DNA transfer between two different species mediated by heterologous cell fusion in Clostridium coculture.

Prokaryotic evolution is driven by random mutations and horizontal gene transfer (HGT). HGT occurs via transformation, transduction, or conjugation. We have previously shown that in syntrophic cocultures of Clostridium acetobutylicum and Clostridium ljungdahlii, heterologous cell fusion leads to a large-scale exchange of proteins and RNA between the two organisms. Here, we present evidence that heterologous cell fusion facilitates the exchange of DNA between the two organisms. Using selective subculturing, we isolated C. acetobutylicum cells which acquired and integrated into their genome portions of plasmid DNA from a plasmid-carrying C. ljungdahlii strain. Limiting-dilution plating and DNA methylation data based on PacBio Single-Molecule Real Time (SMRT) sequencing support the existence of hybrid C. acetobutylicum/C. ljungdahlii cells. These findings expand our understanding of multi-species microbiomes, their survival strategies, and evolution.IMPORTANCEInvestigations of natural multispecies microbiomes and synthetic microbial cocultures are attracting renewed interest for their potential application in biotechnology, ecology, and medical fields. Previously, we have shown the syntrophic coculture of C. acetobutylicum and C. ljungdahlii undergoes heterologous cell-to-cell fusion, which facilitates the exchange of cytoplasmic protein and RNA between the two organisms. We now show that heterologous cell fusion between the two Clostridium organisms can facilitate the exchange of DNA. By applying selective pressures to this coculture system, we isolated clones of wild-type C. acetobutylicum which acquired the erythromycin resistance (erm) gene from the C. ljungdahlii strain carrying a plasmid with the erm gene. Single-molecule real-time sequencing revealed that the erm gene was integrated into the genome in a mosaic fashion. Our data also support the persistence of hybrid C. acetobutylicum/C. ljungdahlii cells displaying hybrid DNA-methylation patterns.

Positive effect of phasin in biohydrogen production of non polyhydroxybutyrate-producing Clostridium acetobutylicum ATCC 824.

In this study, the goal was to enhance the tolerance of Clostridium acetobutylicum ATCC 824 to biomass-based inhibitory compounds for biohydrogen production and evaluate various known genes that enhance the production of biochemicals in various hosts. The introduction of phaP, the major polyhydroxyalkanoate granule-associated protein that has been reported as a chaperone-like protein resulted in increased tolerance to inhibitors and leads to higher levels of hydrogen production, cell growth, and glucose consumption in the presence of these inhibitors. It was observed that the introduction of phaP led to an increase in the transcription of the hydrogenase gene, whereas transcription of the chaperone functional genes decreased compared to the wild type. Finally, the introduction of phaP could significantly enhance biohydrogen production by 2.6-fold from lignocellulosic hydrolysates compared to that of wild type. These findings suggested that the introduction of phaP could enhance growth and biohydrogen production, even in non-polyhydroxyalkanoate-producing strains.

Enhancement of biohydrogen production in Clostridium acetobutylicum ATCC 824 by overexpression of glyceraldehyde-3-phosphate dehydrogenase gene.

In the dark fermentation of hydrogen, development of production host is crucial as bacteria act on substrates and produce hydrogen. The present study aimed to improve hydrogen production through the development of Clostridium acetobutylicum as a superior biohydrogen producer. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH), which produces NADH/NADPH for metabolites and energy in primary pathways, was introduced to enhance hydrogen production. The strain CAC824-G containing gapC that encodes GAPDH showed a 66.3 % higher hydrogen production than the wild-type strain, with increased NADH and NADPH pools. Glucose consumption and other byproducts, such as acetone, butanol, and ethanol, were also high in CAC824-G. Overexpression of gapC resulted in increased hydrogen production with sugars obtained from different biomass, even in the presence of inhibitors such as vanillin, 5-hydroxymethylfufural, acetic acid, and formic acid. Our results imply that overexpression of gapC in Clostridium is possible to expand the production of the reported biochemicals to produce hydrogen.

Differential Binding of NLRP3 to non-oxidized and Ox-mtDNA mediates NLRP3 Inflammasome Activation.

The NLRP3 inflammasome is a key mediator of the innate immune response to sterile tissue injury and is involved in many chronic and acute diseases. Physically and chemically diverse agents activate the NLRP3 inflammasome. Here, we show that NLRP3 binds non-oxidized and Ox-mtDNA differentially, with a half maximum inhibitory concentration (IC50) for non-oxidized and Ox-mtDNA of 4 nM and 247.2 nM, respectively. The NLRP3 Neonatal-Onset Multisystem Inflammatory Disease (NOMIDFCAS) gain of function mutant could bind non-oxidized mtDNA but had higher affinity for Ox-mtDNA compared to WT with an IC50 of 8.1 nM. NLRP3 lacking the pyrin domain can bind both oxidized and non-oxidized mtDNA. Isolated pyrin domain prefers Ox-mtDNA. The NLRP3 pyrin domain shares a protein fold with DNA glycosylases and generate a model for DNA binding based on the structure and sequence alignment to Clostridium acetobutylicum and human OGG1, an inhibitor of Ox-mtDNA generation, 8-oxoguanine DNA glycosylases. We provide a new model for how NLRP3 interacts with Ox-mtDNA supported by DNA binding in the presence of a monoclonal antibody against the pyrin domain. These results give new insights into the mechanism of inflammasome assembly, and into the function of reactive oxygen species in establishing a robust immune response.

Efforts to install a heterologous Wood-Ljungdahl pathway in Clostridium acetobutylicum enable the identification of the native tetrahydrofolate (THF) cycle and result in early induction of solvents.

Here, we report the construction of a Clostridium acetobutylicum strain ATCC 824 (pCD07239) by heterologous expression of carbonyl branch genes (CD630_0723∼CD630_0729) from Clostridium difficile, aimed at installing a heterologous Wood-Ljungdahl pathway (WLP). As part of this effort, in order to validate the methyl branch of the WLP in the C. acetobutylicum, we performed 13C-tracing analysis on knockdown mutants of four genes responsible for the formation of 5-methyl-tetrahydrofolate (5-methyl-THF) from formate: CA_C3201, CA_C2310, CA_C2083, and CA_C0291. While C. acetobutylicum 824 (pCD07239) could not grow autotrophically, in heterotrophic fermentation, it began producing butanol at the early growth phase (OD600 of 0.80; 0.162 g/L butanol). In contrast, solvent production in the parent strain did not begin until the early stationary phase (OD600 of 7.40). This study offers valuable insights for future research on biobutanol production during the early growth phase.

Co­cultivation of anaerobic fungi with Clostridium acetobutylicum bolsters butyrate and butanol production from cellulose and lignocellulose.

A system for co-cultivation of anaerobic fungi with anaerobic bacteria was established based on lactate cross-feeding to produce butyrate and butanol from plant biomass. Several co-culture formulations were assembled that consisted of anaerobic fungi (Anaeromyces robustus, Neocallimastix californiae, or Caecomyces churrovis) with the bacterium Clostridium acetobutylicum. Co-cultures were grown simultaneously (e.g., 'one pot'), and compared to cultures where bacteria were cultured in fungal hydrolysate sequentially. Fungal hydrolysis of lignocellulose resulted in 7-11 mM amounts of glucose and xylose, as well as acetate, formate, ethanol, and lactate to support clostridial growth. Under these conditions, one-stage simultaneous co-culture of anaerobic fungi with C. acetobutylicum promoted the production of butyrate up to 30 mM. Alternatively, two-stage growth slightly promoted solventogenesis and elevated butanol levels (∼4-9 mM). Transcriptional regulation in the two-stage growth condition indicated that this cultivation method may decrease the time required to reach solventogenesis and induce the expression of cellulose-degrading genes in C. acetobutylicum due to relieved carbon-catabolite repression. Overall, this study demonstrates a proof of concept for biobutanol and bio-butyrate production from lignocellulose using an anaerobic fungal-bacterial co-culture system.

Simultaneous production of renewable biohydrogen, biobutanol and biopolymer from phytogenic CoNPs-assisted Clostridial fermentation for sustainable energy and environment.

Considering the environmental impacts, rapid fossil fuel depletion and production costs, sustainable production of clean biofuels from alternative sources is required to meet the increasing demand for energy while avoiding environmental pollution. In this study, phytogenic cobalt nanoparticles (CoNPs)-assisted dark fermentation process was developed for the simultaneous production of biohydrogen, biobutanol and biopolymer from glucose using Clostridium acetobutylicum NCIM 2337. The maximum biohydrogen yield of 2.89 mol H2/mol glucose was achieved at 1.5 mg of CoNPs, which is 1.6 folds higher than that of the control experiment. The high level of soluble metabolites, specifically acetate and butyrate, confirmed the production of biohydrogen through acetate/butyrate pathways. The modified Gompertz model fitted well with experimental results of CoNPs-assisted biohydrogen production. The CoNPs could act as an electron carrier in intracellular metabolism to enhance the activity of ferredoxin and hydrogenase enzymes, thus improving biohydrogen production. Furthermore, biobutanol and biopolymer yields of 975 ± 2.5 mg/L and 1182 ± 1.4 mg/L were achieved, with 2.0 mg and 2.5 mg of CoNP, respectively, which were 1.27 and 1.19 folds higher than the control values. Hence, the inclusion of CoNPs in the fermentation medium seems to be a promising technique for the enhanced simultaneous production of biohydrogen, biobutanol and biopolymer. The environmental perspectives of the obtained renewable biohydrogen, biobutanol and biopolymer are also discussed.

An NADPH-auxotrophic Corynebacterium glutamicum recombinant strain and used it to construct L-leucine high-yielding strain.

The NADPH-regeneration enzymes in Corynebacterium glutamicum were inactivated to construct an NADPH-auxotrophic C. glutamicum strain by gene knockout and gene replacement. The resultant NADPH-auxotrophic C. glutamicum XL-1 ΔZMICg::ISm (i.e., strain Leu-1) grew well in the basic medium only with gluconate as carbon source. Replacement of the native glyceraldehyde 3-phosphate dehydrogenase (NAD-GapDHCg) by NADP-GapDHCa from Clostridium acetobutylicum is an effective strategy for producing L-leucine in NADPH-prototrophic strain XL-1 and NADPH-auxotrophic strain Leu-1, whereas the L-leucine yield did not differ significantly between these strains (14.1 ± 1.8 g/L vs 16.2 ± 1.1 g/L). Enhancing the carbon flux in biosynthetic pathway by recombinant expression plasmid pEC-ABNCE promoted L-leucine production, but the shortage NADPH supply limited the L-leucine yield. The mutated promoters of zwf and icdCg were introduced into C. glutamicum with NADP-GapDHCa and pEC-ABNCE increased L-leucine yield (54.3 ± 2.9 g/L) and improved cell growth (OD562 = 83.4 ± 7.5) in fed-batch fermentation because the resultant strain C. glutamicum XL-1 ΔMICg::ISm GCg::GCa Pzwf-D1 Picd-D2/pEC-ABNCE (i.e., strain Leu-9) exhibited the proper intracellular NADPH and NADH level. This is the first report of constructing an L-leucine high-yielding strain that reasonably supplies NADPH by optimizing the biosynthetic pathway of NADPH from an NADPH-auxotrophic strain.

Biobutanol production from sustainable biomass process of anaerobic ABE fermentation for industrial applications.

The growing population increases the need to develop advanced biological methods for utilizing renewable and sustainable resources to produce environmentally friendly biofuels. Currently, energy resources are limited for global demand and are constantly depleting and creating environmental problems. Some higher chain alcohols, like butanol and ethanol, processing similar properties to gasoline, can be alternate sources of biofuel. However, the industrial production of these alcohols remains challenging because they cannot be efficiently produced by microbes naturally. Therefore, butanol is the most interesting biofuel candidate with a higher octane number produced naturally by microbes through Acetone-Butanol-Ethanol fermentation. Feedstock selection as the substrate is the most crucial step in biobutanol production. Lignocellulosic biomass has been widely used to produce cellulosic biobutanol using agricultural wastes and residue. Specific necessary pretreatments, fermentation strategies, bioreactor designing and kinetics, and modeling can also enhance the efficient production of biobutanol. The recent genetic engineering approaches of gene knock in, knock out, and overexpression to manipulate pathways can increase the production of biobutanol in a user friendly host organism. So far various genetic manipulation techniques like antisense RNA, TargeTron Technology and CRISPR have been used to target Clostridium acetobutylicum for biobutanol production. This review summarizes the recent research and development for the efficient production of biobutanol in various aspects.

Molecular characterization of the missing electron pathways for butanol synthesis in Clostridium acetobutylicum.

Clostridium acetobutylicum is a promising biocatalyst for the renewable production of n-butanol. Several metabolic strategies have already been developed to increase butanol yields, most often based on carbon pathway redirection. However, it has previously demonstrated that the activities of both ferredoxin-NADP+ reductase and ferredoxin-NAD+ reductase, whose encoding genes remain unknown, are necessary to produce the NADPH and the extra NADH needed for butanol synthesis under solventogenic conditions. Here, we purify, identify and partially characterize the proteins responsible for both activities and demonstrate the involvement of the identified enzymes in butanol synthesis through a reverse genetic approach. We further demonstrate the yield of butanol formation is limited by the level of expression of CA_C0764, the ferredoxin-NADP+ reductase encoding gene and the bcd operon, encoding a ferredoxin-NAD+ reductase. The integration of these enzymes into metabolic engineering strategies introduces opportunities for developing a homobutanologenic C. acetobutylicum strain.

A clean in-frame knockout system for gene deletion in Acetobacterium woodii.

Acetogenic bacteria produce acetate following the fixation of CO2 via the Wood-Ljungdahl pathway. As such, they represent excellent process organisms for the production of novel chemicals and fuels from this waste greenhouse gas. Acetobacterium woodii is the model acetogen and numerous studies have been conducted investigating its biochemistry, gas consumption and use as a production chassis. However, there are a dearth of available tools for A. woodii gene modification which limits the research options available for genetic studies. Here, the previously proposed Clostridia Roadmap is implemented in A. woodii leading to the derivation of a knockout system for the generation of clean, in-frame deletions. The replicon of the Gram-positive plasmid pCD6 that originated in Clostridioides difficile was identified as being replication-defective in A. woodii, a property that was exploited to construct a pseudo-suicide knockout plasmid which was used to generate an auxotrophic, pyrE mutant. This allowed the subsequent use of a heterologous pyrE gene (from Clostridium acetobutylicum) as a counter selection marker and the deletion of a number of genes by allelic exchange. Specific mutants generated were affected in growth on glucose, fructose and ethanol as a consequence of deletion of fruA, pstG and adhE, respectively.

Oxidoreduction potential controlling for increasing the fermentability of enzymatically hydrolyzed steam-exploded corn stover for butanol production.

BACKGROUND: Lignocellulosic biomass is recognized as an effective potential substrate for biobutanol production. Though many pretreatment and detoxification methods have been set up, the fermentability of detoxicated lignocellulosic substrate is still far lower than that of starchy feedstocks. On the other hand, the number of recent efforts on rational metabolic engineering approaches to increase butanol production in Clostridium strains is also quite limited, demonstrating the physiological complexity of solventogenic clostridia. In fact, the strain performance is greatly impacted by process control. developing efficient process control strategies could be a feasible solution to this problem. RESULTS: In this study, oxidoreduction potential (ORP) controlling was applied to increase the fermentability of enzymatically hydrolyzed steam-exploded corn stover (SECS) for butanol production. When ORP of detoxicated SECS was controlled at - 350 mV, the period of fermentation was shortened by 6 h with an increase of 27.5% in the total solvent (to 18.1 g/L) and 34.2% in butanol (to 10.2 g/L) respectively. Silico modeling revealed that the fluxes of NADPH, NADH and ATP strongly differed between the different scenarios. Quantitative analysis showed that intracellular concentrations of ATP, NADPH/NADP+, and NADH/NAD+ were increased by 25.1%, 81.8%, and 62.5%. ORP controlling also resulted in a 2.1-fold increase in butyraldehyde dehydrogenase, a 1.2-fold increase in butanol dehydrogenase and 29% increase in the cell integrity. CONCLUSION: ORP control strategy effectively changed the intracellular metabolic spectrum and significantly improved Clostridium cell growth and butanol production. The working mechanism can be summarized into three aspects: First, Glycolysis and TCA circulation pathways were strengthened through key nodes such as pyruvate carboxylase [EC: 6.4.1.1], which provided sufficient NADH and NADPH for the cell. Second, sufficient ATP was provided to avoid "acid crash". Third, the key enzymes activities regulating butanol biosynthesis and cell membrane integrity were improved.

Genome-Wide TSS Distribution in Three Related Clostridia with Normalized Capp-Switch Sequencing.

Transcription initiation is a tightly regulated process that is crucial for many aspects of prokaryotic physiology. High-throughput transcription start site (TSS) mapping can shed light on global and local regulation of transcription initiation, which in turn may help us understand and predict microbial behavior. In this study, we used Capp-Switch sequencing to determine the TSS positions in the genomes of three model solventogenic clostridia: Clostridium acetobutylicum ATCC 824, C. beijerinckii DSM 6423, and C. beijerinckii NCIMB 8052. We first refined the approach by implementing a normalization pipeline accounting for gene expression, yielding a total of 12,114 mapped TSSs across the species. We further compared the distributions of these sites in the three strains. Results indicated similar distribution patterns at the genome scale, but also some sharp differences, such as for the butyryl-CoA synthesis operon, particularly when comparing C. acetobutylicum to the C. beijerinckii strains. Lastly, we found that promoter structure is generally poorly conserved between C. acetobutylicum and C. beijerinckii. A few conserved promoters across species are discussed, showing interesting examples of how TSS determination and comparison can improve our understanding of gene expression regulation at the transcript level. IMPORTANCE Solventogenic clostridia have been employed in industry for more than a century, initially being used in the acetone-butanol-ethanol (ABE) fermentation process for acetone and butanol production. Interest in these bacteria has recently increased in the context of green chemistry and sustainable development. However, our current understanding of their genomes and physiology limits their optimal use as industrial solvent production platforms. The gene regulatory mechanisms of solventogenesis are still only partly understood, impeding efforts to increase rates and yields. Genome-wide mapping of transcription start sites (TSSs) for three model solventogenic Clostridium strains is an important step toward understanding mechanisms of gene regulation in these industrially important bacteria.

Post-hydrolysis of cellulose oligomers by cellulase immobilized on chitosan-grafted magnetic nanoparticles: A key stage of butanol production from waste textile.

The recently developed technologies for immobilization of cellulase may address the challenges in costly hydrolysis of cellulose for cellulosic butanol production. In this study, a "hybrid" hydrolysis was developed based on chemical hydrolysis of cellulose to its oligomers followed by enzymatic post-hydrolysis of the resulting "soluble oligomers" by cellulase immobilized on chitosan-coated Fe3O4 nanoparticles. This hybrid hydrolysis stage was utilized in the process of biobutanol production from a waste textile, jeans waste, leading to selective formation of glucose and high yield of butanol production by Clostridium acetobutylicum. After validating the immobilization process, the optimum immobilization parameters including enzyme concentration and time were achieved on 8 h and 15.0 mg/mL, respectively. The reusability of immobilized enzyme showed that immobilized cellulase could retain 51.5% of its initial activity after three times reuses. Dilute acid hydrolysis of regenerated cellulose at 120-180 °C for 60 min 0.5-1.0% phosphoric acid led to less than 10 g/L glucose production, and enzymatic post-hydrolysis of the oligomers resulted in up to 51.5 g/L glucose. Fermentation of the hydrolysate was accompanied by 5.3 g/L acetone-butanol-ethanol (ABE) production. The simultaneous co-saccharification and fermentation (SCSF) of soluble and insoluble oligomers of cellulose led to 17.4 g/L ABE production.

Engineering Acetobacterium woodii for the production of isopropanol and acetone from carbon dioxide and hydrogen.

The capability of four genetically modified Acetobacterium woodii strains for improved production of acetone from CO2 and hydrogen was tested. The acetone biosynthesis pathway was constructed by combining genes from Clostridium acetobutylicum and Clostridium aceticum. Expression of acetone production genes was demonstrated in all strains. In bioreactors with continuous gas supply, all produced acetic acid, acetone, and, surprisingly, isopropanol. The production of isopropanol was caused by an endogenous secondary alcohol dehydrogenase (SADH) activity at low gas-feeding rate. Although high amounts of the natural end product acetic acid of A. woodii were formed,14.5 mM isopropanol and 7.6 mM acetone were also detected, showing that this is a promising approach for the production of new solvents from C1 gases. The highest acetic acid, acetone, and isopropanol production was detected in the recombinant A. woodii [pJIR750_ac1t1] strain, with final concentrations of 438 mM acetic acid, 7.6 mM acetone, and 14.5 mM isopropanol. The engineered strain A. woodii [pJIR750_ac1t1] was found to be the most promising strain for acetone production from a gas mixture of CO2 and H2 and the formation of isopropanol in A. woodii was shown for the first time.

Effects of orphan histidine kinases on clostridial sporulation progression and metabolism.

Solventogenesis and sporulation of clostridia are the main responsive adaptations to the acidic environment during acetone-butanol-ethanol (ABE) fermentation. It was hypothesized that five orphan histidine kinases (HKs) including Cac3319, Cac0323, Cac0903, Cac2730, and Cac0437 determined the cell fates between sporulation and solventogenesis. In this study, the comparative genomic analysis revealed that a mutation in cac0437 appeared to contribute to the nonsporulating feature of ATCC 55025. Hence, the individual and interactive roles of five HKs in regulating cell growth, metabolism, and sporulation were investigated. The fermentation results of mutants with different HK expression levels suggested that cac3319 and cac0437 played critical roles in regulating sporulation and acids and butanol biosynthesis. Morphological analysis revealed that cac3319 knockout abolished sporulation (Stage 0) whereas cac3319 overexpression promoted spore development (Stage VII), and cac0437 knockout initiated but blocked sporulation before Stage II, indicating the progression of sporulation was altered through engineering HKs. By combinatorial HKs knockout, the interactive effects between two different HKs were investigated. This study elucidated the regulatory roles of HKs in clostridial differentiation and demonstrated that HK engineering can be effectively used to control sporulation and enhance butanol biosynthesis.

Transcriptomic studies of solventogenic clostridia, Clostridium acetobutylicum and Clostridium beijerinckii.

Solventogenic clostridia are not a strictly defined group within the genus Clostridium but its representatives share some common features, i.e. they are anaerobic, non-pathogenic, non-toxinogenic and endospore forming bacteria. Their main metabolite is typically 1-butanol but depending on species and culture conditions, they can form other metabolites such as acetone, isopropanol, ethanol, butyric, lactic and acetic acids, and hydrogen. Although these organisms were previously used for the industrial production of solvents, they later fell into disuse, being replaced by more efficient chemical production. A return to a more biological production of solvents therefore requires a thorough understanding of clostridial metabolism. Transcriptome analysis, which reflects the involvement of individual genes in all cellular processes within a population, at any given (sampling) moment, is a valuable tool for gaining a deeper insight into clostridial life. In this review, we describe techniques to study transcription, summarize the evolution of these techniques and compare methods for data processing and visualization of solventogenic clostridia, particularly the species Clostridium acetobutylicum and Clostridium beijerinckii. Individual approaches for evaluating transcriptomic data are compared and their contributions to advancements in the field are assessed. Moreover, utilization of transcriptomic data for reconstruction of computational clostridial metabolic models is considered and particular models are described. Transcriptional changes in glucose transport, central carbon metabolism, the sporulation cycle, butanol and butyrate stress responses, the influence of lignocellulose-derived inhibitors on growth and solvent production, and other respective topics, are addressed and common trends are highlighted.

Enforcing ATP hydrolysis enhanced anaerobic glycolysis and promoted solvent production in Clostridium acetobutylicum.

BACKGROUND: The intracellular ATP level is an indicator of cellular energy state and plays a critical role in regulating cellular metabolism. Depletion of intracellular ATP in (facultative) aerobes can enhance glycolysis, thereby promoting end product formation. In the present study, we examined this s trategy in anaerobic ABE (acetone-butanol-ethanol) fermentation using Clostridium acetobutylicum DSM 1731. RESULTS: Following overexpression of atpAGD encoding the subunits of water-soluble, ATP-hydrolyzing F1-ATPase, the intracellular ATP level of 1731(pITF1) was significantly reduced compared to control 1731(pIMP1) over the entire batch fermentation. The glucose uptake was markedly enhanced, achieving a 78.8% increase of volumetric glucose utilization rate during the first 18 h. In addition, an early onset of acid re-assimilation and solventogenesis in concomitant with the decreased intracellular ATP level was evident. Consequently, the total solvent production was significantly improved with remarkable increases in yield (14.5%), titer (9.9%) and productivity (5.3%). Further genome-scale metabolic modeling revealed that many metabolic fluxes in 1731(pITF1) were significantly elevated compared to 1731(pIMP1) in acidogenic phase, including those from glycolysis, tricarboxylic cycle, and pyruvate metabolism; this indicates significant metabolic changes in response to intracellular ATP depletion. CONCLUSIONS: In C. acetobutylicum DSM 1731, depletion of intracellular ATP significantly increased glycolytic rate, enhanced solvent production, and resulted in a wide range of metabolic changes. Our findings provide a novel strategy for engineering solvent-producing C. acetobutylicum, and many other anaerobic microbial cell factories.

A division of labor between two biotin protein ligase homologs.

Group I biotin protein ligases (BPLs) catalyze the covalent attachment of biotin to its cognate acceptor proteins. In contrast, Group II BPLs have an additional N-terminal DNA-binding domain and function not only in biotinylation but also in transcriptional regulation of genes of biotin biosynthesis and transport. Most bacteria contain only a single biotin protein ligase, whereas Clostridium acetobutylicum contains two biotin protein ligase homologs: BplA and BirA'. Sequence alignments showed that BplA is a typical group I BPL, whereas BirA' lacked the C-terminal domain conserved throughout extant BPL proteins. This raised the questions of why two BPL homologs are needed and why the apparently defective BirA' has been retained. We have used in vivo and in vitro assays to show that BplA is a functional BPL whereas BirA' acts as a biotin sensor involved in transcriptional regulation of biotin transport. We also successfully converted BirA' into a functional biotin protein ligase with regulatory activity by fusing it to the C-terminal domain from BplA. Finally, we provide evidence that BplA and BirA' interact in vivo.

Improved CRISPR/Cas9 Tools for the Rapid Metabolic Engineering of Clostridium acetobutylicum.

Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas (CRISPR-associated proteins)9 tools have revolutionized biology-several highly efficient tools have been constructed that have resulted in the ability to quickly engineer model bacteria, for example, Escherichia coli. However, the use of CRISPR/Cas9 tools has lagged behind in non-model bacteria, hampering engineering efforts. Here, we developed improved CRISPR/Cas9 tools to enable efficient rapid metabolic engineering of the industrially relevant bacterium Clostridium acetobutylicum. Previous efforts to implement a CRISPR/Cas9 system in C. acetobutylicum have been hampered by the lack of tightly controlled inducible systems along with large plasmids resulting in low transformation efficiencies. We successfully integrated the cas9 gene from Streptococcuspyogenes into the genome under control of the xylose inducible system from Clostridium difficile, which we then showed resulted in a tightly controlled system. We then optimized the length of the editing cassette, resulting in a small editing plasmid, which also contained the upp gene in order to rapidly lose the plasmid using the upp/5-fluorouracil counter-selection system. We used this system to perform individual and sequential deletions of ldhA and the ptb-buk operon.