Analysis of the Mechanism and Regulation of Lactose Transport and Metabolism in Clostridium acetobutylicum ATCC 824

Abstract

Although the acetone-butanol-ethanol fermentation of Clostridium acetobutylicum is currently uneconomic, the ability of the bacterium to metabolize a wide range of carbohydrates offers the potential for revival based on the use of cheap, low-grade substrates. We have investigated the uptake and metabolism of lactose, the major sugar in industrial whey waste, by C. acetobutylicum ATCC 824. Lactose is taken up via a phosphoenolpyruvate-dependent phosphotransferase system (PTS) comprising both soluble and membrane-associated components, and the resulting phosphorylated derivative is hydrolyzed by a phospho-␤-galactosidase. These activities are induced during growth on lactose but are absent in glucose-grown cells. Analysis of the C. acetobutylicum genome sequence identified a gene system, lacRFEG, encoding a transcriptional regulator of the DeoR family, IIA and IICB components of a lactose PTS, and phospho-␤-galactosidase. During growth in medium containing both glucose and lactose, C. acetobutylicum exhibited a classical diauxic growth, and the lac operon was not expressed until glucose was exhausted from the medium. The presence upstream of lacR of a potential catabolite responsive element (cre) encompassing the transcriptional start site is indicative of the mechanism of carbon catabolite repression characteristic of low-GC gram-positive bacteria. A pathway for the uptake and metabolism of lactose by this industrially important organism is proposed.The acetone-butanol-ethanol (ABE) fermentation of Clostridium acetobutylicum was a classical method to produce the commercially important solvents acetone and butanol, which operated successfully at an industrial scale in many countries during the first half of the 20th century. After the Second World War, the fermentation process declined because of the emergence of the competitive petrochemical-based synthesis of the solvents (18). Nevertheless, oil is a finite commodity and global oil prices are on the rise, and this, coupled with a general worldwide interest in exploiting renewable resources, has stimulated research on the biochemistry and physiology of solventogenic clostridia in recent years, with the aim of exploring the potential for revival of the ABE fermentation (17, 22). A major consideration in any bioconversion process is the availability of a cost-effective and high-product-yielding growth medium, whereby there is maximal conversion of the available carbon into the commercial end product. The substrate of choice in the traditional industrial ABE fermentation, molasses, accounted for more than 60% of the overall cost of the process (19). However, the clostridia are metabolically versatile with respect to carbohydrate utilization and the potential therefore exists to exploit alternative, cheaper, low-grade substrates.Several clostridial strains are able to produce solvents by fermentation of whey (21), which has been shown to be economically superior to the traditional process (19). Despite low reactor productivity, an attractive feature of these fermenta...

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“…Consequently, interest in acetone-butanolacetone (ABE) fermentation, which dwindled following the advent of the petrochemical industry, has been revived (Qureshi and Blaschek 2000;Qureshi and Maddox 2005;Yu et al 2007). However, a major challenge hampering re-commercialization of the ABE process is lack of economic competitiveness, stemming in part from the absence of inexpensive, readily available, and easily fermentable substrates capable of generating high ABE yields (Qureshi and Blaschek 2000;Yu et al 2007).…”

“…Consequently, interest in acetone-butanolacetone (ABE) fermentation, which dwindled following the advent of the petrochemical industry, has been revived (Qureshi and Blaschek 2000;Qureshi and Maddox 2005;Yu et al 2007). However, a major challenge hampering re-commercialization of the ABE process is lack of economic competitiveness, stemming in part from the absence of inexpensive, readily available, and easily fermentable substrates capable of generating high ABE yields (Qureshi and Blaschek 2000;Yu et al 2007). Interestingly, solventogenic Clostridium species are capable of fermenting a wide range of carbohydrates (Ezeji and Blaschek 2008), and lignocellulosic biomass has been identified as a potential substrate for inexpensive production of ABE and other fine chemicals (Ezeji and Blaschek 2008;Zhang and Ezeji 2013).…”

“…However, bioconversion of lignocellulosic biomass is currently plagued by a number of limitations, notably generation of microbial inhibitory compounds during pretreatment and hydrolysis of lignocellulose to mixed sugars (Almeida et al 2007), and inefficient utilization of the generated mixed sugars by fermenting microorganisms due to carbon catabolite repression (Ren et al 2010). Therefore, given the broad substrate spectrum of solventogenic Clostridium species (Ezeji and Blaschek 2008;Servinsky et al 2010;Yu et al 2007), other cheap and readily utilizable substrates, whose applications in fermentation do not require pretreatment, may prove to be more cost-effective and efficient substrates than lignocellulose.…”

“…Although some success has been recorded with whey, its use in large scale fermentation is plagued by a number of challenges, particularly its low sugar content (5%), which often warrants an initial concentration step prior to fermentation (Maddox 1980). Although glucose is the preferred sugar for solventogenic Clostridium species, different researchers have shown that lactose (the sugar content of whey) is utilized by these microorganisms when supplied as the sole carbon source (Bahl et al 1986;Maddox 1980;Yu et al 2007). Further, lactose metabolism favors butanol production over acetone (Bahl et al 1986;Maddox 1980), an added economic incentive in light of the current impetus for butanol production.…”

“…However, most of these studies were centered on C. acetobutylicum, perhaps due to higher ABE concentrations obtained with this species relative to C. beijerinckii. Consequently, molecular characterization of lactose transport and utilization, and the underlying regulatory machineries in C. acetobutylicum ATCC 824 has been vigorously pursued (Servinsky et al 2010;Yu et al 2007). Albeit subject to full characterization, genomic information on C. beijerinckii NCIMB 8052 shows the presence of many (about 47) phosphoenol-pyruvate (PEP)-dependent phosphotransferase system (PTS) genes apparently involved in the metabolism of complex carbohydrates (Shi et al 2010), including multiple genes whose protein products are putatively involved in lactose transport and metabolism.…”