The Laboratory Automation Protocol (LAP) Format and Repository: A Platform for Enhancing Workflow Efficiency in Synthetic Biology.

Laboratory automation deals with eliminating manual tasks in high-throughput protocols. It therefore plays a crucial role in allowing fast and reliable synthetic biology. However, implementing open-source automation solutions often demands experimental scientists to possess scripting skills, and even when they do, there is no standardized toolkit available for their use. To address this, we present the Laboratory Automation Protocol (LAP) Format and Repository. LAPs adhere to a standardized script-based format, enhancing end-user implementation and simplifying further development. With a modular design, LAPs can be seamlessly combined to create customized, target-specific workflows. Furthermore, all LAPs undergo experimental validation, ensuring their reliability. Detailed information is provided within each repository entry, allowing users to validate the LAPs in their own laboratory settings. We advocate for the adoption of the LAP Format and Repository as a community resource, which will continue to expand, improving the reliability and reproducibility of the automation processes.

pBLAM1-x: standardized transposon tools for high-throughput screening.

The engineering of pre-defined functions in living cells requires increasingly accurate tools as synthetic biology efforts become more ambitious. Moreover, the characterization of the phenotypic performance of genetic constructs demands meticulous measurements and extensive data acquisition for the sake of feeding mathematical models and matching predictions along the design-build-test lifecycle. Here, we developed a genetic tool that eases high-throughput transposon insertion sequencing (TnSeq): the pBLAM1-x plasmid vectors carrying the Himar1 Mariner transposase system. These plasmids were derived from the mini-Tn5 transposon vector pBAMD1-2 and built following modular criteria of the Standard European Vector Architecture (SEVA) format. To showcase their function, we analyzed sequencing results of 60 clones of the soil bacterium Pseudomonas putida KT2440. The new pBLAM1-x tool has already been included in the latest SEVA database release, and here we describe its performance using laboratory automation workflows. Graphical Abstract.

A conserved SH3-like fold in diverse putative proteins tetramerizes into an oxidoreductase providing an antimicrobial resistance phenotype.

We present a potential mechanism for emergence of catalytic activity that is essential for survival, from a non-catalytic protein fold. The type B dihydrofolate reductase (DfrB) family of enzymes were first identified in pathogenic bacteria because their dihydrofolate reductase activity is sufficient to provide trimethoprim (TMP) resistance. DfrB enzymes are described as poorly evolved as a result of their unusual structural and kinetic features. No characterized protein shares sequence homology with DfrB enzymes; how they evolved to emerge in the modern resistome is unknown. In this work, we identify DfrB homologues from a database of putative and uncharacterized proteins. These proteins include an SH3-like fold homologous to the DfrB enzymes, embedded in a variety of additional structural domains. By means of functional, structural and biophysical characterization, we demonstrate that these distant homologues and their extracted SH3-like fold can display dihydrofolate reductase activity and confer TMP resistance. We provide evidence of tetrameric assembly and catalytic mechanism analogous to that of DfrB enzymes. These results contribute, to our knowledge, the first insights into a potential evolutionary path taken by this SH3-like fold to emerge in the modern resistome following introduction of TMP. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

Tuning Selectivity in CalA Lipase: Beyond Tunnel Engineering.

Engineering studies of Candida (Pseudozyma) antarctica lipase A (CalA) have demonstrated the potential of this enzyme in the selective hydrolysis of fatty acid esters of different chain lengths. CalA has been shown to bind substrates preferentially through an acyl-chain binding tunnel accessed via the hydrolytic active site; it has also been shown that selectivity for substrates of longer or shorter chain length can be tuned, for instance by modulating steric hindrance within the tunnel. Here we demonstrate that, whereas the tunnel region is certainly of paramount importance for substrate recognition, residues in distal regions of the enzyme can also modulate substrate selectivity. To this end, we investigate variants that carry one or more substitutions within the substrate tunnel as well as in distal regions. Combining experimental determination of the substrate selectivity using natural and synthetic substrates with computational characterization of protein dynamics and of tunnels, we deconvolute the effect of key substitutions and demonstrate that epistatic interactions contribute to procuring selectivity toward either long-chain or short/medium-chain fatty acid esters. We demonstrate that various mechanisms contribute to the diverse selectivity profiles, ranging from reshaping tunnel morphology and tunnel stabilization to obstructing the main substrate-binding tunnel, highlighting the dynamic nature of the substrate-binding region. This work provides important insights into the versatility of this robust lipase toward diverse applications.

SEVA 4.0: an update of the Standard European Vector Architecture database for advanced analysis and programming of bacterial phenotypes.

The SEVA platform (https://seva-plasmids.com) was launched one decade ago, both as a database (DB) and as a physical repository of plasmid vectors for genetic analysis and engineering of Gram-negative bacteria with a structure and nomenclature that follows a strict, fixed architecture of functional DNA segments. While the current update keeps the basic features of earlier versions, the platform has been upgraded not only with many more ready-to-use plasmids but also with features that expand the range of target species, harmonize DNA assembly methods and enable new applications. In particular, SEVA 4.0 includes (i) a sub-collection of plasmids for easing the composition of multiple DNA segments with MoClo/Golden Gate technology, (ii) vectors for Gram-positive bacteria and yeast and [iii] off-the-shelf constructs with built-in functionalities. A growing collection of plasmids that capture part of the standard-but not its entirety-has been compiled also into the DB and repository as a separate corpus (SEVAsib) because of its value as a resource for constructing and deploying phenotypes of interest. Maintenance and curation of the DB were accompanied by dedicated diffusion and communication channels that make the SEVA platform a popular resource for genetic analyses, genome editing and bioengineering of a large number of microorganisms.

Methods for enzyme library creation: Which one will you choose?: A guide for novices and experts to introduce genetic diversity.

Enzyme engineering allows to explore sequence diversity in search for new properties. The scientific literature is populated with methods to create enzyme libraries for engineering purposes, however, choosing a suitable method for the creation of mutant libraries can be daunting, in particular for the novices. Here, we address both novices and experts: how can one enter the arena of enzyme library design and what guidelines can advanced users apply to select strategies best suited to their purpose? Section I is dedicated to the novices and presents an overview of established and standard methods for library creation, as well as available commercial solutions. The expert will discover an up-to-date tool to freshen up their repertoire (Section I) and learn of the newest methods that are likely to become a mainstay (Section II). We focus primarily on in vitro methods, presenting the advantages of each method. Our ultimate aim is to offer a selection of methods/strategies that we believe to be most useful to the enzyme engineer, whether a first-timer or a seasoned user.

Development of sulfahydantoin derivatives as ß-lactamase inhibitors.

Sulfahydantoin-based molecules may provide a means to counteract antibiotic resistance, which is on the rise. These molecules may act as inhibitors of ß-lactamase enzymes, which are key in some resistance mechanisms. In this paper, we report on the synthesis of 6 novel sulfahydantoin derivatives by the key reaction of chlorosulfonyl isocyanate to form α-amino acid derived sulfamides, and their cyclization into sulfahydantoins. The synthesis is rapid and provides the target compounds in 8 steps. We investigated their potential as ß-lactamase inhibitors using two common Class A ß-lactamases, TEM-1 and the prevalent extended-spectrum TEM-15. Two compounds, 3 and 6, show substantial inhibition of the ß-lactamases with IC50 values between 130 and 510 µM and inferred Ki values between 32 and 55 µM.

Known Evolutionary Paths Are Accessible to Engineered ß-Lactamases Having Altered Protein Motions at the Timescale of Catalytic Turnover.

The evolution of new protein functions is dependent upon inherent biophysical features of proteins. Whereas, it has been shown that changes in protein dynamics can occur in the course of directed molecular evolution trajectories and contribute to new function, it is not known whether varying protein dynamics modify the course of evolution. We investigate this question using three related ß-lactamases displaying dynamics that differ broadly at the slow timescale that corresponds to catalytic turnover yet have similar fast dynamics, thermal stability, catalytic, and substrate recognition profiles. Introduction of substitutions E104K and G238S, that are known to have a synergistic effect on function in the parent ß-lactamase, showed similar increases in catalytic efficiency toward cefotaxime in the related ß-lactamases. Molecular simulations using Protein Energy Landscape Exploration reveal that this results from stabilizing the catalytically-productive conformations, demonstrating the dominance of the synergistic effect of the E014K and G238S substitutions in vitro in contexts that vary in terms of sequence and dynamics. Furthermore, three rounds of directed molecular evolution demonstrated that known cefotaximase-enhancing mutations were accessible regardless of the differences in dynamics. Interestingly, specific sequence differences between the related ß-lactamases were shown to have a higher effect in evolutionary outcomes than did differences in dynamics. Overall, these ß-lactamase models show tolerance to protein dynamics at the timescale of catalytic turnover in the evolution of a new function.

Holistic engineering of Cal-A lipase chain-length selectivity identifies triglyceride binding hot-spot.

Through the application of a region-focused saturation mutagenesis and randomization approach, protein engineering of the Cal-A enzyme was undertaken with the goal of conferring new triglyceride selectivity. Little is known about the mode of triglyceride binding to Cal-A. Engineering Cal-A thus requires a systemic approach. Targeted and randomized Cal-A libraries were created, recombined using the Golden Gate approach and screened to detect variants able to discriminate between long-chain (olive oil) and short-chain (tributyrin) triglyceride substrates using a high-throughput in vivo method to visualize hydrolytic activity. Discriminative variants were analyzed using an in-house script to identify predominant substitutions. This approach allowed identification of variants that exhibit strong discrimination for the hydrolysis of short-chain triglycerides and others that discriminate towards hydrolysis of long-chain triglycerides. A clear pattern emerged from the discriminative variants, identifying the 217-245 helix-loop-helix motif as being a hot-spot for triglyceride recognition. This was the consequence of introducing the entire mutational load in selected regions, without putting a strain on distal parts of the protein. Our results improve our understanding of the Cal-A lipase mode of action and selectivity. This holistic perspective to protein engineering, where parts of the gene are individually mutated and the impact evaluated in the context of the whole protein, can be applied to any protein scaffold.

Integron-Associated DfrB4, a Previously Uncharacterized Member of the Trimethoprim-Resistant Dihydrofolate Reductase B Family, Is a Clinically Identified Emergent Source of Antibiotic Resistance.

Whole-genome sequencing of trimethoprim-resistant Escherichia coli clinical isolates identified a member of the trimethoprim-resistant type II dihydrofolate reductase gene family (dfrB). The dfrB4 gene was located within a class I integron flanked by multiple resistance genes. This arrangement was previously reported in a 130.6-kb multiresistance plasmid. The DfrB4 protein conferred a >2,000-fold increased trimethoprim resistance on overexpression in E. coli Our results are consistent with the finding that dfrB4 contributes to clinical trimethoprim resistance.

G-Equivalent Acceleration Tolerance in the Eutardigrade Species Hypsibius dujardini.

Tardigrades are microscopic organisms renowned for their ability to survive extreme environmental conditions. Tardigrade extreme-tolerance research has centered on the ability to withstand desiccation, low and high temperatures, and high hydrostatic pressure and radiation levels. Tardigrade tolerance to hypergravity, however, has yet to be described. We used the eutardigrade species Hypsibius dujardini to investigate short-term tolerance to g-equivalent accelerations (i.e., mimicking g-forces). Data obtained from specimens centrifuged between 3421g and 16,060g for 1 min inclusively reveal tolerance in an acceleration-dependent relation, with lower survivorship and egg production at higher accelerations. This is the first study to demonstrate tardigrade potential for tolerance to hypergravity and describe expected effects on tardigrade survival and reproduction. These findings will prove to be useful in lithopanspermia research (i.e., viable spread in meteoritic rocks). Key Words: Astrobiology-Extreme tolerance-Hypergravity-Tardigrade. Astrobiology 17, 55-60.

Insights into Laccase Engineering from Molecular Simulations: Toward a Binding-Focused Strategy.

Understanding the molecular determinants of enzyme performance is of primary importance for the rational design of ad hoc mutants. A novel approach, which combines efficient conformational sampling and quick reactivity scoring, is used here to shed light on how substrate oxidation was improved during the directed evolution experiment of a fungal laccase (from Pycnoporus cinnabarinus), an industrially relevant class of oxidoreductases. It is found that the enhanced activity of the evolved enzyme is mainly the result of substrate arrangement in the active site, with no important change in the redox potential of the T1 copper. Mutations at the active site shift the binding mode into a more buried substrate position and provide a more favorable electrostatic environment for substrate oxidation. As a consequence, engineering the binding event seems to be a viable way to in silico evolution of oxidoreductases.