Non-natural Functions Research Articles

One-Step Purification and N-Terminal Functionalization of Bioactive Proteins via Atypically Split Inteins.

Site-specific installation of non-natural functionality onto proteins has enabled countless applications in biotechnology, chemical biology, and biomaterials science. Though the N-terminus is an attractive derivatization location, prior methodologies targeting this site have suffered from low selectivity, a limited selection of potential chemical modifications, and/or challenges associated with divergent protein purification/modification steps. In this work, we harness the atypically split VidaL intein to simultaneously N-functionalize and purify homogeneous protein populations in a single step. Our method─referred to as VidaL-tagged expression and protein ligation (VEPL)─enables modular and scalable production of N-terminally modified proteins with native bioactivity. Demonstrating its flexibility and ease of use, we employ VEPL to combinatorially install 4 distinct (multi)functional handles (e.g., biotin, alkyne, fluorophores) to the N-terminus of 4 proteins that span three different classes: fluorescent (Enhanced Green Fluorescent Protein, mCherry), enzymatic (β-lactamase), and growth factor (epidermal growth factor). Moving forward, we anticipate that VEPL's ability to rapidly generate and isolate N-modified proteins will prove useful across the growing fields of applied chemical biology.

Synthetic biology: from "build-for-use" to commercialization

The convergence of advances in chemistry, physics, mathematics, computer science, and engineering into life science research gives rise to synthetic biology. Synthetic biology adopts the concept and strategy of engineering science research, aiming at redesigning and reprogramming the existing biological systems, designing and constructing new bio-bricks such as enzymatic parts, genetic circuits, and chassis cells, or even creating non-natural functions of "artificial life". Synthetic biology promotes the leap from understanding of life to design of life, and is revolutionizing biotechnology and sustainable development of bioeconomy. Via this retrospective review of Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, the most representative research entity focusing on "build-for-use" of synthetic biology in China, this article summarizes the important scientific and technological breakthroughs and industry impacts in the past decade, and prospects future development of synthetic biology in China.

Controlling the Orientation and Viscoelasticity of Materials-Binding Peptides on Hexagonal Boron Nitride Using Fatty Acids.

The adsorption of materials-binding peptides to technologically relevant 2D nanosheets of h-BN could be transformative for both property modulation and materials applications. To enhance binding, integration of non-natural functionalities into the biomolecule could prove to be important. However, very little is understood regarding the impact of these biomolecular structural alterations on the binding, which could influence the affinity and surface-adsorbed structures. Here, the effect of fatty acid incorporation site and carbon chain length is investigated using the BP7 peptide, previously identified with affinity for h-BN. The peptide was modified at either the N- or C-terminus with a fatty acid chain length of 6-12 carbons long. The binding affinity and bio-overlayer viscoelasticity are quantified using quartz crystal microbalance analysis. While fatty acid conjugation did not substantially affect the affinity of the resultant biomolecules, it did alter the viscoelasticity of the biomolecular overlayer on the h-BN surface based upon the carbon chain length and incorporation site. Molecular dynamics simulations demonstrate interplay between enthalpic and entropic effects in modifying the overlayer viscoelasticity. The simulations predict that C-terminal conjugation promotes the enhancement of upright adsorbed states, compared with the N-terminal case, with this effect most pronounced for the 10-carbon chain.

Affinity-Based Methods for Site-Specific Conjugation of Antibodies.

Conjugation of various reagents to antibodies has long been an elegant way to combine the superior binding features of the antibody with other desired but non-natural functions. Applications range from labels for detection in different analytical assays to the creation of new drugs by conjugation to molecules which improves the pharmaceutical effect. In many of these applications, it has been proven advantageous to control both the site and the stoichiometry of the conjugation to achieve a homogeneous product with predictable, and often also improved, characteristics. For this purpose, many research groups have, during the latest decade, reported novel methods and techniques, based on small molecules, peptides, and proteins with inherent affinity for the antibody, for site-specific conjugation of antibodies. This review provides a comprehensive overview of these methods and their applications and also describes a historical perspective of the field.

Abiological catalysis by myoglobin mutant with a genetically incorporated unnatural amino acid.

To inculcate biocatalytic activity in the oxygen-storage protein myoglobin (Mb), a genetically engineered myoglobin mutant H64DOPA (DOPA = L-3,4-dihydroxyphenylalanine) has been created. Incorporation of unnatural amino acids has already demonstrated their ability to accomplish many non-natural functions in proteins efficiently. Herein, the presence of redox-active DOPA residue in the active site of mutant Mb presumably stabilizes the compound I in the catalytic oxidation process by participating in an additional hydrogen bonding (H-bonding) as compared to the WT Mb. Specifically, a general acid-base catalytic pathway was achieved due to the availability of the hydroxyl moieties of DOPA. The reduction potential values of WT (E° = -260 mV) and mutant Mb (E° = -300 mV), w.r.t. Ag/AgCl reference electrode, in the presence of hydrogen peroxide, indicated an additional H-bonding in the mutant protein, which is responsible for the peroxidase activity of the mutant Mb. We observed that in the presence of 5 mM H2O2, H64DOPA Mb oxidizes thioanisole and benzaldehyde with a 10 and 54 folds higher rate, respectively, as opposed to WT Mb. Based on spectroscopic, kinetic, and electrochemical studies, we deduce that DOPA residue, when present within the distal pocket of mutant Mb, alone serves the role of His/Arg-pair of peroxidases.

Synthetic Biology Toolkits for Metabolic Engineering of Cyanobacteria.

Cyanobacteria are of great importance to Earth's ecology. Due to their capability in photosynthesis and C1 metabolism, they are ideal microbial chassis that can be engineered for direct conversion of carbon dioxide and solar energy into biofuels and biochemicals. Facilitated by the elucidation of the basic biology of the photoautotrophic microbes and rapid advances in synthetic biology, genetic toolkits have been developed to enable implementation of nonnatural functionalities in engineered cyanobacteria. Hence, cyanobacteria are fast becoming an emerging platform in synthetic biology and metabolic engineering. Herein, the progress made in the synthetic biology toolkits for cyanobacteria and their utilization for transforming cyanobacteria into microbial cell factories for sustainable production of biofuels and biochemicals is outlined. Current techniques in heterologous gene expression, strategies in genome editing, and development of programmable regulatory parts and modules for engineering cyanobacteria towards biochemical production are discussed and prospected. As cyanobacteria synthetic biology is still in its infancy, apart from the achievements made, the difficulties and challenges in applying and developing genetic toolkits in cyanobacteria for biochemical production are also evaluated.

Improved rate of substrate oxidation catalyzed by genetically-engineered myoglobin

This study showcases the potential of unnatural amino acids to enable non-natural functions when incorporated in the protein scaffold of heme metalloproteins. For this purpose, a genetically-engineered myoglobin (Mb) mutant was created by incorporating redox-active 3-amino-l-tyrosine (NH2Tyr) into its active site, replacing the distal histidine (H64) with NH2Tyr. In peroxide-shunt assays, this variant exhibits an increased rate of turnover for thioanisole and benzaldehyde oxidation as compared to the wild-type (WT) Mb. Indeed, in the presence of excess hydrogen peroxide (H2O2), a 9-fold and 81-fold increase in activity was observed over multiple turnovers for thioanisole sulfoxidation and benzoic acid formation, respectively. The increased oxidation activity in the H64NH2Tyr Mb mutant underlined the role of NH2Tyr in the distal active-site scaffold in peroxide activation. Kinetic, electrochemical, and EPR spectroscopic experiments were performed. On the basis of these studies, it is argued that the single NH2Tyr residue within the Mb variant simultaneously serves the role of the conserved His/Arg-pair within the distal pocket of horseradish peroxidase.

Heterogeneous Systems Biocatalysis: The Path to the Fabrication of Self-Sufficient Artificial Metabolic Cells.

Industrial biocatalysis is playing a key role in the development of the global bio-economy that must change our current productive model to pair the socio-economical development with the preservation of our already harmed planet. The exploitation of isolated multi-enzyme systems and the discovery of novel biocatalytic activities are leading us to manufacture chemicals that were inaccessible through biological routes in the early past. These endeavors have been grouped under the concept of systems biocatalysis. However, by using isolated biological machineries, fundamental features underlying the protein confinement found inside the living cells are missed. To re-gain these properties, such concepts can be expanded to a new concept; heterogeneous systems biocatalysis. This new concept is based on the fabrication of heterogeneous biocatalysts inspired by the spatial organization and compartmentalization that orchestrate metabolic pathways within cells. By assembling biological machineries (including enzymes and cofactors) into artificial solid chassis, one can fabricate self-sufficient and robust cell-free systems able to catalyze orchestrated chemical processes. Furthermore, the confinement of enzymes and and "artificial cofactor" inside solid materials has also attracted our attention because these self-sufficient systems exert de novo and non-natural functionalities. Here, we intend to go beyond immobilization of multi-enzyme systems, discussing only those enzymatic systems that have been co-immobilized with their cofactor or exogenous partners to enhance their cooperative action. In this article, we review the latest architectures developed to fabricate self-sufficient heterogeneous biocatalysts with application in chemical manufacturing, biosensing or energy production.

Efficient laboratory evolution of computationally designed enzymes with low starting activities using fluorescence-activated droplet sorting

De novo biocatalysts with non-natural functionality are accessible by computational enzyme design. The catalytic activities obtained for the initial designs are usually low, but can be optimized significantly by directed evolution. Nevertheless, rate accelerations approaching the level of natural enzymes can only be achieved over many rounds of tedious and time-consuming laboratory evolution. In this work, we show that microfluidic-based screening using fluorescence-activated droplet sorting (FADS) is ideally suited for efficient optimization of designed enzymes with low starting activity, essentially straight out of the computer. We chose the designed retro-aldolase RA95.0, which had been previously evolved by conventional microtiter plate screening, as an example and reoptimized it using the microfluidic-based assay. Our results show that FADS is sufficiently sensitive to detect enzyme activities as low as kcat/Km=0.5 M(-1)s(-1) The ultra-high throughput of this system makes screening of large mutant libraries possible in which clusters of up to five residues are randomized simultaneously. Thus, combinations of beneficial mutations can be identified directly, leading to large jumps in catalytic activity of up to 80-fold within a single round of evolution. By exploring several evolutionary trajectories in parallel, we identify alternative active site arrangements that exhibit comparably enhanced efficiency but opposite enantioselectivity.

ChemInform Abstract: A General Scheme for Incorporating Nonnatural Functionality into Peptides.

Abstract We describe a convergent synthesis of ΔGCN4 229–248 terpyridyl 1 , which contains a terpyridyl ligand attached covalently through a disulfide bond to the proposed DNA binding domain of the yeast transcriptional activator protein GCN4.

Mechanism of Accumulation and Incorporation of Organometallic Pd Complexes into the Protein Nanocage of apo-Ferritin

Hybridization of metal complexes and protein scaffolds is an important subject in bioinorganic chemistry and materials science. Efforts to provide non-natural functions to proteins will likely lead to advances in development of catalysts, sensors, and so on. Mechanistic investigations of the process of binding of metal complexes within protein scaffolds and characterization of the resulting coordination structures will help us to design and control coordination structures of metal complexes for construction of hybrid proteins containing metal complexes. In this work, the processes of accumulation and incorporation of organometallic palladium complexes within the cage of the iron storage protein apo-ferritin (apo-Fr) are elucidated by analysis of X-ray crystal structures of apo-Fr and selected mutants thereof, in the presence of the metal complexes. The crystal structure of apo-Fr containing Pd(allyl) (allyl = eta(3)-C(3)H(5)) complexes shows that thiolato-bridged dinuclear Pd(allyl) complexes are formed at two binding sites within the cage of apo-Fr. The crystal structures of apo-Fr and its Cys- and His-deletion mutants containing Pd(allyl) complexes indicate that Cys126 accelerates the incorporation of Pd(allyl) complexes into the cage. In addition, Cys48 and Cys126 are essential for accumulation of Pd(allyl) complexes and stabilizing the square planar coordination structure.

One-pot three-enzyme chemoenzymatic approach to the synthesis of sialosides containing natural and non-natural functionalities

Chemoenzymatic synthesis, which combines the flexibility of chemical synthesis and the high selectivity of enzymatic synthesis, is a powerful approach to obtain complex carbohydrates. It is a preferred method for synthesizing sialic acid-containing structures, including those with diverse naturally occurring and non-natural sialic acid forms, different sialyl linkages and different glycans that link to the sialic acid. Starting from N-acetylmannosamine, mannose or their chemically or enzymatically modified derivatives, sialic acid aldolase-catalyzed condensation reaction leads to the formation of sialic acids and their derivatives. These compounds are subsequently activated by a CMP-sialic acid synthetase and transferred to a wide range of suitable acceptors by a suitable sialyltransferase for the formation of sialosides containing natural and non-natural functionalities. The three-enzyme coupled synthesis of sialosides can be carried out in one pot without the isolation of intermediates. The time for synthesis is 4-18 h. Purification and characterization of the product can be completed within 2-3 d.

Directed Evolution: An Approach to Engineer Enzymes

ABSTRACTDirected evolution is being used increasingly in industrial and academic laboratories to modify and improve commercially important enzymes. Laboratory evolution is thought to make its biggest contribution in explorations of non-natural functions, by allowing us to distinguish the properties nurtured by evolution. In this review we report the significant advances achieved with respect to the methods of biocatalyst improvement and some critical properties and applications of the modified enzymes. The application of directed evolution has been elaborately demonstrated for protein solubility, stability and catalytic efficiency. Modification of certain enzymes for their application in enantioselective catalysis has also been elucidated. By providing a simple and reliable route to enzyme improvement, directed evolution has emerged as a key technology for enzyme engineering and biocatalysis.

A general scheme for incorporating nonnatural functionality into peptides

We describe a convergent synthesis of ΔGCN4 229–248 terpyridyl 1, which contains a terpyridyl ligand attached covalently through a disulfide bond to the proposed DNA binding domain of the yeast transcriptional activator protein GCN4.