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The functionalization of proteins with DNA through the formation of covalent bonds enables a wide range of biotechnology advancements. For example, single-molecule analytical methods rely on bioconjugated DNA as elastic biolinkers for protein immobilization. Labeling proteins with DNA enables facile protein identification, as well as spatial and temporal organization and control of protein within DNA–protein networks. Bioconjugation reactions can target native, engineered, and non-canonical amino acids (NCAAs) within proteins. In addition, further protein engineering via the incorporation of peptide tags and self-labeling proteins can also be used for conjugation reactions. The selection of techniques will depend on application requirements such as yield, selectivity, conjugation position, potential for steric hindrance, cost, commercial availability, and potential impact on protein function. The formation of covalent bonds between proteins and DNA has become an important step in a number of emerging biotechnological applications. Native amino acids can be targeted for DNA conjugation, however, this approach can suffer from poor selectivity. Protein engineering, ranging from single site-directed amino acid changes, to non-canonical amino acid incorporation, to the creation of fusion proteins, can be used to increase conjugation selectivities. More advanced conjugation strategies have been developed but these can require expensive oligonucleotide modifications and can suffer from lower yields. The optimal conjugation technique for each application may differ based on limiting factors such as yield, selectivity, flexibility in conjugation position, steric hindrance, cost, commercial availability of oligonucleotides, and risk of altering native protein properties. [ABSTRACT FROM AUTHOR]

Synthetic biology's multiscale approaches to biosensing facilitate biological detection across diverse environments, leading to the development of advanced bio/electronic devices that convert biological events into electrical signals. Electrochemical biosensors are particularly promising due to their cost-effectiveness, ease of fabrication, and potential for miniaturization. Cell-free synthetic biology (CFSB) utilizes cell-free biological components to engineer and mimic the behavior, functions, and characteristics of cell systems, providing versatile approaches to bioengineering beyond cellular contexts. Engineering electrode surface with the principles of CFSB systems may represent a cutting-edge research direction for electrochemical biosensing, potentially solving bioanalytical issues of selectivity, sensitivity, and biocompatibility. This review highlights the latest trends in creating biosensing electrodes with CFSB components, as categorized by transcriptional, translational, and novel CFSB types. It provides a comprehensive analysis of the benefits and challenges of engineering CFSB components on electrode surfaces, aiming to inspire the future integration of CFSB technologies into electrochemical biosensing research. • Description of the role of cell-free synthetic biology (CFBS) system for bioelectronic surface engineering. • Categorizing and discussing advantages and challenges the CFSB systems for the development of electrochemical biosensors. • Providing new insights on biomolecular engineering of electrode surface for enhanced bioanalytical performance. [ABSTRACT FROM AUTHOR]

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. Highlights The antibiotic penicillin revolutionized human medicine, and now fungal natural products (NPs) play a prominent role in the pharmaceutical and healthcare industries. Fungal genomes host a wealth of biosynthetic gene clusters (BGCs) that are silent under conventional laboratory culture conditions due to their tight regulation. Widely applicable methods have been developed to activate or modify these BGCs in both native and heterologous hosts and scalable expression platforms have been established. Fungal BGCs can be engineered to reveal biosynthetic intermediates or increase titers of NPs or combined with biosynthetic pathways from other species and/or other organisms to generate 'unnatural' NPs. Investigation of fungal BGCs often reveals unique enzymes with novel activities that can be applied to a plethora of unnatural substrates and developed as biocatalysts. [ABSTRACT FROM AUTHOR]

Abstract: The engineering of biological systems requires a set of molecular tools that can be predictably applied to the design of sophisticated genetic systems. Recent advances in the field of RNA synthetic biology, particularly in the design of synthetic regulatory RNAs for the static and dynamic control of gene expression, have increased our ability to efficiently build various biological systems capable of performing programmed cellular behaviors. Furthermore, implementing these synthetic regulatory RNAs in biological systems highlights the potential for designing synthetic cell systems for chemical synthesis, environmental, agricultural, and medical applications. In this paper, we review current developments in synthetic regulatory RNAs for the static and dynamic control of gene expression and the potential applications of these tools. [Copyright &y& Elsevier]

Highlights: [•] Helix 190 (H190) from influenza virus modularized onto unrelated polyomavirus VLP. [•] Antigen module design affected response quality (level of binding to HA1). [•] Flanking H190 element with GCN4 gave a low-quality response (low HA1 binding). [•] Modularizing multiple tandem repeats of H190 gave a high-quality antibody response. [•] Biomolecular engineering offers a future alternative to whole pathogen vaccines. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]