Biocatalysis has undergone revolutionary progress in the past century. Benefited by the integration of multidisciplinary technologies, natural enzymatic reactions are constantly being explored. Protein engineering gives birth to robust biocatalysts that are widely used in industrial production. These research achievements have gradually constructed a network containing natural enzymatic synthesis pathways and artificially designed enzymatic cascades. Nowadays, the development of artificial intelligence, automation, and ultra-high-throughput technology provides infinite possibilities for the discovery of novel enzymes, enzymatic mechanisms and enzymatic cascades, and gradually complements the lack of remaining key steps in the pathway design of enzymatic total synthesis. Therefore, the research of biocatalysis is gradually moving towards the era of novel technology integration, intelligent manufacturing and enzymatic total synthesis.

Biocatalysis has found numerous applications in various fields as an alternative to chemical catalysis. The use of enzymes in organic synthesis, especially to make chiral compounds for pharmaceuticals as well for the flavors and fragrance industry, are the most prominent examples. In addition, biocatalysts are used on a large scale to make specialty and even bulk chemicals. This review intends to give illustrative examples in this field with a special focus on scalable chemical production using enzymes. It also discusses the opportunities and limitations of enzymatic syntheses using distinct examples and provides an outlook on emerging enzyme classes.

Self-assembling peptides are attracting wide interest as biodegradable building blocks to achieve functional nanomaterials that do not persist in the environment. Amongst the many applications, biocatalysis is gaining momentum, although a clear structure-to-activity relationship is still lacking. This work applied emerging design rules to the heterochiral octapeptide sequence His-Leu-DLeu-Ile-His-Leu-DLeu-Ile for self-assembly into nanofibrils that, at higher concentration, give rise to a supramolecular hydrogel for the mimicry of esterase-like activity. The peptide was synthesized by solid-phase and purified by HPLC, while its identity was confirmed by 1H-NMR and electrospray ionization (ESI)-MS. The hydrogel formed by this peptide was studied with oscillatory rheometry, and the supramolecular behavior of the peptide was investigated with transmission electron microscopy (TEM) analysis, circular dichroism (CD) spectroscopy, thioflavin T amyloid fluorescence assay, and attenuated total reflectance (ATR) Fourier-transform infrared (FT-IR) spectroscopy. The biocatalytic activity was studied by monitoring the hydrolysis of p-nitrophenyl acetate (pNPA) at neutral pH, and the reaction kinetics followed an apparent Michaelis-Menten model, for which a Lineweaver-Burk plot was produced to determine its enzymatic parameters for a comparison with the literature. Finally, LC-MS analysis was conducted on a series of experiments to evaluate the extent of, if any, undesired peptide acetylation at the N-terminus. In conclusion, we provide new insights that allow gaining a clearer picture of self-assembling peptide design rules for biocatalysis.

Biocatalysis in flow reactor systems is of increasing importance for the transformation of the chemical industry. However, the necessary immobilization of biocatalysts remains a challenge. We here demonstrate that biogenic magnetic nanoparticles, so-called magnetosomes, represent an attractive alternative for the development of nanoscale particle formulations to enable high and stable conversion rates in biocatalytic flow processes. In addition to their intriguing material characteristics, such as high crystallinity, stable magnetic moments, and narrow particle size distribution, magnetosomes offer the unbeatable advantage over chemically synthesized nanoparticles that foreign protein "cargo" can be immobilized on the enveloping membrane via genetic engineering and thus, stably presented on the particle surface. To exploit these advantages, we develop a modular connector system in which abundant magnetosome membrane anchors are genetically fused with SpyCatcher coupling groups, allowing efficient covalent coupling with complementary SpyTag-functionalized proteins. The versatility of this approach is demonstrated by immobilizing a dimeric phenolic acid decarboxylase to SpyCatcher magnetosomes. The functionalized magnetosomes outperform similarly functionalized commercial particles by exhibiting stable substrate conversion during a 60 h period, with an average space-time yield of 49.2 mmol L-1 h-1. Overall, our results demonstrate that SpyCatcher magnetosomes significantly expand the genetic toolbox for particle surface functionalization and increase their application potential as nano-biocatalysts.

Biocatalysis is becoming an increasingly impactful method in contemporary synthetic chemistry for target molecule synthesis. The selectivity imparted by enzymes has been leveraged to complete previously intractable chemical transformations and improve synthetic routes toward complex molecules. However, the implementation of biocatalysis in mainstream organic chemistry has been gradual to this point. This is partly due to a set of historical and technological barriers that have prevented chemists from using biocatalysis as a synthetic tool with utility that parallels alternative modes of catalysis. In this Perspective, we discuss these barriers and how they have hindered the adoption of enzyme catalysts into synthetic strategies. We also summarize tools and resources that already enable organic chemists to use biocatalysts. Furthermore, we discuss ways to further lower the barriers for the adoption of biocatalysis by the broader synthetic organic chemistry community through the dissemination of resources, demystifying biocatalytic reactions, and increasing collaboration across the field.

As more and more biological reaction data become available, the full exploration of the enzymatic potential for the synthesis of valuable products opens up exciting new opportunities but is becoming increasingly complex. The manual design of multi-step biosynthesis routes involving enzymes from different organisms is very challenging. To harness the full enzymatic potential, we developed a computational tool for the directed design of biosynthetic production pathways for multi-step catalysis with in vitro enzyme cascades, cell hydrolysates and permeabilized cells.

Microbial communication can drive coordinated functions through sensing, analyzing and processing signal information, playing critical roles in biomanufacturing and life evolution. However, it is still a great challenge to develop effective methods to construct a microbial communication system with coordinated behaviors. Here, we report an electron transfer triggered redox communication network consisting of three building blocks including signal router, optical verifier and bio-actuator for microbial metabolism regulation and coordination. In the redox communication network, the Fe3+/Fe2+ redox signal can be dynamically and reversibly transduced, channeling electrons directly and specifically into bio-actuator cells through iron oxidation pathway. The redox communication network drives gene expression of electron transfer proteins and simultaneously facilitates the critical reducing power regeneration in the bio-actuator, thus enabling regulation of microbial metabolism. In this way, the redox communication system efficiently promotes the biomanufacturing yield and CO2 fixation rate of bio-actuator. Furthermore, the results demonstrate that this redox communication strategy is applicable both in co-culture and microbial consortia. The proposed electron transfer triggered redox communication strategy in this work could provide an approach for reducing power regeneration and metabolic optimization and could offer insights into improving biomanufacturing efficiency.

Oxygenases are ubiquitous enzymes that catalyze the introduction of one or two oxygen atoms to unreactive chemical compounds. They require reduction equivalents from NADH or NADPH and comprise metal ions, metal ion complexes, or coenzymes in their active site. Thus, for industrial purposes, oxygenases are most commonly employed using whole cell catalysis, to alleviate the need for co-factor regeneration. Biotechnological applications include bioremediation, chiral synthesis, biosensors, fine chemicals, biofuels, pharmaceuticals, food ingredients and polymers. Controlling activity and selectivity of oxygenases is therefore of great importance and of growing interest to the scientific community. This review focuses on protein engineering of non-heme monooxygenases and dioxygenases for generating improved or novel functionalities. Rational mutagenesis based on x-ray structures and sequence alignment, as well as random methods such as directed evolution, have been utilized. It is concluded that knowledge-based protein engineering accompanied with targeted libraries, is most efficient for the design and tuning of biocatalysts towards novel substrates and enhanced catalytic activity while minimizing the screening efforts.

Enzymes are nature's catalyst of choice for the highly selective and efficient coupling of carbohydrates. Enzymatic sugar coupling is a competitive technology for industrial glycosylation reactions, since chemical synthetic routes require extensive use of laborious protection group manipulations and often lack regio- and stereoselectivity. The application of Leloir glycosyltransferases has received considerable attention in recent years and offers excellent control over the reactivity and selectivity of glycosylation reactions with unprotected carbohydrates, paving the way for previously inaccessible synthetic routes. The development of nucleotide recycling cascades has allowed for the efficient production and reuse of nucleotide sugar donors in robust one-pot multi-enzyme glycosylation cascades. In this way, large glycans and glycoconjugates with complex stereochemistry can be constructed. With recent advances, LeLoir glycosyltransferases are close to being applied industrially in multi-enzyme, programmable cascade glycosylations.

Enzymatic reactions in living cells are highly dynamic but simultaneously tightly regulated. Enzyme engineers seek to construct multienzyme complexes to prevent intermediate diffusion, to improve product yield, and to control the flux of metabolites. Here we choose a pair of short peptide tags (RIAD and RIDD) to create scaffold-free enzyme assemblies to achieve these goals. In vitro, assembling enzymes in the menaquinone biosynthetic pathway through RIAD-RIDD interaction yields protein nanoparticles with varying stoichiometries, sizes, geometries, and catalytic efficiency. In Escherichia coli, assembling the last enzyme of the upstream mevalonate pathway with the first enzyme of the downstream carotenoid pathway leads to the formation of a pathway node, which increases carotenoid production by 5.7 folds. The same strategy results in a 58% increase in lycopene production in engineered Saccharomyces cerevisiae. This work presents a simple strategy to impose metabolic control in biosynthetic microbe factories.

The use of microbial biomasses, such as fungal biomass, to catalyze the transesterification of triglycerides (TG) for biodiesel production provides a sustainable, economical alternative while still having the main advantages of expensive immobilized enzymes.

The practice of computational biocatalysis in ionic liquids (ILs) is still in its infancy, and thus best simulation practices are still developing. Herein, we examine the computational and experimental literature to date featuring systems of enzymes in aqueous and neat ILs. The many different approaches taken to parameterize ILs and set up simulations of enzymes in ILs are discussed, and common analysis techniques are reviewed. We also shed light on potential drawbacks and limitations to simulating enzymes in ILs, which include a lack of experimental data with which to validate computational models and inadequate sampling arising from the slow dynamics of many ILs that can lead to inaccurate descriptions of transport and equilibrium thermodynamic properties. A small case study illustrates the effects of scaling IL partial charges, which is a common practice in the field, on the conformational transitions of alanine dipeptide. The degree of charge scaling has a significant effect on the transition times between states of the biomolecule and highlights the importance of carefully setting up systems of enzymes in ILs. Finally, we discuss means to overcome these challenges and briefly consider possible new directions for the field.

Many crucial biochemical reactions in the cell require not only enzymes for catalysis but also organic cofactors or metal ions. Here, we analyse the physicochemical properties, chemical structures and functions of organic cofactors. Based on a thorough analysis of the literature complemented by our quantitative characterisation and classification, we found that most of these molecules are constructed from nucleotide and amino-acid-type building blocks, as well as some recurring cofactor-specific chemical scaffolds. We show that, as expected, organic cofactors are on average significantly more polar and slightly larger than other metabolites in the cell, yet they cover the full spectrum of physicochemical properties found in the metabolome. Furthermore, we have identified intrinsic groupings among the cofactors, based on their molecular properties, structures and functions, that represent a new way of considering cofactors. Although some classes of cofactors, as defined by their physicochemical properties, exhibit clear structural communalities, cofactors with similar structures can have diverse functional and physicochemical profiles. Finally, we show that the molecular functions of the cofactors not only may duplicate reactions performed by inorganic metal cofactors and amino acids, the cell's other catalytic tools, but also provide novel chemistries for catalysis.

As the University of Minnesota Biocatalysis/Biodegradation Database (UM-BBD, http://umbbd.ahc.umn.edu/) starts its second decade, it includes information on over 900 compounds, over 600 enzymes, nearly 1000 reactions and about 350 microorganism entries. Its Biochemical Periodic Tables have grown to include biological information for almost all stable, non-noble-gas elements (http://umbbd.ahc.umn.edu/periodic/). Its Pathway Prediction System (PPS) (http://umbbd.ahc.umn.edu/predict/) is now an internationally recognized, open system for predicting microbial catabolism of organic compounds. Graphical display of PPS rules, a stand-alone version of the PPS and guidance for PPS users are being developed. The next decade should see the PPS, and the UM-BBD on which it is based, find increasing use by national and international government agencies, commercial organizations and educational institutions.

Whole-cell biocatalysis has been exploited to convert a variety of substrates into high-value bulk or chiral fine chemicals. However, the traditional whole-cell biocatalysis typically utilizes the heterotrophic microbes as the biocatalyst, which requires carbohydrates to power the cofactor (ATP, NAD (P)H) regeneration.

Selenium is a trace element essential for human health that has received considerable attention due to its nutritional value. Selenium's bioactivity and toxicity are closely related to its chemical form, and several studies have suggested that the organic form of selenium (i.e., selenomethionine) is more bioavailable and less toxic than its inorganic form (i.e., sodium selenite). Probiotics, especially Bifidobacteriium and Lactobacillus spp., have received increasing attention in recent years, due to their intestinal microbial balancing effects and nutraceutical benefits. Recently, the bioconversion (a.k.a biotransformation) of various bioactive molecules (e.g., minerals, primary and secondary metabolites) using probiotics has been investigated to improve substrate biofunctional properties. However, there have been few reports of inorganic selenium conversion into its organic form using Bifidobacterium and Lactobacillus spp. Here we report that the biosynthesis of organic selenium was accomplished using the whole cell bioconversion of sodium selenite under controlled Bifidobacterium bifidum BGN4 culture conditions. The total amount of organic and inorganic selenium was quantified using an inductively coupled plasma-atomic emission spectrometer (ICP-AES). The selenium species were separated via anion-exchange chromatography and analyzed with inductively coupled plasma-mass spectrometry (ICP-MS). Our findings indicated that the maximum level of organic selenium was 207.5 µg/g in selenium-enriched B. bifidum BGN4. Selenomethionine was the main organic selenium in selenium-enriched B. bifidum BGN4 (169.6 µg/g). Considering that B. bifidum BGN4 is a commercial probiotic strain used in the functional food industry with clinically proven beneficial effects, selenium-enriched B. bifidum BGN4 has the potential to provide dual healthy functions as a daily supplement of selenium and regulator of intestinal bacteria. This is the first report on the production of organic selenium using B. bifidum spp.

A continuous packed bed reactor for NADH-dependent biocatalysis using enzymes co-immobilized on a simple carbon support was optimized to 100% conversion in a residence time of 30 min. Conversion of pyruvate to lactate was achieved by co-immobilized lactate dehydrogenase and formate dehydrogenase, providing in situ cofactor recycling. Other metrics were also considered as optimization targets, such as low E factors between 2.5-11 and space-time yields of up to 22.9 g L-1 h-1. The long-term stability of the biocatalytic reactor was also demonstrated, with full conversion maintained over more than 30 h of continuous operation.

Ionic liquids have unique chemical properties that have fascinated scientists in many fields. The effects of adding ionic liquids to biocatalysts are many and varied. The uses of ionic liquids in biocatalysis include improved separations and phase behaviour, reduction in toxicity, and stabilization of protein structures. As the ionic liquid state of the art has progressed, concepts of what can be achieved in biocatalysis using ionic liquids have evolved and more beneficial effects have been discovered. In this review ionic liquids for whole-cell and isolated enzyme biocatalysis will be discussed with an emphasis on the latest developments, and a look to the future.

The efficient engineering of iminium biocatalysis has drawn considerable attention, with many applications in pharmaceutical synthesis. Here, we report a tailor-made iminium-activated colorimetric "turn-on" probe, specifically designed as a prescreening tool to facilitate engineering of iminium biocatalysis. Upon complexation of the probe with the catalytic Pro-1 residue of the model enzyme 4-oxalocrotonate tautomerase (4-OT), a brightly colored merocyanine-dye-type structure is formed. 4-OT mutants that formed this brightly colored species upon incubation with the probe proved to have a substantial activity for the iminium-based Michael-type addition of nitromethane to cinnamaldehyde, whereas mutants that showed no staining by the probe exhibited no or very low-level "Michaelase" activity. This system was exploited in a solid-phase prescreening assay termed as activated iminium colony staining (AICS) to enrich libraries for active mutants. AICS prescreening reduced the screening effort up to 20-fold. After two rounds of directed evolution, two artificial Michaelases were identified with up to 39-fold improvement in the activity for the addition of nitromethane to cinnamaldehyde, yielding the target γ-nitroaldehyde product with excellent isolated yield (up to 95%) and enantiopurity (up to >99% ee). The colorimetric activation of the turn-on probe could be extended to the class I aldolase 2-deoxy-d-ribose 5-phosphate aldolase, implicating a broader application of AICS in engineering iminium biocatalysis.

Anaerobic digestion (AD) of waste substrates, and renewable biomass and crop residues offers a means to generate energy-rich biogas. However, at present, AD-derived biogas is primarily flared or used for combined heat and power (CHP), in part due to inefficient gas-to-liquid conversion technologies. Methanotrophic bacteria are capable of utilizing methane as a sole carbon and energy source, offering promising potential for biological gas-to-liquid conversion of AD-derived biogas. Here, we report cultivation of three phylogenetically diverse methanotrophic bacteria on biogas streams derived from AD of a series of energy crop residues. Strains maintained comparable central metabolic activity and displayed minimal growth inhibition when cultivated under batch configuration on AD biogas streams relative to pure methane, although metabolite analysis suggested biogas streams increase cellular oxidative stress. In contrast to batch cultivation, growth arrest was observed under continuous cultivation configuration, concurrent with increased biosynthesis and excretion of lactate. We examined the potential for enhanced lactate production via the employ of a pyruvate dehydrogenase mutant strain, ultimately achieving 0.027 g lactate/g DCW/h, the highest reported lactate specific productivity from biogas to date.