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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.

Since its first appearance in the 1960s, solid support-free liquid-liquid chromatography has played an ever-growing role in the field of natural products research. The use of the two phases of a liquid biphasic system, the mobile and stationary phases, renders the technique highly versatile and adaptable to a wide spectrum of target molecules, from hydrophobic to highly polar small molecules to proteins. Generally considered a niche technique used only for small-scale preparative separations, liquid-liquid chromatography currently lags far behind conventional liquid-solid chromatography and liquid-liquid extraction in process modeling and industrial acceptance. This review aims to expose a broader audience to this high-potential separation technique by presenting the wide variety of available operating modes and solvent systems as well as structured, model-based design approaches. Topics currently offering opportunities for further investigation are also addressed. Expected final online publication date for the Annual Review of Chemical and Biomolecular Engineering, Volume 12 is June 2021. Please see http://www.annualreviews.org/page/journal/pubdates for revised estimates.

Chromatin adopts different configurations that are regulated by reversible covalent modifications, referred to as epigenetic marks. Epigenetic inhibitors have been approved for clinical use to restore epigenetic aberrations that result in silencing of tumor-suppressor genes, oncogene addictions, and enhancement of immune responses. However, these drugs suffer from major limitations, such as a lack of locus selectivity and potential toxicities. Technological advances have opened a new era of precision molecular medicine to reprogram cellular physiology. The locus-specificity of CRISPR/dCas9/12a to manipulate the epigenome is rapidly becoming a highly promising strategy for personalized medicine. This review focuses on new state-of-the-art epigenome editing approaches to modify the epigenome of neoplasms and other disease models towards a more ‘normal-like state’, having characteristics of normal tissue counterparts. We highlight biomolecular engineering methodologies to assemble, regulate, and deliver multiple epigenetic effectors that maximize the longevity of the therapeutic effect, and we discuss limitations of the platforms such as targeting efficiency and intracellular delivery for future clinical applications.

© 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.

Aromatic compounds have broad applications and have been the target of biosynthetic processes for several decades. New biomolecular engineering strategies have been applied to improve production of aromatic compounds in recent years, some of which are expected to set the stage for the next wave of innovations. Here, we will briefly complement existing reviews on microbial production of aromatic compounds by focusing on a few recent trends where considerable work has been performed in the last 5 years. The trends we highlight are pathway modularization and compartmentalization, microbial co-culturing, non-traditional host engineering, aromatic polymer feedstock utilization, engineered ring cleavage, aldehyde stabilization, and biosynthesis of non-standard amino acids. Throughout this review article, we will also touch on unmet opportunities that future research could address.

Diseases are caused by mutations in DNA sequences. DNA tends to form double-helix structures but in more recent times, it has been found that it can also form non-canonical structures in response to changes in the environment around it. Japanese researchers have now unearthed some exciting findings about the chain reaction that results, potentially leading to diseases such as cancer. Associate Professor Hisae Tateishi-Karimata and Professor Naoki Sugimoto are based at the Frontier Institute for Biomolecular Engineering Research (FIBER), Konan University in Japan, and are collaborating on important work to better understand the complex inner workings of cells in disease. An area of focus for this research is the elucidation of mechanisms of diseases caused by non-canonical structures of DNA and regulation for DNA function.

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.

Background:Drug design and development is a vast field that requires huge investment along with a long duration for providing approval to suitable drug candidates. With the advancement in the field of genomics, the information about druggable targets is being updated at a fast rate which is helpful in finding a cure for various diseases.Methods:There are certain biochemicals as well as physiological advantages of using peptide-based therapeutics. Additionally, the limitations of peptide-based drugs can be overcome by modulating the properties of peptide molecules through various biomolecular engineering techniques. Recent advances in computational approaches have been helpful in studying the effect of peptide drugs on the biomolecular targets. Receptor – ligand-based molecular docking studies have made it easy to screen compatible inhibitors against a target.Furthermore, there are simulation tools available to evaluate stability of complexes at the molecular level. Machine learning methods have added a new edge by enabling accurate prediction of therapeutic peptides.Results:Peptide-based drugs are expected to take over many popular drugs in the near future due to their biosafety, lower off-target binding chances and multifunctional properties.Conclusion:This article summarises the latest developments in the field of peptide-based therapeutics related to their usage, tools, and databases.

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.

Regulatory mechanisms in biological systems are analogous to process control exerted in many chemical and biomolecular engineering processes. Ribonucleic acid (RNA) has been identified as a ubiquit...

The halogen bond (or X-bond) is a noncovalent interaction that is increasingly recognized as an important design tool for engineering protein-ligand interactions and controlling the structures of proteins and nucleic acids. In the past decade, there have been significant efforts to characterize the structure-energy relationships of this interaction in macromolecules. Progress in the computational modeling of X-bonds in biological molecules, however, has lagged behind these experimental studies, with most molecular mechanics/dynamics-based simulation methods not properly treating the properties of the X-bond. We had previously derived a force field for biological X-bonds (ffBXB) based on a set of potential energy functions that describe the anisotropic electrostatic and shape properties of halogens participating in X-bonds. Although fairly accurate for reproducing the energies within biomolecular systems, including X-bonds engineered into a DNA junction, the ffBXB with its seven variable parameters was considered to be too unwieldy for general applications. In the current study, we have generalized the ffBXB by reducing the number of variables to just one for each halogen type and show that this remaining electrostatic variable can be estimated for any new halogenated molecule through a standard restricted electrostatic potential calculation of atomic charges. In addition, we have generalized the ffBXB for both nucleic acids and proteins. As a proof of principle, we have parameterized this reduced and more general ffBXB against the AMBER force field. The resulting parameter set was shown to accurately recapitulate the quantum mechanical landscape and experimental interaction energies of X-bonds incorporated into DNA junction and T4 lysozyme model systems. Thus, this reduced and generalized ffBXB is more readily adaptable for incorporation into classical molecular mechanics/dynamics algorithms, including those commonly used to design inhibitors against therapeutic targets in medicinal chemistry and materials in biomolecular engineering.

Classical hydrogen bonds have, for many decades, been the dominant non-covalent interaction in the toolbox that chemists and chemical engineers have used to design and control the structures of compounds and molecular assemblies as novel materials. Recently, a set of non-classical non-covalent (NC-NC) interactions have emerged that exploit the properties of the Group IV, V, VI, and VII elements of the periodic table (the tetrel, pnictogen, chalcogen, and halogen bonds, respectively). Our research group has been characterizing the prevalence, geometric constraints, and structure-function relationship specifically of the halogen bond in biological systems. We have been particularly interested in exploiting the biological halogen bonds (or BXBs) to control the structures, stabilities, and activities of biomolecules, including the DNA Holliday junction and enzymes. In this review, we first provide a set of criteria for how to determine whether BXBs or any other NC-NC interactions would have biological relevance. We then navigate the trail of studies that had led us from an initial, very biological question to our current point in the journey to establish BXBs as a tool for biomolecular engineering. Finally, we close with a perspective on future directions for this line of research.

Proteins are one of the most fundamental biomolecules present in living organisms. They are indispensable to the sustenance of these organisms owing to the multitude of functions that they perform. They serve as structural constituents of cells and are involved in cell signaling, transport of biological components, cell adhesion, eliciting immune responses, catalyzing chemical reactions, etc. In order to get better insights into these functional aspects, it is quintessential to understand not only the physicochemical features of proteins, but also their structural organization (starting from primary sequences and ending in quaternary structures), and the various interactions that stabilize the higher-order structures. Many biological functions in the cell are mediated when a protein interacts with one or two other proteins in a phenomenon known as protein–protein interaction (PPI). The chapter concludes by providing an overview of PPIs including their functional importance.

Advanced diagnostic techniques, such as PCR and ELISA, are widely used in well-equipped laboratories. However, they are expensive and are not accessible in resource-limited settings. With growing evidence that point-of-care testing (POCT) improves clinical outcomes, there exists a need for simple, fast, low cost, yet sensitive POCT technique. We present a single pot “molecular adaptor”, which we name DNA split proximity circuit (SPC), that can be used as an isothermal, enzyme-free and wash-free diagnostics platform. SPC consists of a pair of “plug-and-play” DNA initiators readily adaptable to various biomarkers ranging from nucleic acids to protein complexes, which then triggers a signal amplification readout based on Hybridization Chain Reaction (HCR). We first improved the kinetics and the sensitivity of HCR by refining HCR hairpin designs. Using biomarkers related to breast cancer as model systems, we applied the optimized HCR to our SPC system and demonstrated its capability to detect microRNA (miRNA) and cell surface receptors. SPC demonstrated good limit of detection in the femtomolar range within 30 min for all miRNA targets tested. It also showed high specificity and an ability to differentiate between different cell surface protein markers. Being isothermal and enzyme-free, our system is less sensitive to temperature and buffer conditions, making it highly robust, thus showing great potential for bedside use. With applications in biomarker analysis, cell visualization and disease diagnostics, herein we improved and established the potential of SPC as a powerful next-generation POCT assay.

Despite the vital role of vaccines in fighting viral pathogens, effective vaccines are still unavailable for many infectious diseases. The importance of vaccines cannot be overstated during the outbreak of a pandemic, such as the coronavirus disease 2019 (COVID-19) pandemic. The understanding of genomics, structural biology, and innate/adaptive immunity have expanded the toolkits available for current vaccine development. However, sudden outbreaks and the requirement of population-level immunization still pose great challenges in today's vaccine designs. Well-established vaccine development protocols from previous experiences are in place to guide the pipelines of vaccine development for emerging viral diseases. Nevertheless, vaccine development may follow different paradigms during a pandemic. For example, multiple vaccine candidates must be pushed into clinical trials simultaneously, and manufacturing capability must be scaled up in early stages. Factors from essential features of safety, efficacy, manufacturing, and distributions to administration approaches are taken into consideration based on advances in materials science and engineering technologies. In this review, we present recent advances in vaccine development by focusing on vaccine discovery, formulation, and delivery devices enabled by alternative administration approaches. We hope to shed light on developing better solutions for faster and better vaccine development strategies through the use of biomaterials, biomolecular engineering, nanotechnology, and microfabrication techniques.

Biosurfactants are surface-active molecules originated from renewable resources, which are produced by microbial fermentation or chemical/enzymatic catalysis. These molecules present important advantages as compared to petrochemical surfactants, given their resistance to extreme conditions, biodegradability, specificity, and environmental compatibility. Besides that, the high production costs hinder its commercialization. In this way, this article aimed to analyze microbial biosurfactants production, focusing on the optimization of metabolic pathways and production processes, to identify key aspects and provide alternatives to allow a cost-effective production at industrial scale. This was achieved by a broad analysis of biosurfactants properties, applications, and biosynthetic pathways (in terms of yield, cofactors, and energy), in addition to an assessment of production-associated costs. As a result of the present extensive data survey and analysis, key production aspects are disclosed. The metabolic pathway yield analysis demonstrated that production of biosurfactants can be significantly improved (highest theoretical yield was 0.47 gbiosurfactant /gsubstrate ) by the use of biomolecular engineering techniques to generate optimized synthetic pathways. With an alternative proposed pathway for surfactin, yield was improved and imbalance in cofactors and ATP was reduced. Analysis of productive costs indicated that to make rhamnolipids commercial production feasible, the main efforts should focus on lowering substrate costs as well as the identification of energy-efficient unit operations to lower electricity cost, since these parameters accounted for 19.36 and 78.22%, respectively, of the production costs. The data generated by this analysis highlight the need for multidisciplinary collaboration to make rhamnolipids economically feasible, including biomolecular engineering and process intensification.

Many cell behaviors are significantly affected by cell culture geometry, though it remains unclear which geometry from two- to three-dimensional (2D to 3D) culture is appropriate for probing a specific cell function and mimicking native microenvironments. Toward addressing this, we established a 2.5D culture geometry, enabling initial cell spreading while reducing polarization to bridge between 2D and 3D geometries, and examined the responses of wound healing cells, human pulmonary fibroblasts, within it. To achieve this, we used engineered biomimetic hydrogels formed by photopolymerization, creating robust layered hydrogels with spread fibroblasts at the interface. We found that fibroblast responses were similar between 2D and 2.5D culture and different from 3D culture, with some underlying differences in mechanotransduction. These studies established the 2.5D cell culture geometry in conjunction with biomimetic synthetic matrices as a useful tool for investigations of fibroblast activation with relevance to the study of other cell functions and types.

The rational design of a multi-responsive protein-based supramolecular system that can predictably respond to more than one stimulus remains an essential but highly challenging goal in biomolecular engineering. Herein, we report a novel chemical method for the construction of multi-responsive supramolecular nanoassemblies using custom-designed facially amphiphilic monodisperse protein-dendron bioconjugates. The macromolecular synthons contain a globular hydrophilic protein domain site-specifically conjugated to photo-responsive hydrophobic benzyl-ether dendrons of different generations through oligo(ethylene glycol) linkers of defined length. The size of the protein nanoassemblies can be systematically tuned by choosing an appropriate dendron or linker of defined length. Exposure of protein nanoassemblies to light results in partial rather than complete disassembly of the complex. The newly formed protein nanoparticle no longer responds to light but could be disassembled into constitutive monomers under acidic conditions or by further treatment with a small molecule. More interestingly, the distribution ratio of the assembled versus disassembled states of protein nanoassemblies after photochemical reaction does not depend on dendron generation, the nature of the linker functionality or the identity of the protein, but is heavily influenced by the linker length. In sum, this work discloses a new chemical method for the rational design of a monodisperse multi-responsive protein-based supramolecular system with exquisite control over the disassembly process.

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.

One of the central themes of biomolecular engineering is the challenge of exploiting the properties of biological materials. Part of this challenge has been uncovering and harnessing properties of biological components that only emerge following their ordered self-assembly. One biomolecular building block that has received significant interest in the past decade is the M13 bacteriophage. There have been a number of recent attempts to trigger the ordered assembly of M13 bacteriophage into multivirion structures, relying on the innate tendency of M13 to form liquid crystals at high concentrations. These, in general, yield planar two-dimensional materials. Presented here is the production of multivirion assemblies of M13 bacteriophage via the chemical modification of its surface by the covalent attachment of the xanthene-based dye tetramethylrhodamine (TMR) isothiocyanate (TRITC). We show that TMR induces the formation of three-dimensional aster-like assemblies of M13 by providing "adhesive" action between bacteriophage particles through the formation of H-aggregates (face-to-face stacking of dye molecules). We also show that the H-aggregation of TMR is greatly enhanced by covalent attachment to M13 and is enhanced further still upon the ordered self-assembly of M13, leading to the suggestion that M13 could be used to promote the self-assembly of dyes that form J-aggregates, a desirable arrangement of fluorescent dye, which has interesting optical properties and potential applications in the fields of medicine and light harvesting technology.

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

Artificial multiprotein complexes are sought after reagents for biomolecular engineering. A current limiting factor is the paucity of molecular scaffolds which allow for site-specific multicomponent assembly. Here, we address this limitation by synthesizing bioorthogonal elastin-like polypeptide (ELP) scaffolds containing periodic noncanonical l-azidohomoalanine amino acids in the guest residue position. The nine azide ELP guest residues served as conjugation sites for site-specific modification with dibenzocyclooctyne (DBCO)-functionalized single-domain antibodies (SdAbs) through strain-promoted alkyne-azide cycloaddition (SPAAC). Sortase A and ybbR tags at the C- and N-termini of the ELP scaffold provided two additional sites for derivatization with small molecules and peptides by Sortase A and 4`-phosphopantetheinyl transferase (Sfp), respectively. These functional groups are chemically bioorthogonal, mutually compatible, and highly efficient, thereby enabling synthesis of multi-antibody ELP complexes in a one-pot reaction. We demonstrate application of this material for enhancing the performance of sandwich immunoassays of the recombinant protein mCherry. In undiluted human plasma, surfaces modified with multi-antibody ELP complexes showed between 2.3- and 14.3-fold improvement in sensitivity and ∼30-40% lower limits of detection as compared with nonspecifically adsorbed antibodies. Dual-labeled multi-antibody ELP complexes were further used for cytometric labeling and analysis of live eukaryotic cells. These results demonstrate how multiple antibodies complexed onto bioorthogonal protein-based polymers can be used to enhance immunospecific binding interactions through multivalency effects.

Nanostructures built from biomolecules such as proteins, DNA and RNA are attracting attention in many areas of biological and materials sciences. Such nanoscale engineering was pioneered with proteins, yet the use of DNA is rapidly gaining traction. What are the advantages of the different biopolymers and which is best suited for a given molecular structure, function or application? In this Review, we evaluate the different structural properties of proteins and nucleic acids, as well as possible designs and synthetic routes for functional nanostructures. By comparing protein engineering and DNA nanotechnology, we highlight molecular architectures that are relevant in biotechnology, biomedicine and synthetic biology research, and identify emerging areas for research such as hybrid materials composed of protein and DNA/RNA. Proteins, DNA and RNA can be used to build functional nanostructures. This Review compares protein and DNA/RNA in terms of biochemical properties and ease of engineering in three major areas of application: biomolecular recognition, biocatalysis and structural support.

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.

Synthetic biologists have applied biomolecular engineering approaches toward the goal of novel biological devices and have shown progress in diverse areas of medicine and biotechnology. Especially promising is the application of synthetic biological devices towards a novel class of molecular diagnostics. As an example, a de-novo-designed riboregulator called toehold switch, with its programmability and compatibility with field-deployable devices showed promising in vitro applications for viral RNA detection such as Zika and Corona viruses. However, the in vivo application of high-performance RNA sensors remains challenging due to the secondary structure of long mRNA species. Here, we introduced ‘Helper RNAs’ that can enhance the functionality of toehold switch sensors by mitigating the effect of secondary structures around a target site. By employing the helper RNAs, previously reported mCherry mRNA sensor showed improved fold-changes in vivo. To further generalize the Helper RNA approaches, we employed automatic design pipeline for toehold sensors that target the essential genes within the pks island, an important target of biomedical research in connection with colorectal cancer. The toehold switch sensors showed fold-changes upon the expression of full-length mRNAs that apparently depended sensitively on the identity of the gene as well as the predicted local structure within the target region of the mRNA. Still, the helper RNAs could improve the performance of toehold switch sensors in many instances, with up to 10-fold improvement over no helper cases. These results suggest that the helper RNA approaches can further assist the design of functional RNA devices in vivo with the aid of the streamlined automatic design software developed here. Further, our solutions for screening and stabilizing single-stranded region of mRNA may find use in other in vivo mRNA-sensing applications such as cas13 crRNA design, transcriptome engineering, and trans-cleaving ribozymes.

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.

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.

Molecular hydrogen is a promising currency in the future energy economy due to the uncertain availability of finite fossil fuel resources and environmental effects from their combustion. It also has important uses in the production of fertilizers and platform chemicals as well as in upgrading conventional fuels. Conventional methods for producing molecular hydrogen from natural gas produce carbon dioxide and use a finite resource as feedstock. However, these issues can be overcome by using light energy from the Sun combined with microorganisms and their molecular machinery capable of photosynthesis. In the presence of light, the proteins involved in photosynthesis coupled with appropriate catalysts in higher plants, algae, and cyanobacteria can produce molecular hydrogen, and optimization via genetic modifications and biomolecular engineering further improves production rates. In this review, we will discuss techniques that have been utilized to improve rates of hydrogen production in biological systems based on the protein machinery of photosynthesis coupled with appropriate catalysts. We will also suggest areas for improvement and future directions for work in the field.

Peptide nucleic acids (PNAs) are polyamide analogues of nucleic acids, very effective in terms of affinity and selectivity in DNA/RNA recognition. As other supramolecular entities, the PNA structure has been an interesting scaffold for the development of new molecules, aimed to DNA and RNA recognition, with improved or completely new properties. This review describes recent work, with the aim of describing how the design of these molecules has evolved in recent years, using increasingly effective tools, from simple crystal structure analysis to molecular dynamics and metadynamics. Modified PNA with additional modules appended, either on the backbone or on the nucleobase, are described. Polyfunctional molecules with both backbone and nucleobase modification are then considered. Finally, recent examples of architectures obtained by conjugation of PNAs to inorganic nanostructures as cargo systems for diagnostics and nano-biotechnology are presented.

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.

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.

Materials with novel and enhanced functionalities can be obtained by modifying cellulose with a range of biomolecules. This functionalization can deliver tailored cellulose-based materials with enhanced physical and chemical properties and control of biological interactions that match specific applications. One of the foundations for the success of such biomaterials is to efficiently control the capacity to combine relevant biomolecules into cellulose materials in such a way that the desired functionality is attained. In this context, our main goal was to develop bi-functional biomolecular constructs for the precise modification of cellulose hydrogels with bioactive molecules of interest. The main idea was to use biomolecular engineering techniques to generate and purify different recombinant fusions of carbohydrate binding modules (CBMs) with significant biological entities. Specifically, CBM-based fusions were designed to enable the bridging of proteins or oligonucleotides with cellulose hydrogels. The work focused on constructs that combine a family 3 CBM derived from the cellulosomal-scaffolding protein A from Clostridium thermocellum (CBM3) with the following: (i) an N-terminal green fluorescent protein (GFP) domain (GFP-CBM3); (ii) a double Z domain that recognizes IgG antibodies; and (iii) a C-terminal cysteine (CBM3C). The ability of the CBM fusions to bind and/or anchor their counterparts onto the surface of cellulose hydrogels was evaluated with pull-down assays. Capture of GFP-CBM3 by cellulose was first demonstrated qualitatively by fluorescence microscopy. The binding of the fusion proteins, the capture of antibodies (by ZZ-CBM3), and the grafting of an oligonucleotide (to CBM3C) were successfully demonstrated. The bioactive cellulose platform described here enables the precise anchoring of different biomolecules onto cellulose hydrogels and could contribute significatively to the development of advanced medical diagnostic sensors or specialized biomaterials, among others.

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.

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.

Prelude The early chapters of this book introduce some of the chemicals that are important in human biology. In fact, it is possible to think of the human body as an elaborate bag of chemicals. In the subspecialty called biomolecular engineering (or bio-technology), biomedical engineers examine the changes in chemical components within a biological system and develop methods for modifying these chemicals or their interactions. The concept of introducing chemicals to induce a change in a biological system is familiar; for example, we all have some experience with taking purified chemicals such as acetaminophen or ibuprofen as drugs to relieve pain. But new biological tools now make it possible to consider more complex chemical interventions such as gene therapy (in which a new deoxyribonucleic acid [DNA] sequence is introduced to allow expression of a new genetic activity). The concept of the human body as a bag of chemicals is convenient and comforting. Chemicals can be produced; chemicals can be added or replaced; chemicals are (sometimes) inexpensive. In fact, several decades ago, it was widely reported that the chemicals in the human body were worth about $1. Other investigators estimate the value at closer to $6 million (particularly if the chemicals are purchased from scientific suppliers). Regardless of the dollar value, we often imagine that our bodies can be supplemented, mended, and improved through the addition of chemicals. But the body contains large numbers of many different kinds of chemicals, and as in many complex systems, unexpected patterns can emerge from the sum of the parts. Although the body is composed of chemicals, we don't understand the identity of all of the chemicals in the system, the forces that hold these chemicals together, or the network of interactions that mold the chemicals into an organism. Yet we often attempt to heal with chemicals. This is the challenge of drug delivery (Figure 13.1). Biomolecular engineering includes drug delivery, but also many similar areas of inquiry in which chemical engineering principles are applied to biomedical engineering (BME) problems.

Antibodies are vital for human health because of their ability to function as nature’s drugs by protecting the body from infection. In recent decades, antibodies have been used as pharmaceutics for targeted therapy in patients with cancer, autoimmune diseases, and cardiovascular diseases. Capturing the dynamic structure of antibodies and characterizing antibody fluctuation is critical for gaining a deeper understanding of their structural characteristics and for improving drug development. Current techniques for studying three-dimensional (3D) structural heterogeneity and variability of proteins have limitations in ascertaining the dynamic structural behavior of antibodies and antibody-antigen complexes. Here, we review current techniques used to study antibody structures with a focus on the recently developed individual-particle electron tomography (IPET) technique. IPET, as a particle-by-particle methodology for 3D structural characterization, has shown advantages in studying structural variety and conformational changes of antibodies, providing direct imaging data for biomolecular engineering to improve development and clinical application of synthetic antibodies.

A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes. Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications. Here we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix-loop-helix-loop structural motif. Eighty-three designs with sequences unrelated to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 °C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 Å. Our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering.

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.

Cellular reprogramming using cocktails of transcription factors (TFs) affirms the epigenetic and developmental plasticity of mammalian cells. It demonstrates the ability of TFs to 'read' genetic information and to rewire regulatory networks in different cellular contexts. Silenced chromatin is not an impediment to the genome engagement by ectopically expressed TFs. Reprogramming TFs have been identified in diverse structural families that lack shared domains or sequence motifs. Interestingly, the reprogramming activity of non-redundant paralogous TFs can be switched with a few point mutations. These findings revealed that the sequence-function relationships influencing reprogramming are tied to subtle features directing genome wide binding. Therefore, endogenous reprogramming TFs are amenable to directed biomolecular engineering that opens up new avenues to optimize cell fate conversions.

In nature, assembled protein structures offer the most complex functional structures. The understanding of the mechanisms ruling protein-protein interactions opens the door to manipulate protein assemblies in a rational way. Proteins are versatile scaffolds with great potential as tools in nanotechnology and biomedicine because of their chemical, structural, and functional versatility. Currently, bottom-up self-assembly based on biomolecular interactions of small and well-defined components, is an attractive approach to biomolecular engineering and biomaterial design. Specifically, repeat proteins are simplified systems for this purpose. In this work, we provide an overview of fundamental concepts of the design of new protein interfaces. We describe an experimental approach to form higher order architectures by a bottom-up assembly of repeated building blocks. For this purpose, we use designed consensus tetratricopeptide repeat proteins (CTPRs). CTPR arrays contain multiple identical repeats that interact through a single inter-repeat interface to form elongated superhelices. Introducing a novel interface along the CTPR superhelix allows two CTPR molecules to assemble into protein nanotubes. We apply three approaches to form protein nanotubes: electrostatic interactions, hydrophobic interactions, and π-π interactions. We isolate and characterize the stability and shape of the formed dimers and analyze the nanotube formation considering the energy of the interaction and the structure in the three different models. These studies provide insights into the design of novel protein interfaces for the control of the assembly into more complex structures, which will open the door to the rational design of nanostructures and ordered materials for many potential applications in nanotechnology.

Virus-like particles (VLPs) are repetitive organizations of viral proteins assembled in an appropriate physicochemical environment. VLPs can stimulate both innate and adaptive immune responses, due to their particulate structure enabling uptake by antigen presenting cells. These characteristics have led to successful development of VLP-vaccine products, and will ensure their vast potential in years to come. Future success of VLP therapeutic products will be determined by advances in their bioengineering, and also by the development of tools to design for their stability, function and application. This review focuses on approaches for VLP assembly in controlled chemical environments in vivo and in vitro, and the application of computational tools for improved chemical sequence design, and fundamental understanding of assembly.

This paper outlines the re-design of learning of Water Quality Analysis using problem-based learning (PBL) in a Year 2 module Chemical Engineering Laboratory (CEL) in Chemical and Biomolecular Engineering (CBE) diploma. By constructing an authentic problem in partnership with National Parks Board (NParks), the PBL approach has been shown to be constructive in multiple ways, ranging from creating integrative learning in a real-world context to shaping students to be self-directed and confident problem solvers. This integrative learning design avoids the compartmentalization of teaching of knowledge and concepts, hands-on technical skills and non-engineering “softskills” in separate lecture, practical and general education sessions, respectively. At the same time, it would be critically useful in preparing graduates to handle complex, industry-real issues and situations.