform was 3.5-fold more efficient in identification of beneficial variants than the standard cofactor (NADPH) screening system.