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© 2020 Wiley Periodicals LLC Today's Biochemical Engineer may contribute to advances in a wide range of technical areas. The recent Biochemical and Molecular Engineering XXI conference focused on “The Next Generation of Biochemical and Molecular Engineering: The role of emerging technologies in tomorrow's products and processes”. On the basis of topical discussions at this conference, this perspective synthesizes one vision on where investment in research areas is needed for biotechnology to continue contributing to some of the world's grand challenges.

The majority of clinically approved therapeutics target membrane proteins (MPs), highlighting the need for tools to study this important category of proteins. To overcome limitations with recombinant MP expression, whole cell screening techniques have been developed that present MPs in their native conformations. Whereas many such platforms utilize adherent cells, here we introduce a novel suspension cell-based platform termed “biofloating” that enables quantitative analysis of interactions between proteins displayed on yeast and MPs expressed on mammalian cells, without need for genetic fusions. We characterize and optimize biofloating and illustrate its sensitivity advantage compared to an adherent cell-based platform (biopanning). We further demonstrate the utility of suspension cell-based approaches by iterating rounds of magnetic-activated cell sorting (MACS) selections against MP-expressing mammalian cells to enrich for a specific binder within a yeast-displayed antibody library. Overall, biofloating represents a promising new technology that can be readily integrated into protein discovery and development workflows.

The combination of cell culture models with microscale technology has fostered emergence of in vitro cell-based microphysiological models, also known as organ-on-a-chip systems. Body-on-a-chip systems, which are multi-organ systems on a chip to mimic physiological relations, enable recapitulation of organ-organ interactions and potentially whole-body response to drugs, as well as serve as models of diseases. Chemical reaction engineering principles can be applied to understanding complex reactions inside the cell or human body, which can be treated as a multi-reactor system. These systems use physiologically-based pharmacokinetic (PBPK) models to guide the development of microscale systems of the body where organs or tissues are represented by living cells or tissues, and integrated into body-on-a-chip systems. Here, we provide a brief overview on the concept of chemical reaction engineering and how its principles can be applied to understanding and predicting the behavior of body-on-a-chip systems.

The alkyne functionality has attracted much interest due to its diverse chemical and biological applications. We recently elucidated an acyl carrier protein (ACP)-dependent alkyne biosynthetic pathway, however, little is known about ACP interactions with the alkyne biosynthetic enzymes, an acyl-ACP ligase (JamA) and a membrane-bound bi-functional desaturase/acetylenase (JamB). Here, we showed that JamB has a more stringent interaction with ACP than JamA. In addition, site directed mutagenesis of a non-cognate ACP significantly improved its compatibility with JamB, suggesting a possible electrostatic interaction at the ACP-JamB interface. Finally, error-prone PCR and screening of a second non-cognate ACP identified hot spots on the ACP that are important for interacting with JamB and yielded mutants which were better recognized by JamB. Our data thus not only provide insights into the ACP interactions in alkyne biosynthesis, but it also potentially aids in future combinatorial biosynthesis of alkyne-tagged metabolites for chemical and biological applications.Topical HeadingBiomolecular Engineering, Bioengineering, Biochemicals, Biofuels, and Food

The increased availability of user-friendly and accessible computational tools for biomolecular modeling would expand the reach and application of biomolecular engineering and design. For protein modeling, one key challenge is to reduce the complexities of 3D protein folds to sets of parametric equations that nonetheless capture the salient features of these structures accurately. At present, this is possible for a subset of proteins, namely, repeat proteins. The α-helical coiled coil provides one such example, which represents ≈ 3-5% of all known protein-encoding regions of DNA. Coiled coils are bundles of α helices that can be described by a small set of structural parameters. Here we describe how this parametric description can be implemented in an easy-to-use web application, called CCBuilder 2.0, for modeling and optimizing both α-helical coiled coils and polyproline-based collagen triple helices. This has many applications from providing models to aid molecular replacement for X-ray crystallography, in silico model building and engineering of natural and designed protein assemblies, and through to the creation of completely de novo "dark matter" protein structures. CCBuilder 2.0 is available as a web-based application, the code for which is open-source and can be downloaded freely. http://coiledcoils.chm.bris.ac.uk/ccbuilder2. Lay summary We have created CCBuilder 2.0, an easy to use web-based application that can model structures for a whole class of proteins, the α-helical coiled coil, which is estimated to account for 3-5% of all proteins in nature. CCBuilder 2.0 will be of use to a large number of protein scientists engaged in fundamental studies, such as protein structure determination, through to more-applied research including designing and engineering novel proteins that have potential applications in biotechnology.

We used the molecular modeling program Rosetta to identify clusters of amino acid substitutions in antibody fragments (scFvs and scAbs) that improve global protein stability and resistance to thermal deactivation. Using this methodology, we increased the melting temperature (T(m)) and resistance to heat treatment of an antibody fragment that binds to the Clostridium botulinum hemagglutinin protein (anti-HA33). Two designed antibody fragment variants with two amino acid replacement clusters, designed to stabilize local regions, were shown to have both higher T(m) compared to the parental scFv and importantly, to retain full antigen binding activity after 2 hours of incubation at 70 °C. The crystal structure of one thermostabilized scFv variants was solved at 1.6 Å and shown to be in close agreement with the RosettaAntibody model prediction.

Engineering materials using supramolecular principles enables generalizable and modular platforms that have tunable chemical, mechanical, and biological properties. Applying this bottom‐up, molecular engineering‐based approach to therapeutic design affords unmatched control of emergent properties and functionalities. In preparing responsive materials for biomedical applications, the dynamic character of typical supramolecular interactions facilitates systems that can more rapidly sense and respond to specific stimuli through a fundamental change in material properties or characteristics, as compared to cases where covalent bonds must be overcome. Several supramolecular motifs have been evaluated toward the preparation of “smart” materials capable of sensing and responding to stimuli. Triggers of interest in designing materials for therapeutic use include applied external fields, environmental changes, biological actuators, applied mechanical loading, and modulation of relative binding affinities. In addition, multistimuli‐responsive routes can be realized that capture combinations of triggers for increased functionality. In sum, supramolecular engineering offers a highly functional strategy to prepare responsive materials. Future development and refinement of these approaches will improve precision in material formation and responsiveness, seek dynamic reciprocity in interactions with living biological systems, and improve spatiotemporal sensing of disease for better therapeutic deployment.

Collagen-based biomedical materials have developed into important, clinically effective materials used in a range of devices that have gained wide acceptance. These devices come with collagen in various formats, including those based on stabilized natural tissues, those that are based on extracted and purified collagens, and designed composite, biosynthetic materials. Further knowledge on the structure and function of collagens has led to on-going developments and improvements. Among these developments has been the production of recombinant collagen materials that are well defined and are disease free. Most recently, a group of bacterial, non-animal collagens has emerged that may provide an excellent, novel source of collagen for use in biomaterials and other applications. These newer collagens are discussed in detail. They can be modified to direct their function, and they can be fabricated into various formats, including films and sponges, while solutions can also be adapted for use in surface coating technologies.

Azobenzene has been widely used as a photoregulator due to its reversible photoisomerization, large structural change between E and Z isomers, high photoisomerization yield, and high chemical stability. On the other hand, some azobenzene derivatives can be used as universal quenchers for many fluorophores. Nucleic acid is a good candidate to be modified because it is not only the template of gene expression but also widely used for building well-organized nanostructures and nanodevices. Because the size and polarity distribution of the azobenzene molecule is similar to a nucleobase pair, the introduction of azobenzene into nucleic acids has been shown to be an ingenious molecular design for constructing light-switching biosystems or light-driven nanomachines. Here we review recent advances in azobenzene-modified nucleic acids and their applications for artificial regulation of gene expression and enzymatic reactions, construction of photoresponsive nanostructures and nanodevices, molecular beacons, as well as obtaining structural information using the introduced azobenzene as an internal probe. In particular, nucleic acids bearing multiple azobenzenes can be used as a novel artificial nanomaterial with merits of high sequence specificity, regular duplex structure, and high photoregulation efficiency. The combination of functional groups with biomolecules may further advance the development of chemical biotechnology and biomolecular engineering.

Understanding the microscopic structure and macroscopic properties of condensed matter from a molecular perspective is important for both traditional and modern chemical engineering. A cornerstone of such understanding is provided by statistical mechanics, which bridges the gap between molecular events and the structural and physiochemical properties of macro- and mesoscopic systems. With ever-increasing computer power, molecular simulations and ab initio quantum mechanics are promising to provide a nearly exact route to accomplishing the full potential of statistical mechanics. However, in light of their versatility for solving problems involving multiple length and timescales that are yet unreachable by direct simulations, phenomenological and semiempirical methods remain relevant for chemical engineering applications in the foreseeable future. Classical density functional theory offers a compromise: on the one hand, it is able to retain the theoretical rigor of statistical mechanics and, on the other hand, similar to a phenomenological method, it demands only modest computational cost for modeling the properties of uniform and inhomogeneous systems. Recent advances are summarized of classical density functional theory with emphasis on applications to quantitative modeling of the phase and interfacial behavior of condensed fluids and soft materials, including colloids, polymer solutions, nanocomposites, liquid crystals, and biological systems. Attention is also given to some potential applications of density functional theory to material fabrications and biomolecular engineering. © 2005 American Institute of Chemical Engineers AIChE J, 2006

Natural selection has created optimal catalysts that exhibit their convincing performance even with a number of sometimes counteracting constraints. Optimal performance of enzyme catalysis does not refer necessarily to maximum reaction rate. Rather, it may involve a compromise between specificity, rate, stability, and other chemical constraints ; in some cases, it may involve aintelligento control of rate and specificity. Because enzymes are capable of catalyzing reactions under mild conditions and with high substrate specificity that often is accompanied by high regioand enantioselectivity, it is not surprising that a continually increasing number of industrial and academic reports concern the use of enzyme catalysts in chemical synthesis as well as in biochemical and biomedical applications. However, the demands of modern synthesis and their commercial application were obviously not targeted during the natural evolution of enzymes. Considering a specific, nonnatural application, any property (or combination of properties) of an enzyme may therefore need to be improved. Of course, scientists desired to mimick nature's powerful concepts for tailoring specific enzymatic properties: Following pioneering experiments for evolving molecules in the test tube, evolutionary engineering of biomolecules was successfully realized with first selections of functional nucleic acids (ribozymes) by using the SELEX (systematic evolution of ligands by exponential enrichment with integrated optimization by non-linear analysis) procedure, 8] and with the development of high-affinity ligands (aptamers) by using similar techniques. Meanwhile, evolutionary engineering, also termed adirected evolutiono, has emerged as a key technology for biomolecular engineering and generated impressive results in the functional adaptation of enzymes to artificial environments. Certainly, evolution in the laboratory does not come to a halt at the optimization of single genes and proteins. Recent results excitingly demonstrate the success of amolecular breedingo of metabolic pathways, and even of complete genomes, and the end is not in sight yet. Directed evolution in the laboratory is highly attractive because its principles are simple and do not require detailed knowledge of structure, function, or mechanism. Essentially like natural evolution, directed evolution comprises the iterative implementation of (1) the generation of a alibraryo of mutated genes, (2) its functional expression, and (3) a sensitive assay to identify individuals showing the desired properties, either by selection or by screening (Figure 1). After each round, the genes of improved variants are deciphered and subsequently serve as parents for another round of optimization. This review covers the most important aspects of directed evolution and summarizes key solutions to problems of optimizing and understanding enzyme function.

During the next decade or so, there will be significant and impressive advances in biomolecular engineering, especially in our understanding of the biological roles of various biomolecules inside the cell. The advances in high throughput screening technology for discovery of target molecules and the accumulation of functional genomics and proteomics data at accelerating rates will enable us to design and discover novel biomolecules and proteins on a rational basis in diverse areas of pharmaceutical, agricultural, industrial, and environmental applications. As an applied molecular evolution technology, DNA shuffling will play a key role in biomolecular engineering. In contrast to the point mutation techniques, DNA shuffling exchanges large functional domains of sequences to search for the best candidate molecule, thus mimicking and accelerating the process of sexual recombination in the evolution of life. The phage-display system of combinatorial peptide libraries will be extensively exploited to design and create many novel proteins, as a result of the relative ease of screening and identifying desirable proteins. Even though this system has so far been employed mainly in screening the combinatorial antibody libraries, its application will be extended further into the science of protein-receptor or protein-ligand interactions. The bioinformatics for genome and proteome analyses will contribute substantially toward ever more accelerated advances in the pharmaceutical industry. Biomolecular engineering will no doubt become one of the most important scientific disciplines, because it will enable systematic and comprehensive analyses of gene expression patterns in both normal and diseased cells, as well as the discovery of many new high-value molecules. When the functional genomics database, EST and SAGE techniques, microarray technique, and proteome analysis by 2-dimensional gel electrophoresis or capillary electrophoresis in combination with mass spectrometer are all put to good use, biomolecular engineering research will yield new drug discoveries, improved therapies, and significantly improved or new bioprocess technology. With the advances in biomolecular engineering, the rate of finding new high-value peptides or proteins, including antibodies, vaccines, enzymes, and therapeutic peptides, will continue to accelerate. The targets for the rational design of biomolecules will be broad, diverse, and complex, but many application goals can be achieved through the expansion of knowledge based on biomolecules and their roles and functions in cells and tissues. Some engineered biomolecules, including humanized Mab's, have already entered the clinical trials for therapeutic uses. Early results of the trials and their efficacy are positive and encouraging. Among them, Herceptin, a humanized Mab for breast cancer treatment, became the first drug designed by a biomolecular engineering approach and was approved by the FDA. Soon, new therapeutic drugs and high-value biomolecules will be designed and produced by biomolecular engineering for the treatment or prevention of not-so-easily cured diseases such as cancers, genetic diseases, age-related diseases, and other metabolic diseases. Many more industrial enzymes, which will be engineered to confer desirable properties for the process improvement and manufacturing of high-value biomolecular products at a lower production cost, are also anticipated. New metabolites, including novel antibiotics that are active against resistant strains, will also be produced soon by recombinant organisms having de novo engineered biosynthetic pathway enzyme systems. The biomolecular engineering era is here, and many of benefits will be derived from this field of scientific research for years to come if we are willing to put it to good use.

Natural selection has created optimal catalysts that exhibit their convincing performance even with a number of sometimes counteracting constraints. Optimal performance of enzyme catalysis does not refer necessarily to maximum reaction rate. Rather, it may involve a compromise between specificity, rate, stability, and other chemical constraints ; in some cases, it may involve aintelligento control of rate and specificity. Because enzymes are capable of catalyzing reactions under mild conditions and with high substrate specificity that often is accompanied by high regioand enantioselectivity, it is not surprising that a continually increasing number of industrial and academic reports concern the use of enzyme catalysts in chemical synthesis as well as in biochemical and biomedical applications. However, the demands of modern synthesis and their commercial application were obviously not targeted during the natural evolution of enzymes. Considering a specific, nonnatural application, any property (or combination of properties) of an enzyme may therefore need to be improved. Of course, scientists desired to mimick nature's powerful concepts for tailoring specific enzymatic properties: Following pioneering experiments for evolving molecules in the test tube, evolutionary engineering of biomolecules was successfully realized with first selections of functional nucleic acids (ribozymes) by using the SELEX (systematic evolution of ligands by exponential enrichment with integrated optimization by non-linear analysis) procedure, 8] and with the development of high-affinity ligands (aptamers) by using similar techniques. Meanwhile, evolutionary engineering, also termed adirected evolutiono, has emerged as a key technology for biomolecular engineering and generated impressive results in the functional adaptation of enzymes to artificial environments. Certainly, evolution in the laboratory does not come to a halt at the optimization of single genes and proteins. Recent results excitingly demonstrate the success of amolecular breedingo of metabolic pathways, and even of complete genomes, and the end is not in sight yet. Directed evolution in the laboratory is highly attractive because its principles are simple and do not require detailed knowledge of structure, function, or mechanism. Essentially like natural evolution, directed evolution comprises the iterative implementation of (1) the generation of a alibraryo of mutated genes, (2) its functional expression, and (3) a sensitive assay to identify individuals showing the desired properties, either by selection or by screening (Figure 1). After each round, the genes of improved variants are deciphered and subsequently serve as parents for another round of optimization. This review covers the most important aspects of directed evolution and summarizes key solutions to problems of optimizing and understanding enzyme function.