Back to the Future of Metabolism-Advances in the Discovery and Characterization of Unknown Biocatalytic Functions and Pathways.

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.

Synthesis of Metabolites and Metabolite-like Compounds Using Biocatalytic Systems.

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.

Advances in the Synthesis and Analysis of Biologically Active Phosphometabolites.

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.

Simplified Enzymatic Synthesis of 2-Keto-3-Deoxy-D-Gluconate from D-Gluconate Using the Gluconate Dehydratase from Thermoproteus tenax.

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.

Complexity reduction and opportunities in the design, integration and intensification of biocatalytic processes for metabolite synthesis.

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.

Preface to Special Issue on Biocatalysis as Key to Sustainable Industrial Chemistry.

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.

Biocatalysis as Key to Sustainable Industrial Chemistry.

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.

Selective Biocatalytic Defunctionalization of Raw Materials.

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.

Biocatalysis as Key to Sustainable Industrial Chemistry.

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.

Biocatalysis - Key enabling tools from biocatalytic one-step and multi-step reactions to biocatalytic total synthesis.

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.

Key advances in biocatalytic phosphorylations in the last two decades: Biocatalytic syntheses in vitro and biotransformations in vivo (in humans).

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.

Bioeconomy moving forward step by step - A global journey.

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.

Corrigendum: Enzymatic Synthesis of 2-Keto-3-Deoxy-6-Phosphogluconate by the 6-Phosphogluconate-Dehydratase From Caulobacter crescentus.

[This corrects the article DOI: 10.3389/fbioe.2020.00185.].

Building Bridges between Biotechnology and Chemistry - Oreste Ghisalba's Pioneering Activities, Publications and Programs.

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.

Enzymatic Synthesis of 2-Keto-3-Deoxy-6-Phosphogluconate by the 6-Phosphogluconate-Dehydratase From Caulobacter crescentus.

The availability of metabolic intermediates is a prerequisite in many fields ranging from basic research, to biotechnological and biomedical applications as well as diagnostics. 2-keto-3-deoxy-6-phosphogluconate (KDPG) is the key intermediate of the Entner-Doudoroff (ED) pathway for sugar degradation and of sugar acid and sugar polymer breakdown in many organisms including human and plant pathogens. However, so far KDPG is hardly available due to missing efficient synthesis routes. We here report the efficient biocatalytic KDPG production through enzymatic dehydration of 6-phosphogluconate (6PG) up to gram scale using the 6PG dehydratase/Entner-Doudoroff dehydratase (EDD) from Caulobacter crescentus (CcEDD). The enzyme was recombinantly produced in Escherichia coli, purified to apparent homogeneity in a simple one-step procedure using nickel ion affinity chromatography, and characterized with respect to molecular and kinetic properties. The homodimeric CcEDD catalyzed the irreversible 6PG dehydration to KDPG with a Vmax of 61.6 U mg-1 and a KM of 0.3 mM for 6PG. Most importantly, the CcEDD showed sufficient long-term stability and activity to provide the enzyme in amounts and purity required for the efficient downstream synthesis of KDPG. CcEDD completely converted 1 g 6PG and a straight forward purification method yielded 0.81 g of stereochemically pure KDPG corresponding to a final yield of 90% as shown by HPLC-MS and NMR analyses.

A combined experimental and modelling approach for the Weimberg pathway optimisation.

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.

Bioeconomy for Sustainable Development.

Bioeconomy is an emerging paradigm under which the creation, development, and revitalization of economic systems based on a sustainable use of renewable biological resources in a balanced way is rapidly spreading globally. Bioeconomy is building bridges between biotechnology and economy as well as between science, industry, and society. Biotechnology, from its ancient origins up to the present is at the core of the scientific and innovative foundation of bioeconomy policies developed in numerous countries. The challenges and perspectives of bioeconomies are immense, from resource-efficient large-scale manufacturing of products such as chemicals, materials, food, pharmaceuticals, polymers, flavors, and fragrances to the production of new biomaterials and bioenergy in a sustainable and economic way for a growing world population. Key success factors for different countries working on the bioeconomy vary widely from high-tech bioeconomies, emerging diversified or diversified bioeconomies to advanced and basic primary sector bioeconomies. Despite the large variety of bioeconomies, several common elements are identified, which are simultaneously needed altogether.