Paredes Barrada, M. | Search bioRxiv

bioRxiv2026

Systematic engineering and machine learning analysis of intrinsic terminators reveal crucial nucleotides directly upstream of the terminator hairpin.

Transcriptional termination efficiency is considered an important parameter for finetuning bacterial gene expression. Still, the design principles that determine transcription termination efficiency remain poorly understood. In this study, we aimed to investigate the impact of the 3 untranslated region (3UTR) on gene expression in Escherichia coli and other bacteria. First, 3UTR variant sequences were generated, with randomized 30 bp sequences inserted between the STOP-codon and an intrinsic terminator, consisting of a GC-rich hairpin and a downstream poly(U)-tail. Using three reporter genes, it was found that different 3UTR sequences resulted in an up to five-fold difference in protein production, independent of the upstream coding sequence. The highest protein production was achieved when an adenosine was present directly upstream of the terminator hairpin. This was consolidated by systematic substitution of key nucleotides of the terminator and assessing their effect on mRNA and protein levels. Subsequently, we developed a predictive random forest machine learning model trained on the termination efficiency of different natural and synthetic terminator sequences, revealing an important role for the nucleotides directly upstream of the terminator hairpin. Altogether, this study showed that an additional adenosine nucleotide upstream of the terminator hairpin leads to improved protein production while reducing terminator read-through. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/736697v1_ufig1.gif" ALT="Figure 1"> View larger version (11K): org.highwire.dtl.DTLVardef@d8a976org.highwire.dtl.DTLVardef@5d8269org.highwire.dtl.DTLVardef@11cc3e3org.highwire.dtl.DTLVardef@180a305_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2025

Parageobacillus thermoglucosidasius and the reductive glycine pathway: A journey of Almosts and Maybes.

We aimed to engineer the reductive glycine pathway (rGlyP) in Parageobacillus thermoglucosidasius for synthetic assimilation of formate and methanol via a mixed rational and evolutionary approach. We attempted to obtain glycine or serine auxotrophic strains in P. thermoglucosidasius as a starting point to impose a selective pressure and engineer formate assimilation via the rGlyP. While serine auxotrophy was partially achieved via gene deletion of serA, alternative glycine biosynthesis routes limited its stringency, and glycine auxotrophy could not be obtained due to unsuccessful genome modification. Even though formate supplementation improved the growth of the serA deficient strain, we confirmed that formate was not assimilated into biomass via 13C-labeling experiments. Thus, we hypothesized the increase in growth of the serA deficient mutant was likely due to extra energy provided by formate oxidation, rather than biomass incorporation. We also attempted heterologous overexpression of the C1 module from Moorella thermoacetica, but this did not yield any detectable effect in growth nor formate assimilation. These findings highlight the challenges in rGlyP implementation in non-model organisms, the need for better metabolic annotation in P. thermoglucosidasius, and provide valuable insights for future engineering of synthetic C1 metabolism in thermophiles via the rGlyP.

bioengineering↗