Evaluation of individual and combined effect of lactic acid-consuming bacteria on mesophilic hydrogen production from lactic acid effluent from food waste treatment.

Lactic acid has been applied as a precursor for hydrogen (H2) production from substrates rich in lactic acid bacteria (LAB), focusing on microbial interactions between producing and consuming LAB tested with model substrates. Therefore, this study evaluated the effect of single and combined lactic acid-consuming bacteria on mesophilic H2 production in batch tests from lactic acid from fermented food waste (FW). Megasphaera elsdenii, Clostridium beijerinckii, and Clostridium butyricum were inoculated at different ratios (v/v). Additionally, thermal pretreated sludge (TPS) was added to the strain mixtures. The highest production was obtained with M. elsdenii, C. beijerinckii, and C. butyricum (17:66:17 ratio), obtaining 1629.0 mL/Lreactor. The optimal mixture (68:32:0 of M. elsdenii and C. beijerinckii) enriched with TPS reached 1739.3 ± 98.6 mL H2/Lreactor, consuming 98 % of lactic acid added. M. elsdenii and Clostridium strains enhance H2 production from lactic acid as they persist in a microbial community initially dominated by LAB.

Pseudocin 196, a novel lantibiotic produced by Bifidobacterium pseudocatenulatum elicits antimicrobial activity against clinically relevant pathogens.

Bacteriocins are broad or narrow-spectrum antimicrobial compounds that have received significant scientific attention due to their potential to treat infections caused by antibiotic-resistant pathogenic bacteria. The genome of Bifidobacterium pseudocatenulatum MM0196, an antimicrobial-producing, fecal isolate from a healthy pregnant woman, was shown to contain a gene cluster predicted to encode Pseudocin 196, a novel lantibiotic, in addition to proteins involved in its processing, transport and immunity. Following antimicrobial assessment against various indicator strains, protease-sensitive Pseudocin 196 was purified to homogeneity from cell-free supernatant. MALDI TOF mass spectrometry confirmed that the purified antimicrobial compound corresponds to a molecular mass of 2679 Da, which is consistent with that deduced from its genetic origin. Pseudocin 196 is classified as a lantibiotic based on its similarity to lacticin 481, a lanthionine ring-containing lantibiotic produced by Lactococcus lactis. Pseudocin 196, the first reported bacteriocin produced by a B. pseudocatenulatum species of human origin, was shown to inhibit clinically relevant pathogens, such as Clostridium spp. and Streptococcus spp. thereby highlighting the potential application of this strain as a probiotic to treat and prevent bacterial infections.

Strain-dependent induction of primary bile acid 7-dehydroxylation by cholic acid.

BACKGROUND: Bile acids (BAs) are steroid-derived molecules with important roles in digestion, the maintenance of host metabolism, and immunomodulation. Primary BAs are synthesized by the host, while secondary BAs are produced by the gut microbiome through transformation of the former. The regulation of microbial production of secondary BAs is not well understood, particularly the production of 7-dehydroxylated BAs, which are the most potent agonists for host BA receptors. The 7-dehydroxylation of cholic acid (CA) is well established and is linked to the expression of a bile acid-inducible (bai) operon responsible for this process. However, little to no 7-dehydroxylation has been reported for other host-derived BAs (e.g., chenodeoxycholic acid, CDCA or ursodeoxycholic acid, UDCA). RESULTS: Here, we demonstrate that the 7-dehydroxylation of CDCA and UDCA by the human isolate Clostridium scindens is induced when CA is present, suggesting that CA-dependent transcriptional regulation is required for substantial 7-dehydroxylation of these primary BAs. This is supported by the finding that UDCA alone does not promote expression of bai genes. CDCA upregulates expression of the bai genes but the expression is greater when CA is present. In contrast, the murine isolate Extibacter muris exhibits a distinct response; CA did not induce significant 7-dehydroxylation of primary BAs, whereas BA 7-dehydroxylation was promoted upon addition of germ-free mouse cecal content in vitro. However, E. muris was found to 7-dehydroxylate in vivo. CONCLUSIONS: The distinct expression responses amongst strains indicate that bai genes are regulated differently. CA promoted bai operon gene expression and the 7-dehydroxylating activity in C. scindens strains. Conversely, the in vitro activity of E. muris was promoted only after the addition of cecal content and the isolate did not alter bai gene expression in response to CA. The accessory gene baiJ was only upregulated in the C. scindens ATCC 35704 strain, implying mechanistic differences amongst isolates. Interestingly, the human-derived C. scindens strains were also capable of 7-dehydroxylating murine bile acids (muricholic acids) to a limited extent. This study shows novel 7-dehydroxylation activity in vitro resulting from the presence of CA and suggests distinct bai gene expression across bacterial species.

Lactate-mediated mixotrophic co-cultivation of Clostridium drakei and recombinant Acetobacterium woodii for autotrophic production of volatile fatty acids.

BACKGROUND: Acetogens, a diverse group of anaerobic autotrophic bacteria, are promising whole-cell biocatalysts that fix CO2 during their growth. However, because of energetic constraints, acetogens exhibit slow growth and the product spectrum is often limited to acetate. Enabling acetogens to form more valuable products such as volatile fatty acids during autotrophic growth is imperative for cementing their place in the future carbon neutral industry. Co-cultivation of strains with different capabilities has the potential to ease the limiting energetic constraints. The lactate-mediated co-culture of an Acetobacterium woodii mutant strain, capable of lactate production, with the Clostridium drakei SL1 type strain can produce butyrate and hexanoate. In this study, the preceding co-culture is characterized by comparison of monocultures and different co-culture approaches. RESULTS: C. drakei grew with H2 + CO2 as main carbon and energy source and thrived when further supplemented with D-lactate. Gas phase components and lactate were consumed in a mixotrophic manner with acetate and butyrate as main products and slight accumulation of hexanoate. Formate was periodically produced and eventually consumed by C. drakei. A lactate-mediated co-culture of the A. woodii [PbgaL_ldhD_NFP] strain, engineered for autotrophic lactate production, and C. drakei produced up to 4 ± 1.7 mM hexanoate and 18.5 ± 5.8 mM butyrate, quadrupling and doubling the respective titers compared to a non-lactate-mediated co-culture. Further co-cultivation experiments revealed the possible advantage of sequential co-culture over concurrent approaches, where both strains are inoculated simultaneously. Scanning electron microscopy of the strains revealed cell-to-cell contact between the co-culture partners. Finally, a combined pathway of A. woodii [PbgaL_ldhD_NFP] and C. drakei for chain-elongation with positive ATP yield is proposed. CONCLUSION: Lactate was proven to be a well-suited intermediate to combine the high gas uptake capabilities of A. woodii with the chain-elongation potential of C. drakei. The cell-to-cell contact observed here remains to be further characterized in its nature but hints towards diffusive processes being involved in the co-culture. Furthermore, the metabolic pathways involved are still speculatory for C. drakei and do not fully explain the consumption of formate while H2 + CO2 is available. This study exemplifies the potential of combining metabolically engineered and native bacterial strains in a synthetic co-culture.

Synbiotic therapy with Clostridium sporogenes and xylan promotes gut-derived indole-3-propionic acid and improves cognitive impairments in an Alzheimer's disease mouse model.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized primarily by cognitive impairment. Recent investigations have highlighted the potential of nutritional interventions that target the gut-brain axis, such as probiotics and prebiotics, in forestalling the onset of AD. In this study, whole-genome sequencing was employed to identify xylan as the optimal carbon source for the tryptophan metabolism regulating probiotic Clostridium sporogenes (C. sporogenes). Subsequent in vivo studies demonstrated that administration of a synbiotic formulation comprising C. sporogenes (1 × 1010 CFU per day) and xylan (1%, w/w) over a duration of 30 days markedly enhanced cognitive performance and spatial memory faculties in the 5xFAD transgenic AD mouse model. The synbiotic treatment significantly reduced amyloid-ß (Aß) accumulation in the cortex and hippocampus of the brain. Importantly, synbiotic therapy substantially restored the synaptic ultrastructure in AD mice and suppressed neuroinflammatory responses. Moreover, the intervention escalated levels of the microbial metabolite indole-3-propionic acid (IPA) and augmented the relative prevalence of IPA-synthesizing bacteria, Lachnospira and Clostridium, while reducing the dominant bacteria in AD, such as Aquabacterium, Corynebacterium, and Romboutsia. Notably, synbiotic treatment also prevented the disruption of gut barrier integrity. Correlation analysis indicated a strong positive association between gut microbiota-generated IPA levels and behavioral changes. In conclusion, this study demonstrates that synbiotic supplementation significantly improves cognitive and intellectual deficits in 5xFAD mice, which could be partly attributed to enhanced IPA production by gut microbiota. These findings provide a theoretical basis for considering synbiotic therapy as a novel microbiota-targeted approach for the treatment of metabolic and neurodegenerative diseases.

Identification of volatile metabolites produced from levodopa metabolism by different bacteria strains of the gut microbiome.

Interspecies pathways in the gut microbiome have been shown to metabolize levodopa, the primary treatment for Parkinson's disease, and reduce its bioavailability. While the enzymatic reactions have been identified, the ability to establish the resulting macromolecules as biomarkers of microbial metabolism remains technically challenging. In this study, we leveraged an untargeted mass spectrometry-based approach to investigate volatile organic compounds (VOCs) produced during levodopa metabolism by Enterococcus faecalis, Clostridium sporogenes, and Eggerthella lenta. We cultured these organisms with and without their respective bioactive metabolites and detected levodopa-induced shifts in VOC profiles. We then utilized bioinformatics to identify significant differences in 2,6-dimethylpyrazine, 4,6-dimethylpyrimidine, and 4,5-dimethylpyrimidine associated with its biotransformation. Supplementing cultures with inhibitors of levodopa-metabolizing enzymes revealed specific modulation of levodopa-associated diazines, verifying their relationship to its metabolism. Furthermore, functional group analysis depicts strain-specific VOC profiles that reflect interspecies differences in metabolic activity that can be leveraged to assess microbiome functionality in individual patients. Collectively, this work identifies previously uncharacterized metabolites of microbe-mediated levodopa metabolism to determine potential indicators of this activity and further elucidate the metabolic capabilities of different gut bacteria.

Exploiting the Thermotolerance of Clostridium Strain M1NH for Efficient Caproic Acid Fermentation from Ethanol and Acetic Acid.

A novel thermotolerant caproic acid-producing bacterial strain, Clostridium M1NH, was successfully isolated from sewage sludge. Ethanol and acetic acid at a molar ratio of 4:1 proved to be the optimal substrates, yielding a maximum caproic acid production of 3.5 g/L. Clostridium M1NH exhibited remarkable tolerance to high concentrations of ethanol (up to 5% v/v), acetic acid (up to 5% w/v), and caproic acid (up to 2% w/v). The strain also demonstrated a wide pH tolerance range (pH 5.5-7.5) and an elevated temperature optimum between 35 and 40 °C. Phylogenetic analysis based on 16S rRNA gene sequences revealed that Clostridium M1NH shares a 98% similarity with Clostridium luticellarii DSM 29923 T. The robustness of strain M1NH and its efficient caproic acid production from low-cost substrates highlight its potential for sustainable bio-based chemical production. The maximum caproic acid yield achieved by Clostridium M1NH was 1.6-fold higher than that reported for C. kluyveri under similar fermentation conditions. This study opens new avenues for valorizing waste streams and advancing a circular economy model in the chemical industry.

Simultaneous Formate and Syngas Conversion Boosts Growth and Product Formation by Clostridium ragsdalei.

Electrocatalytic CO2 reduction to CO and formate can be coupled to gas fermentation with anaerobic microorganisms. In combination with a competing hydrogen evolution reaction in the cathode in aqueous medium, the in situ, electrocatalytic produced syngas components can be converted by an acetogenic bacterium, such as Clostridium ragsdalei, into acetate, ethanol, and 2,3-butanediol. In order to study the simultaneous conversion of CO, CO2, and formate together with H2 with C. ragsdalei, fed-batch processes were conducted with continuous gassing using a fully controlled stirred tank bioreactor. Formate was added continuously, and various initial CO partial pressures (pCO0) were applied. C. ragsdalei utilized CO as the favored substrate for growth and product formation, but below a partial pressure of 30 mbar CO in the bioreactor, a simultaneous CO2/H2 conversion was observed. Formate supplementation enabled 20-50% higher growth rates independent of the partial pressure of CO and improved the acetate and 2,3-butanediol production. Finally, the reaction conditions were identified, allowing the parallel CO, CO2, formate, and H2 consumption with C. ragsdalei at a limiting CO partial pressure below 30 mbar, pH 5.5, n = 1200 min-1, and T = 32 °C. Thus, improved carbon and electron conversion is possible to establish efficient and sustainable processes with acetogenic bacteria, as shown in the example of C. ragsdalei.

Pooled CRISPR Interference Screening Identifies Crucial Transcription Factors in Gas-Fermenting Clostridium ljungdahlii.

Gas-fermenting Clostridium species hold tremendous promise for one-carbon biomanufacturing. To unlock their full potential, it is crucial to unravel and optimize the intricate regulatory networks that govern these organisms; however, this aspect is currently underexplored. In this study, we employed pooled CRISPR interference (CRISPRi) screening to uncover a wide range of functional transcription factors (TFs) in Clostridium ljungdahlii, a representative species of gas-fermenting Clostridium, with a special focus on TFs associated with the utilization of carbon resources. Among the 425 TF candidates, we identified 75 and 68 TF genes affecting the heterotrophic and autotrophic growth of C. ljungdahlii, respectively. We focused our attention on two of the screened TFs, NrdR and DeoR, and revealed their pivotal roles in the regulation of deoxyribonucleoside triphosphates (dNTPs) supply, carbon fixation, and product synthesis in C. ljungdahlii, thereby influencing the strain performance in gas fermentation. Based on this, we proceeded to optimize the expression of deoR in C. ljungdahlii by adjusting its promoter strength, leading to an improved growth rate and ethanol synthesis of C. ljungdahlii when utilizing syngas. This study highlights the effectiveness of pooled CRISPRi screening in gas-fermenting Clostridium species, expanding the horizons for functional genomic research in these industrially important bacteria.

Dietary fibre directs microbial tryptophan metabolism via metabolic interactions in the gut microbiota.

Tryptophan is catabolized by gut microorganisms resulting in a wide range of metabolites implicated in both beneficial and adverse host effects. How gut microbial tryptophan metabolism is directed towards indole, associated with chronic kidney disease, or towards protective indolelactic acid (ILA) and indolepropionic acid (IPA) is unclear. Here we used in vitro culturing and animal experiments to assess gut microbial competition for tryptophan and the resulting metabolites in a controlled three-species defined community and in complex undefined human faecal communities. The generation of specific tryptophan-derived metabolites was not predominantly determined by the abundance of tryptophan-metabolizing bacteria, but rather by substrate-dependent regulation of specific metabolic pathways. Indole-producing Escherichia coli and ILA- and IPA-producing Clostridium sporogenes competed for tryptophan within the three-species community in vitro and in vivo. Importantly, fibre-degrading Bacteroides thetaiotaomicron affected this competition by cross-feeding monosaccharides to E. coli. This inhibited indole production through catabolite repression, thus making more tryptophan available to C. sporogenes, resulting in increased ILA and IPA production. The fibre-dependent reduction in indole was confirmed using human faecal cultures and faecal-microbiota-transplanted gnotobiotic mice. Our findings explain why consumption of fermentable fibres suppresses indole production but promotes the generation of other tryptophan metabolites associated with health benefits.

Clostridium sporogenes increases fat accumulation in mice by enhancing energy absorption and adipogenesis.

Gut bacteria belonging to the Clostridium family play a pivotal role in regulating host energy balance and metabolic homeostasis. As a commensal bacterium, Clostridium sporogenes has been implicated in modulating host energy homeostasis, albeit the underlying mechanism remains elusive. Therefore, this study aimed to investigate the impact of C. sporogenes supplementation on various physiological parameters, intestinal morphology, particularly adipose tissue accumulation, and glucolipid metabolism in mice. The findings reveal that mice supplemented with C. sporogenes for 6 weeks exhibited a notable increase in body weight, fat mass, adipocyte size, and serum triglyceride (TG) levels. Notably, the increased fat accumulation is observed despite consistent feed intake in treated mice. Mechanistically, C. sporogenes supplementation significantly improved the structure integrity of intestinal villi and enhanced energy absorption efficiency while reducing excretion of carbohydrates and fatty acids in feces. This was accompanied by upregulation of glucose and fatty acid transporter expression. Furthermore, supplementation with C. sporogenes promoted adipogenesis in both liver and adipose tissues, as evidenced by increased levels of hepatic pyruvate, acetyl-CoA, and TG, along with elevated expression levels of genes associated with lipid synthesis. Regarding the microbiological aspect, C. sporogenes supplementation correlated with an increased abundance of Clostridium genus bacteria and enhanced carbohydrate enzyme activity. In summary, C. sporogenes supplementation significantly promotes fat accumulation in mice by augmenting energy absorption and adipogenesis, possibly mediated by the expansion of Clostridium bacteria population with robust glycolipid metabolic ability. IMPORTANCE: The Clostridia clusters have been implicated in energy metabolism, the specific species and underlying mechanisms remain unclear. This present study is the first to report Clostridium sporogenes is able to affect fat accumulation and glycolipid metabolism. We indicated that gavage of C. sporogenes promoted the adipogenesis and fat accumulation in mice by not only increasing the abundance of Clostridium bacteria but by also enhancing the metabolic absorption of carbohydrates and fatty acids significantly. Obviously, changes of gut microbiota caused by the C. sporogenes, especially the significant increase of Clostridium bacteria, contributed to the fat accumulation of mice. In addition, the enhancement of Clostridium genus bacteria remarkably improved the synthesis of hepatic pyruvate, acetyl-CoA, and triglyceride levels, as well as reduced the excretion of fecal carbohydrates, short-chain fatty acids, and free fatty acids remarkably. These findings will help us to understand the relationship of specific bacteria and host energy homeostasis.

Microbial cell factory for butyl butyrate production: Knowledges and perspectives.

Butyl butyrate is a short-chain fatty acid ester (C8) with a fruity aroma. It has broad prospects in the fields of foods, cosmetics and biofuels. At present, butyl butyrate is produced by chemical synthesis in the industry, but it is highly dependent on petroleum-based products. The growing concerns regarding the future scarcity of fossil fuels have been strongly promoted the transition from traditional fossil fuels and products to renewable bioenergy and biochemicals. Therefore, it is necessary to develop a green biochemical technology to replace traditional petroleum-based materials. In recent years, microorganisms such as Escherichia coli and Clostridium have been engineered to serve as cell factories for the sustainable one-pot production of short-chain fatty acid esters, including butyl butyrate. This opinion highlights the recent development in the use of lipases and alcohol acyltransferases (AATs) for butyl butyrate production in microbial fermentation, as well as future perspectives.

Production of deoxycholic acid by low-abundant microbial species is associated with impaired glucose metabolism.

Alterations in gut microbiota composition are suggested to contribute to cardiometabolic diseases, in part by producing bioactive molecules. Some of the metabolites are produced by very low abundant bacterial taxa, which largely have been neglected due to limits of detection. However, the concentration of microbially produced metabolites from these taxa can still reach high levels and have substantial impact on host physiology. To explore this concept, we focused on the generation of secondary bile acids by 7α-dehydroxylating bacteria and demonstrated that addition of a very low abundant bacteria to a community can change the metabolic output dramatically. We show that Clostridium scindens converts cholic acid into the secondary bile acid deoxycholic acid (DCA) very efficiently even though the abundance of C. scindens is low, but still detectable by digital droplet PCR. We also show that colonization of germ-free female mice with a community containing C. scindens induces DCA production and affects host metabolism. Finally, we show that DCA correlates with impaired glucose metabolism and a worsened lipid profile in individuals with type 2 diabetes, which implies that this metabolic pathway may contribute to the development of cardiometabolic disease.

Hydrogen production pathways in Clostridia and their improvement by metabolic engineering.

Biological production of hydrogen has a tremendous potential as an environmentally sustainable technology to generate a clean fuel. Among the different available methods to produce biohydrogen, dark fermentation features the highest productivity and can be used as a means to dispose of organic waste biomass. Within this approach, Clostridia have the highest theoretical H2 production yield. Nonetheless, most strains show actual yields far lower than the theoretical maximum: improving their efficiency becomes necessary for achieving cost-effective fermentation processes. This review aims at providing a survey of the metabolic network involved in H2 generation in Clostridia and strategies used to improve it through metabolic engineering. Together with current achievements, a number of future perspectives to implement these results will be illustrated.

Integration of biocompatible hydrogen evolution catalyst developed from metal-mix solutions with microbial electrosynthesis.

Microbial conversion of CO2 to multi-carbon compounds such as acetate and butyrate is a promising valorisation technique. For those reactions, the electrochemical supply of hydrogen to the biocatalyst is a viable approach. Earlier we have shown that trace metals from microbial growth media spontaneously form in situ electro-catalysts for hydrogen evolution. Here, we show biocompatibility with the successful integration of such metal mix-based HER catalyst for immediate start-up of microbial acetogenesis (CO2 to acetate). Also, n-butyrate formation started fast (after twenty days). Hydrogen was always produced in excess, although productivity decreased over the 36 to 50 days, possibly due to metal leaching from the cathode. The HER catalyst boosted microbial productivity in a two-step microbial community bioprocess: acetogenesis by a BRH-c20a strain and acetate elongation to n-butyrate by Clostridium sensu stricto 12 (related) species. These findings provide new routes to integrate electro-catalysts and micro-organisms showing respectively bio and electrochemical compatibility.

Influence of fumarate on interspecies electron transfer and the metabolic shift induced in Clostridium pasteurianum by Geobacter sulfurreducens.

AIMS: In previous studies, it was demonstrated that co-culturing Clostridium pasteurianum and Geobacter sulfurreducens triggers a metabolic shift in the former during glycerol fermentation. This shift, attributed to interspecies electron transfer and the exchange of other molecules, enhances the production of 1,3-propanediol at the expense of the butanol pathway. The aim of this investigation is to examine the impact of fumarate, a soluble compound usually used as an electron acceptor for G. sulfurreducens, in the metabolic shift previously described in C. pasteurianum. METHODS AND RESULTS: Experiments were conducted by adding along with glycerol, acetate, and different quantities of fumarate in co-cultures of G. sulfurreducens and C. pasteurianum. A metabolic shift was exhibited in all the co-culture conditions. This shift was more pronounced at higher fumarate concentrations. Additionally, we observed G. sulfurreducens growing even in the absence of fumarate and utilizing small amounts of this compound as an electron donor rather than an electron acceptor in the co-cultures with high fumarate addition. CONCLUSIONS: This study provided evidence that interspecies electron transfer continues to occur in the presence of a soluble electron acceptor, and the metabolic shift can be enhanced by promoting the growth of G. sulfurreducens.

Advancing two-stage hydrogen production from corn stover via dark fermentation: Contributions of thermally modified maifanite to microbial proliferation and pH self-regulation.

This study introduces a two-stage hydrogen production enhancement mechanism using natural particle additives, with a focus on the effects of thermally modified maifanite (TMM) and pH self-regulation on dark fermentation (DF). Initial single-factor experiments identified preliminary parameters for the addition of TMM, which were further optimized using a Box-Behnken design. The established optimal conditions which include mass of 5.5 g, particle size of 120 mesh, and temperature of 324 °C, resulted in a 28.7 % increase in cumulative hydrogen yield (CHY). During the primary hydrogen production stage, TMM significantly boosted the growth and activity of Clostridium_sensu_stricto_1, enhancing hydrogen output. Additionally, a pH self-regulating phenomenon was observed, capable of initiating secondary hydrogen production and further augmenting CHY. These findings presented a novel and efficient approach for optimizing biohydrogen production, offering significant implications for future research and application in sustainable energy technologies.

Hexanoic acid production and microbial community in anaerobic fermentation: Effects of inorganic carbon addition.

Carbon dioxide (CO2) plays a crucial role in carbon chain elongation with ethanol serving as an electron donor. In this study, the impacts of various carbonates on CO2 concentration, hexanoic acid production, and microbial communities during ethanol-butyric acid fermentation were explored. The results showed that the addition of MgCO3 provided sustained inorganic carbon and facilitated interspecific electron transfer, thereby increasing hexanoic acid yield by 58%. MgCO3 and NH4HCO3 inhibited the excessive ethanol oxidation and decreased the yield of acetic acid by 51% and 42%, respectively. The yields of hexanoic acid and acetic acid in the CaCO3 group increased by 19% and 15%, respectively. The NaHCO3 group exhibited high headspace CO2 concentration, promoting acetogenic bacteria enrichment while reducing the abundance of Clostridium_sensu_stricto_12. The batch addition of NaHCO3 accelerated the synthesis of hexanoic acid and increased its production by 26%. The relative abundance of Clostridium_sensus_stricto_12 was positively correlated with hexanoic acid production.

Multiplex genetic manipulations in Clostridium butyricum and Clostridium sporogenes to secrete recombinant antigen proteins for oral-spore vaccination.

BACKGROUND: Clostridium spp. has demonstrated therapeutic potential in cancer treatment through intravenous or intratumoral administration. This approach has expanded to include non-pathogenic clostridia for the treatment of various diseases, underscoring the innovative concept of oral-spore vaccination using clostridia. Recent advancements in the field of synthetic biology have significantly enhanced the development of Clostridium-based bio-therapeutics. These advancements are particularly notable in the areas of efficient protein overexpression and secretion, which are crucial for the feasibility of oral vaccination strategies. Here, we present two examples of genetically engineered Clostridium candidates: one as an oral cancer vaccine and the other as an antiviral oral vaccine against SARS-CoV-2. RESULTS: Using five validated promoters and a signal peptide derived from Clostridium sporogenes, a series of full-length NY-ESO-1/CTAG1, a promising cancer vaccine candidate, expression vectors were constructed and transformed into C. sporogenes and Clostridium butyricum. Western blotting analysis confirmed efficient expression and secretion of NY-ESO-1 in clostridia, with specific promoters leading to enhanced detection signals. Additionally, the fusion of a reported bacterial adjuvant to NY-ESO-1 for improved immune recognition led to the cloning difficulties in E. coli. The use of an AUU start codon successfully mitigated potential toxicity issues in E. coli, enabling the secretion of recombinant proteins in C. sporogenes and C. butyricum. We further demonstrate the successful replacement of PyrE loci with high-expression cassettes carrying NY-ESO-1 and adjuvant-fused NY-ESO-1, achieving plasmid-free clostridia capable of secreting the antigens. Lastly, the study successfully extends its multiplex genetic manipulations to engineer clostridia for the secretion of SARS-CoV-2-related Spike_S1 antigens. CONCLUSIONS: This study successfully demonstrated that C. butyricum and C. sporogenes can produce the two recombinant antigen proteins (NY-ESO-1 and SARS-CoV-2-related Spike_S1 antigens) through genetic manipulations, utilizing the AUU start codon. This approach overcomes challenges in cloning difficult proteins in E. coli. These findings underscore the feasibility of harnessing commensal clostridia for antigen protein secretion, emphasizing the applicability of non-canonical translation initiation across diverse species with broad implications for medical or industrial biotechnology.

Elevated caproic acid production from one-stage anaerobic fermentation of organic waste and its selective recovery by electro-membrane process.

A constructed microbial consortia-based strategy to enhance caproic acid production from one-stage mixed-fermentation of glucose was developed, which incubated with acidogens (Clostridium sensu stricto 1, 11 dominated) and chain elongators (including Clostridium sensu stricto 12, Sporanaerobacter, and Caproiciproducens) acclimated from anaerobic sludge. Significant product upgrading toward caproic acid (8.31 g‧L-1) and improved substrate degradation was achieved, which can be greatly attributed to the lactic acid platform. Whereas, a small amount of caproic acid was observed in the control incubating with acidogens, with an average concentration of 2.09 g‧L-1. The strategy accelerated the shape and cooperation of the specific microbial community dominated by Clostridium sensu stricto and Caproiciproducens, which thereby contributed to caproic acid production via the fatty acid biosynthesis pathway. Moreover, the tailored electrodialysis with bipolar membrane enabled progressive up-concentration and acidification, allowing selective separation of caproic acid as an immiscible product with a purity of 82.58 % from the mixture.