c Eubacterium limosum KIST612 is one of the few acetogens that can produce butyrate from carbon monoxide. We have used a genome-guided analysis to delineate the path of butyrate formation, the enzymes involved, and the potential coupling to ATP synthesis. Oxidation of CO is catalyzed by the acetyl-coenzyme A (CoA) synthase/CO dehydrogenase and coupled to the reduction of ferredoxin. Oxidation of reduced ferredoxin is catalyzed by the Rnf complex and Na ؉ dependent. Consistent with the finding of a Na ؉ -dependent Rnf complex is the presence of a conserved Na ؉ -binding motif in the c subunit of the ATP synthase. Butyrate formation is from acetyl-CoA via acetoacetyl-CoA, hydroxybutyryl-CoA, crotonyl-CoA, and butyryl-CoA and is consistent with the finding of a gene cluster that encodes the enzymes for this pathway. The activity of the butyryl-CoA dehydrogenase was demonstrated. Reduction of crotonyl-CoA to butyryl-CoA with NADH as the reductant was coupled to reduction of ferredoxin. We postulate that the butyryl-CoA dehydrogenase uses flavin-based electron bifurcation to reduce ferredoxin, which is consistent with the finding of etfA and etfB genes next to it. The overall ATP yield was calculated and is significantly higher than the one obtained with H 2 ؉ CO 2 . The energetic benefit may be one reason that butyrate is formed only from CO but not from H 2 ؉ CO 2 .A cetogenic bacteria are a phylogenetically diverse group of strictly anaerobic bacteria able to reduce two molecules of CO 2 to acetate by the Wood-Ljungdahl pathway (WLP) (1-4). Electrons may derive from molecular hydrogen (autotrophic growth), from carbon monoxide, or from organic donors (heterotrophic growth) such as hexoses, pentoses, formate, lactate, alcohols, or methyl group donors (1). Not only does the WLP provide the cell with organic material for biomass formation, but it is also coupled to energy conservation for ATP supply by a chemiosmotic mechanism (2, 5). Every acetogen examined to date uses reduced ferredoxin (Fd) as the electron donor for an ion-translocating membrane protein complex, and acetogens can have either an Fd:NAD ϩ oxidoreductase (Rnf) or an Fd:H ϩ oxidoreductase (Ech) complex for generation of an ion motive force (5). In both cases, the ion gradient can be either an H ϩ or an Na ϩ gradient. The electrochemical ion gradient thus established is then used by a membrane bound, H ϩ -or Na ϩ -translocating F 1 F o ATP synthase (2).Acetate production from CO 2 proceeds via formate that is converted to formyl-tetrahydrofolate (THF) in an ATP-consuming reaction (6). Water is split off from formyl-THF to yield methenyl-THF, which is reduced via methylene-THF to methyl-THF. The latter is condensed with CO (derived from another molecule of CO 2 ) and coenzyme A (CoA) to acetyl-CoA, which is the starting molecule for biosynthetic reactions (4,7,8). Acetyl-CoA is also the precursor of the end product, acetate, that is produced by the enzymes acetyltransferase and acetate kinase. ATP production in the acetate kinase reaction is of special...
Eubacterium limosum KIST612 is an anaerobic acetogenic bacterium that uses CO as the sole carbon/energy source and produces acetate, butyrate, and ethanol. To evaluate its potential as a syngas microbial catalyst, we have sequenced the complete 4.3-Mb genome of E. limosum KIST612.Synthesis gas (syngas) (H 2 , CO 2 , and CO) has been highlighted for use as a potential feedstock for the production of biofuels and valuable chemicals (9, 16). Eubacterium limosum KIST612 isolated from an anaerobic digester has been considered a microbial syngas catalyst due to its rapid growth under high CO pressure (Ͼ1 atm) and production of acetate and butyrate and ethanol from CO (5-7). To understand its physiological properties (e.g., a high tolerance to CO and production of ethanol) and provide metabolic engineering principles, we attempted to obtain the complete genome sequence information for this microorganism.The genome of E. limosum KIST612 was sequenced by a combination of Illumina Genome Analyzer IIx (GAIIx) and Roche 454 GS FLX (454 GS FLX) platforms. We obtained two libraries of 643,326 single-end (SE) reads and 291,735 pairedend (PE) reads containing 3-kb inserts from 454 GS FLX. The third genomic library of 35,235,888 PE reads containing 400-bp inserts was obtained from GAIIx. To combine these three libraries (454 GS FLX SE and PE and GAIIx PE) into a single procedure, we first assembled GAIIx PE reads into 296 contigs (4,635,997 bases) by the ABySS 1.20 assembler (15) and split into overlapping ϳ1.5-kb fake reads (45,221 reads). We merged these fake reads with 454 SE and PE reads (total 935,061 reads) and assembled into 9 scaffolds (34 contigs) by the Newbler gsAssembler 2.3 (454 Life Sciences, Branford, CT). We determined the actual order of 9 scaffolds in a single contig with a series of PCRs based on a permutation table of scaffolds. The genome was finished by filling gaps with sequencing and primer walking of PCR products using an ABI 3730 capillary sequencer (Applied Biosystems, CA).The complete genome of E. limosum KIST612 consisted of 4,276,902 bp in a single circular chromosome with an average GϩC content of 47.5%. Approximately 91% of the nucleotides were predicted as 4,516 protein-coding regions by the union of Glimmer (8), GeneMarkS (3), and Prodigal (10). The predicted proteins were annotated by BLAST (1) and the RAST server (2). Seventy-eight percent (3,541) of the open reading frames were annotated with known proteins. Five copies of the 16S-23S-5S rRNA operon and a separate 5S rRNA locus were predicted by RNAmmer 1.2 (12), and the 58 tRNA genes were identified by tRNAscan-SE 1.23 (13).Metabolic pathway analysis revealed that E. limosum KIST612 uses the Wood-Ljungdahl pathway to fix CO (or CO 2 ) and converts it into acetyl coenzyme A (acetyl-CoA), like other syngas-utilizing acetogens such as Moorella thermoacetica (14), Clostridium ljungdahlii (11), and Clostridium carboxidivorans strain P7 T (4). E. limosum KIST612 also contains 10 genes annotated as subunits of hydrogenases that may provide reducing...
Acetic acid inhibits the metabolic activities of Saccharomyces cerevisiae. Therefore, a better understanding of how S. cerevisiae cells acquire the tolerance to acetic acid is of importance to develop robust yeast strains to be used in industry. To do this, we examined the transcriptional changes that occur at 12 h post-exposure to acetic acid, revealing that 56 and 58 genes were upregulated and downregulated, respectively. Functional categorization of them revealed that 22 protein synthesis genes and 14 stress response genes constituted the largest portion of the upregulated and downregulated genes, respectively. To evaluate the association of the regulated genes with acetic acid tolerance, 3 upregulated genes (DBP2, ASC1, and GND1) were selected among 34 non-protein synthesis genes, and 54 viable mutants individually deleted for the downregulated genes were retrieved from the non-essential haploid deletion library. Strains overexpressing ASC1 and GND1 displayed enhanced tolerance to acetic acid, whereas a strain overexpressing DBP2 was sensitive. Fifty of 54 deletion mutants displayed enhanced acetic acid tolerance. Three chosen deletion mutants (hsps82Δ, ato2Δ, and ssa3Δ) were also tolerant to benzoic acid but not propionic and sorbic acids. Moreover, all those five (two overexpressing and three deleted) strains were more efficient in proton efflux and lower in membrane permeability and internal hydrogen peroxide content than controls. Individually or in combination, those physiological changes are likely to contribute at least in part to enhanced acetic acid tolerance. Overall, information of our transcriptional profile was very useful to identify molecular factors associated with acetic acid tolerance.
The development of high-throughput technology using RNA-seq has allowed understanding of cellular mechanisms and regulations of bacterial transcription. In addition, transcriptome analysis with RNA-seq has been used to accelerate strain improvement through systems metabolic engineering. Synechococcus elongatus PCC 7942, a photosynthetic bacterium, has remarkable potential for biochemical and biofuel production due to photoautotrophic cell growth and direct CO2 conversion. Here, we performed a transcriptome analysis of S. elongatus PCC 7942 using RNA-seq to understand the changes of cellular metabolism and regulation for nitrogen starvation responses. As a result, differentially expressed genes (DEGs) were identified and functionally categorized. With mapping onto metabolic pathways, we probed transcriptional perturbation and regulation of carbon and nitrogen metabolisms relating to nitrogen starvation responses. Experimental evidence such as chlorophyll a and phycobilisome content and the measurement of CO2 uptake rate validated the transcriptome analysis. The analysis suggests that S. elongatus PCC 7942 reacts to nitrogen starvation by not only rearranging the cellular transport capacity involved in carbon and nitrogen assimilation pathways but also by reducing protein synthesis and photosynthesis activities.
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