SeParate: multiway fluorescence-activated droplet sorting based on integration of serial and parallel triaging concepts.

Fluorescence-activated droplet sorting (FADS) has emerged as a versatile high-throughput sorting tool that is, unlike most fluorescence-activated cell sorting (FACS) platforms, capable of sorting droplet-compartmentalized cells, cell secretions, entire enzymatic reactions and more. Recently, multiplex FADS platforms have been developed for the sorting of multi-fluorophore populations towards different outlets in addition to the standard, more commonly used, 2-way FADS platform. These multiplex FADS platforms consist of either multiple 2-way junctions one after the other (i.e. serial sorters) or of one junction sorting droplets in more than 2 outlets (i.e. parallel sorters). In this work, we present SeParate, a novel platform based on integrating s̲e̲rial and p̲a̲r̲allel sorting principles for accura̲t̲e̲ multiplex droplet sorting that is able to mitigate limitations of current multiplex sorters. We show the SeParate platform and its capability in highly accurate 4-way sorting of a multi-fluorophore population into four subpopulations with the potential to expand to more. More specifically, the SeParate platform was thoroughly validated using mixed populations of fluorescent beads and picoinjected droplets, yielding sorting accuracies up to 100% and 99.9%, respectively. Finally, transfected HEK-293T cells were sorted employing two different optical setups, resulting in an accuracy up to 99.5%. SeParate's high accuracy for a diverse set of samples, including highly variable biological specimens, together with its scalability beyond the demonstrated 4-way sorting, warrants a broad applicability for multi-fluorophore studies in life sciences, environmental sciences and others.

Bacillus subtilis as a host for natural product discovery and engineering of biosynthetic gene clusters.

Covering: up to October 2023Many bioactive natural products are synthesized by microorganisms that are either difficult or impossible to cultivate under laboratory conditions, or that produce only small amounts of the desired compound. By transferring biosynthetic gene clusters (BGCs) into alternative host organisms that are more easily cultured and engineered, larger quantities can be obtained and new analogues with potentially improved biological activity or other desirable properties can be generated. Moreover, expression of cryptic BGCs in a suitable host can facilitate the identification and characterization of novel natural products. Heterologous expression therefore represents a valuable tool for natural product discovery and engineering as it allows the study and manipulation of their biosynthetic pathways in a controlled setting, enabling innovative applications. Bacillus is a genus of Gram-positive bacteria that is widely used in industrial biotechnology as a host for the production of proteins from diverse origins, including enzymes and vaccines. However, despite numerous successful examples, Bacillus species remain underexploited as heterologous hosts for the expression of natural product BGCs. Here, we review important advantages that Bacillus species offer as expression hosts, such as high secretion capacity, natural competence for DNA uptake, and the increasing availability of a wide range of genetic tools for gene expression and strain engineering. We evaluate different strain optimization strategies and other critical factors that have improved the success and efficiency of heterologous natural product biosynthesis in B. subtilis. Finally, future perspectives for using B. subtilis as a heterologous host are discussed, identifying research gaps and promising areas that require further exploration.

Distinct mode of action of a highly stable, engineered phage lysin killing Gram-negative bacteria.

IMPORTANCE: Engineered lysins are considered as highly promising alternatives for antibiotics. Our previous screening study using VersaTile technology identified 1D10 as a possible lead compound with activity against Acinetobacter baumannii strains under elevated human serum concentrations. In this manuscript, we reveal an unexpected mode of action and exceptional thermoresistance for lysin 1D10. Our findings shed new light on the development of engineered lysins, providing valuable insights for future research in this field.

The Specific Capsule Depolymerase of Phage PMK34 Sensitizes Acinetobacter baumannii to Serum Killing.

The rising antimicrobial resistance is particularly alarming for Acinetobacter baumannii, calling for the discovery and evaluation of alternatives to treat A. baumannii infections. Some bacteriophages produce a structural protein that depolymerizes capsular exopolysaccharide. Such purified depolymerases are considered as novel antivirulence compounds. We identified and characterized a depolymerase (DpoMK34) from Acinetobacter phage vB_AbaP_PMK34 active against the clinical isolate A. baumannii MK34. In silico analysis reveals a modular protein displaying a conserved N-terminal domain for anchoring to the phage tail, and variable central and C-terminal domains for enzymatic activity and specificity. AlphaFold-Multimer predicts a trimeric protein adopting an elongated structure due to a long α-helix, an enzymatic ß-helix domain and a hypervariable 4 amino acid hotspot in the most ultimate loop of the C-terminal domain. In contrast to the tail fiber of phage T3, this hypervariable hotspot appears unrelated with the primary receptor. The functional characterization of DpoMK34 revealed a mesophilic enzyme active up to 50 °C across a wide pH range (4 to 11) and specific for the capsule of A. baumannii MK34. Enzymatic degradation of the A. baumannii MK34 capsule causes a significant drop in phage adsorption from 95% to 9% after 5 min. Although lacking intrinsic antibacterial activity, DpoMK34 renders A. baumannii MK34 fully susceptible to serum killing in a serum concentration dependent manner. Unlike phage PMK34, DpoMK34 does not easily select for resistant mutants either against PMK34 or itself. In sum, DpoMK34 is a potential antivirulence compound that can be included in a depolymerase cocktail to control difficult to treat A. baumannii infections.

Combinatorial assembly and optimisation of designer cellulosomes: a galactomannan case study.

BACKGROUND: Designer cellulosomes are self-assembled chimeric enzyme complexes that can be used to improve lignocellulosic biomass degradation. They are composed of a synthetic multimodular backbone protein, termed the scaffoldin, and a range of different chimeric docking enzymes that degrade polysaccharides. Over the years, several functional designer cellulosomes have been constructed. Since many parameters influence the efficiency of these multi-enzyme complexes, there is a need to optimise designer cellulosome architecture by testing combinatorial arrangements of docking enzyme and scaffoldin variants. However, the modular cloning procedures are tedious and cumbersome. RESULTS: VersaTile is a combinatorial DNA assembly method, allowing the rapid construction and thus comparison of a range of modular proteins. Here, we present the extension of the VersaTile platform to facilitate the construction of designer cellulosomes. We have constructed a tile repository, composed of dockerins, cohesins, linkers, tags and enzymatically active modules. The developed toolbox allows us to efficiently create and optimise designer cellulosomes at an unprecedented speed. As a proof of concept, a trivalent designer cellulosome able to degrade the specific hemicellulose substrate, galactomannan, was constructed and optimised. The main factors influencing cellulosome efficiency were found to be the selected dockerins and linkers and the docking enzyme ratio on the scaffoldin. The optimised designer cellulosome was able to hydrolyse the galactomannan polysaccharide and release mannose and galactose monomers. CONCLUSION: We have eliminated one of the main technical hurdles in the designer cellulosome field and anticipate the VersaTile platform to be a starting point in the development of more elaborate multi-enzyme complexes.

Rapid and High-Throughput Evaluation of Diverse Configurations of Engineered Lysins Using the VersaTile Technique.

Bacteriophage-encoded lysins are an emerging class of antibacterial enzymes based on peptidoglycan degradation. The modular composition of lysins is a hallmark feature enabling optimization of antibacterial and pharmacological properties by engineering of lysin candidates based on lysin and non-lysin modules. In this regard, the recent introduction of the VersaTile technique allows the rapid construction of large modular lysin libraries based on a premade repository of building blocks. In this study, we perform a high-throughput construction and screening of five combinatorial lysin libraries with different configurations, targeting Klebsiella pneumoniae. An elaborate analysis of the activity distribution of 940 variants and sequencing data of 74 top hits inhibiting the growth of Klebsiella pneumoniae could be associated with specific design rules. Specific outer membrane permeabilizing peptides (OMPs) and enzymatically active domains (EADs) are significantly overrepresented among the top hits, while cell wall binding domains (CBDs) are equally represented. Especially libraries with the configuration (OMP-linker-CBD-EAD) and the inverse configuration (CBD-EAD-linker-OMP) yield the most active variants, with discernible clusters of variants that emerge above the remaining variants. The approach implemented here provides a blueprint for discovery campaigns of engineered lysins starting from libraries with different configurations and compositions.

Predicting bacteriophage hosts based on sequences of annotated receptor-binding proteins.

Nowadays, bacteriophages are increasingly considered as an alternative treatment for a variety of bacterial infections in cases where classical antibiotics have become ineffective. However, characterizing the host specificity of phages remains a labor- and time-intensive process. In order to alleviate this burden, we have developed a new machine-learning-based pipeline to predict bacteriophage hosts based on annotated receptor-binding protein (RBP) sequence data. We focus on predicting bacterial hosts from the ESKAPE group, Escherichia coli, Salmonella enterica and Clostridium difficile. We compare the performance of our predictive model with that of the widely used Basic Local Alignment Search Tool (BLAST). Our best-performing predictive model reaches Precision-Recall Area Under the Curve (PR-AUC) scores between 73.6 and 93.8% for different levels of sequence similarity in the collected data. Our model reaches a performance comparable to that of BLASTp when sequence similarity in the data is high and starts outperforming BLASTp when sequence similarity drops below 75%. Therefore, our machine learning methods can be especially useful in settings in which sequence similarity to other known sequences is low. Predicting the hosts of novel metagenomic RBP sequences could extend our toolbox to tune the host spectrum of phages or phage tail-like bacteriocins by swapping RBPs.

Quality control and statistical evaluation of combinatorial DNA libraries using nanopore sequencing.

Protein engineering and synthetic biology applications increasingly rely on the assembly of modular libraries composed of thousands of different combinations of DNA building blocks. At present, the validation of such libraries is performed by Sanger sequencing analysis on a small subset of clones on an ad hoc basis. Here, we implement a systematic procedure for the comprehensive evaluation of combinatorial libraries, immediately after their creation in vitro, using long reads sequencing technology. After an initial step of nanopore sequencing, we use straightforward bioinformatics tools to tabulate the composition and synteny of the building blocks in each read. We subsequently use exploratory statistics to assess the library and validate its diversity before carrying downstream cloning and screening assays.

The Preclinical and Clinical Progress of Bacteriophages and Their Lytic Enzymes: The Parts are Easier than the Whole.

The therapeutic potential of phages has been considered since their first identification more than a century ago. The evident concept of using a natural predator to treat bacterial infections has, however, since then been challenged considerably. Initially, the vast success of antibiotics almost eliminated the study of phages for therapy. Upon the renaissance of phage therapy research, the most provocative and unique properties of phages such as high specificity, self-replication and co-evolution prohibited a rapid preclinical and clinical development. On the one hand, the typical trajectory followed by small molecule antibiotics could not be simply translated into the preclinical analysis of phages, exemplified by the need for complex broad spectrum or personalized phage cocktails of high purity and the more complex pharmacokinetics. On the other hand, there was no fitting regulatory framework to deal with flexible and sustainable phage therapy approaches, including the setup and approval of adequate clinical trials. While significant advances are incrementally made to eliminate these hurdles, phage-inspired antibacterials have progressed in the slipstream of phage therapy, benefiting from the lack of hurdles that are typically associated with phage therapy. Most advanced are phage lytic enzymes that kill bacteria through peptidoglycan degradation and osmotic lysis. Both phages and their lytic enzymes are now widely considered as safe and have now progressed to clinical phase II to show clinical efficacy as pharmaceutical. Yet, more initiatives are needed to fill the clinical pipeline to beat the typical attrition rates of clinical evaluation and to come to a true evaluation of phages and phage lytic enzymes in the clinic.

Target Confinement in Small Reaction Volumes Using Microfluidic Technologies: A Smart Approach for Single-Entity Detection and Analysis.

Over the last decades, the study of cells, nucleic acid molecules, and proteins has evolved from ensemble measurements to so-called single-entity studies. The latter offers huge benefits, not only as biological research tools to examine heterogeneities among individual entities within a population, but also as biosensing tools for medical diagnostics, which can reach the ultimate sensitivity by detecting single targets. Whereas various techniques for single-entity detection have been reported, this review focuses on microfluidic systems that physically confine single targets in small reaction volumes. We categorize these techniques as droplet-, microchamber-, and nanostructure-based and provide an overview of their implementation for studying single cells, nucleic acids, and proteins. We furthermore reflect on the advantages and limitations of these techniques and highlight future opportunities in the field.

Synthetic biology of modular endolysins.

Endolysins and their derivatives have emerged in recent years as a novel class of antibacterials, which have now entered the clinical phases. Their rapid mode-of-action and proteinaceous nature differentiates them from any other class of antibiotics. A key feature of endolysins is their modularity and the opportunities that emerge thereof to customize properties such as specificity, activity, stability and solubility. Extensive protein engineering efforts have expanded the activity spectrum to (pan)drug-resistant Gram-negative bacteria or have improved the activity against Gram-positive pathogens. In addition, specific cell wall binding domains derived from endolysins are exploited for the development of diagnostics.

DUF3380 Domain from a Salmonella Phage Endolysin Shows Potent N-Acetylmuramidase Activity.

UNLABELLED: Bacteriophage-encoded endolysins are highly diverse enzymes that cleave the bacterial peptidoglycan layer. Current research focuses on their potential applications in medicine, in food conservation, and as biotechnological tools. Despite the wealth of applications relying on the use of endolysin, little is known about the enzymatic properties of these enzymes, especially in the case of endolysins of bacteriophages infecting Gram-negative species. Automated genome annotations therefore remain to be confirmed. Here, we report the biochemical analysis and cleavage site determination of a novel Salmonella bacteriophage endolysin, Gp110, which comprises an uncharacterized domain of unknown function (DUF3380; pfam11860) in its C terminus and shows a higher specific activity (34,240 U/µM) than that of 14 previously characterized endolysins active against peptidoglycan from Gram-negative bacteria (corresponding to 1.7- to 364-fold higher activity). Gp110 is a modular endolysin with an optimal pH of enzymatic activity of pH 8 and elevated thermal resistance. Reverse-phase high-performance liquid chromatography (RP-HPLC) analysis coupled to mass spectrometry showed that DUF3380 has N-acetylmuramidase (lysozyme) activity cleaving the ß-(1,4) glycosidic bond between N-acetylmuramic acid and N-acetylglucosamine residues. Gp110 is active against directly cross-linked peptidoglycans with various peptide stem compositions, making it an attractive enzyme for developing novel antimicrobial agents. IMPORTANCE: We report the functional and biochemical characterization of the Salmonella phage endolysin Gp110. This endolysin has a modular structure with an enzymatically active domain and a cell wall binding domain. The enzymatic activity of this endolysin exceeds that of all other endolysins previously characterized using the same methods. A domain of unknown function (DUF3380) is responsible for this high enzymatic activity. We report that DUF3380 has N-acetylmuramidase activity against directly cross-linked peptidoglycans with various peptide stem compositions. This experimentally verified activity allows better classification and understanding of the enzymatic activities of endolysins, which mostly are inferred by sequence similarities. Three-dimensional structure predictions for Gp110 suggest a fold that is completely different from that of known structures of enzymes with the same peptidoglycan cleavage specificity, making this endolysin quite unique. All of these features, combined with increased thermal resistance, make Gp110 an attractive candidate for engineering novel endolysin-based antibacterials.

From endolysins to Artilysin®s: novel enzyme-based approaches to kill drug-resistant bacteria.

One of the last untapped reservoirs in nature for the identification of new anti-microbials is bacteriophages, the natural killers of bacteria. Lytic bacteriophages encode peptidoglycan (PG) lytic enzymes able to degrade the PG layer in different steps of their infection cycle. Endolysins degrade the bacterial cell wall at the end of the infection cycle, causing lysis of the host to release the viral progeny. Recombinant endolysins have been successfully applied as anti-bacterial agent against antibiotic-resistant Gram-positive pathogens. This has boosted the study of these enzymes as new anti-microbials in different fields (e.g. medical, food technology). A key example is the recent development of endolysin-based anti-bacterials against Gram-negative pathogens in which the exogenous application of endolysins is hindered by the outer membrane (OM). These novel anti-microbials, termed Artilysin®s, are able to pass through the OM and reach the PG where they exert their action. In addition, mycobacteria whose cell wall is structurally different from both Gram-positive and Gram-negative bacteria have also been reported to be inhibited by mycobacteriophage-encoded endolysins. Endolysins and endolysin-based anti-microbials can be considered as ideal candidates for an alternative to antibiotics for several reasons: (1) their unique mode of action and activity against bacterial persisters (independent of an active host metabolism), (2) their selective activity against both Gram-positive and Gram-negative pathogens (including antibiotic resistant strains) and mycobacteria, (3) the limited resistance development reported so far. The present review summarizes and discusses the potential applications of endolysins as new anti-microbials.