RMH

Antisense oligonucleotides (ASOs) are a powerful class of drugs with the potential to treat a wide range of human diseases. However, the prediction of ASO efficacy remains challenging, as large-scale and costly experimental screens are typically required to identify optimal candidates for a specific therapeutic target. To address this challenge, we compiled ASO Atlas, a database comprising 188,521 RNase H-mediated ASO sequences targeting 334 unique genes with corresponding knockdown efficacy measurements extracted from published patents. Using ASO Atlas, we trained OligoAI, a deep learning model capable of jointly modelling RNA target context, ASO sequence, sugar and backbone chemistries, and dosage to predict in vitro efficacy. We experimentally validated OligoAI by targeting KCNT2, achieving a 5.72-fold reduction in screening effort compared to random selection. ASO Atlas provides the first systematic resource to rigorously evaluate hypotheses regarding key parameters in ASO design, including sequence composition, chemical modifications, and target region selection. Both ASO Atlas and OligoAI have been made freely accessible through an online web-tool with the aim of facilitating the accelerated optimisation of ASO design.

The Wood–Ljungdahl (WL) pathway, which is widely distributed in both archaea and bacteria, is an ancient carbon fixation pathway from CO2. CO2 fixation proceeds via two branches of pathway: progressive reduction to the methyl group in the methyl branch and one-step reduction to CO in the carbonyl branch. In the final step of the pathway, the methyl group, CO, and CoA are combined into a carbon monoxide dehydrogenase (CODH)/acetyl-CoA synthase (ACS) complex to form acetyl-CoA. Here, we show direct CO fixation to the carbonyl group of acetyl-CoA in both archaeal and bacterial WL pathways under hydrogenogenic growth conditions using 13C tracer-based metabolomics. A combination of metabolomics and proteomics suggested that the hydrogenotrophically grown Thermodesulfatator indicus and Archaeoglobus sp. strain MCR cells, directly fixed CO using free-form ACS in the carbonyl branch with relatively low CO availability. In contrast, carboxydotrophically grown Archaeoglobus cells utilize the CODH/ACS complex for CO2 fixation rather than CO fixation. Direct CO fixation by free-form ACS is more advantageous for conserving reduced ferredoxin compared with the thermodynamically challenged CO2 reduction by CODH. These findings provide further insight into the origin and evolution of the most ancient inorganic carbon fixation pathway and geochemical cycles on early Earth.

With the development of synthetic biology and biotechnology, chassis engineering has become the main means of industrial protein production, but it has been limited by the lack of efficient gene editing methods and effective engineering strategies. Bacillus amyloliquefaciens shows potential for expressing heterologous proteins, but its cells undergo early autolysis, hindering further application. In this study, an autolysis-related prophage gene cluster was rationally deleted by establishing an efficient CRISPR-nCas9 editing process, and the prophage mutant strain was constructed, which prevented cell lysis. Based on the prophage mutant strain, we screened secondary metabolite biosynthetic gene clusters that hindered the expression of heterologous proteins, and we made reasonable deletions to further improve their efficient expression. Finally, an optimized yield of acid-stable α amylase (2,46,089.21 U/mL) was obtained in a 5-L fed-batch fermentation. Therefore, we successfully constructed an ideal candidate strain for the expression of heterologous proteins, which provides an important research basis for the development of more chassis strains.

The lack of robust genetic tools has hindered the precise engineering of Zymomonas mobilis, a key organism for the bioproduction of many commodity chemicals. Here, we developed a library of synthetic promoters with a wide range of expression, not described in Z. mobilis to date, strong transcriptional terminators to enable fine transcriptional control, and bicistronic designs (BCDs) for predictable translation across diverse genetic contexts. Promoter activity in Z. mobilis was shown to be unaffected by oxygen availability, indicating stable performance under both aerobic and anaerobic conditions. The characterised molecular tools were made compatible with Start-Stop assembly, a high-throughput combinatorial assembly method, and their functionality demonstrated with the rapid construction of a library of plasmids expressing a 2,3-butanediol biosynthetic pathway. Together, these molecular tools lay the foundation for the high-resolution and predictable engineering of multi-enzyme pathways in Z. mobilis. 

Trehalose is a widely prevalent, abundant disaccharide that acts as a cellular stress protectant, and functions as an energy source that enters central carbon metabolism when broken down. The evolution and distribution of trehalose breakdown pathways across kingdoms of life have not been studied, and therefore the ability of different organisms to consume trehalose as a carbon source is unknown. In this study, we build a comprehensive evolutionary analysis of the four known trehalose breakdown pathways - trehalase (acid, neutral, glycosyl hydrolase 15), trehalose phosphorylases (TP, treP), and trehalose specific phosphotransferases (PTS), by studying their distributions across ~4000 prokaryotic and eukaryotic genomes. Our study suggests the presence of trehalase in the Last Eukaryotic Common Ancestor (LECA), and reveals near-universal presence of trehalase in eukaryotes, except in all birds where trehalase was lost in the first bird ancestor. Fungi alone retain additional trehalose phosphorylases (TP) in addition to trehalase. In contrast, trehalose breakdown in prokaryotes is highly sporadic but can occur via multiple, independently evolved pathways, including trehalase, the trehalose-specific PTS and trehalose phosphorylase. Finally, we observe that a subset of fast-growing Gammaproteobacteria retain the trehalose specific PTS, the loss of which reduces growth in Escherichia coli. Overall, our findings uncover the evolutionary landscape of trehalose breakdown, and use of this versatile disaccharide as an energy reserve in different kingdoms of life.

Microbial chain elongation via reverse β-oxidation offers a more sustainable route to produce medium-chain fatty acids like caproate, commodity chemicals typically produced via (petro)chemical processes. Thermophilic anaerobic microbiomes allow production at a high rate and selectivity but remain poorly understood due to the limited cultivability of their members. To better access functional taxa from a thermophilic chain-elongating reactor community, we applied multiple isolation strategies: conventional anaerobic plating, dilution-to-extinction (DTE), droplet-based microfluidics, and fluorescence-activated cell sorting (FACS). We evaluated the taxonomic range and cultivation success of each method using 16S rRNA gene sequencing. Each method yielded a distinct subset of microbial taxa. While Clostridium acetireducens-related strains were consistently isolated across all strategies, key thermophilic chain elongators (e.g., Thermocaproicibacter melissae-like organisms) only appeared in DTE. Droplet microfluidics enriched the most unique taxa in total, mostly rare taxa, including Caproicibacter and Thermoanaerobacterium spp. Plating yielded the lowest diversity, recovering only dominant taxa. FACS-based approaches failed to yield isolates, likely due to stress during processing. Comparing droplet-based isolation to DTE revealed critical insights: although droplets offer higher throughput, which intrinsically increases the chance of capturing rare taxa, not all DTE-cultivated organisms grew in droplets. This suggests additional contributing factors (apart from an increased throughput), such as encapsulation stress and droplet-specific microenvironments. These findings clarify the advantages and limitations of droplet cultivation strategies, allowing a more informed application of these techniques to access the so-called “microbial dark matter.”  

Mammalian cell engineering offers the opportunity to uncover biological principles and develop next-generation biotechnologies. However, epigenetic silencing of transgenes hinders the control of gene expression in mammalian cells. Here, we use chromatin editing of an integrated reporter in both CHO-K1 and human induced pluripotent stem cells to dissect how histone H3 lysine 9 trimethylation (H3K9me3) and DNA methylation cooperate to maintain the silenced state and how this state can be reversed. After transient induction of either DNA methylation or H3K9me3, stable silencing was exclusively observed with both marks. Our chemical reaction model of chromatin modifications predicts that DNA methylation drives silencing maintenance. Accordingly, targeted DNA demethylation reactivated the reporter irrespective of whether silencing was achieved by inducing DNA methylation, H3K9me3, or by the endogenous cellular machinery. These results shed light on molecular mechanisms at play during silencing and provide engineering tools for potent and specific transgene reactivation in mammalian cells.