Design of Artificial Enzymes: Insights into Protein Scaffolds.

The design of artificial enzymes has emerged as a promising tool for the generation of potent biocatalysts able to promote new-to-nature reactions with improved catalytic performances, providing a powerful platform for wide-ranging applications and a better understanding of protein functions and structures. The selection of an appropriate protein scaffold plays a key role in the design process. This review aims to give a general overview of the most common protein scaffolds that can be exploited for the generation of artificial enzymes. Several examples are discussed and categorized according to the strategy used for the design of the artificial biocatalyst, namely the functionalization of natural enzymes, the creation of a new catalytic site in a protein scaffold bearing a wide hydrophobic pocket and de novo protein design. The review is concluded by a comparison of these different methods and by our perspective on the topic.

Protein Mediated Enzyme Immobilization.

Enzyme immobilization is an essential technology for commercializing biocatalysis. It imparts stability, recoverability, and other valuable features that improve the effectiveness of biocatalysts. While many avenues to join an enzyme to solid phases exist, protein-mediated immobilization is rapidly developing and has many advantages. Protein-mediated immobilization allows for the binding interaction to be genetically coded, can be used to create artificial multienzyme cascades, and enables modular designs that expand the variety of enzymes immobilized. By designing around binding interactions between protein domains, they can be integrated into functional materials for protein immobilization. These materials are framed within the context of biocatalytic performance, immobilization efficiency, and stability of the materials. In this review, supports composed entirely of protein are discussed first, with systems such as cellulosomes and protein cages being discussed alongside newer technologies like spore-based biocatalysts and forizymes. Protein-composite materials such as polymersomes and protein-inorganic supraparticles are then discussed to demonstrate how protein-mediated strategies are applied to many classes of solid materials. Critical analysis and future directions of protein-based immobilization are then discussed, with a particular focus on both computational and design strategies to advance this area of research and make it more broadly applicable to many classes of enzymes.

Construction of cell factory through combinatorial metabolic engineering for efficient production of itaconic acid.

BACKGROUND: Itaconic acid, an unsaturated C5 dicarbonic acid, has significant market demand and prospects. It has numerous biological functions, such as anti-cancer, anti-inflammatory, and anti-oxidative in medicine, and is an essential renewable platform chemical in industry. However, the development of industrial itaconic acid production by Aspergillus terreus, the current standard production strain, is hampered by the unavoidable drawbacks of that species. Developing a highly efficient cell factory is essential for the sustainable and green production of itaconic acid. RESULTS: This study employed combinatorial engineering strategies to construct Escherichia coli cells to produce itaconic acid efficiently. Two essential genes (cis-aconitate decarboxylase (CAD) encoding gene cadA and aconitase (ACO) encoding gene acn) employed various genetic constructs and plasmid combinations to create 12 recombination E. coli strains to be screened. Among them, E. coli BL-CAC exhibited the highest titer with citrate as substrate, and the induction and reaction conditions were further systematically optimized. Subsequently, employing enzyme evolution to optimize rate-limiting enzyme CAD and synthesizing protein scaffolds to co-localize ACO and CAD were used to improve itaconic acid biosynthesis efficiency. Under the optimized reaction conditions combined with the feeding control strategy, itaconic acid titer reached 398.07 mM (51.79 g/L) of engineered E. coli BL-CAR470E-DS/A-CS cells as a catalyst with the highest specific production of 9.42 g/g(DCW) among heterologous hosts at 48 h. CONCLUSIONS: The excellent catalytic performance per unit biomass shows the potential for high-efficiency production of itaconic acid and effective reduction of catalytic cell consumption. This study indicates that it is necessary to continuously explore engineering strategies to develop high-performance cell factories to break through the existing bottleneck and achieve the economical commercial production of itaconic acid.

A "push-pull-restrain" strategy to improve citronellol production in Saccharomyces cerevisiae.

Microbial production of monoterpenes has attracted increasing attention in recent years. Up to date, there are only few reports on the biosynthesis of the monoterpene alcohol citronellol that is widely used as fragrant and pharmaceutical intermediates. Here, we engineered Saccharomyces cerevisiae by employing a "push-pull-restrain" strategy to improve citronellol production based on the reduction of geraniol. Starting from a engineered geraniol-producing strain, different reductases were investigated and the best performing iridoid synthase from Catharanthus roseus (CrIS) resulted in 285.89 mg/L enantiomerically pure S-citronellol in shake flasks. Geranyl diphosphate (GPP), the most important precursor for monoterpenes, was enhanced by replacing the wild farnesyl diphosphate synthase (Erg20) with the mutant Erg20F96W, increasing the citronellol titer to 406.01 mg/L without negative influence on cell growth. Moreover, we employed synthetic protein scaffolds and protein fusion to colocalize four sequential enzymes to achieve better substrate channeling along with the deletion of an intermediate degradation pathway gene ATF1, which elevated the citronellol titer to 972.02 mg/L with the proportion of 97.8% of total monoterpenes in YPD medium. Finally, the engineered strain with complemented auxotrophic markers produced 8.30 g/L of citronellol by fed-batch fermentation, which was the highest citronellol titer reported to date. The multi-level engineering strategies developed here demonstrate the potential of monoterpenes overproduction in yeast, which can serve as a generally applicable platform for overproduction of other monoterpenes.

Antibodies as Biosensors' Key Components: State-of-the-Art in Russia 2020-2021.

The recognition of biomolecules is crucial in key areas such as the timely diagnosis of somatic and infectious diseases, food quality control, and environmental monitoring. This determines the need to develop highly sensitive display devices based on the achievements of modern science and technology, characterized by high selectivity, high speed, low cost, availability, and small size. Such requirements are met by biosensor systems-devices for reagent-free analysis of compounds that consist of a biologically sensitive element (receptor), a transducer, and a working solution. The diversity of biological material and methods for its immobilization on the surface or in the volume of the transducer and the use of nanotechnologies have led to the appearance of an avalanche-like number of different biosensors, which, depending on the type of biologically sensitive element, can be divided into three groups: enzyme, affinity, and cellular/tissue. Affinity biosensors are one of the rapidly developing areas in immunoassay, where the key point is to register the formation of an antigen-antibody complex. This review analyzes the latest work by Russian researchers concerning the production of molecules used in various immunoassay formats as well as new fundamental scientific data obtained as a result of their use.

Development of a Protein Scaffold for Arginine Sensing Generated through the Dissection of the Arginine-Binding Protein from Thermotoga maritima.

Arginine is one of the most important nutrients of living organisms as it plays a major role in important biological pathways. However, the accumulation of arginine as consequence of metabolic defects causes hyperargininemia, an autosomal recessive disorder. Therefore, the efficient detection of the arginine is a field of relevant biomedical/biotechnological interest. Here, we developed protein variants suitable for arginine sensing by mutating and dissecting the multimeric and multidomain structure of Thermotoga maritima arginine-binding protein (TmArgBP). Indeed, previous studies have shown that TmArgBP domain-swapped structure can be manipulated to generate simplified monomeric and single domain scaffolds. On both these stable scaffolds, to measure tryptophan fluorescence variations associated with the arginine binding, a Phe residue of the ligand binding pocket was mutated to Trp. Upon arginine binding, both mutants displayed a clear variation of the Trp fluorescence. Notably, the single domain scaffold variant exhibited a good affinity (~3 µM) for the ligand. Moreover, the arginine binding to this variant could be easily reverted under very mild conditions. Atomic-level data on the recognition process between the scaffold and the arginine were obtained through the determination of the crystal structure of the adduct. Collectively, present data indicate that TmArgBP scaffolds represent promising candidates for developing arginine biosensors.

Building a toolbox of protein scaffolds for future immobilization of biocatalysts.

Biological materials that are genetically encoded and can self-assemble offer great potential as immobilization platforms in industrial biocatalysis. Protein-based scaffolds can be used for the spatial organization of enzymes, to stabilize the catalysts and provide optimal microenvironments for reaction sequences. In our previous work, we created a protein scaffold for enzyme localization by engineering the bacterial microcompartment shell protein EutM from Salmonella enterica. Here, we sought to expand this work by developing a toolbox of EutM proteins with different properties, with the potential to be used for future immobilization of enzymes. We describe the bioinformatic identification of hundreds of homologs of EutM from diverse microorganisms. We specifically select 13 EutM homologs from extremophiles for characterization, based on phylogenetic analyses. We synthesize genes encoding the novel proteins, clone and express them in E. coli, and purify the proteins. In vitro characterization shows that the proteins self-assemble into robust nano- and micron-scale architectures including protein nanotubes, filaments, and scaffolds. We explore the self-assembly characteristics from a sequence-based approach and create a synthetic biology platform for the coexpression of different EutM homologs as hybrid scaffolds with integrated enzyme attachment points. This work represents a step towards our goal of generating a modular toolbox for the rapid production of self-assembling protein-based materials for enzyme immobilization.

Small-Molecule-Induced and Cooperative Enzyme Assembly on a 14-3-3 Scaffold.

Scaffold proteins regulate cell signalling by promoting the proximity of putative interaction partners. Although they are frequently applied in cellular settings, fundamental understanding of them in terms of, amongst other factors, quantitative parameters has been lagging behind. Here we present a scaffold protein platform that is based on the native 14-3-3 dimeric protein and is controllable through the action of a small-molecule compound, thus permitting study in an in vitro setting and mathematical description. Robust small-molecule regulation of caspase-9 activity through induced dimerisation on the 14-3-3 scaffold was demonstrated. The individual parameters of this system were precisely determined and used to develop a mathematical model of the scaffolding concept. This model was used to elucidate the strong cooperativity of the enzyme activation mediated by the 14-3-3 scaffold. This work provides an entry point for the long-needed quantitative insights into scaffold protein functioning and paves the way for the optimal use of reengineered 14-3-3 proteins as chemically inducible scaffolds in synthetic systems.

The future of protein scaffolds as affinity reagents for purification.

Affinity purification is one of the most powerful separation techniques extensively employed both at laboratory and production scales. While antibodies still represent the gold standard affinity reagents, others derived from non-immunoglobulin scaffolds emerged as interesting alternatives in particular for affinity purification. The lower costs of production, fast ligand development, and high robustness are appealing advantages of non-immunoglobulin scaffolds. These have successfully been used in the affinity purification of relevant targets as antibodies, human serum albumin, transferrin, and other biomarkers, as reviewed in this work. Furthermore, a critical assessment on the strengths, weaknesses, opportunities, and threats related with the implementation of non-immunoglobulin scaffolds as ligands in affinity purification are discussed. Biotechnol. Bioeng. 2017;114: 481-491. © 2016 Wiley Periodicals, Inc.

Achieving single-nucleotide resolution of 5-methylcytosine detection with TALEs.

We report engineered transcription-activator-like effectors (TALEs) as the first DNA-binding molecules that detect 5-methylcytosine (mC) at single-nucleotide resolution with fully programmable sequence selectivity. This is achieved by a design strategy such that a single cytosine (C) in a DNA sequence is selectively interrogated for its mC-modification level by targeting with a discriminatory TALE repeat; other Cs are ignored by targeting with universal-binding TALE repeats.

Improvement of catechin production in Escherichia coli through combinatorial metabolic engineering.

Reconstruction of highly efficient biosynthesis pathways is essential for the production of valuable plant secondary metabolites in recombinant microorganisms. In order to improve the titer of green tea catechins in Escherichia coli, combinatorial strategies were employed using the ePathBrick vectors to express the committed catechin pathway: flavanone 3ß-hydroxylase (F3H), dihydroflavonol 4-reductase (DFR), and leucoanthocyanidin reductase (LAR). Three F3H, three DFR, and two LAR genes originating from different plant species were selected and synthesized, to create 18 pathway variants to be screened in E. coli. Constructs containing F3H(syn) originally from Camellia sinensis, DFR(syn) from Anthurium andraeanum, C. sinensis, or Fragaria ananass, and LAR(syn) from Desmodium uncinatum (p148, p158 and p168) demonstrated high conversion efficiency with either eriodictyol or naringenin as substrate. A highly efficient construct was created by assembling additional copies of DFR(syn) and LAR(syn) enabling a titer of 374.6 ± 43.6 mg/L of (+)-catechin. Improving the NADPH availability via the ΔpgiΔppc mutation, BLΔpgiΔppc-p148 produced the highest titer of catechin at 760.9 ± 84.3 mg/L. After utilizing a library of scaffolding proteins, the strain BLΔpgiΔppc-p168-759 reached the highest titer of (+)-catechin of 910.9 ± 61.3 mg/L from 1.0 g/L of eriodictyol in batch culture with M9 minimal media. The impact of oxygen availability on the biosynthesis of catechin was also investigated.

Bioengineering strategies to generate artificial protein complexes.

For many applications, increasing synergy between distinct proteins through organization is important for the specificity, regulation, and overall reaction efficiency. Although there are many examples of protein complexes in nature, a generalized method to create these complexes remains elusive. Many conventional techniques such as random chemical conjugation, physical adsorption onto surfaces, and encapsulation within matrices are imprecise approaches and can lead to deactivation of protein native functionalities. More "bio-friendly" approaches such as genetically fused proteins and biological scaffolds often can result in low yields and low complex stability. Alternatively, site-specific protein conjugation or ligation can generate artificial protein complexes that preserve the native functionalities of protein domains and maintain stability through covalent bonds. In this review, we describe three distinct methods to synthesize artificial protein complexes (genetic incorPoration of unnatural amino acids to introduce bio-orthogonal azide and alkyne groups to proteins, split-intein based expressed protein ligation, and sortase mediated ligation) and highlight interesting applications for each technique.

Programmable and highly resolved in vitro detection of 5-methylcytosine by TALEs.

Gene expression is extensively regulated by specific patterns of genomic 5-methylcytosine (mC), but the ability to directly detect this modification at user-defined genomic loci is limited. One reason is the lack of molecules that discriminate between mC and cytosine (C) and at the same time provide inherent, programmable sequence-selectivity. Programmable transcription-activator-like effectors (TALEs) have been observed to exhibit mC-sensitivity in vivo, but to only a limited extent in vitro. We report an mC-detection assay based on TALE control of DNA replication that displays unexpectedly strong mC-discrimination ability in vitro. The status and level of mC modification at single positions in oligonucleotides can be determined unambiguously by this assay, independently of the overall target sequence. Moreover, discrimination is reliably observed for positions bound by N-terminal and central regions of TALEs. This indicates the wide scope and robustness of the approach for highly resolved mC detection and enabled the detection of a single mC in a large, eukaryotic genome.

Study on the properties of a dual-system-based protein scaffold for orthogonal self-assembly.

Protein scaffolds possessing the ability to efficiently organize enzymes to improve the catalytic performance, enzyme stability and provide an optimal micro-environment for biocatalysis. Here, SpyCatcher fused to the C-terminus of Treptavidin (a variant of streptavidin) to construct a chimeric tetramers protein scaffold (Tr-SC) with dual orthogonal conjugation moieties. The results showed that the expressed Tr-SC scaffold was an active tetramer with good stability under 80 °C and pH 6.5-8.5, which could bind 4 SpyTag-mCherry and 4 Biotin-EGFP. Tr-SC scaffold can bind 1-4 ligands alone under different conditions. The order in which protein scaffolds bind to proteins has little effect on the final complex structure. It is more difficult for SpyTag-mCherry than Biotin-EGFP to bind to Tr-SC, so incomplete conjugates of a hexameric complex composed of 2 SpyTag-mCherry and 4 Biotin-EGFP form when the molar ratio of scaffold and two ligands is 1:4:4. Therefore, it was suggest that the Tr-SC can first bind to excess SpyTag-protein and mixed with Biotin-protein to promote the formation of higher multimers. The results can be important reference for more extensive use of Tr-SC to construct heterologous protein polymers and assembly of heterologous enzyme molecular machine in vitro to carry on efficient cascade reaction in the future.

Spatially resolved proteomics of the Arabidopsis stomatal lineage identifies polarity complexes for cell divisions and stomatal pores.

Cell polarity is used to guide asymmetric divisions and create morphologically diverse cells. We find that two oppositely oriented cortical polarity domains present during the asymmetric divisions in the Arabidopsis stomatal lineage are reconfigured into polar domains marking ventral (pore-forming) and outward-facing domains of maturing stomatal guard cells. Proteins that define these opposing polarity domains were used as baits in miniTurboID-based proximity labeling. Among differentially enriched proteins, we find kinases, putative microtubule-interacting proteins, and polar SOSEKIs with their effector ANGUSTIFOLIA. Using AI-facilitated protein structure prediction models, we identify potential protein-protein interaction interfaces among them. Functional and localization analyses of the polarity protein OPL2 and its putative interaction partners suggest a positive interaction with mitotic microtubules and a role in cytokinesis. This combination of proteomics and structural modeling with live-cell imaging provides insights into how polarity is rewired in different cell types and cell-cycle stages.

Engineering bio-brick protein scaffolds for organizing enzyme assemblies.

Enzyme scaffolding is an emerging approach for enhancing the catalytic efficiency of multi-enzymatic cascades by controlling their spatial organization and stoichiometry. This study introduces a novel family of engineered SCAffolding Bricks, named SCABs, utilizing the consensus tetratricopeptide repeat (CTPR) domain for organized multi-enzyme systems. Two SCAB systems are developed, one employing head-to-tail interactions with reversible covalent disulfide bonds, the other relying on non-covalent metal-driven assembly via engineered metal coordinating interfaces. Enzymes are directly fused to SCAB modules, triggering assembly in a non-reducing environment or by metal presence. A proof-of-concept with formate dehydrogenase (FDH) and L-alanine dehydrogenase (AlaDH) shows enhanced specific productivity by 3.6-fold compared to free enzymes, with the covalent stapling outperforming the metal-driven assembly. This enhancement likely stems from higher-order supramolecular assembly and improved NADH cofactor regeneration, resulting in more efficient cascades. This study underscores the potential of protein engineering to tailor scaffolds, leveraging supramolecular spatial-organizing tools, for more efficient enzymatic cascade reactions.

Inhibition of a malaria host-pathogen interaction by a computationally designed inhibitor.

Malaria is a substantial global health burden with 229 million cases in 2019 and 450,000 deaths annually. Plasmodium vivax is the most widespread malaria-causing parasite putting 2.5 billion people at risk of infection. P. vivax has a dormant liver stage and therefore can exist for long periods undetected. Its blood-stage can cause severe reactions and hospitalization. Few treatment and detection options are available for this pathogen. A unique characteristic of P. vivax is that it depends on the Duffy antigen/receptor for chemokines (DARC) on the surface of host red blood cells for invasion. P. vivax employs the Duffy binding protein (DBP) to bind to DARC. We first de novo designed a three helical bundle scaffolding database which was screened via protease digestions for stability. Protease-resistant scaffolds highlighted thresholds for stability, which we utilized for selecting DARC mimetics that we subsequentially designed through grafting and redesign of these scaffolds. The optimized design small helical protein disrupts the DBP:DARC interaction. The inhibitor blocks the receptor binding site on DBP and thus forms a strong foundation for a therapeutic that will inhibit reticulocyte infection and prevent the pathogenesis of P. vivax malaria.

Membrane curvature as a signal to ensure robustness of diverse cellular processes.

An increasing corpus of research has demonstrated that membrane shape, generated either by the external environment of the cell or by intrinsic mechanisms such as cytokinesis and vesicle or organelle formation, is an important parameter in the control of diverse cellular processes. In this review we discuss recent findings that demonstrate how membrane curvature (from nanometer to micron length-scales) alters protein function. We describe an expanding toolkit for experimentally modulating membrane curvature to reveal effects on protein function, and discuss how membrane curvature - far from being a passive consequence of the physical environment and the internal protein activity of a cell - is an important signal that controls protein affinity and enzymatic activity to ensure robust forward progression of key processes within the cell.

Combined in vitro and cell-based selection display method producing specific binders against IL-9 receptor in high yields.

We combined cell-free ribosome display and cell-based yeast display selection to build specific protein binders to the extracellular domain of the human interleukin 9 receptor alpha (IL-9Rα). The target, IL-9Rα, is the receptor involved in the signalling pathway of IL-9, a pro-inflammatory cytokine medically important for its involvement in respiratory diseases. The successive use of modified protocols of ribosome and yeast displays allowed us to combine their strengths-the virtually infinite selection power of ribosome display and the production of (mostly) properly folded and soluble proteins in yeast display. The described experimental protocol is optimized to produce binders highly specific to the target, including selectivity to common proteins such as BSA, and proteins potentially competing for the binder such as receptors of other cytokines. The binders were trained from DNA libraries of two protein scaffolds called 57aBi and 57bBi developed in our laboratory. We show that the described unconventional combination of ribosome and yeast displays is effective in developing selective small protein binders to the medically relevant molecular target.

Multivalent protein-drug conjugates - An emerging strategy for the upgraded precision and efficiency of drug delivery to cancer cells.

With almost 20 million new cases per year, cancer constitutes one of the most important challenges for public health systems. Unlike traditional chemotherapy, targeted anti-cancer strategies employ sophisticated therapeutics to precisely identify and attack cancer cells, limiting the impact of drugs on healthy cells and thereby minimizing the unwanted side effects of therapy. Protein drug conjugates (PDCs) are a rapidly growing group of targeted therapeutics, composed of a cancer-recognition factor covalently coupled to a cytotoxic drug. Several PDCs, mainly in the form of antibody-drug conjugates (ADCs) that employ monoclonal antibodies as cancer-recognition molecules, are used in the clinic and many PDCs are currently in clinical trials. Highly selective, strong and stable interaction of the PDC with the tumor marker, combined with efficient, rapid endocytosis of the receptor/PDC complex and its subsequent effective delivery to lysosomes, is critical for the efficacy of targeted cancer therapy with PDCs. However, the bivalent architecture of contemporary clinical PDCs is not optimal for tumor receptor recognition or PDCs internalization. In this review, we focus on multivalent PDCs, which represent a rapidly evolving and highly promising therapeutics that overcome most of the limitations of current bivalent PDCs, enhancing the precision and efficiency of drug delivery to cancer cells. We present an expanding set of protein scaffolds used to generate multivalent PDCs that, in addition to folding into well-defined multivalent molecular structures, enable site-specific conjugation of the cytotoxic drug to ensure PDC homogeneity. We provide an overview of the architectures of multivalent PDCs developed to date, emphasizing their efficacy in the targeted treatment of various cancers.