Autotrophic conversion of CO2 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 CO2 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.

Eubacterium limosum is one of the important bacteria in C 1 feedstock utilization as well as in human gut microbiota. Although E. limosum has recently garnered much attention and investigation on a genome-wide scale, a bottleneck for systematic engineering in E. limosum is the lack of available genetic tools and an efficient genome editing platform. To overcome this limitation, we here report expanded genetic tools and the CRISPR-Cas9 system. We have developed an inducible promoter system that enables implementation of the CRISPR-Cas9 system to precisely manipulate target genes of the Wood-Ljungdahl pathway with 100% efficiency. Furthermore, we exploited the effectiveness of CRISPR interference to reduce the expression of target genes, exhibiting substantial repression of several genes in the Wood-Ljungdahl pathway and fructose-PTS system. These expanded genetic tools and CRISPR-Cas9 system comprise powerful and widely applicable genetic tools to accelerate functional genomic study and genome engineering in E. limosum.

BackgroundAcetogenic bacteria constitute promising biocatalysts for the conversion of CO2/H2 or synthesis gas (H2/CO/CO2) 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.ResultsBy integrating genome-scale transcriptomic and translatomic data, we explored the regulatory logic of the acetogenesis to convert CO2 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (10.1186/s12864-018-5238-0) contains supplementary material, which is available to authorized users.

Among CO 2 -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 CO 2 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, 13 C 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 CO 2 , subsequently converting CO 2 into acetyl-CoA, acetyl-phosphate, and serine. Moreover, the functional cooperation of the pathways enhances CO 2 consumption and cellular growth rates via bypassing reducing power required reactions for cellular metabolism during autotrophic growth of acetogens.CO 2 fixation | acetogen | Wood-Ljungdahl pathway | systems biology | glycine synthase-reductase pathway T he linear Wood-Ljungdahl pathway (WLP) in anaerobic acetogens is considered the most energetically efficient pathway to convert CO 2 to formate and then into acetyl-CoA. With this advantage, acetogens are considered to be the most promising industrial platform to produce biofuels and chemical commodities through synthesis gas fermentation (1-4). Although gene composition and arrangement of the WLP vary among acetogens, the WLP-coding genes are well-conserved, along with two genes encoding a partial glycine synthase: the glycine cleavage system H protein (gcvH) and dihydrolipoyl dehydrogenase (lpdA) genes (5-8). The glycine synthase pathway was initially proposed for the utilization of CO 2 under autotrophic growth conditions (2). While the two genes are well-conserved in the gene cluster, other genes in the glycine synthase pathway are missing in many acetogen genomes, which raises questions regarding a potential functional role of these enzymes under autotrophic growth conditions. Following synthesis, glycine can be reduced to acetyl-phosphate (acetyl-P), which is likely to be converted into acetate by acetate kinase (ackA), thereby producing one ATP, termed the glycine synthasereductase pathway (GSRP). Alternatively, serine hydroxymethyltransferase (SHMT) converts the produced glycine to serine, which then becomes transformed to pyruvate and biomass (9-11). Recently, an artificial metabolic pathway constructed with glycine synthase and SHMT, termed the reductive glycine pathway (RGP), has shown the capability of fixing CO 2 using alternative electron donors (12,13). Despite sharing common reactions and the presence of genes encoding a partial glycine synthase, the functional role of the pathway in the presence of intact WLP has remained uncertain.In this study, we elucidated the role of the GSRP a...

Proteins containing a placenta-specific 8 domain (PLAC8) function as major organ size regulators in Solanum lycopersicum and Zea may, and putative metal ion transporters in Arabidopsis thaliana, Oryza sativa and Brassica juncea. However, it is unknown how PLAC8 domain-containing proteins fulfill such diverse roles. Here, we found that plant cadmium resistance 1 (PCR1) influences both zinc (Zn) accumulation and grain weight in rice. OsPCR1 knockout and knockdown lines produced lighter grains than the wild type, while OsPCR1 overexpression lines produced heavier grains. Furthermore, the grains of OsPCR1 knockdown lines exhibited substantially higher Zn and lower cadmium (Cd) concentrations than the control, as did yeast heterologously expressing OsPCR1. Through sequence analysis, we showed that the amino acid sequence of japonica-type PCR1 was distinct from that of indica-type and wild rice accessions. This difference was correlated with distinct Zn-related phenotypes. Japonica-type PCR1 had a shorter N-terminus than did PCR1 in the other rice types, and yeast heterologously expressing japonica-type PCR1 was more sensitive to Zn than was yeast expressing indica-type PCR1. Furthermore, japonica-type grains accumulated less Zn than did indica-type grains. Our study suggests that rice PCR1 maintains metal ion homeostasis and grain weight and might have been selected for during domestication.

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.

Acetogens are anaerobic bacteria that utilise gaseous feedstocks such as carbon monoxide (CO) and carbon dioxide (CO2) 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 CO2 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 CO2 fixation and will help in the designing of strains for the desired biochemical production.

Acetogens are naturally capable of metabolizing carbon monoxide (CO), a component of synthesis gas (syngas), for autotrophic growth in order to produce biomass and metabolites such as acetyl-CoA via the Wood-Ljungdahl pathway. However, the autotrophic growth of acetogens is often inhibited by the presence of high CO concentrations because of CO toxicity, thus limiting their biosynthetic potential for industrial applications. Herein, we implemented adaptive laboratory evolution (ALE) for growth improvement of Eubacterium limosum ATCC 8486 under high CO conditions. The strain evolved under syngas conditions with 44% CO over 150 generations, resulting in a significant increased optical density (600 nm) and growth rate by 2.14 and 1.44 folds, respectively. In addition, the evolved populations were capable of proliferating under CO concentrations as high as 80%. These results suggest that cell growth is enhanced as beneficial mutations are selected and accumulated, and the metabolism is altered to facilitate the enhanced phenotype. To identify the causal mutations related to growth improvement under high CO concentrations, we performed whole genome resequencing of each population at 50-generation intervals. Interestingly, we found key mutations in CO dehydrogenase/acetyl-CoA synthase (CODH/ACS) complex coding genes, acsA and cooC. To characterize the mutational effects on growth under CO, we isolated single clones and confirmed that the growth rate and CO tolerance level of the single clone were comparable to those of the evolved populations and wild type strain under CO conditions. Furthermore, the evolved strain produced 1.34 folds target metabolite acetoin when compared to the parental strain while introducing the biosynthetic pathway coding genes to the strains. Consequently, this study demonstrates that the mutations in the CODH/ACS complex affect autotrophic growth enhancement in the presence of CO as well as the CO tolerance of E. limosum ATCC 8486.