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Comparative genomic and transcriptomic analysis revealed genetic characteristics related to solvent formation and xylose utilization in Clostridium acetobutylicum EA 2018
Shiyuan Hu, Huajun Zheng, Yang Gu, Jingbo Zhao, Weiwen Zhang, Yunliu Yang, Shengyue Wang, Guoping Zhao, Sheng Yang, Weihong Jiang
BMC Genomics , 2011, DOI: 10.1186/1471-2164-12-93
Abstract: Complete genome of C. acetobutylicum EA 2018 was sequenced using Roche 454 pyrosequencing. Genomic comparison with ATCC 824 identified many variations which may contribute to the hyper-butanol producing characteristics in the EA 2018 strain, including a total of 46 deletion sites and 26 insertion sites. In addition, transcriptomic profiling of gene expression in EA 2018 relative to that of ATCC824 revealed expression-level changes of several key genes related to solvent formation. For example, spo0A and adhEII have higher expression level, and most of the acid formation related genes have lower expression level in EA 2018. Interestingly, the results also showed that the variation in CEA_G2622 (CAC2613 in ATCC 824), a putative transcriptional regulator involved in xylose utilization, might accelerate utilization of substrate xylose.Comparative analysis of C. acetobutylicum hyper-butanol producing strain EA 2018 and type strain ATCC 824 at both genomic and transcriptomic levels, for the first time, provides molecular-level understanding of non-sporulation, higher solvent production and enhanced xylose utilization in the mutant EA 2018. The information could be valuable for further genetic modification of C. acetobutylicum for more effective butanol production.High oil prices, growing concerns over national security and climate change are driving investment and innovation in the renewable alternative fuels [1,2]. Among various potentially alternatives, butanol has been proposed as an excellent substitute or supplement for gasoline, and has been demonstrated to work in some vehicles designed for use with gasoline without any engine modification [1]. In addition to manufacture from petroleum through chemical refinery process, industry production of butanol is typically through a so-called ABE fermentation process employing gram-positive, spore forming and anaerobic organism Clostridium acetobutylicum [2]. C. acetobutylicum is capable of producing a mixture of acetone (A)
Pleiotropic functions of catabolite control protein CcpA in Butanol-producing Clostridium acetobutylicum  [cached]
Ren Cong,Gu Yang,Wu Yan,Zhang Weiwen
BMC Genomics , 2012, DOI: 10.1186/1471-2164-13-349
Abstract: Background Clostridium acetobutylicum has been used to produce butanol in industry. Catabolite control protein A (CcpA), known to mediate carbon catabolite repression (CCR) in low GC gram-positive bacteria, has been identified and characterized in C. acetobutylicum by our previous work (Ren, C. et al. 2010, Metab Eng 12:446–54). To further dissect its regulatory function in C. acetobutylicum, CcpA was investigated using DNA microarray followed by phenotypic, genetic and biochemical validation. Results CcpA controls not only genes in carbon metabolism, but also those genes in solvent production and sporulation of the life cycle in C. acetobutylicum: i) CcpA directly repressed transcription of genes related to transport and metabolism of non-preferred carbon sources such as d-xylose and l-arabinose, and activated expression of genes responsible for d-glucose PTS system; ii) CcpA is involved in positive regulation of the key solventogenic operon sol (adhE1-ctfA-ctfB) and negative regulation of acidogenic gene bukII; and iii) transcriptional alterations were observed for several sporulation-related genes upon ccpA inactivation, which may account for the lower sporulation efficiency in the mutant, suggesting CcpA may be necessary for efficient sporulation of C. acetobutylicum, an important trait adversely affecting the solvent productivity. Conclusions This study provided insights to the pleiotropic functions that CcpA displayed in butanol-producing C. acetobutylicum. The information could be valuable for further dissecting its pleiotropic regulatory mechanism in C. acetobutylicum, and for genetic modification in order to obtain more effective butanol-producing Clostridium strains.
Identification and Characterization of Two Functionally Unknown Genes Involved in Butanol Tolerance of Clostridium acetobutylicum  [PDF]
Kaizhi Jia, Yanping Zhang, Yin Li
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0038815
Abstract: Solvents toxicity is a major limiting factor hampering the cost-effective biotechnological production of chemicals. In Clostridium acetobutylicum, a functionally unknown protein (encoded by SMB_G1518) with a hypothetical alcohol interacting domain was identified. Disruption of SMB_G1518 and/or its downstream gene SMB_G1519 resulted in increased butanol tolerance, while overexpression of SMB_G1518-1519 decreased butanol tolerance. In addition, SMB_G1518-1519 also influences the production of pyruvate:ferredoxin oxidoreductase (PFOR) and flagellar protein hag, the maintenance of cell motility. We conclude that the system of SMB_G1518-1519 protein plays a role in the butanol sensitivity/tolerance phenotype of C. acetobutylicum, and can be considered as potential targets for engineering alcohol tolerance.
Optimization of butanol production from corn straw hydrolysate by Clostridium acetobutylicum using response surface method
YouSheng Lin,Jing Wang,XuMing Wang,XiaoHong Sun
Chinese Science Bulletin , 2011, DOI: 10.1007/s11434-010-4186-0
Abstract: Butanol is a new kind of very potential biofuels. Enzymatic hydrolysis of corn stalk was utilized in this study to produce butanol by Clostridium acetobutylicum CICC 8008. Plackett-Burman (P-B) design and Central Composite Design (CCD) were adopted to screen crucial factors during fermentation as well as the optimization of experimental conditions. The result demonstrated that among the seven factors, namely, Yeast extract, (NH4)2SO4, KH2PO4, MgSO4, FeSO4, CuSO4 and CaCO3, only CaCO3 was selected as the most critical factor. The optimization experiment results for CaCO3 usage, temperature and reaction time by CCD were determined to be 5.04 g/L, 35°C and 70 h, respectively. A corresponding mathematical model was established to predict the fermentation experiment and maximum butanol yield of 6.57 g/L was acquired. The result of verification experiment under the optimum conditions showed that 6.20 g/L was the maximum butanol yield. This demonstrated that statistical method was a powerful tool for the optimization of butanol production from enzymatic hydrolysis of corn stalk.
A comparison of three pH control methods for revealing effects of undissociated butyric acid on specific butanol production rate in batch fermentation of Clostridium acetobutylicum
Xuepeng Yang, Maobing Tu,, Rui Xie, Sushil Adhikari and Zhaohui Tong
AMB Express , 2013, DOI: 10.1186/2191-0855-3-3
Abstract: pH control has been essential for butanol production with Clostridium acetobutylicum. However, it is not very clear at what pH level the acid crash will occur, at what pH level butanol production will be dominant, and at what pH level butyric acid production will be prevailing. Furthermore, contradictory results have been reported about required acidic conditions for initiation of solventogenesis. In this study, with the aim of further understanding the role of undissociated butyric acid in butanol production, we investigated the correlation between undissociated butyric acid concentration and specific butanol production rate in batch fermentation of Clostridium acetobutylicum by comparing three pH control approaches: NaOH neutralization (at 12, 24 or 36 h), CaCO3 supplementation (2, 5, or 8 g/l) and NaOAc buffering (pH 4.6, 5.0 or 5.6). By neutralizing the fermentation pH to ~5.0 at different time, we observed that neutralization should take place at the beginning of exponential phase (12 h), and otherwise resulting in lower concentrations of undissociated butyric acid, cell biomass and final butanol. CaCO3 supplementation extended cell growth to 36 h and resulted in higher butyrate yield under 8 g/L of CaCO3. In the NaOAc buffering, the highest specific butanol rate (0.58 h 1) was associated with the highest undissociated butyric acid (1.92 g/L). The linear correlation of the undissociated butyric acid with the specific butanol production rates suggested the undissociated butyric acid could be the major driving force for butanol production.
Butanol production from hydrolysate of Jerusalem artichoke juice by Clostridium acetobutylicum L7

Lijie Chen,Chengxun Xin,Pan Deng,Jiangang Ren,Huanhuan Liang,Fengwu Bai,

生物工程学报 , 2010,
Abstract: Butanol production from acid hydrolysate of Jerusalem artichoke juice by Clostridium acetobutylicum L7 was investigated, and it was found that natural components of the hydrolysate were suitable for solvent production with the species. With batch fermentation using the medium containing 48.36 g/L total sugars, 8.67 g/L butanol was produced at 60 h, and the ratio of butanol to acetone to ethanol was 0.58:0.36:0.06, which were similar to the fermentation with fructose as carbon source, but both of these two fermentations were slower than that with glucose as carbon source, indicating the fructose transport of the species might not be effective as that for glucose. When the total sugars of the medium were increased to 62.87 g/L, the residual sugars increased slightly from 3.09 g/L to 3.26 g/L, but butanol production of the fermentation system was improved significantly, with 11.21 g/L butanol produced and the ratio of butanol to acetone to ethanol at 0.64:0.29:0.05, which illustrated that an excess in sugars enhanced the butanol biosynthesis of the species by compromising its acetone production. When the sugar concentration of the medium was further increased, much more sugars were remained unconsumed, making the process economically unfavourable.
Reconstruction of xylose utilization pathway and regulons in Firmicutes
Yang Gu, Yi Ding, Cong Ren, Zhe Sun, Dmitry A Rodionov, Weiwen Zhang, Sheng Yang, Chen Yang, Weihong Jiang
BMC Genomics , 2010, DOI: 10.1186/1471-2164-11-255
Abstract: A comparative genomic approach was used to reconstruct the xylose and xyloside utilization pathway and analyze its regulatory mechanisms in 24 genomes of the Firmicutes. A novel xylose isomerase that is not homologous to previously characterized xylose isomerase, was identified in C. acetobutylicum and several other Clostridia species. The candidate genes for the xylulokinase, xylose transporters, and the transcriptional regulator of xylose metabolism (XylR), were unambiguously assigned in all of the analyzed species based on the analysis of conserved chromosomal gene clustering and regulons. The predicted functions of these genes in C. acetobutylicum were experimentally confirmed through a combination of genetic and biochemical techniques. XylR regulons were reconstructed by identification and comparative analysis of XylR-binding sites upstream of xylose and xyloside utilization genes. A novel XylR-binding DNA motif, which is exceptionally distinct from the DNA motif known for Bacillus XylR, was identified in three Clostridiales species and experimentally validated in C. acetobutylicum by an electrophoretic mobility shift assay.This study provided comprehensive insights to the xylose catabolism and its regulation in diverse Firmicutes bacteria especially Clostridia species, and paved ways for improving xylose utilization capability in C. acetobutylicum by genetic engineering in the future.The Firmicutes (Bacilli/Clostridia) are a diverse group of Gram-positive bacteria that includes a large number of species that produce lactic acid, acetone, butanol, and ethanol through fermentation of a variety of carbon sources. Many of these bacteria were originally isolated from the plant environments such as garden soil, fruits, and vegetables [1,2]. Among them, Clostridium acetobutylicum is one of the best-studied clostridia and was used to develop an industrial fermentation process for producing solvents [3,4]. This strain is known to utilize a broad range of monosaccharide
Introducing a single secondary alcohol dehydrogenase into butanol-tolerant Clostridium acetobutylicum Rh8 switches ABE fermentation to high level IBE fermentation
zongjie dai, hongjun dong, yan zhu, yanping zhang, yin li, yanhe ma
Biotechnology for Biofuels , 2012, DOI: 10.1186/1754-6834-5-44
Abstract: The heterogenous gene sADH was functionally expressed in C. acetobutylicum Rh8. This simple, one-step engineering approach switched the traditional ABE (acetone-butanol-ethanol) fermentation to IBE (isopropanol-butanol-ethanol) fermentation. The total alcohol titer reached 23.88?g/l (7.6?g/l isopropanol, 15?g/l butanol, and 1.28?g/l ethanol) with a yield to glucose of 31.42%. The acid (butyrate and acetate) assimilation rate in isopropanol producing strain Rh8(psADH) was increased.The improved butanol tolerance and the enhanced solvent biosynthesis machinery in strain Rh8 is beneficial for production of high concentration of mixed alcohols. Strain Rh8 can thus be considered as a good host for further engineering of solvent/alcohol production.
Genetic modification systems for Clostridium acetobutylicum

Hongjun Dong,Yanping Zhang,Yin Li,

生物工程学报 , 2010,
Abstract: Clostridium acetobutylicum, a biofuel-butanol producer, has attracted worldwide interests. Strain improvement is important for the process of biobutanol industrialization where efficient genetic modification systems are essential. In this review, the history of genetic modification systems of C. acetobutylicum was introduced, and the types and principles of these systems and their disadvantages are summarized and analysed. The development of updated genetic modification systems for C. acetobutylicum is also proposed.
Date Fruit as Carbon Source in RCM-Modified Medium to Produce Biobutanol by Clostridium acetobutylicum NCIMB 13357  [PDF]
Emran I. Khamaiseh,Mohd Sahaid Kalil,Olujimi Dada,Ibrahim El-Shawabkeh
Journal of Applied Sciences , 2012,
Abstract: Date fruit can be used as an organic carbohydrate source in modified fermentation media to produce biobutanol. Filtrate from date fruit contains high concentration of simple sugars ranging from 65 to 75 %. Reinforced Clostridial Medium (RCM) supplemented with filtrate of date fruit was investigated under anaerobic fermentation conditions using Clostridium acetobutylicum NCIMB 13357 for production of Acetone, Butanol and ethanol (ABE). The effects of temperature, pH and concentration of date fruit filtrate on the efficiency of C. acetobutylicum showed that 40 g L-1 date fruit filtrate at initial pH 7 and temperature 35°C were the optimal fermentation condition that gave the highest amount of butanol- 4.4 g L-1. The yield and productivity of Biobutanol were 0. 3 g g-1 and 0.07 g L-1 h-1 respectively while the Yield and Productivity of ABE were 0.41 g g-1 and 0.09 g L-1 h-1, respectively.
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