Artificial (beta / alpha)8 barrel enzymes by in vitro evolution

Natural evolution has yielded countless enzymes of the (β/α)8 barrel fold (TIM barrel). This versatile fold is highly favored by natural enzymes to catalyze a wide array of reactions and appears in five of the six enzymatic classes. Therefore, the (β/α)8 barrel fold is an attractive starting point for enzyme engineering. Several examples of modified TIM barrel enzymes have been reported that accept different substrates or even catalyze a different reaction. However, engineering new activities into the (β/α)8 barrel fold is apparently still far more challenging than its ubiquitous role in nature suggests. Please click Additional Files below to see the full abstract

Nature-inspired engineering of an artificial RNA ligase created by in vitro selection

Please click Additional Files below to see the full abstrac

Cell Survival Enabled by Leakage of a Labile Metabolic Intermediate

Many metabolites are generated in one step of a biochemical pathway and consumed in a subsequent step. Such metabolic intermediates are often reactive molecules which, if allowed to freely diffuse in the intracellular milieu, could lead to undesirable side reactions and even become toxic to the cell. Therefore, metabolic intermediates are often protected as protein-bound species and directly transferred between enzyme active sites in multi-functional enzymes, multi-enzyme complexes, and metabolons. Sequestration of reactive metabolic intermediates thus contributes to metabolic efficiency. It is not known, however, whether this evolutionary adaptation can be relaxed in response to challenges to organismal survival. Here, we report evolutionary repair experiments on Escherichia coli cells in which an enzyme crucial for the biosynthesis of proline has been deleted. The deletion makes cells unable to grow in a culture medium lacking proline. Remarkably, however, cell growth is efficiently restored by many single mutations (12 at least) in the gene of glutamine synthetase. The mutations cause the leakage to the intracellular milieu of a highly reactive phosphorylated intermediate common to the biosynthetic pathways of glutamine and proline. This intermediate is generally assumed to exist only as a protein-bound species. Nevertheless, its diffusion upon mutation-induced leakage enables a new route to proline biosynthesis. Our results support that leakage of sequestered metabolic intermediates can readily occur and contribute to organismal adaptation in some scenarios. Enhanced availability of reactive molecules may enable the generation of new biochemical pathways and the potential of mutation-induced leakage in metabolic engineering is noted.This work was supported by Human Frontier Science Program Grant RGP0041/2017 (J.M.S.R. and B.S.), Spanish Ministry of Science and Innovation FEDER Funds Grants RTI2018-097142-B-100 and PID2021-124534OB-100 (J.M.S.R.), National Aeronautics and Space Administration (NASA) Grant 80NSSC18K1277 (B.S.) Grant RYC2021-031155-I (E.M.C.) from Spanish Ministry of Science and Innovation and NextGenerationEU/PRTR and Grant E-BIO-464-UGR-20 (E.M.C.) from FEDER Funds and Consejería de Economía, Conocimiento, Empresas y Universidad de la Junta de Andalucia. E.A.L. was a recipient of a postdoctoral fellowship from the regional Andalusian Government (2020_DOC_00541). We thank the “Centro de Supercomputacion” (ALHAMBRA-CSIRC) of the University of Granada for providing computational resources and Dr Valeria A. Risso for useful discussions, comments on the manuscript and help with the evolutionary and structural analyses of the rescuing mutations.Peer reviewe

Facile immobilization of pyridoxal 5′-phosphate using p-diazobenzoyl-derivatized Sepharose 4B

Pyridoxal 5′-phosphate (PLP) is a ubiquitous and versatile cofactor utilized by numerous enzymes involved in amino acid biosynthetic pathways. Immobilized PLP is a valuable tool to isolate unknown PLP-dependent enzymes in nature or to perform in vitro selection or directed evolution on existing or de novo PLP-dependent enzymes. The C-6 position is preferred for covalent immobilization of PLP because it maintains all important functional groups in their native, unmodified form. Previously reported diazonium derivatization methods for C-6 immobilization utilized an azide linker compound that is hazardous and not readily available. Here we report a safer and more accessible method to synthesize p-diazobenzoyl-derivatized Sepharose 4B using the N-hydroxysuccinimide (NHS) ester chemistry. The derivative was used to immobilize PLP, and the resulting C-6 immobilized PLP had a loading of ∼2.6 µmol PLP per mL of resin, comparable to commercially available products of other immobilized cofactors

EasyDIVER: A Pipeline for Assembling and Counting High-Throughput Sequencing Data from In Vitro Evolution of Nucleic Acids or Peptides.

In vitro evolution is a well-established technique for the discovery of functional RNA and peptides. Increasingly, these experiments are analyzed by high-throughput sequencing (HTS) for both scientific and engineering objectives, but computational analysis of HTS data, particularly for peptide selections, can present a barrier to entry for experimentalists. We introduce EasyDIVER (Easy pre-processing and Dereplication of In Vitro Evolution Reads), a simple, user-friendly pipeline for processing high-throughput sequencing data from in vitro selections and directed evolution experiments. The pipeline takes as input raw, paired-end, demultiplexed Illumina read files. For each sample provided, EasyDIVER outputs a dereplicated list of unique nucleic acid and/or peptide sequences and their count reads

Thermostable Artificial Enzyme Isolated by <i>In Vitro</i> Selection

<div><p>Artificial enzymes hold the potential to catalyze valuable reactions not observed in nature. One approach to build artificial enzymes introduces mutations into an existing protein scaffold to enable a new catalytic activity. This process commonly results in a simultaneous reduction of protein stability as an undesired side effect. While protein stability can be increased through techniques like directed evolution, care needs to be taken that added stability, conversely, does not sacrifice the desired activity of the enzyme. Ideally, enzymatic activity and protein stability are engineered simultaneously to ensure that stable enzymes with the desired catalytic properties are isolated. Here, we present the use of the <i>in vitro</i> selection technique mRNA display to isolate enzymes with improved stability and activity in a single step. Starting with a library of artificial RNA ligase enzymes that were previously isolated at ambient temperature and were therefore mostly mesophilic, we selected for thermostable active enzyme variants by performing the selection step at 65°C. The most efficient enzyme, ligase 10C, was not only active at 65°C, but was also an order of magnitude more active at room temperature compared to related enzymes previously isolated at ambient temperature. Concurrently, the melting temperature of ligase 10C increased by 35 degrees compared to these related enzymes. While low stability and solubility of the previously selected enzymes prevented a structural characterization, the improved properties of the heat-stable ligase 10C finally allowed us to solve the three-dimensional structure by NMR. This artificial enzyme adopted an entirely novel fold that has not been seen in nature, which was published elsewhere. These results highlight the versatility of the <i>in vitro</i> selection technique mRNA display as a powerful method for the isolation of thermostable novel enzymes.</p></div

High throughput sequencing of in vitro selections of mRNA-displayed peptides: data analysis and applications.

In vitro selection using mRNA display is currently a widely used method to isolate functional peptides with desired properties. The analysis of high throughput sequencing (HTS) data from in vitro evolution experiments has proven to be a powerful technique but only recently has it been applied to mRNA display selections. In this Perspective, we introduce aspects of mRNA display and HTS that may be of interest to physical chemists. We highlight the potential of HTS to analyze in vitro selections of peptides and review recent advances in the application of HTS analysis to mRNA display experiments. We discuss some possible issues involved with HTS analysis and summarize some strategies to alleviate them. Finally, the potential for future impact of advancing HTS analysis on mRNA display experiments is discussed

High throughput sequencing of in vitro selections of mRNA-displayed peptides: data analysis and applications.

In vitro selection using mRNA display is currently a widely used method to isolate functional peptides with desired properties. The analysis of high throughput sequencing (HTS) data from in vitro evolution experiments has proven to be a powerful technique but only recently has it been applied to mRNA display selections. In this Perspective, we introduce aspects of mRNA display and HTS that may be of interest to physical chemists. We highlight the potential of HTS to analyze in vitro selections of peptides and review recent advances in the application of HTS analysis to mRNA display experiments. We discuss some possible issues involved with HTS analysis and summarize some strategies to alleviate them. Finally, the potential for future impact of advancing HTS analysis on mRNA display experiments is discussed

<i>In vitro</i> selection of artificial ligase enzymes with increased stability.

<p>(<b>A</b>) Schematic of the isolation of ligase enzymes. The DNA library encodes the library of proteins that resulted from the original selection of ligase enzymes at 23°C <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112028#pone.0112028-Seelig1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112028#pone.0112028-Seelig2" target="_blank">[22]</a>. The DNA is transcribed into RNA, modified with puromycin at the 3′-end and translated <i>in vitro</i> yielding a library of mRNA-displayed proteins <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112028#pone.0112028-Seelig2" target="_blank">[22]</a>. Reverse transcription with a primer containing one RNA substrate shown in red results in a complex of protein, mRNA, cDNA and substrate. This complex is incubated at 65°C with the second RNA substrate (red) and the complementary splint as highlighted in the orange box. The cDNA of ligases active at this temperature is immobilized on streptavidin beads and amplified for subsequent rounds of selection, or identified by cloning and sequencing. (<b>B</b>) Detailed view of ligation reaction substrates in complex with the mRNA-displayed protein. The two strands of RNA in red, the 5′-triphosphate RNA (PPP-substrate) and 3′-hydroxyl RNA (HO-substrate), are joined in a template-dependent ligation reaction. The PPP-substrate is part of the reverse transcription primer. The photocleavable site (PC) is used to release the cDNA that encodes active enzymes from streptavidin immobilization by irradiation at 365 nm. The splint acts as template of the ligation and base pairs with 8 nucleotides of each RNA substrate during the previously published selection at 23°C <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112028#pone.0112028-Seelig1" target="_blank">[17]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112028#pone.0112028-Seelig2" target="_blank">[22]</a>, and with (<b>C</b>) 20 nucleotides of each substrate during the current selection at 65°C. HEG₄ represents the linker of four hexaethylene glycol units (red wavy line).</p