Background
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), butanol (B) and ethanol (E) from a variety of carbohydrate substrates such as starch [3]. According to an estimate in 2008, butanol accounted for a 7-8.4 billon US dollar market worldwide and has a projected market expansion of 3% per year in the near future [4].
Significant efforts have been spent on physiological and genetic characterization of solvent-producing C. acetobutylicum in the past decades [5-8], and tools for genetic manipulation of C. acetobutylicum were also developed [9-11]. In 2001, the whole genome of well studied C. acetobutylicum ATCC 824 was sequenced, revealing a 3.94 Mb chromosome which encodes 3740 open reading frames (ORF), and a 192 Kb megaplasmid which encodes 178 ORFs [12]. Afterwards, a series of studies employing global approaches have been performed [13-16], and the genome-scale metabolic model of C. acetobutylicum was also constructed [17-19]. These efforts have improved the understanding of regulatory and metabolic networks of this industry significant species.
However, most of the C. acetobutylicum strains are not optimized systems for butanol production because their spore-forming life cycle decreases the efficiency of industrial fermentation, and the ABE fermentation process also creates a number of by-products, such as H2, acetic, lactic and propionic acids, acetone, isopropanol and ethanol [20]. As a result, the butanol yield is difficult to control and a significant amount of energy is wasted in these by-products. Moreover, it also increases the cost of downstream butanol purification. To address these issues, various modification approaches, such as mutagenesis by chemical or radiation agents, and genetic engineering, have been performed to improve the butanol production [10,21]. Our laboratory has previously obtained a high butanol producing strain, C. acetobutylicum EA 2018, through butanol resistance screening of N-methyl-N-nitro-N-nitrosoguanidine (NTG) treated Clostridium strain isolated from soil [22]. Preliminary results in a 100-ton continuous fermenter showed that butanol ratio and starch conversion rates of EA 2018 strain were 10% and 5% higher than those reported in recent literature [23]. To explore the genetic difference between EA 2018 and ATCC 824, in this study, the C. acetobutylicum EA 2018 genome was sequenced using Roche 454 pyrosequencing together with traditional Sanger sequencing. In addition, comparative genomic and transcriptomic analyses of EA 2018 and ATCC 824 were also performed. The study, for the first time, provides a molecular-level understanding of higher solvent production, enhanced xylose utilization and non-sporulation in the mutant EA 2018. The information could be valuable for further genetic modification of C. acetobutylicum for more effective butanol production.