Clostridium tyrobutyricum in Combination with Chito-oligosaccharides Modulate Inflammation and Gut Microbiota for Inflammatory Bowel Disease Treatment.

Synbiotics, the combination of probiotics and prebiotics, are thought to be a pragmatic approach for the treatment of various diseases, including inflammatory bowel disease (IBD). The synergistic therapeutic effects of probiotics and prebiotics remain underexplored. Clostridium tyrobutyricum, a short-chain fatty acid (SCFA) producer, has been recognized as a promising probiotic candidate that can offer health benefits. In this study, the treatment effects of synbiotics containing C. tyrobutyricum and chitooligosaccharides (COSs) on IBD were evaluated. The results indicated that the synbiotic supplement effectively relieved inflammation and restored intestinal barrier function. Additionally, the synbiotic supplement could contribute to the elimination of reactive oxygen species (ROS) and improve the production of SCFAs through the SCFAs-producer of C. tyrobutyricum. Furthermore, such the synbiotic could also regulate the composition of gut microbiota. These findings underscore the potential of C. tyrobutyricum and COSs as valuable living biotherapeutics for the treatment of intestinal-related diseases.

Integration of food raw materials, food microbiology, and food additives: systematic research and comprehensive insights into sweet sorghum juice, Clostridium tyrobutyricum TGL-A236 and bio-butyric acid.

Introduction: Sweet sorghum juice is a typical production feedstock for natural, eco-friendly sweeteners and beverages. Clostridium tyrobutyricum is one of the widely used microorganisms in the food industry, and its principal product, bio-butyric acid is an important food additive. There are no published reports of Clostridium tyrobutyricum producing butyric acid using SSJ as the sole substrate without adding exogenous substances, which could reach a food-additive grade. This study focuses on tailoring a cost-effective, safe, and sustainable process and strategy for their production and application. Methods: This study modeled the enzymolysis of non-reducing sugars via the first/second-order kinetics and added food-grade diatomite to the hydrolysate. Qualitative and quantitative analysis were performed using high-performance liquid chromatography, gas chromatography-mass spectrometer, full-scale laser diffraction method, ultra-performance liquid chromatography-tandem mass spectrometry, the cell double-staining assay, transmission electron microscopy, and Oxford nanopore technology sequencing. Quantitative real-time polymerase chain reaction, pathway and process enrichment analysis, and homology modeling were conducted for mutant genes. Results: The treated sweet sorghum juice showed promising results, containing 70.60 g/L glucose and 63.09 g/L fructose, with a sucrose hydrolysis rate of 98.29% and a minimal sucrose loss rate of 0.87%. Furthermore, 99.62% of the colloidal particles and 82.13% of the starch particles were removed, and the concentrations of hazardous substances were effectively reduced. A food microorganism Clostridium tyrobutyricum TGL-A236 with deep utilization value was developed, which showed superior performance by converting 30.65% glucose and 37.22% fructose to 24.1364 g/L bio-butyric acid in a treated sweet sorghum juice (1:1 dilution) fermentation broth. This titer was 2.12 times higher than that of the original strain, with a butyric acid selectivity of 86.36%. Finally, the Genome atlas view, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and evolutionary genealogy of genes: Non-supervised Orthologous (eggNOG) functional annotations, three-dimensional structure and protein cavity prediction of five non-synonymous variant genes were obtained. Conclusion: This study not only includes a systematic process flow and in-depth elucidation of relevant mechanisms but also provides a new strategy for green processing of food raw materials, improving food microbial performance, and ensuring the safe production of food additives.

Targeted microbial collaboration to enhance key flavor metabolites by inoculating Clostridium tyrobutyricum and Saccharomyces cerevisiae in the strong-flavor Baijiu simulated fermentation system.

Ethyl hexanoate and ethyl butyrate are indispensable flavor metabolites in strong-flavor Baijiu (SFB), but batch production instability in fermenting grains can reduce the quality of distilled Baijiu. Biofortification of the fermentation process by designing a targeted microbial collaboration pattern is an effective method to stabilize the quality of Baijiu. In this study, we explored the metabolism under co-culture liquid fermentation with Clostridium tyrobutyricum DB041 and Saccharomyces cerevisiae YS219 and investigated the effects of inoculation with two functional microorganisms on physicochemical factors, flavor metabolites, and microbial communities in solid-state simulated fermentation of SFB for the first time. The headspace solid-phase microextraction-gas chromatography-mass spectrometry results showed that ethyl butyrate and ethyl hexanoate significantly increased in fermented grain. High-throughput sequencing analysis showed that Pediococcus, Lactobacillus, Weissella, Clostridium_sensu_stricto_12, and Saccharomyces emerged as the dominant microorganisms at the end of fermentation. Co-occurrence analysis showed that ethyl hexanoate and ethyl butyrate were significantly correlated (|r| > 0.5, P < 0.05) with a cluster of interactions dominated by lactic acid bacteria (Pediococcus, Lactobacillus, Weissella, and Lactococcus), which was driven by the functional C. tyrobutyricum and S. cerevisiae. Mantel test showed that moisture and reducing sugars were the main physicochemical factor affecting microbial collaboration (|r| > 0.7, P < 0.05). Taken together, the collaborative microbial pattern of inoculation with C. tyrobutyricum and S. cerevisiae showed positive results in enhancing typical flavor metabolites and the synergistic effects of microorganisms in SFB.

Microbiome dysbiosis in patients with chronic endometritis and Clostridium tyrobutyricum ameliorates chronic endometritis in mice.

Chronic endometritis is associated with the imbalance of female reproductive tract microbiota and pathogenic microbial infection. This study aimed to identify the specific changes in the endometrial microbiome in patients with endometritis and to explore how Clostridium tyrobutyricum (C.t) influences the progression of endometritis in mice for further elucidating endometritis pathogenesis. For this purpose, endometrial tissues from 100 participants were collected and divided into positive, weakly positive, and negative groups based on CD138 levels, while endometrial microbiome differences were detected and analyzed using 16S rRNA gene sequencing. Staphylococcus aureus (S. aureus)-induced endometritis mouse model was established, followed by treatment with C.t, and inflammatory response, epithelial barrier, and TLR4/NF-κB pathway were evaluated. Results showed that α- and ß-diversity was significantly lower in the positive group compared with the weakly positive or negative groups, where the negative group had more unique operational taxonomic units. The abundance of Proteobacteria was found to be increased, while that of Actinobacteria, Firmicutes, and Bacteroidetes was found to be reduced in the positive group, while the area under the curve value was found to be 0.664. Furthermore, C.t treatment resulted in the alleviation of S. aureus-induced inflammatory response, epithelial barrier damage, and activation of the TLR4/NF-κB pathway in mice. Clinical samples analysis revealed that the diversity and abundance of microbiota were altered in patients with endometritis having positive CD138 levels, while mechanistic investigations revealed C.t alleviated S. aureus-induced endometritis by inactivating TLR4/NF-κB pathway. The findings of this study are envisaged to provide a diagnostic and therapeutic potential of microbiota in endometritis.

Metabolic and Bioprocess Engineering of Clostridium tyrobutyricum for Butyl Butyrate Production on Xylose and Shrimp Shell Waste.

Microbial conversion of agri-food waste to valuable compounds offers a sustainable route to develop the bioeconomy and contribute to sustainable biorefinery. Clostridium tyrobutyricum displays a series of native traits suitable for high productivity conversion of agri-food waste, which make it a promising host for the production of various compounds, such as the short-chain fatty acids and their derivative esters products. In this study, a butanol synthetic pathway was constructed in C. tyrobutyricum, and then efficient butyl butyrate production through in situ esterification was achieved by the supplementation of lipase into the fermentation. The butyryl-CoA/acyl-CoA transferase (cat1) was overexpressed to balance the ratio between precursors butyrate and butanol. Then, a suitable fermentation medium for butyl butyrate production was obtained with xylose as the sole carbon source and shrimp shell waste as the sole nitrogen source. Ultimately, 5.9 g/L of butyl butyrate with a selectivity of 100%, and a productivity of 0.03 g/L·h was achieved under xylose and shrimp shell waste with batch fermentation in a 5 L bioreactor. Transcriptome analyses exhibited an increase in the expression of genes related to the xylose metabolism, nitrogen metabolism, and amino acid metabolism and transport, which reveal the mechanism for the synergistic utilization of xylose and shrimp shell waste. This study presents a novel approach for utilizing xylose and shrimp shell waste to produce butyl butyrate by using an anaerobic fermentative platform based on C. tyrobutyricum. This innovative fermentation medium could save the cost of nitrogen sources (~97%) and open up possibilities for converting agri-food waste into other high-value products.

Specificity of the AMP-6000 Method for Enumerating Clostridium Endospores in Milk.

Enumeration of endospores of butyric acid-forming clostridia in cheese milk is an essential part of milk quality monitoring for cheese producers to avoid late blowing, severe spoilage caused by clostridia during ripening. However, due to the lack of an internationally standardized method, different methods are used and it is important to consider how the choice of method affects the results. This is particularly relevant when clostridial spore counts in milk are considered for quality payments. The aim of this study was to evaluate the specificity of the AMP-6000 method for the enumeration of endospores of cheese spoiling clostridia in milk. First, to assess the prevalence of Clostridium diversity and to determine potential non-target species, we identified isolates from positive reactions of the AMP-6000 method used to quantify clostridial endospores in raw milk and teat skin samples by MALDI-TOF MS. Based on these results, a strain library was designed to evaluate method inclusivity and exclusivity using pure cultures of target and non-target strains according to ISO 16140-2:2016. Most target Clostridium tyrobutyricum strains, as well as all tested C. butyricum and C. sporogenes strains were inclusive. However, C. beijerinckii may be underestimated as only some strains gave positive results. All non-target strains of bacilli and lysinibacilli, but not all paenibacilli, were confirmed to be exclusive. This study provides performance data to better understand the results of microbiological enumeration of butyric acid-forming clostridia in milk and serves as a basis for future methodological considerations and improvements.

Development of inducible promoters for regulating gene expression in Clostridium tyrobutyricum for biobutanol production.

Clostridium tyrobutyricum is an anaerobe known for its ability to produce short-chain fatty acids, alcohols, and esters. We aimed to develop inducible promoters for fine-tuning gene expression in C. tyrobutyricum. Synthetic inducible promoters were created by employing an Escherichia coli lac operator to regulate the thiolase promoter (PCathl) from Clostridium acetobutylicum, with the best one (LacI-Pto4s) showing a 5.86-fold dynamic range with isopropyl ß- d-thiogalactoside (IPTG) induction. A LT-Pt7 system with a dynamic range of 11.6-fold was then created by combining LacI-Pto4s with a T7 expression system composing of RNA polymerase (T7RNAP) and Pt7lac promoter. Furthermore, two inducible expression systems BgaR-PbgaLA and BgaR-PbgaLB with a dynamic range of ~40-fold were developed by optimizing a lactose-inducible expression system from Clostridium perfringens with modified 5' untranslated region (5' UTR) and ribosome-binding site (RBS). BgaR-PbgaLB was then used to regulate the expressions of a bifunctional aldehyde/alcohol dehydrogenase encoded by adhE2 and butyryl-CoA/acetate Co-A transferase encoded by cat1 in C. tyrobutyricum wild type and Δcat1::adhE2, respectively, demonstrating its efficient inducible gene regulation. The regulated cat1 expression also confirmed that the Cat1-catalyzed reaction was responsible for acetate assimilation in C. tyrobutyricum. The inducible promoters offer new tools for tuning gene expression in C. tyrobutyricum for industrial applications.

Clostridium strain FAM25158, a unique endospore-forming bacterium related to Clostridium tyrobutyricum and isolated from Emmental cheese shows low tolerance to salt.

The genus Clostridium is a large and diverse group of species that can cause food spoilage, including late blowing defect (LBD) in cheese. In this study, we investigated the taxonomic status of strain FAM25158 isolated from Emmental cheese with LBD using a polyphasic taxonomic and comparative genomic approach. A 16S rRNA gene sequence phylogeny suggested affiliation to the Clostridium sensu stricto cluster, with Clostridium tyrobutyricum DSM 2637T being the closest related type strain (99.16% sequence similarity). Average Nucleotide Identity (ANI) analysis revealed that strain FAM25158 is at the species threshold with C. tyrobutyricum, with ANI values ranging from 94.70 to 95.26%, while the digital DNA-DNA hybridization values were below the recommended threshold, suggesting that FAM25158 is significantly different from C. tyrobutyricum at the genomic level. Moreover, comparative genomic analysis between FAM25158 and its four closest C. tyrobutyricum relatives revealed a diversity of metabolic pathways, with FAM25158 differing from other C. tyrobutyricum strains by the presence of genes such as scrA, srcB, and scrK, responsible for sucrose utilization, and the absence of many important functional genes associated with cold and osmolality adaptation, which was further supported by phenotypic analyses. Surprisingly, strain FAM25158 exhibited unique physiologic traits, such as an optimal growth temperature of 30°C, in contrast to its closest relatives, C. tyrobutyricum species with an optimal growth temperature of 37°C. Additionally, the growth of FAM25158 was inhibited at NaCl concentrations higher than 0.5%, a remarkable observation considering its origin from cheese. While the results of this study provide novel information on the genetic content of strain FAM25158, the relationship between its genetic content and the observed phenotype remains a topic requiring further investigation.

Efficient production of butyric acid from lignocellulosic biomass by revealing the mechanisms of Clostridium tyrobutyricum tolerance to phenolic inhibitors.

Phenolic compounds (PCs) generated during pretreatment of lignocellulosic biomass severely hinder the biorefinery by Clostridia. As a hyperbutyrate-producing strain, Clostridium tyrobutyricum has excellent tolerance to PCs, but its tolerance mechanism is poorly understood. In this study, a comprehensive transcriptome analysis was applied to elucidate the response of C. tyrobutyricum to four typical PCs. The findings revealed that the expression levels of genes associated with PC reduction, HSPs, and membrane transport were significantly altered under PC stress. Due to PCs being reduced to low-toxicity alcohols/acids by C. tyrobutyricum, enhancing the reduction of PCs by overexpressing reductase genes could enhance the strain's tolerance to PCs. Under 1.0 g/L p-coumaric acid stress, compared with the wild-type strain, ATCC 25755/sdr1 exhibited a 31.2 % increase in butyrate production and a 38.5 % increase in productivity. These insights contribute to the construction of PC-tolerant Clostridia, which holds promise for improving biofuel and chemical production from lignocellulosic biomass.

Update of the list of qualified presumption of safety (QPS) recommended microbiological agents intentionally added to food or feed as notified to EFSA 19: Suitability of taxonomic units notified to EFSA until September 2023.

The qualified presumption of safety (QPS) process was developed to provide a safety assessment approach for microorganisms intended for use in food or feed chains. The QPS approach is based on an assessment of published data for each taxonomic unit (TU), with respect to its taxonomic identity, the body of relevant knowledge and safety concerns. Safety concerns identified for a TU are, where possible, confirmed at the species/strain or product level and reflected by 'qualifications'. In the period covered by this Statement, no new information was found that would change the status of previously recommended QPS TUs. Of 71 microorganisms notified to EFSA between April and September 2023 (30 as feed additives, 22 as food enzymes or additives, 7 as novel foods and 12 from plant protection products [PPP]), 61 were not evaluated because: 26 were filamentous fungi, 1 was Enterococcus faecium, 5 were Escherichia coli, 1 was a bacteriophage (all excluded from the QPS evaluation) and 28 were TUs that already have a QPS status. The other 10 notifications belonged to 9 TUs which were evaluated for a possible QPS status: Ensifer adhaerens and Heyndrickxia faecalis did not get the QPS recommendation due to the limited body of knowledge about their occurrence in the food and/or feed chains and Burkholderia ubonensis also due to its ability to generate biologically active compounds with antimicrobial activity; Klebsiella pneumoniae, Serratia marcescens and Pseudomonas putida due to safety concerns. K. pneumoniae is excluded from future QPS evaluations. Chlamydomonas reinhardtii is recommended for QPS status with the qualification 'for production purposes only'; Clostridium tyrobutyricum is recommended for QPS status with the qualification 'absence of genetic determinants for toxigenic activity'; Candida oleophila has been added as a synonym of Yarrowia lipolytica. The Panel clarifies the extension of the QPS status for genetically modified strains.

Clostridium butyricum and Clostridium tyrobutyricum: angel or devil for necrotizing enterocolitis?

IMPORTANCE: This study sheds light on that treatment with Clostridium tyrobutyricum but not Clostridium butyricum is entitled to protect against necrotizing enterocolitis (NEC) development potentially. The mechanisms behind the opposite effect on NEC may result in different modulation on the level of Akkermansia muciniphila, which is deeply associated with intestinal homoeostasis. Briefly, through improving the abundance of A. muciniphila to alleviate intestinal inflammation and enhance intestinal barrier integrity, C. tyrobutyricum supplement may become a promising therapy for NEC.

Clostridium tyrobutyricum occurrence in silages and cattle feed: Use of molecular and simulation data to optimize predictive models.

Introduction: Poor quality silage can derive from the presence of deleterious microorganisms such as clostridia. Their dissemination along the food chain, especially in milk, causes issues such as the cheese late-blowing defect, particularly triggered by Clostridium tyrobutyricum. The scope of our study was to determine the C. tyrobutyricum occurrence in three different farms across four time periods in relation to the animal diets, specifically the Total Mixed Ration (TMR), by using real-time PCR. Methods: For this purpose, molecular-derived data were exploited to optimize a predictive model that simulated the farm conditions favoring the growth of butyric acid bacteria such as C. tyrobutyricum. Results: Our results showed that the originally utilized predictive model strongly underestimated the growth of C. tyrobutyricum in comparison to the molecular data. At the same time, our findings uncovered an additional source of contamination in the TMR related to silage and dietary residues that represent a reservoir of microbial contamination during successive TMR preparation. Based on these findings, the optimization of the model parameters such as growth rate range and the inclusion of the residues in the model, allowed a more accurate prediction of the contamination levels. Therefore, this study revealed that proper hygiene practices such as the removal of silage and TMR residues within the farm environment is essential to control the contamination by C. tyrobutyricum and avoid food waste and economic losses.

De novo biosynthesis of butyl butyrate in engineered Clostridium tyrobutyricum.

Butyl butyrate has broad applications in foods, cosmetics, solvents, and biofuels. Microbial synthesis of bio-based butyl butyrate has been regarded as a promising approach recently. Herein, we engineered Clostridium tyrobutyricum ATCC 25755 to achieve de novo biosynthesis of butyl butyrate from fermentable sugars. Through introducing the butanol synthetic pathway (enzyme AdhE2), screening alcohol acyltransferases (AATs), adjusting transcription of VAAT and adhE2 (i.e., optimizing promoter), and efficient supplying butyryl-CoA, an excellent engineered strain, named MUV3, was obtained with ability to produce 4.58 g/L butyl butyrate at 25 °C with glucose in serum bottles. More NADH is needed for butyl butyrate synthesis, thus mannitol (the more reduced substrate) was employed to produce butyl butyrate. Ultimately, 62.59 g/L butyl butyrate with a selectivity of 95.97%, and a yield of 0.21 mol/mol was obtained under mannitol with fed-batch fermentation in a 5 L bioreactor, which is the highest butyl butyrate titer reported so far. Altogether, this study presents an anaerobic fermentative platform for de novo biosynthesis of butyl butyrate in one step, which lays the foundation for butyl butyrate biosynthesis from renewable biomass feedstocks.

Construction of Clostridium tyrobutyricum strain and ionic membrane technology combination pattern for refinery final molasses recovery and butyric acid production.

Introduction: Clostridium tyrobutyricum has considerable prospect in the production of organic acids. Globally, refinery final molasses is rich in sugar and reported to have high levels of accumulation and high emission costs, recognized as an excellent substrate for C. tyrobutyricum fermentation, but there is no suitable method available at present. Methods: In this study, an acid-base treatment combined with a new green membrane treatment technology - a dynamic ion-exchange membrane -was used to pretreat refinery final molasses, so that it could be used for C. tyrobutyricum to produce butyric acid. A high-performance liquid chromatography method was established to determine the conversion of a large amount of sucrose into fermentable sugars (71.88 g/L glucose and 38.06 g/L fructose) in the treated refinery final molasses. The process of sequential filtration with 3, 1, and 0.45 µm-pore diameter dynamic ion-exchange membranes could remove impurities, pigments, and harmful substances from the refinery final molasses, and retain the fermentable sugar. Results and discussion: This means that refinery final molasses from the sugar industry could be utilized as a high-value by-product and used for the growth of C. tyrobutyricum, with industrial feasibility and economic competitiveness. Using the treated refinery final molasses as a carbon source, C. tyrobutyricum was screened by the method of adaptive evolution. The strain with butyric acid yielded 52.54 g/L, and the yield of the six carbon sugar was increased from 0.240 to 0.478 g/g. The results showed that combination of C. tyrobutyricum and ionic membrane technology broke through the bottleneck of its utilization of refinery final molasses. This study provided an innovative idea for the C. tyrobutyricum fermentation to produce butyric acid.

Bioinformatics and metabolic flux analysis highlight a new mechanism involved in lactate oxidation in Clostridium tyrobutyricum.

Climate change and environmental issues compel us to find alternatives to the production of molecules of interest from petrochemistry. This study aims at understanding the production of butyrate, hydrogen, and CO2 from the oxidation of lactate with acetate in Clostridium tyrobutyricum and thus proposes an alternative carbon source to glucose. This specie is known to produce more butyrate than the other butyrate-producing clostridia species due to a lack of solvent genesis phase. The recent discoveries on flavin-based electron bifurcation and confurcation mechanism as a mode of energy conservation led us to suggest a new metabolic scheme for the formation of butyrate from lactate-acetate co-metabolism. While searching for genes encoding for EtfAB complexes and neighboring genes in the genome of C. tyrobutyricum, we identified a cluster of genes involved in butyrate formation and another cluster involved in lactate oxidation homologous to Acetobacterium woodii. A phylogenetic approach encompassing other butyrate-producing and/or lactate-oxidizing species based on EtfAB complexes confirmed these results. A metabolic scheme on the production of butyrate, hydrogen, and CO2 from the lactate-acetate co-metabolism in C. tyrobutyricum was constructed and then confirmed with data of steady-state continuous culture. This in silico metabolic carbon flux analysis model showed the coherence of the scheme from the carbon recovery, the cofactor ratio, and the ATP yield. This study improves our understanding of the lactate oxidation metabolic pathways and the role of acetate and intracellular redox balance, and paves the way for the production of molecules of interest as butyrate and hydrogen with C. tyrobutyricum.

Isolation and characterization of new bacteriophages active against Clostridium tyrobutyricum and their role in preventing the late blowing defect of cheese.

Lytic bacteriophages (phages) offer a great potential as biocontrol agents for spoilage Clostridium tyrobutyricum, responsible for butyric acid fermentation in semi-hard and hard ripened cheeses, resulting in late gas blowing defect. With this aim, we have isolated, identified and characterized new lytic phages of C. tyrobutyricum, and have evaluated their efficacy to control cheese late blowing by adding them to manufacture milk. Silage, soil, milk and cheese from dairy farms were screened for anti-clostridial phages, obtaining 96 isolates active against C. tyrobutyricum. According to host range, source and plaque morphology, we obtained 20 phage profiles, 8 of them (represented by phages FA3, FA21, FA29, FA52, FA58, FA67, FA70 and FA88) showing a wider host range and high quality lysis, which were further characterized. Selected isolates showed a non-contractile tail, belonging to the Siphoviridae family, and were grouped into 3 restriction profiles. Viable phages were detected after storage in sodium-magnesium buffer (SM buffer), skim milk and acidified skim milk (pH 5) for 7 d at 4 °C, 12 °C and 37 °C, although a decline in infectivity was observed in some cases. Good phage survival was also detected during semi-hard cheese manufacture and ripening (60 d), and cheese lactococci counts, pH, dry matter values, and volatile compounds were not affected by phage addition. In semi-hard cheese, phage FA67 impaired the early germination of C. tyrobutyricum spores and caused a significant decrease in clostridial vegetative cells counts at 14 d of ripening, delaying by 2 weeks the consumption of lactic acid, formation of butyric acid and appearance of late blowing symptoms, compared to the spoilt control cheese without the phage. This is the first report on the application of phage to control C. tyrobutyricum in cheese.

Transcriptome analysis reveals reasons for the low tolerance of Clostridium tyrobutyricum to furan derivatives.

Lignocellulosic biomass is considered the most abundant and renewable feedstock for biobased butyric acid production. However, the furan derivatives (FAs, mainly furfural and 5-hydroxymethylfurfural) generated from the pretreatment of lignocellulose severely inhibit the growth of Clostridium tyrobutyricum, which is the best strain for producing butyric acid. The tolerance mechanism of C. tyrobutyricum to FAs has not been investigated thus far. Here, the response of C. tyrobutyricum ATCC 25755 to FA challenge was first evaluated by using comprehensive transcriptional analysis. The results indicated that the genes related to membrane transport, heat shock proteins, and transcriptional regulation were upregulated under FA stress. However, the expression of almost all genes encoding reductases was not changed, and only the ad gene CTK_RS02625 and the bud gene CTK_RS07810 showed a significant increase of ~ 1.05-fold. Then, the enzyme activity assays indicated that BUD could catalyze the reduction of FAs with relatively low activity and that AD could not participate in the conversion of FAs, indicating that the inability to rapidly convert FAs to their low-toxicity alcohols may be the main reason for the low FA tolerance of C. tyrobutyricum. This research provides insights into the development of FA-tolerant strains, thereby enhancing the bioconversion of lignocellulosic biomass to butyric acid. KEY POINTS: • The response of C. tyrobutyricum to FAs was evaluated for the first time. • Genes encoding membrane transporters and heat shock proteins were triggered by FAs. • A lack of effective FA reductases leads to low FA tolerance in C. tyrobutyricum.

Deciphering of the Mannitol Metabolism Pathway in Clostridium tyrobutyricum ATCC 25755 by Comparative Transcriptome Analysis.

Clostridium tyrobutyricum has great potential for bio-based chemicals and biofuel production from mannitol; however, the mannitol metabolic pathway and its metabolic regulatory mechanism have not been elucidated. To this end, the RNA-seq analysis on the mid-log growth phase of C. tyrobutyricum grown on mannitol or xylose was performed. Comparative transcriptome analysis and co-transcription experiment indicated that mtlARFD, which encodes the mannitol-specific IIA component, transcription activator, mannitol-specific IIBC components, and mannitol-1-phosphate 5-dehydrogenase, respectively, formed a polycistronic operon and could be responsible for mannitol uptake and metabolism. In addition, comparative genomic analysis of the mtlARFD organization and the MtlR protein structural domain among various Firmicutes strains identified the putative cre (catabolite-responsive element) sites and conserved phosphorylation sites, but whether the expression of mannitol operon was affected by CcpA- and MtlR-mediated metabolic regulation during mixed substrate fermentation needs to be further verified experimentally. Based on the gene knockout and complementation results, the predicted mannitol operon mtlARFD was confirmed to be responsible for mannitol utilization in C. tyrobutyricum. The results of this study could be used to enhance the mannitol metabolic pathway and explore the potential metabolic regulation mechanism of mannitol during mixed substrate fermentation.

The Physiological Functions of AbrB on Sporulation, Biofilm Formation and Carbon Source Utilization in Clostridium tyrobutyricum.

As a pleiotropic regulator, Antibiotic resistant protein B (AbrB) was reported to play important roles in various cellular processes in Bacilli and some Clostridia strains. In Clostridium tyrobutyricum, abrB (CTK_C 00640) was identified to encode AbrB by amino acid sequence alignment and functional domain prediction. The results of abrB deletion or overexpression in C. tyrobutyricum showed that AbrB not only exhibited the reported characteristics such as the negative regulation on sporulation, positive effects on biofilm formation and stress resistance but also exhibited new functions, especially the negative regulation of carbon metabolism. AbrB knockout strain (Ct/ΔabrB) could alleviate glucose-mediated carbon catabolite repression (CCR) and enhance the utilization of xylose compared with the parental strain, resulting in a higher butyrate titer (14.79 g/L vs. 7.91 g/L) and xylose utilization rate (0.19 g/L·h vs. 0.02 g/L·h) from the glucose and xylose mixture. This study confirmed the pleiotropic regulatory function of AbrB in C. tyrobutyricum, suggesting that Ct/ΔabrB was the potential candidate for butyrate production from abundant, renewable lignocellulosic biomass mainly composed of glucose and xylose.

Functional Characterization of Clostridium tyrobutyricum L319: A Promising Next-Generation Probiotic for Short-Chain Fatty Acid Production.

Probiotics contribute a lot to human health and the occurrence of diseases. Correspondingly, probiotics' safety evaluation and probiotic properties have received increasing attention in the food industry and disease treatment. Clostridium tyrobutyricum L319 is a short-chain fatty acid (SCFA)-producing strain isolated from Grana Padano cheese with a blowing defect. Our previous study has shown its safety at the genomic level. This study focused more on the safety evaluation and probiotic properties in vitro. According to the results, this strain has no potential virulence factors or the possibility of antibiotic resistance genes propagation. It also fulfilled several criteria to be used as a probiotic, including significant hydrophobicity under an acidic condition (pH 5.0) and resistance to simulate gastric juice and intestinal juice. Additionally, this strain was found to be tolerant to the harsh conditions of the external environment, including resistance to low (20°C) and high (50°C) temperatures, high salts (3% NaCl), and low pH (pH 5.0). Finally, we found that this strain could ferment prebiotics, such as chito-oligosaccharides, to produce SCFAs. It exhibited excellent growth performance whether using chito-oligosaccharide as a sole carbon source or combining glucose as the mixed carbon source. Furthermore, chito-oligosaccharide and glucose (1:1) mixed carbon sources were the optimal strategy for the production of SCFAs. Our findings demonstrated that this strain might be considered a promising candidate for future use as a probiotic to promote health benefits.