Selection and enrichment of microbial species with an increased lignocellulolytic phenotype from a native soil microbiome by activity-based probing.

Multi-omic analyses can provide information on the potential for activity within a microbial community but often lack specificity to link functions to cell, primarily offer potential for function or rely on annotated databases. Functional assays are necessary for understanding in situ microbial activity to better describe and improve microbiome biology. Targeting enzyme activity through activity-based protein profiling enhances the accuracy of functional studies. Here, we introduce a pipeline of coupling activity-based probing with fluorescence-activated cell sorting, culturing, and downstream activity assays to isolate and examine viable populations of cells expressing a function of interest. We applied our approach to a soil microbiome using two activity-based probes to enrich for communities with elevated activity for lignocellulose-degradation phenotypes as determined by four fluorogenic kinetic assays. Our approach efficiently separated and identified microbial members with heightened activity for glycosyl hydrolases, and by expanding this workflow to various probes for other function, this process can be applied to unique phenotype targets of interest.

Nanobody assemblies with fully flexible topology enabled by genetically encoded tetrazine amino acids.

Assembling nanobodies (Nbs) into polyvalent multimers is a powerful strategy for improving the effectiveness of Nb-based therapeutics and biotechnological tools. However, generally effective approaches to Nb assembly are currently restricted to the amino or carboxyl termini, greatly limiting the diversity of Nb multimer topologies that can be produced. Here, we show that reactive tetrazine groups-site-specifically inserted by genetic code expansion at Nb surface sites-are compatible with Nb folding and function, enabling Nb assembly at any desired point. Using two anti-SARS-CoV-2 Nbs with viral neutralization ability, we created Nb homo- and heterodimers with improved properties compared with conventionally linked Nb homodimers, which, in the case of our tetrazine-conjugated trimer, translated into enhanced viral neutralization. Thus, this tetrazine-based approach is a generally applicable strategy that greatly increases the accessible range of Nb assembly topologies, and thereby adds the optimization of topology as an effective avenue to generate Nb assemblies with improved efficacy.

Creating a Selective Nanobody Against 3-Nitrotyrosine Containing Proteins.

A critical step in developing therapeutics for oxidative stress-related pathologies is the ability to determine which specific modified protein species are innocuous by-products of pathology and which are causative agents. To achieve this goal, technologies are needed that can identify, characterize and quantify oxidative post translational modifications (oxPTMs). Nanobodies (Nbs) represent exquisite tools for intracellular tracking of molecules due to their small size, stability and engineerability. Here, we demonstrate that it is possible to develop a selective Nb against an oxPTM protein, with the key advance being the use of genetic code expansion (GCE) to provide an efficient source of the large quantities of high-quality, homogenous and site-specific oxPTM-containing protein needed for the Nb selection process. In this proof-of-concept study, we produce a Nb selective for a 3-nitrotyrosine (nitroTyr) modified form of the 14-3-3 signaling protein with a lesser recognition of nitroTyr in other protein contexts. This advance opens the door to the GCE-facilitated development of other anti-PTM Nbs.

Overcoming Near-Cognate Suppression in a Release Factor 1-Deficient Host with an Improved Nitro-Tyrosine tRNA Synthetase.

Genetic code expansion (GCE) technologies incorporate non-canonical amino acids (ncAAs) into proteins at amber stop codons. To avoid unwanted truncated protein and improve ncAA-protein yields, genomically recoded strains of Escherichia coli lacking Release Factor 1 (RF1) are becoming increasingly popular expression hosts for GCE applications. In the absence of RF1, however, endogenous near-cognate amber suppressing tRNAs can lead to contaminating protein forms with natural amino acids in place of the ncAA. Here, we show that a second-generation amino-acyl tRNA synthetase (aaRS)/tRNACUA pair for site-specific incorporation of 3-nitro-tyrosine could not outcompete near-cognate suppression in an RF1-deficient expression host and therefore could not produce homogenously nitrated protein. To resolve this, we used Rosetta to target positions in the nitroTyr aaRS active site for improved substrate binding, and then constructed of a small library of variants to subject to standard selection protocols. The top selected variant had an ~2-fold greater efficiency, and remarkably, this relatively small improvement enabled homogeneous incorporation of nitroTyr in an RF1-deficient expression host and thus eliminates truncation issues associated with typical RF1-containing expression hosts. Structural and biochemical data suggest the aaRS efficiency improvement is based on higher affinity substrate binding. Taken together, the modest improvement in aaRS efficiency provides a large practical impact and expands our ability to study the role protein nitration plays in disease development through producing homogenous, truncation-free nitroTyr-containing protein. This work establishes Rosetta-guided design and incremental aaRS improvement as a viable and accessible path to improve GCE systems challenged by truncation and/or near-cognate suppression issues.

Genetically Encoded Protein Tyrosine Nitration in Mammalian Cells.

Tyrosine nitration has served as a major biomarker for oxidative stress and is present in high abundance in over 50 disease pathologies in humans. While data mounts on specific disease pathways from specific sites of tyrosine nitration, the role of these modifications is still largely unclear. Strategies for installing site-specific tyrosine nitration in target proteins in eukaryotic cells, through routes not dependent on oxidative stress, would provide a powerful method to address the consequences of tyrosine nitration. Developed here is a Methanosarcina barkeri aminoacyl-tRNA synthetase/tRNA pair that efficiently incorporates nitrotyrosine site-specifically into proteins in mammalian cells. We demonstrate the utility of this approach to produce nitrated proteins identified in disease conditions by producing site-specific nitroTyr-containing manganese superoxide dismutase and 14-3-3 proteins in eukaryotic cells.