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Summary The generation of a library of variant genes is a prerequisite of directed evolution, a powerful tool for biomolecular engineering. As the number of all possible sequences often far exceeds the diversity of a practical library, methods that allow efficient library diversification in living cells are essential for in vivo directed evolution technologies to effectively sample the sequence space and allow hits to emerge. While traditional whole-genome mutagenesis often results in toxicity and the emergence of “cheater” mutations, recent developments that exploit the targeting and editing abilities of genome editors to facilitate in vivo library diversification have allowed for precise mutagenesis focused on specific genes of interest, higher mutational density, and reduced the occurrence of cheater mutations. This minireview summarizes recent advances in genome editor-directed in vivo library diversification and provides an outlook on their future applications in chemical biology.

Protein interactions communicate critical information from the environment into cells to orchestrate functional responses relevant to health and disease. Whereas the natural repertoire of protein interfaces is finite, biomolecular engineering tools provide access to an unlimited scope of potential interactions that can be custom-designed for affinity, specificity, mechanism, or other properties of interest. This review highlights recent developments in protein interface engineering that offer insight into human physiology to inform the design of new pharmaceuticals, with a particular focus on immunotherapeutics. We cover three innovative and translationally promising approaches: (1) reprogramming receptor oligomerization to manipulate signaling pathways; (2) computational protein interface design strategies; and (3) engineering bioorthogonal protein interaction networks.

Joonbaek Jang received his B.S. from Korea University in 2016. He then moved to the University of Pennsylvania where he obtained his M.S. working with Prof. Raymond J. Gorte. During his master’s degree, Joonbaek used atomic layer deposition to modify metal oxide catalysts for the improvement of industrial catalytic processes. In 2018, he joined the Morales-Guio Lab in the Department of Chemical and Biomolecular Engineering at UCLA. Joonbaek is currently working toward the understanding of electrode/electrolyte interfaces and mass transport effects in electrocatalytic processes for sustainable energy applications. Kangze Shen received his B.S. in Materials Chemistry from University of Science and Technology of China in 2018. His undergraduate research with Prof. Yitai Qian focused on the development of high-performance electrode materials for rechargeable Li-ion and Na-ion batteries. He then joined the Morales-Guio Lab in the Department of Chemical and Biomolecular Engineering at UCLA where he is working toward the understanding of electrocatalytic processes and electrochemical systems engineering in sustainable energy applications. Carlos G. Morales-Guio is an Assistant Professor of Chemical and Biomolecular Engineering at UCLA. The Morales-Guio Lab is interested in electrochemical catalysis, particularly with respect to energy and chemical transformations for sustainable energy applications. The lab is currently focused on understanding fundamentals of mass and charge transport coupled to electrochemical transformations that will guide the scale up of electrocatalytic reactors. Carlos received his B. Eng. degree from Osaka University in 2011 and his M.S. and Ph.D. in Chemistry and Chemical Engineering from EPFL in 2013 and 2016, respectively. Most recently, he was a postdoctoral research fellow at Stanford University before joining UCLA in 2018.

Fundamental advances in the field of biomolecular engineering present researchers with the unprecedented opportunity to apply molecular design strategies to modify naturally occurring proteins and generate new molecules for targeted disease therapy. In particular, the development of immunotherapeutics such as cytokine-based and antibody-based drugs has been empowered by evolving technologies and insights from protein engineering. In this review, we highlight specific examples that demonstrate the growing influence of biomolecular engineering on the discovery and design of immunotherapeutics, with particular emphasis on three strategies in biologics development: (1) Cytokine engineering, (2) Cytokine-targeted antibody design, and (3) Fine-tuning the disease-targeting mechanisms of antibody drugs. These strategies have already yielded significant success in the clinic and promise to continue informing and improving the drug development landscape.

International audience; ERK1/2 involvement in cell death remains unclear, although many studies have demonstrated the importance of ERK1/2 dynamics in determining cellular re- sponses. To untangle how ERK1/2 contributes to two cell death programs, we investigated ERK1/2 signaling dynamics during hFasL-induced apoptosis and TNF-induced necroptosis in L929 cells. We observed that ERK1/2 inhibition sensi- tizes cells to apoptosis while delaying necroptosis. By monitoring ERK1/2 activity by live-cell imaging using an improved ERK1/2 biosensor (EKAR4.0), we reported differential ERK1/2 signaling dynamics between cell survival, apoptosis, and nec- roptosis. We also decrypted a temporally shifted amplitude- and frequency-modu- lated (AM/FM) ERK1/2 activity profile in necroptosis versus apoptosis. ERK1/2 inhibition, which disrupted ERK1/2 signaling dynamics, prevented TNF and IL-6 gene expression increase during TNF-induced necroptosis. Using an inducible cell line for activated MLKL, the final executioner of necroptosis, we showed ERK1/2 and its distinctive necroptotic ERK1/2 activity dynamics to be positioned downstream of MLKL.

Immobilized antibody systems are the key to develop efficient diagnostics and separations tools. In the last decade, developments in the field of biomolecular engineering and crosslinker chemistry have greatly influenced the development of this field. With all these new approaches at our disposal, several new immobilization methods have been created to address the main challenges associated with immobilized antibodies. Few of these challenges that we have discussed in this review are mainly associated to the site-specific immobilization, appropriate orientation, and activity retention. We have discussed the effect of antibody immobilization approaches on the parameters on the performance of an immunoassay.

Halogenation is widespread in Nature, as witnessed by the several thousands of naturally occurring halogenated compounds. This review seeks to provide an overview on the pathways and multiple roles of halogenation in Nature, as a source of inspiration to chemists. Remarkably, several halogenation pathways were recently found in humans, sparking an increasing interest into halogens as biomolecular recognition sites. A description of the molecular mechanisms underlining the role of halogenation in the biomolecular context is provided, highlighting how the strategic use of halogen-driven interactions can be exploited to tune the properties of both small compounds and macromolecules. The complexity, versatility and bio-orthogonality of halogen-driven interactions are indeed key figures of merit in promoting novel opportunities for halogenation in a range of applications, spanning from drug design to self-assembly and biomolecular engineering. Specifically, the most relevant and impactful recent contributions in the field of peptide and protein science are presented and critically discussed, with a particular emphasis on the existing opportunities to control their conformational and self-assembly properties towards novel therapeutics and functional materials.

Fungi are a prolific source of bioactive compounds, some of which have been developed as essential medicines and life-enhancing drugs. Genome sequencing has revealed that fungi have the potential to produce considerably more natural products (NPs) than are typically observed in the laboratory. Recently, there have been significant advances in the identification, understanding, and engineering of fungal biosynthetic gene clusters (BGCs). This review briefly describes examples of the engineering of fungal NP biosynthesis at the global, pathway, and enzyme level using in vivo and in vitro approaches and refers to the range and scale of heterologous expression systems available, developments in combinatorial biosynthesis, progress in understanding how fungal BGCs are regulated, and the applications of these novel biosynthetic enzymes as biocatalysts.

Biomolecular engineering enables synthesis of improved proteins through synergistic fusion of modules from unrelated biomolecules. Modularization of peptide antigen from an unrelated pathogen for presentation on a modular virus-like particle (VLP) represents a new and promising approach to synthesize safe and efficacious vaccines. Addressing a key knowledge gap in modular VLP engineering, this study investigates the underlying fundamentals affecting the ability of induced antibodies to recognize the native pathogen. Specifically, this quality of immune response is correlated to the peptide antigen module structure. We modularized a helical peptide antigen element, helix 190 (H190) from the influenza hemagglutinin (HA) receptor binding region, for presentation on murine polyomavirus VLP, using two strategies aimed to promote H190 helicity on the VLP. In the first strategy, H190 was flanked by GCN4 structure-promoting elements within the antigen module; in the second, dual H190 copies were arrayed as tandem repeats in the module. Molecular dynamics simulation predicted that tandem repeat arraying would minimize secondary structural deviation of modularized H190 from its native conformation. In vivo testing supported this finding, showing that although both modularization strategies conferred high H190-specific immunogenicity, tandem repeat arraying of H190 led to a strikingly higher immune response quality, as measured by ability to generate antibodies recognizing a recombinant HA domain and split influenza virion. These findings provide new insights into the rational engineering of VLP vaccines, and could ultimately enable safe and efficacious vaccine design as an alternative to conventional approaches necessitating pathogen cultivation.

Synthetic Biology offers qualitatively new perspectives on the benefits of industrially harnessed biological processes. The ability to modify and reprogramme natural biology increases the scope of tailored bioprocesses and yields attractive prospects beyond conventional Biotechnology. The present review summarises the major achievements and categorises them according to a hierarchy of system levels. Similar structures are known in the engineering sciences and might prove useful for the future development of Synthetic Biology. The hierarchy encompasses several levels of detail. Biological (macro-)molecules present the most detailed level (parts), followed by compartmentalised or non-compartmentalised modules (devices). In the next level, parts and devices are combined into functional cells and further into cellular communities. The manifold interactions between biological entities of the same hierarchical level or between different levels are accounted for by networks, primarily metabolic pathways and regulatory circuits. Networks of different types are represented as a superordinate hierarchical level that achieves full system integration. On all these levels, extensive and sound scientific foundations exist regarding experimental but also theoretical methods. These have led to diverse manifestations of Synthetic Biology on the parts and devices levels. Investigations involving synthetic components on the systems scale represent the most difficult and remain limited in number. A main challenge lies with the quantitative prediction of interactions between different entities across different scales. Systems-theoretical approaches provide important tools to analyse complex biological behaviour and can support the design of artificial biological systems. A promising strategy is seen in an efficient modularisation that reduces biological systems to a limited set of functional modules with well-characterised interfaces. For the design of synthetic biological systems the interactions across these interfaces should be standardised to reduce complexity. Yet, the identification of modules and standardised interaction routes remains a non-trivial problem. Furthermore, an appropriate platform that efficiently describes replication and evolutionary processes has to be developed in order to extend the achievements of Synthetic Biology into designed biological processes.

Erratum Erratum to ‘‘Pharmacophore modeling strategies for the development of novel nonsteroidal inhibitors of human aromatase (CYP19)” [Bioorg. Med. Chem. Lett. 20 (2010) 3050] Yagmur Muftuoglu , Gabriela Mustata b,* Departments of Biophysics and Chemical Biomolecular Engineering, Johns Hopkins University, Baltmore, MD 21218, United States Department of Computational Biology, School of Medicine, University of Pittsburgh, Pittsburgh, PA 15213, United States

A review of current processing practices in preparation of recombinant single chain antibody fragments is presented. Single chain antibody fragments which are superior to their Fab and IgG counterparts due to their higher affinity for target antigens while imposing minimal antigenicity in recipient hosts, have sparked breakthroughs in immunology and the medical field at large. The rapidly increasing market demand for pure single chain antibodies for research and therapeutic applications, necessitates viable manufacture routes that can produce large amounts of these antibodies efficiently and as cheaply as possible. Medium- to high-producing expression systems reported for recombinant single chain antibody production are reviewed, and their reported or potential success for efficient commercial-viable preparation of pure antibodies discussed. The effects of expression host system choice on product molecular constraints, ease of processing, and flowsheet design and scale-up are compared. It is concluded that there is no unique host system that can consistently yield high expression levels for a wide range of single chain antibodies; instead, product sequence and end application often dictate the optimum choice of expression host. Irrespective of host systems, adequate a priori design and engineering of the molecular construct supported by good biophysical understanding of the single chain fragment molecules, is a crucial pre-requisite for improved product stability and downstream recovery, which will favourably impact final product yield and functionality.

A broad review of technologically focused work concerning biomolecules at interfaces is presented. The emphasis is on developments in interfacial biomolecular engineering that may have a practical impact in bioanalysis, tissue engineering, emulsion processing or bioseparations. We also review methods for fabrication in an attempt to draw out those approaches that may be useful for product manufacture, and briefly review methods for analysing the resulting interfacial nanostructures. From this review we conclude that the generation of knowledge and-innovation at the nanoscale far exceeds our ability to translate this innovation into practical outcomes addressing a market need, and that significant technological challenges exist. A particular challenge in this translation is to understand how the structural properties of biomolecules control the assembled architecture, which in turn defines product performance, and how this relationship is affected by the chosen manufacturing route. This structure-architecture-process-performance (SAPP) interaction problem is the familiar laboratory scale-up challenge in disguise. A further challenge will be to interpret biomolecular self- and directed-assembly reactions using tools of chemical reaction engineering, enabling rigorous manufacturing optimization of self-assembly laboratory techniques. We conclude that many of the technological problems facing this field are addressable using tools of modem chemical and biomolecular engineering, in conjunction with knowledge and skills from the underpinning sciences. (c) 2005 Elsevier Ltd. All rights reserved.

With recent advances in biomolecular engineering, the bioremediation of persistent organic pollutants (POPs) using genetically modified microorganisms has become a rapidly growing area of research for environmental protection. Two main biomolecular approaches, rational design and directed evolution, have been developed to engineer enhanced microorganisms and enzymes for the biodegradation of POPs. This review describes the most recent developments and applications of these biomolecular tools for enhancing the capability of microorganisms to bioremediate three major classes of POPs – polycyclic aromatic hydrocabons (PAHs), polychlorinated biphenyls (PCBs) and pesticides. Most of the examples focused on the redesign of various features of the enzymes involved in the bioremediation of POPs, including the enzyme expression level, enzymatic activity and substrate specificity. Overall, the rapidly expanding potential of biomolecular engineering techniques has created the exciting potential of remediating some of the most recalcitrant and hazardous compounds in the environment.

The advances in high throughput screening technology for discovery of target molecules and the accumulation of functional genomics and proteomics data at an ever-accelerating rate 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. The biomolecular engineering will no doubt become one of the most important scientific disciplines in that it will enable us to comprehensively analyze gene expression patterns in both normal and diseased cells and to discover many new biologically active molecules rationally and systematically. 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 more novel proteins, due to the relative ease of screening and identifying desirable proteins. Its application will be extended further into the science of protein–receptor or protein–ligand interactions. The bioinformatics including EST-based or SAGE-tag-based functional genomics and proteomics will continue to advance rapidly. Its biological knowledge base will expand the scope of biomolecular engineering, and the impact of well-coordinated biomolecular engineering research will be very significant on our understanding of gene expression, upregulation and downregulation, and posttranslational protein processing in healthy and diseased cells. The bioinformatics for genome and proteome analysis will contribute substantially toward ever more accelerated advances in pharmaceutical industry. When the functional genomics database, EST and SAGE techniques, microarray technique, and proteome analysis by 2-dimensional gel electrophoresis or capillary electrophoresis are all put to good use, the biomolecular engineering research will yield new drug discoveries, improved therapies, and new or significantly improved bioprocesses. 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 be accelerated. The targets for rational design of biomolecules will be very broad, diverse, and complex, but many application goals can be achieved through the expansion of knowledge base on biomolecules of interest and their roles and functions in cells and tissues. In the near future, more therapeutic drugs and high-value biomolecules will be designed and produced for the treatment or prevention of not-so-easily-cured diseases such as cancers, genetic diseases, age-related diseases, and other metabolic diseases. Also anticipated are many more industrial enzymes that will be engineered to confer desirable properties for the process improvement and manufacturing of many high-value biomolecular products. Many more new metabolites including novel antibiotics that are active against resistant strains will be also produced by recombinant organisms having de novo engineered biosynthetic pathway enzyme systems. The biomolecular engineering era is here and a great deal of benefits can be derived from this field of scientific research for many years to come if we are willing to put it to good use.

Huimin Zhao is a professor of Chemical and Biomolecular Engineering at the University of Illinois at Urbana-Champaign and the holder of Centennial Chair. His research interests encompass many different aspects of synthetic biology and chemical biology. They include directed evolution and rational design of proteins and pathways, novel approaches for protein, pathway, and genome engineering, the molecular genetics and enzymology of natural product biosynthesis, the fundamental aspects of biocatalysis, and the use of protein engineering and metabolic engineering methods in designing platform organisms including E. coli and S. cerevisiae for the synthesis of important chemicals, antibiotics, and biofuels from renewable feedstocks.

Food materials science is an area of rapid growth in the larger field of food science. It is an applied area overlapping food science and food engineering that is concerned with structure, properties and processing of te materials used in food as well as their production and breakdown. The goals of researchers working in this area include: measuring the size and distribution of elements in a complex mixture, understanding how these elements interact and using this information to develop processes that will maximize the formation of useful structures. Examples are provided of newer methods for generating these structures such as the use of biomolecular engineering to design and produce novel proteins using amino acid sequences not found in nature. The concept of composite materials has been applied to foods and would appear to offer some advantages in dealing with these complex structures. An example is given dealing with the production of a food material composite, mixed and filled dairy gels, in which casein micelles are absorbed at the surface of protein-coated fat globules, leading to the formation of a copolymer network with overall greater strength. Of equal interest to the development of new food materials is how they behave under mechanical stress, and the idea of fracture behaviour, in particular notch sensitivity, is illustrated.

On 9 and 10 October 1984 the European Communities' program in biomolecular engineering of crop plants and associated microorganisms met at the Carlsberg Laboratory in order to assess research progress, discuss future plans and coordinate efforts. Major trends and highlights apparent from the presentations are discussed here 1,2 .