Biology subjects

Galindo, J. L.

Publications and source records attributed to Galindo, J. L..

5 recordsLinked to original sources

bioRxiv2026

Development of a CRISPR-Cas9 genome editing system for the extremely thermophilic bacterium Anaerocellum bescii

Anaerocellum (f. Caldicellulosiruptor) bescii is an extremely thermophilic (Topt 75-78 {degrees}C) anaerobic bacterium and is also a highly efficient degrader of lignocellulose, making it a promising platform for metabolic engineering. To date, chromosomal genetic manipulations in A. bescii have required multistep procedures involving homologous recombination with integrating vectors and counterselection. Here, we demonstrate the first implementation of CRISPR-Cas9 genome editing in A. bescii facilitated by a thermostable Cas9 enzyme from Geobacillus stearothermophilus (GeoCas9). Delivery of heterologous CRISPR machinery via a replicating vector enabled us to individually and sequentially delete genes for lactate dehydrogenase (ldh) and pyrE in A. bescii. We then deployed this system to delete a novel target, tryptophan synthase (trpBA), generating a tryptophan auxotrophic strain of A. bescii. Using this strain, we show trpBA complementation enables selection and maintenance of a replicating vector expressing a heterologous enzymatic reporter gene.

microbiology↗

bioRxiv2025

A Straightforward and Robust Enzymatic Reporter System for Anaerobic Thermophiles

Thermophilic anaerobic organisms, particularly species that can naturally degrade lignocellulosic biomass, show great promise for next generation bioprocessing. This has led to the development of nascent genetic systems to metabolically engineer these non-model organisms. However, a major challenge remains a lack of reliable reporter systems compatible with the combination of thermophilic and anaerobic growth conditions. Additionally, native glycoside hydrolases in these organisms limit the usefulness of traditional glycosidic enzyme reporters (e.g. LacZ) because of the native background activity present on para-nitrophenyl glucoside substrates. Here we describe the development of a straightforward and robust enzymatic reporter system that overcomes these challenges in Anaerocellum (f. Caldicellulosiruptor) bescii, an anaerobic, extremely thermophilic (Topt [~]78 {degrees}C), lignocellulolytic bacterium. Our method is based on heterologous expression of hyperthermophilic archaeal galactosidases: an -galactosidase from Pyroccous furiosus (Pfgal), and a {beta}-galactosidase from Caldivirga maquilingensis (Cm{beta}gal). We show that these reporters produce strong, orthogonal signals on colorimetric substrates at high temperatures ([≥]90{degrees}C) that eliminate background activity from endogenous galactosidases. We then demonstrate the capability of Cm{beta}gal, the stronger of the two reporters, to distinguish differences in levels of expression between A. bescii promoter sequences, which we verify through qRT-PCR. With its high signal to noise ratio and ease of use, this reporter system offers a reliable method for assessing protein expression in anaerobic thermophilic organisms, opening doors to improved genetic tools and metabolic engineering applications for industrial biotechnology.

synthetic biology↗

bioRxiv2025

Functional and structural characterization of AtAbf43C: An exo-1,5-α-L-arabinofuranosidase from Acetivibrio thermocellus DSM1313

The Acetivibrio thermocellus DSM1313 genome codes for seven predicted glycoside hydrolase family 43 (GH43) enzymes, four of which remain uncharacterized. This study describes the function and structure of one such enzyme, AtAbf43C, from GH43 subfamily 26 (GH43_26) which acts as an -L-arabinofuranosidase (EC 3.2.1.55). AtAbf43C is active on para-nitrophenol--L-arabinofuranoside (pNPAra), with optimal activity observed at pH 5.5 and 65 . Multiple crystal structures of AtAbf43C were obtained, in which an N-terminal carbohydrate binding module family 42 (CBM42) domain displays a {beta}-trefoil type fold and the C-terminal GH43 domain displays a canonical 5-bladed {beta}-propeller motif. One structure, which was solved with two L-arabinofuranose molecules bound to {beta}- and {gamma}-subdomains of the CBM42, builds upon previous literature suggesting the -binding pocket of the AtAbf43C CBM42 is non-functional. Furthermore, structural alignment with the substrate bound structure of a closely homologous GH43_26 exo--1,5-arabinofuranosidase, SaAraf43A from Streptomyces avermitilis (PDB 3AKH), allowed for identification of the conserved catalytic triad via site-directed mutagenesis in AtAbf43C, as well as insight into the deep-narrow topology of the AtAbf43C binding pocket that suggested it would be active on similar arabino-oligosaccharide (AOS) substrates as SaAraf43A. Subsequent liquid chromatography-mass spectrometry (LC-MS) analysis of polysaccharides and oligosaccharides hydrolyzed by AtAbf43C provides experimental evidence confirming this enzyme acts in an exo manner primarily towards -1,5 linked arabino-oligosaccharides.

molecular biology↗

bioRxiv2023

Novel and improved Caenorhabditis briggsae gene models generated by community curation

BackgroundThe nematode Caenorhabditis briggsae has been used as a model for genomics studies compared to Caenorhabditis elegans because of its striking morphological and behavioral similarities. These studies yielded numerous findings that have expanded our understanding of nematode development and evolution. However, the potential of C. briggsae to study nematode biology is limited by the quality of its genome resources. The reference genome and gene models for the C. briggsae laboratory strain AF16 have not been developed to the same extent as C. elegans. The recent publication of a new chromosome-level reference genome for QX1410, a C. briggsae wild strain closely related to AF16, has provided the first step to bridge the gap between C. elegans and C. briggsae genome resources. Currently, the QX1410 gene models consist of protein-coding gene predictions generated from short- and long-read transcriptomic data. Because of the limitations of gene prediction software, the existing gene models for QX1410 contain numerous errors in their structure and coding sequences. In this study, a team of researchers manually inspected over 21,000 software-derived gene models and underlying transcriptomic data to improve the protein-coding gene models of the C. briggsae QX1410 genome. ResultsWe designed a detailed workflow to train a team of nine students to manually curate genes using RNA read alignments and predicted gene models. We manually inspected the gene models using the genome annotation editor, Apollo, and proposed corrections to the coding sequences of over 8,000 genes. Additionally, we modeled thousands of putative isoforms and untranslated regions. We exploited the conservation of protein sequence length between C. briggsae and C. elegans to quantify the improvement in protein-coding gene model quality before and after curation. Manual curation led to a substantial improvement in the protein sequence length accuracy of QX1410 genes. We also compared the curated QX1410 gene models against the existing AF16 gene models. The manual curation efforts yielded QX1410 gene models that are similar in quality to the extensively curated AF16 gene models in terms of protein-length accuracy and biological completeness scores. Collinear alignment analysis between the QX1410 and AF16 genomes revealed over 1,800 genes affected by spurious duplications and inversions in the AF16 genome that are now resolved in the QX1410 genome. ConclusionsCommunity-based, manual curation using transcriptome data is an effective approach to improve the quality of software-derived protein-coding genes. Comparative genomic analysis using a related species with high-quality reference genome(s) and gene models can be used to quantify improvements in gene model quality in a newly sequenced genome. The detailed protocols provided in this work can be useful for future large-scale manual curation projects in other species. The chromosome-level reference genome for the C. briggsae strain QX1410 far surpasses the quality of the genome of the laboratory strain AF16, and our manual curation efforts have brought the QX1410 gene models to a comparable level of quality to the previous reference, AF16. The improved genome resources for C. briggsae provide reliable tools for the study of Caenorhabditis biology and other related nematodes.

genomics↗

bioRxiv2022

Orthogonal tRNA Expression using Endogenous Machinery in Cell-Free Systems

A wide variety of non-canonical amino acids (ncAAs) can be incorporated into proteins through the coordinated action of a stop codon suppressing tRNA and aminoacyl-tRNA synthetase. However, methods to discover and characterize suppressor tRNAs are generally lacking. In this work, we show that cell-free systems can express functional suppressor tRNAs using endogenous machinery and characterize their activity. This method is compatible with widely used orthogonal tRNAs, such as the Methanocaldococus jannaschii tyrosyl tRNA, the Methanosarcina barkeri pyrrolysyl tRNA, the Methanomethylophilus Alvus pyrrolysyl tRNA, and an engineered Int pyrroysyl tRNA. Modifying the workflow to evaluate TAA suppression revealed that the M. jannaschii and M. alvus are highly functional TAA suppressors in cell-free systems. Finally, we show that we can express two distinct tRNAs simultaneously, enabling the incorporation of multiple, distinct ncAAs. In total, our work shows that cell-free systems are useful platforms to express and characterize tRNAs.

synthetic biology↗