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.

Exploiting the Type I-B CRISPR Genome Editing System in Thermoanaerobacterium aotearoense SCUT27 and Engineering the Strain for Enhanced Ethanol Production.

Thermoanaerobacterium aotearoense strain SCUT27 is a potential industrial biofuel-producing strain because of its broad substrate spectrum, especially the ability to co-use glucose and xylose. The bottleneck hindering the development of strain SCUT27 is the lack of selective markers for polygene manipulation in this thermophilic bacterium. In this study, the endogenous type I-B clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system was developed for multiplex genome editing of strain SCUT27. The protospacer-adjacent motif was identified by in silico analysis and verified with orotidine-5'-phosphate decarboxylase (pyrF) or lactate dehydrogenase (ldh) as the editing target. The type I-B CRISPR/Cas system was functional in strain SCUT27 with 58.3% to 100% editing efficiency. A multiplex genome editing method based on thymidine kinase (tdk) as a negative selection marker was developed, and strain SCUT27/Δtdk/Δldh/ΔargR, in which ldh and the arginine repressor (argR) were knocked out successively, was successfully obtained. Strain SCUT27/Δtdk/Δldh/ΔargR exhibited prominent advantages over wild-type SCUT27 in ethanol production, with significantly improved ability to metabolize xylose. IMPORTANCE Thermophilic microbes have attracted great attention as potential candidates for production of biofuels and chemicals from lignocellulose because of their thermal tolerance and wide substrate spectra. The ability to edit multiple genes using the native type I-B CRISPR/Cas system would speed up engineering of Thermoanaerobacterium aotearoense strain SCUT27 for higher ethanol production from lignocellulosic hydrolysates. Here, we produced a mutant strain, T. aotearoense SCUT27/Δtdk/Δldh/ΔargR, using the native CRISPR/Cas system. The engineered strain showed satisfactory performance with improved ethanol productivity from various lignocellulosic hydrolysates. Our data lay the foundations for development of this thermophilic microbe into an excellent ethanol producer using lignocellulosic hydrolysates. The methods described here may also provide a reference to develop multigene editing methods for other microorganisms.

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.

Isolation and characterization of a newly identified Clostridium butyricum strain SCUT343-4 for 1,3-propanediol production.

A novel 1,3-propanediol (1,3-PDO) producing strain was isolated and identified as Clostridium butyricum with respect to its morphological and physiological characteristics, as well as 16S rDNA. The results of substrates test and stress tolerance indicated that C. butyricum SCUT343-4 could produce 1,3-PDO efficiently from glycerol. The optimal fermentation conditions were determined to be 5 g/L yeast extract at 37 °C and pH 6.5. To fully evaluate its 1,3-PDO production capacity, different cultivation strategies have been implemented. The highest 1,3-PDO concentration obtained for batch and fed-batch fermentation were 51.64 and 61.30 g/L, respectively. Immobilized cell fermentation in fibrous-bed bioreactor was also performed, and the concentration of 1,3-PDO further increased to 86 g/L with a yield of 0.52 g/g. In addition, the 1,3-PDO productivity reached 4.20 g/L h, which is the highest level reported for C. butyricum, demonstrating the potential of C. butyricum SCUT343-4 for 1,3-PDO production from glycerol.

The redox-sensing transcriptional repressor Rex is important for regulating the products distribution in Thermoanaerobacterium aotearoense SCUT27.

The redox-sensing transcriptional repressor Rex (Rex) displayed diverse functions in different microbial species. Nowadays, only part function of rex has been verified in vitro and alcohol dehydrogenase gene (adhE) as the target of Rex has been widely reported. In this study, rex was knocked out in Thermoanaerobacterium aotearoense SCUT27 (GDMCC 60765) and the carbon metabolic distribution analysis was performed. Results showed that the ethanol yield (mol product/mol carbon) of SCUT27(Δrex) had increased by 75.00-90.91%, cell growth improved by 27.27-36.36%, and acetic acid and lactic acid decreased by 58.33-61.54% accompanied with the yield of hydrogen decreased by 46.15-58.35% within different carbon sources. The ability of sugar consumption of SCUT27(Δrex) had improved about 74.19-130.55% with the improvement of total ATP concentration and the cofactors NADH and NAD+ concentrations. In addition, the specific activities of alcohol dehydrogenase of SCUT27(Δrex) with NADH and NADPH as cofactors were improved by 119.26-140.28% and 35.66-47.69%, respectively. After ldh was further knocked out in SCUT27(Δrex), SCUT27(ΔldhΔrex) showed higher ethanol production and yield when various carbon resources were used as substrates (including glucose, xylose, glucose/xylose mixture and three kinds of lignocellulosic hydrolysates). This study confirms that Rex is an important regulator for determining products distribution in SCUT27 and deletion of rex and ldh is a promising strategy for enhanced ethanol 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.

Improving the fermentation performance of Clostridium acetobutylicum ATCC 824 by strengthening the VB1 biosynthesis pathway.

Vitamin B1 (VB1) is an essential coenzyme for carbohydrate metabolism and involved in energy generation in most organisms. In this study, we found that insufficient biosynthesis of VB1 in Clostridium acetobutylicum ATCC 824 is a major limiting factor for efficient acetone-butanol-ethanol (ABE) fermentation. In order to improve the fermentation performance of C. acetobutylicum ATCC 824, the VB1 biosynthesis pathway was strengthened by overexpressing the thiC, thiG, and thiE genes. The engineered strain 824(thiCGE) showed enhanced VB1 and energy synthesis, resulting in better growth, faster sugar consumption, higher solvents production, and lower acids formation than the wild-type strain in both VB1 free and normal P2 medium (1 mg/L). Compared with the wild-type strain, 824(thiCGE) produced 13.0 ± 0.1% or 12.7 ± 1.2% more butanol in VB1 free P2 medium when glucose or xylose was used as the substrate, respectively. When mixed sugar (glucose:xylose = 2:1) was used as the substrate in VB1 free P2 medium, the xylose consumption rate and butanol titer of 824(thiCGE) were 45.8 ± 1.9% and 20.4 ± 0.3% higher than those of the wild-type strain. All these results demonstrated that this metabolic engineering strategy could provide a new and effective way to improve the cellular performance of solventogenic clostridia. In addition, it may have some potential application value in ABE fermentation using simple medium and/or lignocellulosic biomass.

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.

Metabolic engineering of Clostridium tyrobutyricum for enhanced butyric acid production from glucose and xylose.

Clostridium tyrobutyricum is a promising microorganism for butyric acid production. However, its ability to utilize xylose, the second most abundant sugar found in lignocellulosic biomass, is severely impaired by glucose-mediated carbon catabolite repression (CCR). In this study, CCR in C. tyrobutyricum was eliminated by overexpressing three heterologous xylose catabolism genes (xylT, xylA and xlyB) cloned from C. acetobutylicum. Compared to the parental strain, the engineered strain Ct-pTBA produced more butyric acid (37.8g/L vs. 19.4g/L) from glucose and xylose simultaneously, at a higher xylose utilization rate (1.28g/L·h vs. 0.16g/L·h) and efficiency (94.3% vs. 13.8%), resulting in a higher butyrate productivity (0.53g/L·h vs. 0.26g/L·h) and yield (0.32g/g vs. 0.28g/g). When the initial total sugar concentration was ~120g/L, both glucose and xylose utilization rates increased with increasing their respective concentration or ratio in the co-substrates but the total sugar utilization rate remained almost unchanged in the fermentation at pH 6.0. Decreasing the pH to 5.0 significantly decreased sugar utilization rates and butyrate productivity, but the effect was more pronounced for xylose than glucose. The addition of benzyl viologen (BV) as an artificial electron carrier facilitated the re-assimilation of acetate and increased butyrate production to a final titer of 46.4g/L, yield of 0.43g/g sugar consumed, productivity of 0.87g/L·h, and acid purity of 98.3% in free-cell batch fermentation, which were the highest ever reported for butyric acid fermentation. The engineered strain with BV addition thus can provide an economical process for butyric acid production from lignocellulosic biomass.

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

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

Cofactor engineering in Thermoanaerobacterium aotearoense SCUT27 for maximizing ethanol yield and revealing an enzyme complex with high ferredoxin-NAD+ reductase activity.

Thermoanaerobacterium aotearoense SCUT27 is a prominent producer of biofuels from lignocellulosic materials. To provide sufficient NAD(P)H for ethanol production, redox-related genes, including lactate dehydrogenase (ldh), redox-sensing transcriptional repressor (rex), and hydrogenase (hfsB), were knocked out. However, the growth of strain PRH (Δldh/Δrex/ΔhfsB) was suppressed due to the intracellular redox state imbalance with the increased NADH concentration. Coincidentally, when the Bcd-EtfAB (BCD) complex was overexpressed, the resulting strain PRH-B3 (Δldh/Δrex/ΔhfsB::BCD) grew rapidly and produced ethanol with a high yield. With lignocellulosic hydrolysates, PRH-BA (Δldh/Δrex/ΔhfsB::BCD::adhE) demonstrated high ethanol productivity and yield, reaching levels of 0.45-0.51 g/L/h and 0.46-0.53 g/g sugars, respectively. The study results shed light on the cofactor balance for cell stability and the high ferredoxin-NAD+ reductase activity of the BCD complex under an intracellular low redox state. They also provide an essential reference for developing strains for improved biofuel production.

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.

Isolation of Bacillus subtilis and Bacillus pumilus with Anti-Vibrio parahaemolyticus Activity and Identification of the Anti-Vibrio parahaemolyticus Substance.

The adoption of intensive farming has exacerbated disease outbreaks in aquaculture, particularly vibriosis caused by Vibrio parahaemolyticus. The use of probiotics to control V. parahaemolyticus is recognized as a good alternative to antibiotics for avoiding the development of antibiotic-resistant bacteria. In this study, two strains of B. HLJ1 and B. C1 with strong inhibitory activity on V. parahaemolyticus were isolated from aquaculture water and identified as Bacillus subtilis and Bacillus pumilus, respectively. Both B. HLJ1 and B. C1 lacked antibiotic resistance and virulence genes, suggesting that they are safe for use in aquaculture. In addition, these two strains can tolerate acid environments, produce spores, secrete extracellular enzymes, and co-aggregate as well as auto-aggregate with V. parahaemolyticus. B. HLJ1 and B. C1 produced the same anti-V. parahaemolyticus substance, which was identified as AI-77-F and belongs to amicoumacins. Both B. C1 and B. HLJ1 showed inhibitory activity against 11 different V. parahaemolyticus and could effectively control the growth of V. parahaemolyticus in simulated aquaculture wastewater when the concentration of B. C1 and B. HLJ1 reached 1 × 107 CFU/mL. This study shows that B. HLJ1 and B. C1 have great potential as aquaculture probiotics.

Metabolic engineering of Thermoanaerobacterium aotearoense strain SCUT27 for biofuels production from sucrose and molasses.

BACKGROUND: Sucrose-rich sugarcane trash surpasses 28 million tons globally per year. Effective biorefinery systems could convert these biomasses to bioproducts, such as bioethanol from sugarcane sucrose in Brazil. Thermophilic microbes for biofuels have attracted great attention due to their higher fermentation temperature and wide substrate spectrum. However, few thermophiles using sucrose or molasses for biofuels production was reported. Thermoanaerobacterium aotearoense SCUT27 has been considered as an efficient ethanol producer, but it cannot directly utilize sucrose. In this study, various sucrose metabolic pathways were introduced and analyzed in Thermoanaerobaterium. RESULTS: The sucrose-6-phosphate hydrolase (scrB), which was from a screened strain Thermoanaerobacterium thermosaccharolyticum G3-1 was overexpressed in T. aotearoense SCUT27 and endowed this strain with the ability to utilize sucrose. In addition, overexpression of the sucrose-specific PTS system (scrA) from Clostridium acetobutylicum accelerated the sucrose transport. To strengthen the alcohols production and substrates metabolism, the redox-sensing transcriptional repressor (rex) in T. aotearoense was further knocked out. Moreover, with the gene arginine repressor (argR) deleted, the ethanologenic mutant P8S10 showed great inhibitors-tolerance and finally accumulated ~ 34 g/L ethanol (a yield of 0.39 g/g sugars) from pretreated cane molasses in 5 L tank by fed-batch fermentation. When introducing butanol synthetic pathway, 3.22 g/L butanol was produced by P8SB4 with a yield of 0.44 g alcohols/g sugars at 50℃. This study demonstrated the potential application of T. aotearoense SCUT27 for ethanol and butanol production from low cost cane molasses. CONCLUSIONS: Our work provided strategies for sucrose utilization in thermophiles and improved biofuels production as well as stress tolerances of T. aotearoense SCUT27, demonstrating the potential application of the strain for cost-effective biofuels production from sucrose-based feedstocks.

Elimination of carbon catabolite repression in Clostridium tyrobutyricum for enhanced butyric acid production from lignocellulosic hydrolysates.

Clostridium tyrobutyricum, a gram-positive anaerobic bacterium, is recognized as the promising butyric acid producer. But, the existence of carbon catabolite repression (CCR) is the major drawback for C. tyrobutyricum to efficiently use the lignocellulosic biomass. In this study, the xylose pathway genes were first identified and verified. Then, the potential regulatory mechanisms of CCR in C. tyrobutyricum were proposed and the predicted engineering targets were experimental validated. Inactivation of hprK blocked the CcpA-mediated CCR and resulted in simultaneous conversion of glucose and xylose, although xylose consumption was severe lagging behind. Deletion of xylR further shortened the lag phase of xylose utilization. When hprK and xylR were inactivated together, the CCR in C. tyrobutyricum was completely eliminated. Consequently, ATCC 25755/ΔhprKΔxylR showed significant increase in butyrate productivity (1.8 times faster than the control) and excellent butyric acid fermentation performance using both mixed sugars (11.0-11.9 g/L) and undetoxified lignocellulosic hydrolysates (12.4-13.4 g/L).

Model-based driving mechanism analysis for butyric acid production in Clostridium tyrobutyricum.

BACKGROUND: Butyric acid, an essential C4 platform chemical, is widely used in food, pharmaceutical, and animal feed industries. Clostridium tyrobutyricum is the most promising microorganism for industrial bio-butyrate production. However, the metabolic driving mechanism for butyrate synthesis was still not profoundly studied. RESULTS: This study reports a first-generation genome-scale model (GEM) for C. tyrobutyricum, which provides a comprehensive and systematic analysis for the butyrate synthesis driving mechanisms. Based on the analysis in silico, an energy conversion system, which couples the proton efflux with butyryl-CoA transformation by two redox loops of ferredoxin, could be the main driving force for butyrate synthesis. For verifying the driving mechanism, a hydrogenase (HydA) expression was perturbed by inducible regulation and knockout. The results showed that HydA deficiency significantly improved the intracellular NADH/NAD+ rate, decreased acetate accumulation (63.6% in serum bottle and 58.1% in bioreactor), and improved the yield of butyrate (26.3% in serum bottle and 34.5% in bioreactor). It was in line with the expectation based on the energy conversion coupling driving mechanism. CONCLUSIONS: This work show that the first-generation GEM and coupling metabolic analysis effectively promoted in-depth understanding of the metabolic driving mechanism in C. tyrobutyricum and provided a new insight for tuning metabolic flux direction in Clostridium chassis cells.

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.

T-SPOT with CT image analysis based on deep learning for early differential diagnosis of nontuberculous mycobacteria pulmonary disease and pulmonary tuberculosis.

OBJECTIVES: This study aimed to establish a diagnostic algorithm combining T-SPOT with computed tomography image analysis based on deep learning (DL) for early differential diagnosis of nontuberculous mycobacteria pulmonary disease (NTM-PD) and pulmonary tuberculosis (PTB). METHODS: A total of 1049 cases were enrolled, including 467 NTM-PD and 582 PTB cases. A total of 320 cases (160 NTM-PD and 160 PTB) were randomized as the testing set and were analyzed using T-SPOT combined with the DL model. The testing cases were first divided into T-SPOT-positive and -negative groups, and the DL model was then used to separate the cases into four subgroups further. RESULTS: The precision was found to be 91.7% for the subgroup of T-SPOT-negative and DL classified as NTM-PD, and 89.8% for T-SPOT-positive and DL classified as PTB, which covered 66.9% of the total cases, compared with the accuracy rate of 80.3% of T-SPOT alone. In the other two remaining groups, where the T-SPOT prediction was inconsistent with the DL model, the accuracy was 73.0% and 52.2%, separately. CONCLUSION: Our study shows that the new diagnostic system combining T-SPOT with DL based computed tomography image analysis can greatly improve the classification precision of NTM-PD and PTB when the two methods of prediction are consistent.

Butanol production from Saccharina japonica hydrolysate by engineered Clostridium tyrobutyricum: The effects of pretreatment method and heat shock protein overexpression.

Macroalgal biomass is currently considered as a potential candidate for biofuel production. In this study, the effects of pretreatment method and heat shock protein overexpression were investigated for efficient butanol production from Saccharina japonica using engineered Clostridium tyrobutyricum. First, various pretreatment methods including acid hydrolysis, acid hydrolysis and enzymatic saccharification, and ultrasonic-assisted acid hydrolysis were employed to obtain the fermentable sugars, and the resulted hydrolysates were evaluated for butanol fermentation. The results showed that ultrasonic-assisted acid hydrolysate obtained the highest butanol yield (0.26 g/g) and productivity (0.19 g/L⋅h). Then, the effects of homologous or heterologous heat shock protein overexpression on butanol production and tolerance were examined. Among all the engineered strains, Ct-pMA12G exhibited improved butanol tolerance and enhanced butanol production (12.15 g/L butanol with a yield of 0.34 g/g and productivity of 0.15 g/L⋅h) from 1.8-fold concentrated S. japonica hydrolysate, which was the highest level ever reported for macroalgal biomass.