Selection,Addiction and Catalysis: Emerging Trends for the Incorporation of Noncanonical Amino Acids into Peptides and Proteins in Vivo

Expanding the genetic code of organisms by incorporating noncanonical amino acids (ncAAs) into target proteins through the suppression of stop codons in vivo has profoundly impacted how we perform protein modification or detect proteins and their interaction partners in their native environment. Yet, with genetic code expansion strategies maturing over the past 15 years, new applications that make use—or indeed repurpose—these techniques are beginning to emerge. This Concept article highlights three of these developments: 1) The incorporation of ncAAs for the biosynthesis and selection of bioactive macrocyclic peptides with novel ring architectures, 2) synthetic biocontainment strategies based on the addiction of microorganisms to ncAAs, and 3) enzyme design strategies, in which ncAAs with unique functionalities enable the catalysis of new-to-nature reactions. Key advances in all three areas are presented and potential future applications discussed.     

Stronger Together: Multivalent Phage Capsids Inhibit Virus Entry

Antivirals are now more important than ever. To efficiently inhibit virus replication, antiviral multivalent strategies need sufficient affinity to overcome the excellent matching between the virus and its receptor. This report highlights a phage capsid scaffold strategy that can be used to precisely position sialic acid moieties to inhibit influenza A virus replication.     

Synthetic Biology: A New Tool for the Trade

Protein–protein interactions are fundamental to many biological processes. Yet, the weak and transient noncovalent bonds that characterize most protein–protein interactions found in nature impose limits on many bioengineering experiments. Here, a new class of genetically encodable peptide–protein pairs—isopeptag‐N/pilin‐N, isopeptag/pilin‐C, and SpyTag/SpyCatcher—that interact through autocatalytic intermolecular isopeptide bond formation is described. Reactions between peptide–protein pairs are specific, robust, orthogonal, and able to proceed under most biologically relevant conditions both in vitro and in vivo. As fusion constructs, they provide a handle on molecules of interest, both organic and inorganic, that can be grasped with an iron grip. Such stable interactions provide robust post‐translational control over biological processes and open new opportunities in synthetic biology for engineering programmable and self‐assembling protein nanoarchitectures.     

The Quest for Xenobiotic Enzymes: From New Enzymes for Chemistry to a Novel Chemistry of Life

Enzyme engineering has made impressive progress in the past decades, paving the way for the widespread use of enzymes for various purposes. In contrast to “classical” enzyme engineering, which focuses on optimizing specific properties of natural enzymes, a more recent trend towards the creation of artificial enzymes that catalyze fundamentally distinct, new-to-nature reactions is observable. While approaches for creating such enzymes differ significantly, they share the common goal of enabling biocatalytic novelty to broaden the range of applications for enzymes. Although most artificial enzymes reported to date are only moderately active and barely function in vivo, they have the potential to endow cells with capabilities that were previously out of reach and thus herald a new wave of “functional xenobiology”. Herein, we highlight recent developments in the field of artificial enzymes with a particular focus on challenges and opportunities for their use in xenobiology.     

Xenobiology: A Journey towards Parallel Life Forms

Xenobiology is the science of estranged life forms. More specifically, this is an emergent technoscience that combines advances in genetic engineering with the design of biological systems based on unusual biochemistries delivered by chemical compounds of mostly anthropogenic origin. Xenobiology enables us to create and study strange new life forms, “aliens”, not in the way science fiction books do it, but in terms of enlightened science, design, and engineering.     

Metal‐Assisted Folding of Prolinomycin Allows Facile Design of Functional Peptides

Cyclic peptides have been proposed as privileged scaffolds that might mimic the folding and function of natural proteins. However, simple cyclic peptides typically cannot fold into well‐defined structures. Herein, we describe a foldable cyclic peptide scaffold on which functional side chains can be displayed for targeted recognition of biomolecules. The foldable scaffold is based on prolinomycin, a proline‐rich analogue of valinomycin. We report synthetic mutants of prolinomycin that retain the metal‐assisted folding behavior under physiological conditions. The predictable structure formation of prolinomycin makes it a powerful platform to enable the development of synthetic receptors for biomolecules of interest. We demonstrate the potential of this scaffold by creating prolinomycin mutants that selectively bind anionic vesicles and bacterial cells.     

Amyloid assemblies: protein legos at a crossroads in bottom-up synthetic biology

One of the major objectives that bottom-up synthetic biology shares with chemical biology is to engineer extant biological molecules to implement novel functionalities in living systems. Proteins, due to their astonishing structural and functional versatility and to their central roles in the biology of cells, should be cornerstones of synthetic biology. In particular, protein amyloid cross-β assemblies constitute one of the most stable, conceptually simple and universal macromolecular architectures ever found in Nature and thus have enormous potential to be explored. This article focuses on the concepts behind the use of the amyloid cross-β-structural framework as a synthetic biology part, underlining recent basic findings and ideas. The pros and the cons associated with the polymorphism and the cellular toxicity of protein amyloids are also discussed, keeping in mind the possible suitability of these protein assemblies for scaffolding novel orthogonal macromolecular devices in vivo.     

Anion-Exchange Chromatography at the Service of Gene Therapy: Baseline Separation of Full/Empty Adeno-Associated Virus Capsids by Screening of Conditions and Step Gradient Elution Mode

Gene therapy is opening unprecedented opportunities for novel therapeutic approaches. Based on the concept of rescuing function mutations by co-expressing the correct gene to allow biological functions to be restored, it requires the use of viral vectors to ensure the proper delivery of therapeutic genes. In this context, recombinant adeno-associated viruses (rAAV) are the most widely used vectors. Their biomanufacturing process requires the insertion of the therapeutic gene into the rAAV (full capsids). However, a percentage of rAAV that do not contain the desired gene (empty capsids), as well as partly filled capsids, might also be produced, potentially impacting the efficiency of the therapy. Therefore, the determination of the rAAV capsids’ full/empty ratio needs to be monitored to ensure consistent product quality and efficacy. Anion-exchange chromatography (AEX) can serve this need. In this contribution, thorough AEX method development, including a mobile phase, a stationary phase and gradient conditions, has highlighted its potential in supporting gene therapy. Taking advantage of the fact that viral capsids follow an “on/off” retention behavior, the application of a step gradient approach to the rAAV serotype 8 (rAAV8) allowed the unprecedented separation of rAAV8 full/empty capsids, with a resolution gain of 3.7 as compared to the resolution obtained with a fully optimized linear gradient. Finally, the developed analytical approach allowed a precise and accurate baseline separation and quantification of full and empty rAAV8 capsids, with the potential to be applied as a high-throughput quality control (QC) method.     

Modified Histone Peptides Linked to Magnetic Beads Reduce Binding Specificity

Histone post-translational modifications are small chemical changes to the histone protein structure that have cascading effects on diverse cellular functions. Detecting histone modifications and characterizing their binding partners are critical steps in understanding chromatin biochemistry and have been accessed using common reagents such as antibodies, recombinant assays, and FRET-based systems. High-throughput platforms could accelerate work in this field, and also could be used to engineer de novo histone affinity reagents; yet, published studies on their use with histones have been noticeably sparse. Here, we describe specific experimental conditions that affect binding specificities of post-translationally modified histones in classic protein engineering platforms and likely explain the relative difficulty with histone targets in these platforms. We also show that manipulating avidity of binding interactions may improve specificity of binding.     

Alteration of the Route to Menaquinone towards Isochorismate-Derived Metabolites

Chorismate and isochorismate constitute branch-point intermediates in the biosynthesis of many aromatic metabolites in microorganisms and plants. To obtain unnatural compounds, we modified the route to menaquinone in Escherichia coli. We propose a model for the binding of isochorismate to the active site of MenD ((1R,2S, 5S,6S)-2-succinyl-5-enolpyruvyl-6-hydroxycyclohex-3-ene-1-carboxylate (SEPHCHC) synthase) that explains the outcome of the native reaction with α-ketoglutarate. We have rationally designed variants of MenD for the conversion of several isochorismate analogues. The double-variant Asn117Arg–Leu478Thr preferentially converts (5S,6S)-5,6-dihydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHD), the hydrolysis product of isochorismate, with a >70-fold higher ratio than that for the wild type. The single-variant Arg107Ile uses (5S,6S)-6-amino-5-hydroxycyclohexa-1,3-diene-1-carboxylate (2,3-trans-CHA) as substrate with >6-fold conversion compared to wild-type MenD. The novel compounds have been made accessible in vivo (up to 5.3 g L⁻¹). Unexpectedly, as the identified residues such as Arg107 are highly conserved (>94 %), some of the designed variations can be found in wild-type SEPHCHC synthases from other bacteria (Arg107Lys, 0.3 %). This raises the question for the possible natural occurrence of as yet unexplored branches of the shikimate pathway.     

Optical Control of Transcription: Genetically Encoded Photoswitchable Variants of T7 RNA Polymerase

Light-sensing protein domains that link an exogenous light signal to the activity of an enzyme have attracted much attention for the engineering of new regulatory mechanisms into proteins and for studying the dynamic behavior of intracellular reactions and reaction cascades. Light–oxygen–voltage (LOV) photoreceptors are blue-light-sensing modules that have been intensely characterized for this purpose and linked to several proteins of interest. For the successful application of these tools, it is crucial to identify appropriate fusion strategies for combining sensor and enzyme domains that sustain activity and light-induced responsivity. Terminal fusion of LOV domains is the natural strategy; however, this is not transferrable to T7 RNA polymerase because both of its termini are involved in catalysis. It is shown herein that it is possible to covalently insert LOV domains into the polymerase protein, while preserving its activity and generating new light-responsive allosteric coupling.     

Biological Applications of Expanded Genetic Codes

Substantial efforts in the past decade have resulted in the systematic expansion of genetic codes, allowing for the direct ribosomal incorporation of ～100 unnatural amino acids into bacteria, yeast, mammalian cells, and animals. Here, we illustrate the versatility of expanded genetic codes in biology and bioengineering, focusing on the application of expanded genetic codes to problems in protein, cell, synthetic, and experimental evolutionary biology. As the expanded genetic code field continues to develop, its place as a foundational technology in the whole of biological sciences will solidify.     

Biophysical Characterization of Adeno-Associated Virus Vectors Using Ion-Exchange Chromatography Coupled to Light Scattering Detectors

Ion-exchange chromatography coupled to light scattering detectors represents a fast and simple analytical method for the assessment of multiple critical quality attributes (CQA) in one single measurement. The determination of CQAs play a crucial role in Adeno-Associated Virus (AAV)-based gene therapies and their applications in humans. Today, several different analytical techniques, including size-exclusion chromatography (SEC), analytical ultracentrifugation (AUC), qPCR or ELISA, are commonly used to characterize the gene therapy product regarding capsid titer, packaging efficiency, vector genome integrity, aggregation content and other process-related impurities. However, no universal method for the simultaneous determination of multiple CQAs is currently available. Here, we present a novel robust ion-exchange chromatography method coupled to multi-angle light scattering detectors (IEC-MALS) for the comprehensive characterization of empty and filled AAVs concerning capsid titer, full-to-total ratio, absolute molar mass of the protein and nucleic acid, and the size and polydispersity without baseline-separation of both species prior to data analysis. We demonstrate that the developed IEC-MALS assay is applicable to different serotypes and can be used as an orthogonal method to other established analytical techniques.     

From Bugs to Bioplastics: Total (+)-Dihydrocarvide Biosynthesis by Engineered Escherichia coli

The monoterpenoid lactone derivative (+)-dihydrocarvide ((+)-DHCD) can be polymerised to form shape-memory polymers. Synthetic biology routes from simple, inexpensive carbon sources are an attractive, alternative route over chemical synthesis from (R)-carvone. We have demonstrated a proof-of-principle in vivo approach for the complete biosynthesis of (+)-DHCD from glucose in Escherichia coli (6.6 mg L⁻¹). The pathway is based on the Mentha spicata route to (R)-carvone, with the addition of an ′ene′-reductase and Baeyer–Villiger cyclohexanone monooxygenase. Co-expression with a limonene synthesis pathway enzyme enables complete biocatalytic production within one microbial chassis. (+)-DHCD was successfully produced by screening multiple homologues of the pathway genes, combined with expression optimisation by selective promoter and/or ribosomal binding-site screening. This study demonstrates the potential application of synthetic biology approaches in the development of truly sustainable and renewable bioplastic monomers.     

Post-translational Assembly of Protein Parts into Complex Devices by Using SpyTag/SpyCatcher Protein Ligase

Exploiting the innate modularity of proteins has allowed advances across the fields of synthetic biology and biotechnology. By using standardized protein components as building blocks, complex, multiprotein assemblies with sophisticated functions can be generated; feats previously not possible with strictly genetic-engineering approaches. The development of strategies for protein assembly is accelerating, pushing the boundaries of protein architecture. SpyTag and SpyCatcher protein ligase is a recent advance in this field that allows plug-and-play modularity by harnessing post-translational protein assembly. Herein, we review the latest applications of this powerful tool including novel enzyme assemblies, modularizing protein display, and the generation of antibody and antibody-like “devices” by using SpyTag/SpyCatcher technology.