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 β- 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.
High temperature influences rumen and gut health, passage rate, and diet digestibility, with effects on fermentative processes. The main aim of the study was to investigate the effect of hot season ...on hindgut fermentation, the occurrence of Clostridium tyrobutyricum spores in bovine feces, and on their relationship with metabolic conditions in dairy cows producing milk used for Grana Padano cheese. The study was carried out on 7 dairy farms located in the Po Valley (Italy), involving 1,950 Italian Friesian dairy cows. The study was carried out from November 2013 till the end of July 2014. Temperature and relative humidity were recorded daily by weather stations. Constant management conditions were maintained during the experimental period. Feed and diet characteristics, metabolic conditions, and fecal characteristics were recorded in winter (from late November 2013 to the end of January 2014), spring (from April to May 2014), and summer (July 2014) season. In each season, blood samples were collected from 14 multiparous lactating dairy cows per herd to measure biochemical indices related to energy, protein, and mineral metabolism, as well as markers of inflammation and some enzyme activities. Fecal samples were also collected and measurements of moisture, pH and volatile fatty acids (VFA) were performed. The DNA extracted and purified from fecal samples was used to detect Clostridium tyrobutyricum spores in a quantitative real-time PCR assay. The daily mean temperature-humidity index was 40.7 ± 4.6 (range 25 to 55), 61.2 ± 3.7 (range 39 to 77), and 70.8 ± 3.2 (range 54 to 83) in winter, spring, and summer, respectively. Total VFA concentration in feces progressively decreased from winter to summer. The seasonal changes of acetate and propionate followed the same trend of total VFA; conversely, butyrate did not show any difference between seasons, and its molar proportion was greater in summer compared with winter. A greater occurrence of Cl. tyrobutyricum spores in summer compared with the other seasons was observed. The plasma concentrations of glucose, urea, albumin, Ca, Mg, Cl, Zn, and alkaline phosphatase activity were lower in summer compared with winter, whereas the opposite occurred for bilirubin and Na. Our results show that summer season, through direct and indirect effect of heat stress, affected fecal fermentative parameters and hindgut buffering capacity, and was responsible for the increasing occurrence of Cl. tyrobutyricum spores in feces.
In this study, reuterin-producing Lactobacillus reuteri INIA P572 was added to cheese as an adjunct culture together with 50 or 100 mM glycerol (required for reuterin production), with the aim of ...controlling Clostridium tyrobutyricum CECT 4011 growth and preventing the late blowing defect (LBD) of cheese caused by this strain. L. reuteri survived cheese manufacture and produced reuterin in situ, detected at 6 and 24 h. However, the produced reuterin was enough to inhibit the growth of Clostridium, showing undetectable spore counts from day 30 onward and, therefore, to prevent cheese LBD during ripening (60 d, 14 °C). The acidification of these cheeses was not affected, although from day 14 they showed significantly lower lactococci counts than cheese made only with the starter (control cheese). Cheeses with LBD showed lower levels of lactic acid than control cheese and the formation of propionic and butyric acids, but cheeses with reuterin showed the same organic acids profile than control cheese. The cheese made with L. reuteri and 100 mM glycerol showed a light pink colour, not observed in the cheese made with L. reuteri and 50 mM glycerol. These results demonstrated a potent anti-clostridial activity of reuterin produced in an actual food product like cheese, and proved to be a novel approach to prevent LBD of cheese.
•Clostridium tyrobutyricum CECT 4011 caused late blowing defect in cheese.•Reuterin-producing Lactobacillus reuteri was used as adjunct plus glycerol to control cheese blowing.•L. reuteri survived cheese manufacture and produced reuterin in situ.•Reuterin inhibited Clostridium growth and prevented cheese blowing.•Cheeses with L. reuteri and glycerol showed the same organic acid profile than control cheese.
•A complete proteomics profiling of C. tyrobutyricum with 78.1% coverage was established.•Integrated carbon redirections led to high butanol production by engineered strain.•Redistributed redox could ...limit butanol production.•Cellular responses to butanol production were identified.
The acidogenic Clostridium tyrobutyricum has recently been metabolically engineered to produce n-butanol. The objective of this study was to obtain a comprehensive understanding as to how butanol production was regulated in C. tyrobutyricum to guide the engineering of next-generation strains. We performed a comparative proteomics analysis, covering 78.1% of open reading frames and 95% of core enzymes, using wild type, ACKKO mutant (Δack) producing 37.30g/L of butyrate and ACKKO-adhE2 mutant (Δack-adhE2) producing 16.68g/L of butanol. In ACKKO-adhE2, the expression of most glycolytic enzymes was decreased, the thiolase (thl), acetyl-CoA acetyltransferase (ato), 3-hydroxybutyryl-CoA dehydrogenase (hbd) and crotonase (crt) that convert acetyl-CoA to butyryl-CoA were increased, and the heterologous bifunctional acetaldehyde/alcohol dehydrogenase (adhE2) catalyzing butanol formation was highly expressed. The apparent imbalance of energy and redox was observed due to the downregulation of acids production and the addition of butanol synthesis pathway, which also resulted in increased expression of chaperone proteins and glycerol-3-phosphate dehydrogenase (glpA) and the silence of sporulation transcription factor Spo0A (spo0A) as the cellular responses to butanol production. This study revealed the mechanism of carbon redistribution, and limiting factors and rational metabolic cell and process engineering strategies to achieve high butanol production in C. 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.
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.
The overexpression of CoA transferase (ctfAB), which catalyzes the reaction: acetate/butyrate + acetoacetyl-CoA → acetyl/butyryl-CoA + acetoacetate, was studied for its effects on acid reassimilation ...and butanol biosynthesis in Clostridium tyrobutyricum (Δack, adhE2). The plasmid pMTL007 was used to co-express adhE2 and ctfAB from Clostridium acetobutylicum ATCC 824. In addition, the sol operon containing ctfAB, adc (acetoacetate decarboxylase), and ald (aldehyde dehydrogenase) was also cloned from Clostridium beijerinckii NCIMB 8052 and expressed in C. tyrobutyricum (Δack, adhE2). Mutants expressing these genes were evaluated for their ability to produce butanol from glucose in batch fermentations at pH 5.0 and 6.0. Compared to C. tyrobutyricum (Δack, adhE2) without expressing ctfAB, all mutants with ctfAB overexpression produced more butanol, with butanol yield increased to 0.22 − 0.26 g/g (vs. 0.10 − 0.13 g/g) and productivity to 0.35 g/l h (vs. 0.13 g/l h) because of the reduced acetate and butyrate production. The expression of ctfAB also resulted in acetone production from acetoacetate through a non-enzymatic decarboxylation.
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.
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.
•GFP-cell wall-binding domain (CBD) of endolysin CTP1L binds specifically to Clostridium.•Cheeses were made with C. tyrobutyricum spores and with or without reuterin.•Cheese bacteria were extracted and C. tyrobutyricum labelled with GFP-CBD-CTP1L.•Clostridial germinated spores and vegetative cells were detected in cheeses made with spores.•C. tyrobutyricum cells were not detected in cheeses with the antimicrobial reuterin.
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