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Acetogens are anaerobic bacteria that utilise gaseous feedstocks such as carbon monoxide (CO) and carbon dioxide (CO 2 ) to synthesise biomass and various metabolites via the energetically efficient Wood-Ljungdahl pathway. Because of this pathway, acetogens have been considered as a novel platform to produce biochemicals from gaseous feedstocks, potentially replacing the conventional thermochemical processes. Despite their advantages, a lack of systematic understanding of the transcriptional and translational regulation in acetogens during autotrophic growth limits the rational strain design to produce the desired products. To overcome this problem, we presented RNA sequencing and ribosome profiling data of four acetogens cultivated under heterotrophic and autotrophic conditions, providing data on genome-scale transcriptional and translational responses of acetogens during CO 2 fixation. These data facilitate the discovery of regulatory elements embedded in their genomes, which could be utilised to engineer strains to achieve better growth and productivity. We anticipate that these data will expand our understanding of the processes of CO 2 fixation and will help in the designing of strains for the desired biochemical production. Measurement(s) RNA • transcriptome • translatome Technology Type(s) RNA sequencing • ribosomal profiling by sequencing assay Factor Type(s) carbon source • growth conditions Sample Characteristic - Organism Acetobacterium woodii • Clostridium aceticum • Clostridium drakei • Eubacterium limosum Sample Characteristic - Environment anaerobic Machine-accessible metadata file describing the reported data: https://doi.org/10.6084/m9.figshare.13625942

Sporomusa sphaeroides KIAC is a novel acetogen isolated from cattle feces that exhibits rapid CO2 utilization. To investigate the molecular basis of this phenotype, we performed a comprehensive multi-omics analysis, including Genome-seq, RNA-seq, dRNA-seq, and Term-seq, to map its transcriptome architecture. We identified 2,158 transcription start sites and 2,275 transcript 3′ ends, enabling high-resolution reconstruction of the transcriptional landscape and associated regulatory features. This analysis uncovered key cis-regulatory elements and an expanded regulatory role for the alternative sigma factor SigH in controlling acetogenesis-related genes. Notably, KIAC harbors nine functionally diverse hydrogenases—a greater diversity than observed in other acetogens—likely contributing to its rapid CO2 utilization. Heterologous expression of KIAC-derived hydrogenases in Eubacterium limosum led to doubled H2 and CO2 consumption rates, increased growth rates, and notably, the first reported butyrate production under energy-limited H2/CO2 conditions. These improvements stem from enhanced H2 oxidation, which supplies additional reducing equivalents for growth and biochemical production. Our findings provide critical insights into the genetic basis for rapid autotrophic growth in acetogens. The discovery of the expanded regulatory role of SigH and the energetic advantages of diverse hydrogenases offers new strategies for enhanced CO2 bioconversion of acetogens.IMPORTANCEAcetogens offer a promising solution for sustainable CO2 bioconversion into multicarbon biochemicals through the Wood-Ljungdahl pathway, the most energy-efficient carbon fixation route known in nature. However, an incomplete understanding of their metabolism and regulatory systems has limited metabolic engineering efforts to achieve superior CO2 fixation efficiency. In this study, we investigated Sporomusa sphaeroides KIAC, a newly isolated acetogen with rapid CO2 utilization, to uncover the molecular mechanisms underlying its superior performance. By revealing an expanded regulatory role for an alternative sigma factor and a highly diverse set of hydrogenases, our findings provide a foundation for engineering acetogens with enhanced CO2 conversion efficiency under energy-limited conditions.

Background Acetogenic bacteria constitute promising biocatalysts for the conversion of CO 2 /H 2 or synthesis gas (H 2 /CO/CO 2 ) into biofuels and value-added biochemicals. These microorganisms are naturally capable of autotrophic growth via unique acetogenesis metabolism. Despite their biosynthetic potential for commercial applications, a systemic understanding of the transcriptional and translational regulation of the acetogenesis metabolism remains unclear. Results By integrating genome-scale transcriptomic and translatomic data, we explored the regulatory logic of the acetogenesis to convert CO 2 into biomass and metabolites in Eubacterium limosum . The results indicate that majority of genes associated with autotrophic growth including the Wood-Ljungdahl pathway, the reduction of electron carriers, the energy conservation system, and gluconeogenesis were transcriptionally upregulated. The translation efficiency of genes in cellular respiration and electron bifurcation was also highly enhanced. In contrast, the transcriptionally abundant genes involved in the carbonyl branch of the Wood-Ljungdahl pathway, as well as the ion-translocating complex and ATP synthase complex in the energy conservation system, showed decreased translation efficiency. The translation efficiencies of genes were regulated by 5′UTR secondary structure under the autotrophic growth condition. Conclusions The results illustrated that the acetogenic bacteria reallocate protein synthesis, focusing more on the translation of genes for the generation of reduced electron carriers via electron bifurcation, rather than on those for carbon metabolism under autotrophic growth.

Acetogens are capable of reducing CO 2 to multicarbon compounds (e.g., ethanol or 2,3-butanediol) via the Wood-Ljungdahl pathway. Given that protein synthesis in bacteria is highly energy consuming, acetogens living at the thermodynamic limit of life are inevitably under translation control. Acetogens synthesize acetyl-CoA via the CO 2 -fixing Wood-Ljungdahl pathway. Despite their ecological and biotechnological importance, their translational regulation of carbon and energy metabolisms remains unclear. Here, we report how carbon and energy metabolisms in the model acetogen Acetobacterium woodii are translationally controlled under different growth conditions. Data integration of genome-scale transcriptomic and translatomic analyses revealed that the acetogenesis genes, including those of the Wood-Ljungdahl pathway and energy metabolism, showed changes in translational efficiency under autotrophic growth conditions. In particular, genes encoding the Wood-Ljungdahl pathway are translated at similar levels to achieve efficient acetogenesis activity under autotrophic growth conditions, whereas genes encoding the carbonyl branch present increased translation levels in comparison to those for the methyl branch under heterotrophic growth conditions. The translation efficiency of genes in the pathways is differentially regulated by 5′ untranslated regions and ribosome-binding sequences under different growth conditions. Our findings provide potential strategies to optimize the metabolism of syngas-fermenting acetogenic bacteria for better productivity. IMPORTANCE Acetogens are capable of reducing CO 2 to multicarbon compounds (e.g., ethanol or 2,3-butanediol) via the Wood-Ljungdahl pathway. Given that protein synthesis in bacteria is highly energy consuming, acetogens living at the thermodynamic limit of life are inevitably under translation control. Here, we dissect the translational regulation of carbon and energy metabolisms in the model acetogen Acetobacterium woodii under heterotrophic and autotrophic growth conditions. The latter may be experienced when acetogen is used as a cell factory that synthesizes products from CO 2 during the gas fermentation process. We found that the methyl and carbonyl branches of the Wood-Ljungdahl pathway are activated at similar translation levels during autotrophic growth. Translation is mainly regulated by the 5′-untranslated-region structure and ribosome-binding-site sequence. This work reveals novel translational regulation for coping with autotrophic growth conditions and provides the systematic data set, including the transcriptome, translatome, and promoter/5′-untranslated-region bioparts.

The marine dinoflagellate Alexandrium is associated with harmful algal blooms (HABs) worldwide, causing paralytic shellfish poisoning (PSP) in humans. We found that the marine bacterium Pseudoruegeria sp. M32A2M exhibits algicidal activity against Alexandrium catenella (Group I), inhibiting its motility and consequently inducing cell disruption after 24 h of co-culture. To understand the communication between the two organisms, we investigated the time-course cellular responses through genome-wide transcriptome analysis. Functional analysis of differentially expressed genes revealed that the core reactions of the photosystem in A. catenella were inhibited within 2 h, eventually downregulating the entire pathways of oxidative phosphorylation and carbon fixation, as well as associated metabolic pathways. Conversely, Pseudoruegeria upregulated its glycolysis, tricarboxylic acid cycle, and oxidative phosphorylation pathways. Also, the transporters for nutrients such as C3/C4 carbohydrates and peptides were highly upregulated, leading to the speculation that nutrients released by disrupted A. catenella cells affect the central metabolism of Pseudoruegeria . In addition, we analyzed the secondary metabolite-synthesizing clusters of Pseudoruegeria that were upregulated by co-culture, suggesting their potential roles in algicidal activity. Our time-course transcriptome analysis elucidates how A. catenella is affected by algicidal bacteria and how these bacteria obtain functional benefits through metabolic pathways.

Acetogens synthesize acetyl-CoA via the CO2-fixing Wood-Ljungdahl pathway. Despite their ecological and biotechnological importance, their translational regulation of carbon and energy metabolisms remains unclear. Here, we report how carbon and energy metabolisms in the model acetogen Acetobacterium woodii are translationally controlled under different growth conditions. Data integration of genome-scale transcriptomic and translatomic analyses revealed that the acetogenesis genes, including those of the Wood-Ljungdahl pathway and energy metabolism, showed changes in translational efficiency under autotrophic growth conditions. In particular, genes encoding the Wood-Ljungdahl pathway are translated at similar levels to achieve efficient acetogenesis activity under autotrophic growth conditions, whereas genes encoding the carbonyl branch present increased translation levels in comparison to those for the methyl branch under heterotrophic growth conditions. The translation efficiency of genes in the pathways is differentially regulated by 5′ untranslated regions and ribosome-binding sequences under different growth conditions. Our findings provide potential strategies to optimize the metabolism of syngas-fermenting acetogenic bacteria for better productivity. IMPORTANCE Acetogens are capable of reducing CO2 to multicarbon compounds (e.g., ethanol or 2,3-butanediol) via the Wood-Ljungdahl pathway. Given that protein synthesis in bacteria is highly energy consuming, acetogens living at the thermodynamic limit of life are inevitably under translation control. Here, we dissect the translational regulation of carbon and energy metabolisms in the model acetogen Acetobacterium woodii under heterotrophic and autotrophic growth conditions. The latter may be experienced when acetogen is used as a cell factory that synthesizes products from CO2 during the gas fermentation process. We found that the methyl and carbonyl branches of the Wood-Ljungdahl pathway are activated at similar translation levels during autotrophic growth. Translation is mainly regulated by the 5′-untranslated-region structure and ribosome-binding-site sequence. This work reveals novel translational regulation for coping with autotrophic growth conditions and provides the systematic data set, including the transcriptome, translatome, and promoter/5′-untranslated-region bioparts.

Among CO₂-fixing metabolic pathways in nature, the linear Wood– Ljungdahl pathway (WLP) in phylogenetically diverse acetateforming acetogens comprises the most energetically efficient pathway, requires the least number of reactions, and converts CO₂ to formate and then into acetyl-CoA. Despite two genes encoding glycine synthase being well-conserved in WLP gene clusters, the functional role of glycine synthase under autotrophic growth conditions has remained uncertain. Here, using the reconstructed genomescale metabolic model iSL771 based on the completed genome sequence, transcriptomics, 13C isotope-based metabolite-tracing experiments, biochemical assays, and heterologous expression of the pathway in another acetogen, we discovered that the WLP and the glycine synthase pathway are functionally interconnected to fix CO₂, subsequently converting CO₂ into acetyl-CoA, acetyl-phosphate, and serine. Moreover, the functional cooperation of the pathways enhances CO₂ consumption and cellular growth rates via bypassing reducing power required reactions for cellular metabolism during autotrophic growth of acetogens.

SignificanceDespite sharing the first four reactions, coutilization of the Wood–Ljungdahl pathway (WLP) with the glycine synthase-reductase pathway (GSRP) and reductive glycine pathway (RGP) to fix C1 compounds has remained unknown. In this study, using Clostridium drakei, we elucidated the role of the GSRP and RGP in the presence of the WLP, via a genome-scale metabolic model, RNA-seq, 13C isotope-based metabolite-tracing experiments, biochemical assays, and heterologous expression. Overall, the data suggested the pathways are functional under autotrophic conditions. Along with the WLP, GSRP and RGP convert CO2 to glycine and then to acetyl-phosphate and serine, which then obtain ATP by producing acetate and operate with limited reducing power. This is a unique coutilization of the pathways under autotrophic conditions in acetogens. Among CO2-fixing metabolic pathways in nature, the linear Wood–Ljungdahl pathway (WLP) in phylogenetically diverse acetate-forming acetogens comprises the most energetically efficient pathway, requires the least number of reactions, and converts CO2 to formate and then into acetyl-CoA. Despite two genes encoding glycine synthase being well-conserved in WLP gene clusters, the functional role of glycine synthase under autotrophic growth conditions has remained uncertain. Here, using the reconstructed genome-scale metabolic model iSL771 based on the completed genome sequence, transcriptomics, 13C isotope-based metabolite-tracing experiments, biochemical assays, and heterologous expression of the pathway in another acetogen, we discovered that the WLP and the glycine synthase pathway are functionally interconnected to fix CO2, subsequently converting CO2 into acetyl-CoA, acetyl-phosphate, and serine. Moreover, the functional cooperation of the pathways enhances CO2 consumption and cellular growth rates via bypassing reducing power required reactions for cellular metabolism during autotrophic growth of acetogens.

Acetogenic bacteria use cellular redox energy to convert CO₂ to acetate using the Wood–Ljungdahl (WL) pathway. Such redox energy can be derived from electrons generated from H₂ as well as from inorganic materials, such as photoresponsive semiconductors. We have developed a nanoparticle-microbe hybrid system in which chemically synthesized cadmium sulfide nanoparticles (CdS-NPs) are displayed on the cell surface of the industrial acetogen Clostridium autoethanogenum. The hybrid system converts CO₂ into acetate without the need for additional energy sources, such as H₂, and uses only light-induced electrons from CdS-NPs. To elucidate the underlying mechanism by which C. autoethanogenum uses electrons generated from external energy sources to reduce CO₂, we performed transcriptional analysis. Our results indicate that genes encoding the metal ion or flavin-binding proteins were highly up-regulated under CdS-driven autotrophic conditions along with the activation of genes associated with the WL pathway and energy conservation system. Furthermore, the addition of these cofactors increased the CO² fixation rate under light-exposure conditions. Our results demonstrate the potential to improve the efficiency of artificial photosynthesis systems based on acetogenic bacteria integrated with photoresponsive nanoparticles.

Autotrophic conversion of CO 2 to value-added biochemicals has received considerable attention as a sustainable route to replace fossil fuels. Particularly, anaerobic acetogenic bacteria are naturally capable of reducing CO 2 or CO to various metabolites. To fully utilize their biosynthetic potential, an understanding of acetogenesis-related genes and their regulatory elements is required. Here, we completed the genome sequence of the syngas fermenting Eubacterium limosum ATCC 8486 and determined its transcription start sites (TSS). We constructed a 4.4 Mb long circular genome with a GC content of 47.2% and 4,090 protein encoding genes. To understand the transcriptional and translational regulation, the primary transcriptome was augmented, identifying 1,458 TSSs containing a high pyrimidine (T/C) and purine nucleotide (A/G) content at the −1 and +1 position, respectively, along with 1,253 5′-untranslated regions, and principal promoter elements such as −10 (TATAAT) and −35 (TTGACA), and Shine-Dalgarno motifs (GGAGR). Further analysis revealed 93 non-coding RNAs, including one for potential transcriptional regulation of the hydrogenase complex via interaction with molybdenum or tungsten cofactors, which in turn controls formate dehydrogenase activity of the initial step of Wood-Ljungdahl pathway. Our results provide comprehensive genomic information for strain engineering to enhance the syngas fermenting capacity of acetogenic bacteria.