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
mtl
ARFD, 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
mtl
ARFD 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
mtl
ARFD 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.
Multi‐catalytic cascade reactions bear a great potential to minimize downstream and purification steps, leading to a drastic reduction of the produced waste. In many examples, the compatibility of ...chemo‐ and biocatalytic steps could be easily achieved. Problems associated with the incompatibility of the catalysts and their reactions, however, are very frequent. Cascade‐like reactions can hardly occur in this way. One possible solution to combine, in principle, incompatible chemo‐ and biocatalytic reactions is the defined control of the microenvironment by compartmentalization or scaffolding. Current methods for the control of the microenvironment of biocatalysts go far beyond classical enzyme immobilization and are thus believed to be very promising tools to overcome incompatibility issues and to facilitate the synthetic application of cascade reactions. In this Minireview, we will summarize recent synthetic examples of (chemo)enzymatic cascade reactions and outline promising methods for their spatial control either by using bio‐derived or synthetic systems.
Compartmentalize me! Compartmentalization is nature's successful strategy of controlling the microenvironment of catalysts in a defined way, thereby making the combination of different reactions feasible. This Minireview summarizes recent synthetic examples of (chemo)enzymatic cascade reactions and outlines promising methods for their spatial control either by using bio‐derived or synthetic systems.
Microbial consortia are a promising alternative to monocultures of genetically modified microorganisms for complex biotransformations. We developed a versatile consortium-based strategy for the ...direct conversion of lignocellulose to short-chain fatty acids, which included the funneling of the lignocellulosic carbohydrates to lactate as a central intermediate in engineered food chains. A spatial niche enabled in situ cellulolytic enzyme production by an aerobic fungus next to facultative anaerobic lactic acid bacteria and the product-forming anaerobes.
,
, or
were integrated into the lactate platform to produce 196 kilograms of butyric acid per metric ton of beechwood. The lactate platform demonstrates the benefits of mixed cultures, such as their modularity and their ability to convert complex substrates into valuable biochemicals.
► We studied butyric acid production from sugarcane bagasse (SCB) hydrolysate. ► High sugar yields were obtained from SCB after acid pretreatment and enzymatic hydrolysis. ► C. tyrobutyricum was ...immobilized in a FBB for fed-batch fermentation of SCB hydrolysate. ► High butyric acid yield (>0.45g/gsugar) and productivity (0.51g/Lh) were obtained. ► This study demonstrated the feasibility of producing butyric acid from low-cost SCB feedstock.
A fermentation process using Clostridium tyrobutyricum immobilized in a fibrous-bed bioreactor (FBB) was developed for butyric acid production from sugarcane bagasse (SCB) hydrolysate. SCB was first treated with dilute acid and then hydrolyzed with cellulases. The hydrolysate containing glucose and xylose was used as carbon source for the fermentation without detoxification. The bacterium was able to grow at a specific growth rate of ∼0.06h−1 in media containing 15–20% (w/v) SCB in serum bottles. In batch cultures in the FBB, both glucose and xylose in the SCB hydrolysate were simultaneously converted to butyrate with a high yield (0.45–0.54g/gsugar) and productivity (0.48–0.60g/Lh). A final butyrate concentration of 20.9g/L was obtained in a fed-batch culture, with an overall productivity of 0.51g/Lh and butyrate yield of 0.48g/g sugar consumed. This work demonstrated the feasibility of using SCB as a low-cost feedstock to produce butyric acid.
Optimal conditions of hyper thermal (HT) acid hydrolysis of the
Saccharina japonica
was determined to a seaweed slurry content of 12% (w/v) and 144 mM H
2
SO
4
at 160 °C for 10 min. Enzymatic ...saccharification was carried out at 50 °C and 150 rpm for 48 h using the three enzymes at concentrations of 16 U/mL. Celluclast 1.5 L showed the lowest half-velocity constant (
K
m
) of 0.168 g/L, indicating a higher affinity for
S. japonica
hydrolysate. Pretreatment yielded a maximum monosaccharide concentration of 36.2 g/L and 45.7% conversion from total fermentable monosaccharides of 79.2 g/L with 120 g dry weight/L
S. japonica
slurry. High cell densities of
Clostridium acetobutylicum
and
Clostridium tyrobutyricum
were obtained using the retarding agents KH
2
PO
4
(50 mM) and NaHCO
3
(200 mM). Adaptive evolution facilitated the efficient use of mixed monosaccharides. Therefore, adaptive evolution and retarding agents can enhance the overall butanol and butyric acid yields from
S. japonica
.
Scope
This study aims to roundly investigate whether Clostridium tyrobutyricum (Ct) alleviates inflammation via regulating immune cells in the small intestines.
Methods and Results
Mice are ...pre‐treated with different concentrations of Ct follow by LPS stimulation. Ct maintains the mice body weight under inflammation. In response to LPS, 107 CFU mL−1 Ct decreases the mRNA expression of inflammatory cytokines in the duodenum, while 108 CFU mL−1 Ct reduces inflammatory cytokines expression in both duodenum and ileum and protected intestinal morphology. The small intestinal immune cells are analyzed using flow cytometry. Ct decreases the numbers of macrophages and mast cells in the intestines in response to LPS. In the duodenum, Ct enhances dentritic cells (DCs), regulatory T cells (Tregs), and T helper cell 17 (Th17) proportions. Ct decreases DCs and Tregs proportions, while enhances Th17 numbers in the ileum. The underlying mechanism of Ct in preventing inflammation may rely on the physiological immune cell composition of the intestines. In response to LPS, Ct may mainly stimulate Tregs via activating DCs in the duodenum while trigger Th17 cells in the ileum, thereby maintaining the intestinal homeostasis.
Conclusion
Ct alleviates the LPS‐induce inflammation via regulating different immune cell types in the small intestines, highlighting that Ct is a potential prophylactic probiotic in intestinal diseases.
Clostridium tyrobutyricum alleviates LPS‐induced intestinal dysfunction in both duodenum and ileum via regulating intestinal immune cells. In response to LPS, Clostridium tyrobutyricum blocks the over‐activation of macrophages and stimulates the regulatory T cells or T helper cell 17 via dendritic cells in the duodenum, while in the ileum, Clostridium tyrobutyricum inhibits mast cells and activates T helper 17 under inflammation.
Display omitted
•n-Butanol production from lignocellulosic biomass hydrolysates was studied.•Engineered Clostridium tyrobutyricum was immobilized in a fibrous-bed bioreactor.•Co-utilization of ...glucose and xylose in the hydrolysates was achieved.•High-level butanol production was obtained from hydrolysates without detoxification.•Techno-economic analysis shows the potential for industrial application.
Acetone-butanol-ethanol fermentation suffers from high substrate cost and low butanol titer and yield. In this study, engineered Clostridium tyrobutyricum CtΔack-adhE2 immobilized in a fibrous-bed bioreactor was used for butanol production from glucose and xylose present in the hydrolysates of low-cost lignocellulosic biomass including corn fiber, cotton stalk, soybean hull, and sugarcane bagasse. The biomass hydrolysates obtained after acid pretreatment and enzymatic hydrolysis were supplemented with corn steep liquor and used in repeated-batch fermentations. Butanol production with high titer (∼15 g/L), yield (∼0.3 g/g), and productivity (∼0.3 g/L∙h) was obtained from cotton stalk, soybean hull, and sugarcane bagasse hydrolysates, while corn fiber hydrolysate with higher inhibitor contents gave somewhat inferior results. The fermentation process was stable for long-term operation without any noticeable degeneration, demonstrating its potential for industrial application. A techno-economic analysis showed that n-butanol could be produced from lignocellulosic biomass using this novel fermentation process at ∼$2.5/gal for biofuel application.
Clostridium tyrobutyricum has been successfully engineered to produce butyrate, butanol, butyl butyrate, and γ-aminobutyric acid. It would be interesting to produce bio-chemicals and bio-fuels ...directly using starch from non-food crop, e.g., cassava, by engineered C. tyrobutyricum. In this study, heterologous α-amylases were screened and expressed in C. tyrobutyricum, resulting in successfully starch hydrolyzation. Furthermore, α-glucosidase (AgluI) was co-expressed with α-amylases, resulting in enhancement in the capacity of starch hydrolyzation and butyrate production. When increasing the cassava starch concentration to 100 g/L, the engineered strain CTAA05 produced 27.0 g/L butyrate. In addition, when introducing butyl butyrate synthetic pathway, strain MU3-AAV produced 26.8 g/L butyl butyrate with 100 g/L cassava starch as substrate. This study showed a generalizable framework to engineered anaerobes for anaerobic production of bio-chemicals and bio-fuels from starchy biomass.
Clostridium tyrobutyricum is a gram-positive spore-forming anaerobe that is considered as the main causative agent for late blowing in cheese due to butyric acid fermentation. In this study, ...multilocus variable-number of tandem repeat (VNTR) analysis (MLVA) for C. tyrobutyricum was developed to identify the source of contamination by C. tyrobutyricum spores in the cheese production environment. For each contig constructed from the results of a whole genome draft sequence of C. tyrobutyricum JCM11008T based on next-generation sequencing, VNTR loci that were effective for typing were searched using the Tandem Repeat Finder program. Five VNTR loci were amplified by polymerase chain reaction (PCR) to determine their number of repeats by sequencing, and MLVA was conducted. 25 strains of C. tyrobutyricum isolated from the environment, raw milk, and silage were classified into 18 MLVA types (DI=0.963). Of the C. tyrobutyricum strains isolated from raw milk, natural cheese, and blown processed cheese, strains with identical MLVA type were detected, which suggested that these strains might have shifted from natural cheese to blown processed cheese. MLVA could be an effective tool for monitoring contamination of natural cheese with C. tyrobutyricum in the processed cheese production environment because of its high discriminability, thereby allowing the analyst to trace the source of contamination.
•Whole genome draft sequence of C. tyrobutyricum was determined by NGS.•MLVA for C. tyrobutyricum was developed.•This newly developed MLVA method has high D.I. value (0.96).•This MLVA could be an effective tool for monitoring C. tyrobutyricum in cheese.
The effect of pH (between 5.0 and 6.3) on butyric acid fermentation of xylose by
Clostridium tyrobutyricum was studied. At pH 6.3, the fermentation gave a high butyrate production of 57.9
g
l
−1 with ...a yield of 0.38–0.59
g
g
−1 xylose and a reactor productivity up to 3.19
g
l
−1
h
−1. However, at low pHs (<5.7), the fermentation produced more acetate and lactate as the main products, with only a small amount of butyric acid. The metabolic shift from butyrate formation to lactate and acetate formation in the fermentation was found to be associated with changes in the activities of several key enzymes. The activities of phosphotransbutyrylase (PTB), which is the key enzyme controlling butyrate formation, and NAD-independent lactate dehydrogenase (iLDH), which catalyzes the conversion of lactate to pyruvate, were higher in cells producing mainly butyrate at pH 6.3. In contrast, cells at pH 5.0 had higher activities of phosphotransacetylase (PTA), which is the key enzyme controlling acetate formation, and lactate dehydrogenase (LDH), which catalyzes the conversion of pyruvate to lactate. Also, PTA was very sensitive to the inhibition by butyric acid. Difference in the specific metabolic rate of xylose at different pHs suggests that the balance in NADH is a key in controlling the metabolic pathway used by the cells in the fermentation.
PDF
