Thermostable in vitro transcription-translation compatible with microfluidic droplets.

Background: In vitro expression involves the utilization of the cellular transcription and translation machinery in an acellular context to produce one or more proteins of interest and has found widespread application in synthetic biology and in pharmaceutical biomanufacturing. Most in vitro expression systems available are active at moderate temperatures, but to screen large libraries of natural or artificial genetic diversity for highly thermostable enzymes or enzyme variants, it is instrumental to enable protein synthesis at high temperatures.

Objectives: Develop an in vitro expression system operating at high temperatures compatible with enzymatic assays and with technologies that enable ultrahigh-throughput protein expression in reduced volumes, such as microfluidic water-in-oil (w/o) droplets.

Stabilization of Enzymes by Using Thermophiles.

Manufactured steroid compounds have many applications in the pharmaceutical industry. Due to the chemical complexity and chirality of steroids, there is an increasing demand for enzyme-based bioconversion processes to replace those based on chemical synthesis. In this context, thermostability of the involved enzymes is a highly desirable property as both the increased half-life of the enzyme and the enhanced solubility of substrates and products will improve the yield of the reactions.

From accurate genome sequence to biotechnological application: The thermophile Mycolicibacterium hassiacum as experimental model.

Mycobacteria constitute a large group of microorganisms belonging to the phylum Actinobacteria encompassing some of the most relevant pathogenic bacteria and many saprophytic isolates that share a unique and complex cell envelope. Also unique to this group is the extensive capability to use and synthesize sterols, a class of molecules that include active signalling compounds of pharmaceutical use. However, few mycobacterial species and strains have been established as laboratory models to date, Mycolicibacterium smegmatis mc 155 being the most common one.

A Coupled Ketoreductase-Diaphorase Assay for the Detection of Polyethylene Terephthalate-Hydrolyzing Activity.

In the last two decades, several PET-degrading enzymes from already known microorganisms or metagenomic sources have been discovered to face the growing environmental concern of polyethylene terephthalate (PET) accumulation. However, there is a limited number of high-throughput screening protocols for PET-hydrolyzing activity that avoid the use of surrogate substrates. Herein, a microplate fluorescence screening assay was described.

Flow-based method for biofilm microbiota enrichment and exploration of metagenomes.

Most bacteria live in biofilms in their natural habitat rather than the planktonic cell stage that dominates during traditional laboratory cultivation and enrichment schemes. The present study describes the establishment of a flow-based enrichment method based on multispecies biofilm communities for directing biofilm functionality using an environmental inoculum. By controlling flow conditions and physio-chemical properties, the set-up aims to simulate natural conditions ex situ for biofilm formation.

Ultrahigh-Throughput Screening of Metagenomic Libraries Using Droplet Microfluidics.

Droplet microfluidics enables the ultrahigh-throughput screening of the natural or man-made genetic diversity for industrial enzymes, with reduced reagent consumption and lower costs than conventional robotic alternatives. Here we describe an example of metagenomic screening for nucleoside 2'-deoxyribosyl transferases using FACS as a more widespread and accessible alternative than microfluidic on-chip sorters. This protocol can be easily adapted to directed evolution libraries by replacing the library construction steps and to other enzyme activities, e.

Thermostability Engineering of a Class II Pyruvate Aldolase from by Folding Interference.

The use of enzymes in industrial processes is often limited by the unavailability of biocatalysts with prolonged stability. Thermostable enzymes allow increased process temperature and thus higher substrate and product solubility, reuse of expensive biocatalysts, resistance against organic solvents, and better "evolvability" of enzymes. In this work, we have used an activity-independent method for the selection of thermostable variants of any protein in through folding interference at high temperature of a thermostable antibiotic reporter protein at the C-terminus of a fusion protein.

Biochemical and Structural Characterization of a novel thermophilic esterase EstD11 provide catalytic insights for the HSL family.

A novel esterase, EstD11, has been discovered in a hot spring metagenomic library. It is a thermophilic and thermostable esterase with an optimum temperature of 60°C. A detailed substrate preference analysis of EstD11 was done using a library of chromogenic ester substrate that revealed the broad substrate specificity of EstD11 with significant measurable activity against 16 substrates with varied chain length, steric hindrance, aromaticity and flexibility of the linker between the carboxyl and the alcohol moiety of the ester.

Insights into the molecular determinants of thermal stability in halohydrin dehalogenase HheD2.

Halohydrin dehalogenases (HHDHs) are promising enzymes for application in biocatalysis due to their promiscuous epoxide ring-opening activity with various anionic nucleophiles. So far, seven different HHDH subtypes A to G have been reported with subtype D containing the by far largest number of enzymes. Moreover, several characterized members of subtype D have been reported to display outstanding characteristics such as high catalytic activity, broad substrate spectra or remarkable thermal stability.

Hypoxanthine-Guanine Phosphoribosyltransferase/adenylate Kinase From : A Bifunctional Catalyst for the Synthesis of Nucleoside-5'-Mono-, Di- and Triphosphates.

In our search for novel biocatalysts for the synthesis of nucleic acid derivatives, we found a good candidate in a putative dual-domain hypoxanthine-guanine phosphoribosyltransferase (HGPRT)/adenylate kinase (AMPK) from (HGPRT/AMPK). In this respect, we report for the first time the recombinant expression, production, and characterization of a bifunctional HGPRT/AMPK. Biochemical characterization of the recombinant protein indicates that the enzyme is a homodimer, with high activity in the pH range 6-7 and in a temperature interval from 30 to 80°C.

Intraparticle pH Sensing Within Immobilized Enzymes: Immobilized Yellow Fluorescent Protein as Optical Sensor for Spatiotemporal Mapping of pH Inside Porous Particles.

pH is a fundamental variable in enzyme catalysis and its measurement therefore is crucial for understanding and optimizing enzyme-catalyzed reactions. Whereas measurements within homogeneous bulk liquid solution are prominently used, enzymes immobilized inside porous particles often suffer from pH gradients due to partition effects and heterogeneously catalyzed biochemical reactions. Unfortunately, the measurements of intraparticle pH are not available due to the lack of useful suitable methodologies; as a consequence the biocatalyst characterization is hampered.

Stabilization of Multimeric Enzymes via Immobilization and Further Cross-Linking with Aldehyde-Dextran.

Subunit dissociation of multimeric proteins is one of the most important causes of inactivation of proteins having quaternary structure, making these proteins very unstable under diluted conditions. A sequential two-step protocol for the stabilization of this protein is proposed. A multisubunit covalent immobilization may be achieved by performing very long immobilization processes between multimeric enzymes and porous supports composed of large internal surfaces and covered by a very dense layer of reactive groups.

Functional Characterization and Structural Analysis of NADH Oxidase Mutants from HB27: Role of Residues 166, 174, and 194 in the Catalytic Properties and Thermostability.

The strain HB27 NADH-oxidase (Tt27-NOX) catalyzes the oxidation of nicotinamide adenine dinucleotide (NAD(P)H) by reducing molecular oxygen to hydrogen peroxide in a two-electron transfer mechanism. Surprisingly, Tt27-NOX showed significant differences in catalytic properties compared to its counterpart from the strain HB8 (Tt8-NOX), despite a high degree of sequence homology between both variants. The sequence comparison between both enzymes revealed only three divergent amino acid residues at positions 166, 174, and 194.

Thermostability enhancement of the Pseudomonas fluorescens esterase I by in vivo folding selection in Thermus thermophilus.

Prolonged stability is a desired property for the biotechnological application of enzymes since it allows its reutilization, contributing to making biocatalytic processes more economically competitive with respect to chemical synthesis. In this study, we have applied selection by folding interference at high temperature in Thermus thermophilus to obtain thermostable variants of the esterase I from Pseudomonas fluorescens (PFEI). The most thermostable variant (Q11L/A191S) showed a melting temperature (T ) of 77.

A Modular Vector Toolkit with a Tailored Set of Thermosensors To Regulate Gene Expression in .

Modular plasmid architectures have shown to be a very useful resource to standardize, build, share, and compare biological parts and functional vectors, and are being applied in an increasing number of microorganisms. Here, we present a modular plasmid toolkit for , a species considered as a workhorse for biotechnology and a model for high-temperature biology. Apart from integrating improved versions of already existing parts, we have characterized specific promoters and developed a thermosensor-based palette that restricts the expression to and, at the same time, controls protein expression in this organism in a temperature-dependent manner.

Biobased, Internally pH-Sensitive Materials: Immobilized Yellow Fluorescent Protein as an Optical Sensor for Spatiotemporal Mapping of pH Inside Porous Matrices.

The pH is fundamental to biological function and its measurement therefore crucial across all biosciences. Unlike homogenous bulk solution, solids often feature internal pH gradients due to partition effects and confined biochemical reactions. Thus, a full spatiotemporal mapping for pH characterization in solid materials with biological systems embedded in them is essential.

A Brief Guide to the High-Throughput Expression of Directed Evolution Libraries.

The process of protein production optimization requires time and labor, constituting one of the main bottlenecks for the downstream utilization of the proteins. However, once through this bottleneck, the protein production process can be easily standardized and multiplexed to find the fittest variants in large libraries created by random mutagenesis. In this chapter, we present an overview of the most important choices to achieve homogeneous and functional expression of directed evolution libraries in microplate format: (1) choice of induction system and host strain, (2) choice of media and growth conditions, and (3) modifications to the genetic sequence.

Stabilization of Enzymes by Using Thermophiles.

Manufactured steroid compounds have many applications in the pharmaceutical industry. Due to the chemical complexity and chirality of steroids, there is an increasing demand for enzyme-based bioconversion processes to replace those based on chemical synthesis. In this context, thermostability of the involved enzymes is a highly desirable property as both the increased half-life of the enzyme and the enhanced solubility of substrates and products will improve the yield of the reactions.

Are in vivo selections on the path to extinction?

Droplet microfluidics will become a disruptive technology in the field of library screening and replace biological selections if the central dogma of biology and other processes are successfully implemented within microdroplets.

A novel thermostable protein-tag: optimization of the Sulfolobus solfataricus DNA- alkyl-transferase by protein engineering.

In the last decade, a powerful biotechnological tool for the in vivo and in vitro specific labeling of proteins (SNAP-tag™ technology) was proposed as a valid alternative to classical protein-tags (green fluorescent proteins, GFPs). This was made possible by the discovery of the irreversible reaction of the human alkylguanine-DNA-alkyl-transferase (hAGT) in the presence of benzyl-guanine derivatives. However, the mild reaction conditions and the general instability of the mesophilic SNAP-tag™ make this new approach not fully applicable to (hyper-)thermophilic and, in general, extremophilic organisms.

One-pot Simple methodology for CAssette Randomization and Recombination for focused directed evolution (OSCARR).

The OSCARR methodology (One-pot Simple methodology for CAssette Randomization and Recombination) bridges the gap between site-directed mutagenesis and full randomization by making use of carefully designed mutagenic cassettes and an optimized one-pot megaprimer PCR. The method is especially suited to construct libraries of up to ten randomized codons for focused directed evolution, exhibits up to 97 % efficiency in the amplification of mutated over wild-type products, and is sufficiently versatile to allow mutagenesis and recombination of several cassettes within the same gene.

Parallel pathways for nitrite reduction during anaerobic growth in Thermus thermophilus.

Respiratory reduction of nitrate and nitrite is encoded in Thermus thermophilus by the respective transferable gene clusters. Nitrate is reduced by a heterotetrameric nitrate reductase (Nar) encoded along transporters and regulatory signal transduction systems within the nitrate respiration conjugative element (NCE). The nitrite respiration cluster (nic) encodes homologues of nitrite reductase (Nir) and nitric oxide reductase (Nor).

Transferable denitrification capability of Thermus thermophilus.

Laboratory-adapted strains of Thermus spp. have been shown to require oxygen for growth, including the model strains T. thermophilus HB27 and HB8.

Engineering the substrate specificity of a thermophilic penicillin acylase from thermus thermophilus.

A homologue of the Escherichia coli penicillin acylase is encoded in the genomes of several thermophiles, including in different Thermus thermophilus strains. Although the natural substrate of this enzyme is not known, this acylase shows a marked preference for penicillin K over penicillin G. Three-dimensional models were created in which the catalytic residues and the substrate binding pocket were identified.