ABSTRACT
Phosphorus-containing metabolites cover a large molecular diversity and represent an important domain of small molecules which are highly relevant for life and represent essential interfaces between biology and chemistry, between the biological and abiotic world. The large but not unlimited amount of phosphate minerals on our planet is a key resource for living organisms on our planet, while the accumulation of phosphorus-containing waste is associated with negative effects on ecosystems. Therefore, resource-efficient and circular processes receive increasing attention from different perspectives, from local and regional levels to national and global levels. The molecular and sustainability aspects of a global phosphorus cycle have become of much interest for addressing the phosphorus biochemical flow as a high-risk planetary boundary. Knowledge of balancing the natural phosphorus cycle and the further elucidation of metabolic pathways involving phosphorus is crucial. This requires not only the development of effective new methods for practical discovery, identification, and high-information content analysis, but also for practical synthesis of phosphorus-containing metabolites, for example as standards, as substrates or products of enzymatic reactions, or for discovering novel biological functions. The purpose of this article is to review the advances which have been achieved in the synthesis and analysis of phosphorus-containing metabolites which are biologically active.
Subject(s)
Ecosystem , Phosphorus , Minerals/chemistry , Phosphates , Metabolic Networks and PathwaysABSTRACT
This contribution focusses on Oreste Ghisalba's pioneering activities in both fundamental as well as applied research in biocatalysis and his work on building bridges not only between biotechnology and chemistry, but also culturally, geographically and between academia and industry. His scientific work published in journals, books and conferences will be reviewed and his teaching at ETH Zurich and the University of Basel will be highlighted. Furthermore, an appreciation will be given of his broad knowledge and vision in shaping the activities of the Swiss Coordination Committee Biotechnology (SKB), the Swiss-Japanese Meetings in Biotechnology, conferences and research programs such as the Swiss Priority Program Biotechnology (SPP Biotech) of the Swiss National Science Foundation.
Subject(s)
Biotechnology , Industry , BiocatalysisABSTRACT
Rapid biocatalytic process development and intensification continues to be challenging with currently available methods. Chiral amino-alcohols are of particular interest as they represent key industrial synthons for the production of complex molecules and optically pure pharmaceuticals. (2S,3R)-2-amino-1,3,4-butanetriol (ABT), a building block for the synthesis of protease inhibitors and detoxifying agents, can be synthesized from simple, non-chiral starting materials, by coupling a transketolase- and a transaminase-catalyzed reaction. However, until today, full conversion has not been shown and, typically, long reaction times are reported, making process modifications and improvement challenging. In this contribution, we present a novel microreactor-based approach based on free enzymes, and we report for the first time full conversion of ABT in a coupled enzyme cascade for both batch and continuous-flow systems. Using the compartmentalization of the reactions afforded by the microreactor cascade, we overcame inhibitory effects, increased the activity per unit volume, and optimized individual reaction conditions. The transketolase-catalyzed reaction was completed in under 10 min with a volumetric activity of 3.25 U ml-1 . Following optimization of the transaminase-catalyzed reaction, a volumetric activity of 10.8 U ml-1 was attained which led to full conversion of the coupled reaction in 2 hr. The presented approach illustrates how continuous-flow microreactors can be applied for the design and optimization of biocatalytic processes.
Subject(s)
Amino Alcohols/chemical synthesis , Aminoacyltransferases/chemistry , Escherichia coli Proteins/chemistry , Escherichia coli/enzymology , Transketolase/chemistry , Amino Alcohols/chemistry , CatalysisABSTRACT
The efficient synthesis of pure d-glycerate-2-phosphate is of great interest due to its importance as an enzyme substrate and metabolite. Therefore, we investigated a straightforward one-step biocatalytic phosphorylation of glyceric acid. Glycerate-2-kinase from Thermotoga maritima was expressed in Escherichia coli, allowing easy purification. The selective glycerate-2-kinase-catalyzed phosphorylation was followed by 31 P NMR and showed excellent enantioselectivity towards phosphorylation of the d-enantiomer of glyceric acid. This straightforward phosphorylation reaction and subsequent product isolation enabled the preparation of enantiomerically pure d-glycerate 2-phosphate. This phosphorylation reaction, using recombinant glycerate-2-kinase, yielded d-glycerate 2-phosphate in fewer reaction steps and with higher purity than chemical routes.
Subject(s)
Glyceric Acids/chemical synthesis , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Recombinant Fusion Proteins/chemistry , Biocatalysis , Endopeptidases/chemistry , Escherichia coli/genetics , Glyceric Acids/chemistry , Kinetics , Magnetic Resonance Spectroscopy , Maltose-Binding Proteins/genetics , Phosphorus Radioisotopes , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/genetics , Recombinant Fusion Proteins/genetics , Stereoisomerism , Thermotoga maritima/enzymologyABSTRACT
The architecture, organization, and functioning of biocatalytic reaction networks, which are coded in the cell-specific genome and which work together in the small space of biological cells, are a fascinating feature of life evolved over more than 3 billion years. Knowledge about the diversity of biocatalytic functions and metabolic pathways sustaining life on our planet is highly important, especially as the currently occurring loss of biodiversity is considered a planetary boundary that is at high risk, and knowledge about the life of current biological organisms should be gained before they become extinct. In addition to the well-known enzymatic reactions involved in biochemical pathways, the enzyme universe offers numerous opportunities for discovering novel functions and pathways. Maintaining thousands of molecules and reactions functioning properly within biological cells, which may be exposed to various kinds of external hazards, environmental stress, enzymatic side reactions, or non-enzymatic chemical reactions, is key for keeping cellular life healthy. This review aims to outline advances in assigning enzyme functions to protein sequences and the discovery of novel biocatalytic functions and pathways.
ABSTRACT
Biocatalytic synthesis in continuous-flow microreactors is of increasing interest for the production of specialty chemicals. However, the yield of production achievable in these reactors can be limited by the adverse effects of high substrate concentration on the biocatalyst, including inhibition and denaturation. Fed-batch reactors have been developed in order to overcome this problem, but no continuous-flow solution exists. We present the design of a novel multi-input microfluidic reactor, capable of substrate feeding at multiple points, as a first step towards overcoming these problems in a continuous-flow setting. Using the transketolase-(TK) catalysed reaction of lithium hydroxypyruvate (HPA) and glycolaldehyde (GA) to l-erythrulose (ERY), we demonstrate the transposition of a fed-batch substrate feeding strategy to our microfluidic reactor. We obtained a 4.5-fold increase in output concentration and a 5-fold increase in throughput compared with a single input reactor.
ABSTRACT
Methodologies for the synthesis and purification of metabolites, which have been developed following their discovery, analysis, and structural identification, have been involved in numerous life science milestones. The renewed focus on the small molecule domain of biological cells has also created an increasing awareness of the rising gap between the metabolites identified and the metabolites which have been prepared as pure compounds. The design and engineering of resource-efficient and straightforward synthetic methodologies for the production of the diverse and numerous metabolites and metabolite-like compounds have attracted much interest. The variety of metabolic pathways in biological cells provides a wonderful blueprint for designing simplified and resource-efficient synthetic routes to desired metabolites. Therefore, biocatalytic systems have become key enabling tools for the synthesis of an increasing number of metabolites, which can then be utilized as standards, enzyme substrates, inhibitors, or other products, or for the discovery of novel biological functions.
ABSTRACT
Biobased raw materials, such as carbohydrates, amino acids, nucleotides, or lipids contain valuable functional groups with oxygen and nitrogen atoms. An abundance of many functional groups of the same type, such as primary or secondary hydroxy groups in carbohydrates, however, limits the synthetic usefulness if similar reactivities cannot be differentiated. Therefore, selective defunctionalization of highly functionalized biobased starting materials to differentially functionalized compounds can provide a sustainable access to chiral synthons, even in case of products with fewer functional groups. Selective defunctionalization reactions, without affecting other functional groups of the same type, are of fundamental interest for biocatalytic reactions. Controlled biocatalytic defunctionalizations of biobased raw materials are attractive for obtaining valuable platform chemicals and building blocks. The biocatalytic removal of functional groups, an important feature of natural metabolic pathways, can also be utilized in a systemic strategy for sustainable metabolite synthesis.
Subject(s)
Amino Acids , Carbohydrates , Amines , BiocatalysisABSTRACT
The biosynthesis of metabolites from available starting materials is becoming an ever important area due to the increasing demands within the life science research area. Access to metabolites is making essential contributions to analytical, diagnostic, therapeutic and different industrial applications. These molecules can be synthesized by the enzymes of biological systems under sustainable process conditions. The facile synthetic access to the metabolite and metabolite-like molecular space is of fundamental importance. The increasing knowledge within molecular biology, enzyme discovery and production together with their biochemical and structural properties offers excellent opportunities for using modular cell-free biocatalytic systems. This reduces the complexity of synthesizing metabolites using biological whole-cell approaches or by classical chemical synthesis. A systems biocatalysis approach can provide a wealth of optimized enzymes for the biosynthesis of already identified and new metabolite molecules.
ABSTRACT
Many research areas, e.g., basic research but also applied fields of biotechnology, biomedicine, and diagnostics often suffer from the unavailability of metabolic compounds. This is mostly due to missing easy and efficient synthesis procedures. We herein describe the biocatalytic/enzymatic production of 2-keto-3-deoxy-D-gluconate, an intermediate of central metabolic pathways in all three domains of life and also of bacterial polysaccharides, lipopolysaccharides, and cell wall components. The method is based on the gluconate dehydratase from the hyperthermophilic crenarchaeon Thermoproteus tenax, which can be easily recombinantly overproduced in Escherichia coli and-due to its intrinsic thermostability-rapidly be purified by two precipitation steps. The enzyme completely converts D-gluconate to solely stereochemically pure KDG, taking benefits from the enol-keto-tautomerism of the primary reaction product. The final product can then easily be separated from the protein by ultrafiltration. The simple one-step procedure, which is suitable at least for the lab-scale/gram-scale production of KDG, replaces lengthy multi-step reactions and is easily scalable. This approach also illustrates the great application potential of Archaea with their unusual metabolic pathways and enzymes for the synthesis of added value products.
Subject(s)
Thermoproteus , Escherichia coli/metabolism , Gluconates/metabolism , Hydro-Lyases , Lipopolysaccharides/metabolism , Thermoproteus/metabolismABSTRACT
Invited for this month's cover is the Working Group Sustainable Chemistry of the European Society of Applied Biocatalysis (ESAB). The image shows the significant contributions of Biocatalysis to science, industry, society, and environment as a technology of first choice for Sustainable Chemistry in the 21st century. The Perspective itself is available at 10.1002/cssc.202102709.
Subject(s)
Industry , BiocatalysisABSTRACT
In their Editorial for the Special Issue on Biocatalysis as Key to Sustainable Industrial Chemistry, Guest Editors Andrés Alcántara, Pablo Domínguez de María, Jennifer Littlechild, and Roland Wohlgemuth and their co-workers on the European Society of Applied Biocatalysis' (ESAB) Working Group on Sustainable Chemistry Martin Schürmann and Roger Sheldon discuss the Special Issue and the importance of biocatalysis in carrying out cutting-edge industrial chemistry in a sustainable way, as well as the future prospects for the field.
Subject(s)
Biotechnology , Industry , Biocatalysis , Enzymes , HumansABSTRACT
The role and power of biocatalysis in sustainable chemistry has been continuously brought forward step by step to its present outstanding position. The problem-solving capabilities of biocatalysis have been realized by numerous substantial achievements in biology, chemistry and engineering. Advances and breakthroughs in the life sciences and interdisciplinary cooperation with chemistry have clearly accelerated the implementation of biocatalytic synthesis in modern chemistry. Resource-efficient biocatalytic manufacturing processes have already provided numerous benefits to sustainable chemistry as well as customer-centric value creation in the pharmaceutical, food, flavor, fragrance, vitamin, agrochemical, polymer, specialty, and fine chemical industries. Biocatalysis can make significant contributions not only to manufacturing processes, but also to the design of completely new value-creation chains. Biocatalysis can now be considered as a key enabling technology to implement sustainable chemistry.
Subject(s)
Chemical Industry , Industry , Biocatalysis , Green Chemistry TechnologyABSTRACT
In the area of human-made innovations to improve the quality of life, biocatalysis has already had a great impact and contributed enormously to a growing number of catalytic transformations aimed at the detection and analysis of compounds, the bioconversion of starting materials and the preparation of target compounds at any scale, from laboratory small scale to industrial large scale. The key enabling tools which have been developed in biocatalysis over the last decades also provide great opportunities for further development and numerous applications in various sectors of the global bioeconomy. Systems biocatalysis is a modular, bottom-up approach to designing the architecture of enzyme-catalyzed reaction steps in a synthetic route from starting materials to target molecules. The integration of biocatalysis and sustainable chemistry in vitro aims at ideal conversions with high molecular economy and their intensification. Retrosynthetic analysis in the chemical and biological domain has been a valuable tool for target-oriented synthesis while, on the other hand, diversity-oriented synthesis builds on forward-looking analysis. Bioinformatic tools for rapid identification of the required enzyme functions, efficient enzyme production systems, as well as generalized bioprocess design tools, are important for rapid prototyping of the biocatalytic reactions. The tools for enzyme engineering and the reaction engineering of each enzyme-catalyzed one-step reaction are also valuable for coupling reactions. The tools to overcome interaction issues with other components or enzymes are of great interest in designing multi-step reactions as well as in biocatalytic total synthesis.
Subject(s)
Biotechnology , Computational Biology , Enzymes/metabolism , Biocatalysis , Biotechnology/economics , Computational Biology/economics , Enzymes/economics , Humans , Protein Engineering , Quality of LifeABSTRACT
Biocatalytic phosphorylation reactions provide several benefits, such as more direct, milder, more selective, and shorter access routes to phosphorylated products. Favorable characteristics of biocatalytic methodologies represent advantages for in vitro as well as for in vivo phosphorylation reactions, leading to important advances in the science of synthesis towards bioactive phosphorylated compounds in various areas. The scope of this review covers key advances of biocatalytic phosphorylation reactions over the last two decades, for biocatalytic syntheses in vitro and for biotransformations in vivo (in humans). From the origins of probiotic life to in vitro synthetic applications and in vivo formation of bioactive pharmaceuticals, the common purpose is to outline the importance, relevance, and underlying connections of biocatalytic phosphorylations of small molecules. Asymmetric phosphorylations attracting increased attention are highlighted. Phosphohydrolases, phosphotransferases, phosphorylases, phosphomutases, and other enzymes involved in phosphorus chemistry provide powerful toolboxes for resource-efficient and selective in vitro biocatalytic syntheses of phosphorylated metabolites, chiral building blocks, pharmaceuticals as well as in vivo enzymatic formation of biologically active forms of pharmaceuticals. Nature's large diversity of phosphoryl-group-transferring enzymes, advanced enzyme and reaction engineering toolboxes make biocatalytic asymmetric phosphorylations using enzymes a powerful and privileged phosphorylation methodology.
Subject(s)
Biotransformation , Biocatalysis , Humans , PhosphorylationABSTRACT
Continuous, inspiring and interconnected step-by-step changes in thought and understanding, knowhow, actions and behaviour have often been instrumental in transitions from one particular age to the next in human history. This also applies to the present century and its sustainability challenges at the planetary, regional and local levels. Therefore, it is of great importance and relevance to move forward on the journey which has been started globally to address the not insignificant number of challenges. It is however essential to go beyond descriptive work by continuing with novel, inspiring and interconnected steps to find solutions to overcome these challenges. As this huge task also requires multidimensional communication, understanding and actions across different regions, cultures, disciplines and knowledge areas, the development of a common conceptual framework such as the concept of bioeconomy has been accepted globally as very valuable. The momentum which has been created in more than 50 countries around the world by the growing number of activities, initiatives and strategies in bioeconomy is very encouraging. This offers great opportunities to mobilize even more stakeholders in science and industry, as well as society, to join the bioeconomy journey. Strategic high-level concepts such as preserving the value of the natural capital of planet earth, connecting economy and ecology, sustaining the boundary conditions and habitability of our biosphere are highly important. It is also essential on the bioeconomy journey to connect with highly specific and actionable missions, programs and plans towards sustainable economic growth under the boundary conditions of our planet.
Subject(s)
Biotechnology/economics , Conservation of Natural Resources/economics , Economic Development , HumansABSTRACT
A whole-cell catalyst using Escherichia coli BL21(DE3) as a host, co-expressing glycerol dehydrogenase (GlyDH) from Gluconobacter oxydans and glucose dehydrogenase (GDH) from Bacillus subtilis for cofactor regeneration, has been successfully constructed and used for the reduction of aliphatic aldehydes, such as hexanal or glyceraldehyde to the corresponding alcohols. This catalyst was characterized in terms of growth conditions, temperature and pH dependency, and regarding the influence of external cofactor and permeabilization. In the case of external cofactor addition we found a 4.6-fold increase in reaction rate caused by the addition of 1 mM NADP(+). Due to the fact that pH and temperature are also factors which may affect the reaction rate, their effect on the whole-cell catalyst was studied as well. Comparative studies between the whole-cell catalyst and the cell-free system were investigated. Furthermore, the successful application of the whole-cell catalyst in repetitive batch conversions could be demonstrated in the present study. Since the GlyDH was recently characterized and successfully applied in the kinetic resolution of racemic glyceraldehyde, we were now able to transfer and establish the process to a whole-cell system, which facilitated the access to L-glyceraldehyde in high enantioselectivity at 54% conversion. All in all, the whole-cell catalyst shows several advantages over the cell-free system like a higher thermal, a similar operational stability and the ability to recycle the catalyst without any loss-of-activity. The results obtained making the described whole-cell catalyst an improved catalyst for a more efficient production of enantiopure L-glyceraldehyde.
Subject(s)
Bacillus subtilis/enzymology , Escherichia coli/metabolism , Gluconobacter oxydans/enzymology , Glucose 1-Dehydrogenase/metabolism , Glyceraldehyde/metabolism , Sugar Alcohol Dehydrogenases/metabolism , Biotransformation , Escherichia coli/genetics , Glucose 1-Dehydrogenase/genetics , Hydrogen-Ion Concentration , Kinetics , NADP/metabolism , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sugar Alcohol Dehydrogenases/genetics , TemperatureABSTRACT
Taking up the common challenges in biocatalysis, a group of industrialists decided to react with a bottom-up solution, and created the Swiss Industrial Biocatalysis Consortium (SIBC). The Swiss Industrial Biocatalysis Consortium is a pre-competitive working group to better implement and utilize existing know-how and resources in biocatalysis, and to influence and shape the economic and educational political environment. Recent examples of activities are outlined.
Subject(s)
Biocatalysis , Industry , Biotechnology , SwitzerlandABSTRACT
The oxidative Weimberg pathway for the five-step pentose degradation to α-ketoglutarate is a key route for sustainable bioconversion of lignocellulosic biomass to added-value products and biofuels. The oxidative pathway from Caulobacter crescentus has been employed in in-vivo metabolic engineering with intact cells and in in-vitro enzyme cascades. The performance of such engineering approaches is often hampered by systems complexity, caused by non-linear kinetics and allosteric regulatory mechanisms. Here we report an iterative approach to construct and validate a quantitative model for the Weimberg pathway. Two sensitive points in pathway performance have been identified as follows: (1) product inhibition of the dehydrogenases (particularly in the absence of an efficient NAD+ recycling mechanism) and (2) balancing the activities of the dehydratases. The resulting model is utilized to design enzyme cascades for optimized conversion and to analyse pathway performance in C. cresensus cell-free extracts.