Ryan, V. | Search bioRxiv

bioRxiv2024

Engineering a probiotic Bacillus subtilis for acetaldehyde removal: A hag locus integration to robustly express acetaldehyde dehydrogenase

We have identified solutions to key challenges in probiotic design to create a commercially viable strain for the removal of the intestinal toxin acetaldehyde. Here we report engineering of a {sigma}D-dependent flagellin expression locus, hag, as a stable location for robust enzyme production. We demonstrate constitutive gene expression in relevant conditions driven by the endogenous hag promoter following a deletion of the gene encoding a post-translational regulator of {sigma}D, FlgM, and a point mutation to abrogate the binding of the translational inhibitor CsrA. Reporter constructs demonstrate hag locus activity after germination and show a steady increase in heterologous expression throughout outgrowth and resumption of vegetative growth. To test the chassis as a spore-based probiotic solution we identified the physiologically relevant pathway of ethanol metabolism and the buildup of gut-derived acetaldehyde after alcohol consumption. Herein, we describe the integration of a robustly expressed Cupriavidus necator aldehyde dehydrogenase, AcoD, under a flagellin protein promoter at the hag locus and report the rapid reduction of acetaldehyde levels after germination in gut simulated conditions.

microbiology↗

bioRxiv2023

Mis-spliced transcripts generate de novo proteins in TDP-43-related ALS/FTD

Functional loss of TDP-43, an RNA-binding protein genetically and pathologically linked to ALS and FTD, leads to inclusion of cryptic exons in hundreds of transcripts during disease. Cryptic exons can promote degradation of affected transcripts, deleteriously altering cellular function through loss-of-function mechanisms. However, the possibility of de novo protein synthesis from cryptic exon transcripts has not been explored. Here, we show that mRNA transcripts harboring cryptic exons generate de novo proteins both in TDP-43 deficient cellular models and in disease. Using coordinated transcriptomic and proteomic studies of TDP-43 depleted iPSC-derived neurons, we identified numerous peptides that mapped to cryptic exons. Cryptic exons identified in iPSC models were highly predictive of cryptic exons expressed in brains of patients with TDP-43 proteinopathy, including cryptic transcripts that generated de novo proteins. We discovered that inclusion of cryptic peptide sequences in proteins altered their interactions with other proteins, thereby likely altering their function. Finally, we showed that these de novo peptides were present in CSF from patients with ALS. The demonstration of cryptic exon translation suggests new mechanisms for ALS pathophysiology downstream of TDP-43 dysfunction and may provide a strategy for novel biomarker development. One Sentence SummaryLoss of TDP-43 function results in the expression of de novo proteins from mis-spliced mRNA transcripts.

neuroscience↗