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.

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.

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.

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.

Inhibitory activity of aromatic plant extracts against dairy-related Clostridium species and their use to prevent the late blowing defect of cheese.

The aim of the present work was the selection of aromatic plant essential oils (EOs) and/or ethanolic extracts (EEs) to prevent the late blowing defect (LBD) of cheese caused by Clostridium spp. EEs resulted more effective than EOs to inhibit dairy-borne Clostridium spp. in vitro. Savory, hyssop, lavender and tarragon EEs, which showed the lowest minimal inhibitory concentration against Clostridium tyrobutyricum, were selected to study the prevention of LBD caused by this bacterium in cheese. Addition of savory and lavender EEs to cheese milk delayed LBD by 2 weeks, but at the end of ripening these cheeses showed similar clostridial vegetative cells counts, spoilage symptoms and propionic, and butyric acids levels than blown control cheese. Tarragon EE, with the highest content in caffeic acid, also delayed LBD by 2 weeks, but it was more effective to inhibit Clostridium, since cheese with tarragon EE showed minor LBD symptoms, lower vegetative cells count and lower concentrations of propionic and butyric acids than the rest of cheeses made with EEs. This fact could be also attributable to the greater number of antimicrobial terpenes (1,8-cineole, 4-terpineol, α-terpineol, isoelemicin, methyl eugenol, and methyl trans-isoeugenol) detected in this cheese. This is the first report on the application of EEs to control C. tyrobutyricum in cheese.

Biosorption of lead ions from aqueous solution by Clostridium tyrobutyricum immobilized in macroporous Ca-alginate-lignin beads.

AIMS: The adsorption of lead ions from aqueous solution by macroporous Ca-alginate-lignin (MCAL) beads immobilized with Clostridium tyrobutyricum and free strains was evaluated. METHODS AND RESULTS: The effects of different factors including pH, adsorption time, adsorbent dosage and initial concentration of lead ions were explored. Different characterization methods were used to evaluate the adsorption process of lead ions. Meanwhile, the adsorption kinetics models and adsorption isotherm models were applied. The fitting results showed that the adsorption behaviour of C. tyrobutyricum immobilized in MCAL beads and free strains was better described by the pseudo-second-order kinetic model and the adsorption process followed the Langmuir isotherm model. The maximum biosorption of lead ions by C. tyrobutyricum immobilized in MCAL beads and free strains was 144.9 and 106.4 mg/g respectively. CONCLUSIONS: The C. tyrobutyricum immobilized in MCAL beads proved to be practicable and had better adsorption effects on lead ions compared with the free strains. SIGNIFICANCE AND IMPACT OF THE STUDY: The paper demonstrated a new insight and strategy for the effective treatment of lead ions from aqueous solutions by the novel function of C. tyrobutyricum.

Development of a risk assessment model to predict the occurrence of late blowing defect in Gouda cheese and evaluate potential intervention strategies.

Late blowing defect (LBD) is an important spoilage issue in semi-hard cheese, with the outgrowth of Clostridium tyrobutyricum spores during cheese aging considered to be the primary cause. Although previous studies have explored the microbial and physicochemical factors influencing the defect, a risk assessment tool that allows for improved and rational management of LBD is lacking. The purpose of this study was to develop a predictive model to estimate the probability of LBD in Gouda cheese and evaluate different intervention strategies. The spore concentration distribution of butyric acid bacteria (BAB) in bulk tank milk was obtained from 8 dairy farms over 12 mo. The concentration of C. tyrobutyricum from raw milk to the end of aging was simulated based on Gouda brined for 2 d in saturated brine at 8°C and aged at 13°C. Predicted C. tyrobutyricum concentrations during aging and estimated concentration thresholds in cheese at onset of LBD were used to predict product loss due to LBD during a simulated 1-yr production. With the estimated concentration thresholds in cheese ranging from 4.36 to 4.46 log most probable number (MPN)/kg of cheese, the model predicted that 9.2% (±1.7%) of Gouda cheese showed LBD by d 60; cheeses predicted to show LBD at d 60 showed a mean pH of 5.39 and were produced with raw milk with a mean BAB spore count of 143 MPN/L. By d 90, 36.1% (±3.4%) of cheeses were predicted to show LBD, indicating that LBD typically manifests between d 60 and 90, which is consistent with observations from the literature and the cheese industry. Sensitivity analysis indicated that C. tyrobutyricum maximum growth rate as well as concentration threshold in cheese at onset of LBD are the most important variables, identifying key data needs for development of more accurate models. The implementation of microfiltration or bactofugation of raw milk (assumed to show 98% efficiency of spore removal) in our model prevented occurrence of LBD during the first 60 d of aging. Overall, our findings provide a framework for predicting the occurrence of LBD in Gouda as well as other cheeses and illustrate the value of developing digital tools for managing dairy product quality.

Effects of benzyl viologen on increasing NADH availability, acetate assimilation, and butyric acid production by Clostridium tyrobutyricum.

Clostridium tyrobutyricum produces butyric and acetic acids from glucose. The butyric acid yield and selectivity in the fermentation depend on NADH available for acetate reassimilation to butyric acid. In this study, benzyl viologen (BV), an artificial electron carrier that inhibits hydrogen production, was used to increase NADH availability and butyric acid production while eliminating acetic acid accumulation by facilitating its reassimilation. To better understand the mechanism of and find the optimum condition for BV effect on enhancing acetate assimilation and butyric acid production, BV at various concentrations and addition times during the fermentation were studied. Compared with the control without BV, the addition of 1 µM BV increased butyric acid production from glucose by ∼50% in yield and ∼29% in productivity while acetate production was completely inhibited. Furthermore, BV also increased the coutilization of glucose and exogenous acetate for butyric acid production. At a concentration ratio of acetate (g/L) to BV (mM) of 4, both acetate assimilation and butyrate biosynthesis increased with increasing the concentrations of BV (0-6.25 µM) and exogenous acetate (0-25 g/L). In a fed-batch fermentation with glucose and ∼15 g/L acetate and 3.75 µM BV, butyrate production reached 55.9 g/L with productivity 0.93 g/L/h, yield 0.48 g/g, and 97.4% purity, which would facilitate product purification and reduce production cost. Manipulating metabolic flux and redox balance via BV and acetate addition provided a simple to implement metabolic process engineering approach for butyric acid production from sugars and biomass hydrolysates.

Enhancing plasmid transformation efficiency and enabling CRISPR-Cas9/Cpf1-based genome editing in Clostridium tyrobutyricum.

Clostridium tyrobutyricum ATCC 25755 is known as a natural hyper-butyrate producer with great potentials as an excellent platform to be engineered for valuable biochemical production from renewable resources. However, limited transformation efficiency and the lack of genetic manipulation tools have hampered the broader applications of this micro-organism. In this study, the effects of Type I restriction-modification system and native plasmid on conjugation efficiency of C. tyrobutyricum were investigated through gene deletion. The deletion of Type I restriction endonuclease resulted in a 3.7-fold increase in conjugation efficiency, while the additional elimination of the native plasmid further enhanced conjugation efficiency to 6.05 ± 0.75 × 103 CFU/ml-donor, which was 15.3-fold higher than the wild-type strain. Fermentation results indicated that the deletion of those two genetic elements did not significantly influence the end-products production in the resultant mutant ΔRMIΔNP. Thanks to the increased conjugation efficiency, the CRISPR-Cas9/Cpf1 systems, which previously could not be implemented in C. tyrobutyricum, were successfully employed for genome editing in ΔRMIΔNP with an efficiency of 12.5-25%. Altogether, approaches we developed herein offer valuable guidance for establishing efficient DNA transformation methods in nonmodel micro-organisms. The ΔRMIΔNP mutant can serve as a great chassis to be engineered for diverse valuable biofuel and biochemical production.

Genome Sequence Analysis of Clostridium tyrobutyricum, a Promising Microbial Host for Human Health and Industrial Applications.

Clostridium tyrobutyricum is a promising microbial host for the anaerobic production of bio-based chemicals, especially butyric acid. At the same time, it also has great potential as a probiotic for the production of short-chain fatty acids in the intestines. However, due to the insufficient knowledge of the genetic characteristics of this organism, there has been little progress in its genetic engineering to date. Here, we present the complete genome sequence of C. tyrobutyricum L319, which consists of a circular chromosome and a plasmid with a G + C content of 31.69%, encompassing approximately 3.09 Mb with 3052 protein-coding genes. Functional gene annotation revealed better results than previous studies based on KEGG pathway classification. Furthermore, we obtained detailed functional characterization of 93 genes previously annotated as putative proteins. Genomic analysis revealed that this organism contains multiple genes encoding enzymes involved in the CRISPR-Cas systems, substrate utilization, isopeptide and ester bonds, transcriptional regulation, and oxidative stress. The safety evaluation at genetic level indicated that this organism does not possess transferable resistance genes, invasive defensive pathogenicity factors, or harmful enzymes. The genome sequence data analyzed in this study will be available for further research and will facilitate the further understanding and development of potential applications of C. tyrobutyricum.

Production of isopropyl and butyl esters by Clostridium mono-culture and co-culture.

Production of esters from the acetone-butanol-ethanol (ABE) fermentation by Clostridium often focuses on butyl butyrate, leaving acetone as an undesired product. Addition of butyrate is also often needed because ABE fermentation does not produce enough butyrate. Here we addressed the problems using Clostridium beijerinckii BGS1 that preferred to produce isopropanol instead of acetone, and co-culturing it with Clostridium tyrobutyricum ATCC 25,755 that produced butyrate. Unlike acetone, isopropanol could be converted into ester using lipase and acids. C. tyrobutyricum ATCC 25,755 produced acids at pH 6, while C. beijerinckii BGS1 produced mainly solvents at the same pH. When the two strains were co-cultured, more butyrate was produced, leading to a higher titer of esters than the mono-culture of C. beijerinckii BGS1. As the first study reporting the production of isopropyl butyrate from the Clostridium fermentation, this study highlighted the potential use of lipase and co-culture strategy in ester production.

Recent advances in n-butanol and butyrate production using engineered Clostridium tyrobutyricum.

Acidogenic clostridia naturally producing acetic and butyric acids has attracted high interest as a novel host for butyrate and n-butanol production. Among them, Clostridium tyrobutyricum is a hyper butyrate-producing bacterium, which re-assimilates acetate for butyrate biosynthesis by butyryl-CoA/acetate CoA transferase (CoAT), rather than the phosphotransbutyrylase-butyrate kinase (PTB-BK) pathway widely found in clostridia and other microbial species. To date, C. tyrobutyricum has been engineered to overexpress a heterologous alcohol/aldehyde dehydrogenase, which converts butyryl-CoA to n-butanol. Compared to conventional solventogenic clostridia, which produce acetone, ethanol, and butanol in a biphasic fermentation process, the engineered C. tyrobutyricum with a high metabolic flux toward butyryl-CoA produced n-butanol at a high yield of > 0.30 g/g and titer of > 20 g/L in glucose fermentation. With no acetone production and a high C4/C2 ratio, butanol was the only major fermentation product by the recombinant C. tyrobutyricum, allowing simplified downstream processing for product purification. In this review, novel metabolic engineering strategies to improve n-butanol and butyrate production by C. tyrobutyricum from various substrates, including glucose, xylose, galactose, sucrose, and cellulosic hydrolysates containing the mixture of glucose and xylose, are discussed. Compared to other recombinant hosts such as Clostridium acetobutylicum and Escherichia coli, the engineered C. tyrobutyricum strains with higher butyrate and butanol titers, yields and productivities are the most promising hosts for potential industrial applications.

Use of fluorescent CTP1L endolysin cell wall-binding domain to study the evolution of Clostridium tyrobutyricum during cheese ripening.

Clostridium tyrobutyricum is a bacteria of concern in the cheese industry, capable of surviving the manufacturing process and causing butyric acid fermentation and late blowing defect of cheese. In this work, we implement a method based on the cell wall-binding domain (CBD) of endolysin CTP1L, which detects C. tyrobutyricum, to monitor its evolution in cheeses challenged with clostridial spores and in the presence or absence of reuterin, an anti-clostridial agent. For this purpose, total bacteria were extracted from cheese samples and C. tyrobutyricum cells were specifically labelled with the CBD of CTP1L attached to green fluorescent protein (GFP), and detected by fluorescence microscopy. By using this GFP-CBD, germinated spores were visualized on day 1 in all cheeses inoculated with clostridial spores. Vegetative cells of C. tyrobutyricum, responsible for butyric acid fermentation, were detected in cheeses without reuterin from 30 d onwards, when LBD symptoms also became evident. The number of fluorescent Clostridium cells increased during ripening in the blowing cheeses. However, vegetative cells of C. tyrobutyricum were not detected in cheese containing the antimicrobial reuterin, which also did not show LBD throughout ripening. This simple and fast method provides a helpful tool to study the evolution of C. tyrobutyricum during cheese ripening.

Abatement of the clostridial load in the teats of lactating cows with lysozyme derived from donkey milk.

The use of a sterilized product for washing cows' udders before milking may be useful to reduce or prevent Clostridium tyrobutyricum contamination, the main cause of the late-blowing defect in hard and semi-hard cheeses. The aim of this research was to evaluate the antibacterial efficacy of an experimental formula containing 15% condensed donkey milk (lysozyme content 825 mg/L). The antimicrobial activity of condensed milk was first evaluated in vitro, using the disk diffusion method, on the following microorganisms: Bacillus megaterium, Bacillus mojavensis, Clavibacter michiganensis, and Clostridium tyrobutyricum. These results were compared with the effects of 2 antibiotics, ampicillin (100 mg/mL) and kanamycin (50 mg/ mL), and a commercial pre-dipping formula. The results showed that the inhibitory activity of lysozyme from donkey milk on all the considered microorganisms was higher than that of the commercial product and similar to that of the 2 antibiotics. Next, the formula with lysozyme was compared with a commercial pre-dipping formula on 48 lactating cows (24 cows in each group). Skin tests were performed on teats before and after pre-dipping. Results showed that the formula with condensed milk significantly reduced the clostridial load detected on the skin of cows' teats before cleaning (-55.61% vs. -27.99%) and in the bulk milk of the experimental group compared with the control group with commercial product (-52.53% vs. -32.42%).

Dairy farm management practices and the risk of contamination of tank milk from Clostridium spp. and Paenibacillus spp. spores in silage, total mixed ration, dairy cow feces, and raw milk.

The occurrence of Paenibacillus and Clostridium spores in silage is of great concern for dairy producers because their spores can contaminate milk and damage processed milk and semi-hard cheeses. Spoiled silage is considered to be the main contamination source of the total mixed ration (TMR), feces of dairy cows, and consequently bulk tank milk via the contamination of cow teats by dirt during milking. The presence of an anaerobic and facultative anaerobic sporeformer population in different matrices (soil, corn silage, other feeds, TMR, feces, and milk) and its transmission pathway has been studied on 49 dairy farms by coupling plate count data with 16S-DNA identification. The different matrices have shown a high variability in the anaerobic and facultative anaerobic spore count, with the highest values being found in the aerobically deteriorated areas of corn silages. Clostridium tyrobutyricum, Paenibacillus macerans, and Paenibacillus thermophilus were detected in all the matrices. The TMR spore count was influenced by the amount of spoiled corn silage in the TMR and by the care taken when cleaning the spoiled silage before feed-out. Most of the farms that prevent the presence of visible moldy silage in the silo and carefully clean to remove molded spots were able to maintain their TMR spore counts below 4.0 log spores/g. When a level of 4.5 log spores/g of TMR was exceeded, the feces presented a greater contamination than 3.0 log spores/g. Moreover, the higher the number of spores in the feces was, the higher the number of spores in the milk. Most of the farms that presented a feces contamination greater than 5.0 log spores/g had a higher milk spore contamination than 1,000 spores/L. Careful animal cleaning and good milking practices have been found to be essential to maintain low levels of contamination in bulk tank milk, but it has emerged that only by coupling these practices with a correct silage management and cleaning during TMR preparation can the contamination of milk by spores be kept at a low level. It has been found that aerobically deteriorated silage has a great capacity to contaminate TMR and consequently to increase the risk of milk spore contamination, even when routine milking practices are adopted correctly.

Butanol and butyric acid production from Saccharina japonica by Clostridium acetobutylicum and Clostridium tyrobutyricum with adaptive evolution.

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 H2SO4 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 (Km) 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 KH2PO4 (50 mM) and NaHCO3 (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.

Clostridium Tyrobutyricum Protect Intestinal Barrier Function from LPS-Induced Apoptosis via P38/JNK Signaling Pathway in IPEC-J2 Cells.

BACKGROUND/AIMS: The intestinal mucosa forms a physical and metabolic barrier against the diffusion of pathogens, toxins, and allergens from the lumen into the circulatory system. Early weaning, a critical phase in swine production, can compromise intestinal barrier function through mucosal damage and alteration of tight junction integrity Maintenance of intestinal barrier function plays a pivotal role in optimum gastrointestinal health. In this study, we investigated the effects of Clostridium tyrobutyricum (C.t) on intestinal barrier dysfunction induced by lipopolysaccharide (LPS) and the underlying mechanisms involved in intestinal barrier protection. METHODS: A Transwell model of IPEC-J2 cells was used to imitate the intestinal barrier. Fluorescence microscopy and flow cytometry were used to evaluate apoptosis. Real-time PCR was used to detect apoptosis-related genes and the downstream genes of the p38/c-Jun N-terminal kinase (JNK) signaling pathways. Western blotting was used to measure the expressions of tight junction proteins and mitogen-activated protein kinases. RESULTS: C.t efficiently maintained trans-epithelium electrical resistance values and intestinal permeability after LPS-induced intestinal barrier disruption. The expressions of tight junction proteins (ZO-1, claudin-1, and occludin) were promoted when IPEC-J2 cells were treated with C.t. Fluorescence imaging and flow cytometry revealed that C.t qualitatively and quantitatively inhibited LPS-induced cell apoptosis. C.t also increased the relative expression of the anti-apoptotic gene Bcl-2 and decreased that of the apoptotic genes Bax and caspase-3/-8. Moreover, the protective effect of C.t on damaged intestinal cell models was associated with suppression of p38 and JNK phosphorylation, negative regulation of the relative expressions of downstream genes including AP-1, ATF-2, ELK-1, and p53, and activation of Stat3 expression. CONCLUSIONS: These findings indicate that C.t may promote intestinal integrity, suggesting a novel probiotic effect on intestinal barrier function.

Exploiting endogenous CRISPR-Cas system for multiplex genome editing in Clostridium tyrobutyricum and engineer the strain for high-level butanol production.

Although CRISPR-Cas9/Cpf1 have been employed as powerful genome engineering tools, heterologous CRISPR-Cas9/Cpf1 are often difficult to introduce into bacteria and archaea due to their severe toxicity. Since most prokaryotes harbor native CRISPR-Cas systems, genome engineering can be achieved by harnessing these endogenous immune systems. Here, we report the exploitation of Type I-B CRISPR-Cas of Clostridium tyrobutyricum for genome engineering. In silico CRISPR array analysis and plasmid interference assay revealed that TCA or TCG at the 5'-end of the protospacer was the functional protospacer adjacent motif (PAM) for CRISPR targeting. With a lactose inducible promoter for CRISPR array expression, we significantly decreased the toxicity of CRISPR-Cas and enhanced the transformation efficiency, and successfully deleted spo0A with an editing efficiency of 100%. We further evaluated effects of the spacer length on genome editing efficiency. Interestingly, spacers ≤ 20 nt led to unsuccessful transformation consistently, likely due to severe off-target effects; while a spacer of 30-38 nt is most appropriate to ensure successful transformation and high genome editing efficiency. Moreover, multiplex genome editing for the deletion of spo0A and pyrF was achieved in a single transformation, with an editing efficiency of up to 100%. Finally, with the integration of the alcohol dehydrogenase gene (adhE1 or adhE2) to replace cat1 (the key gene responsible for butyrate production and previously could not be deleted), two mutants were created for n-butanol production, with the butanol titer reached historically record high of 26.2 g/L in a batch fermentation. Altogether, our results demonstrated the easy programmability and high efficiency of endogenous CRISPR-Cas. The developed protocol herein has a broader applicability to other prokaryotes containing endogenous CRISPR-Cas systems. C. tyrobutyricum could be employed as an excellent platform to be engineered for biofuel and biochemical production using the CRISPR-Cas based genome engineering toolkit.

Overcoming the Incompatibility Challenge in Chemoenzymatic and Multi-Catalytic Cascade Reactions.

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.

Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production with high butyrate/acetate ratio.

Butyric acid fermentation by Clostridium couples with the synthesis of acetic acid. But the presence of acetic acid reduces butyric acid yield and increases separation and purification costs of butyric acid. Hence, enhancing the butyrate/acetate ratio is important for economical butyric acid production. This study indicated that enhancing the acetyl-CoA to butyrate flux by overexpression of both the butyryl-CoA/acetate CoA transferase (cat1) and crotonase (crt) genes in C. tyrobutyricum could significantly reduce acetic acid concentration. Fed-batch fermentation of ATCC 25755/cat1 + crt resulted in increased butyrate/acetate ratio of 15.76 g/g, which was 2.24-fold higher than that of the wild-type strain. Furthermore, in order to simultaneously increase the butyrate/acetate ratio, butyric acid concentration and productivity, the recombinant strain ATCC 25755/ppcc (co-expression of 6-phosphofructokinase (pfkA) gene, pyruvate kinase (pykA) gene, cat1, and crt) was constructed. Consequently, ATCC 25755/ppcc produced more butyric acid (46.8 vs. 35.0 g/L) with a higher productivity (0.83 vs. 0.49 g/L·h) and butyrate/acetate ratio (13.22 vs. 7.22 g/g) as compared with the wild-type strain in batch fermentation using high glucose concentration (120 g/L). This study demonstrates that enhancing the acetyl-CoA to butyrate flux is an effective way to reduce acetic acid production and increase butyrate/acetate ratio.