C. acetobutylicum was the first completely sequenced Clostridium. The genome sequence of the type-strain ATCC 824 (Weyer and Rettger, 1927) was already released in 2001 by Nolling et al. This provided valuable information and helped in further understanding of solventogenic clostridia. Meanwhile, the genome sequence of another major solventogenic Clostridium species, C. beijerinckii NCIMB 8052, is available too (JGI, 2005). While the genome of C. acetobutylicum consists of a 3.94-Mbp chromosome and a 192-kbp megaplasmid pSOLl, C. beijerinckii contains no megaplasmid but has a significantly larger chromosome with a size of 6.0 Mbp.

One surprising finding in the genome sequence of C. acetobutylicum was the presence of 11 genes, whose products were unambiguously identified as cellulosome components (Nolling et al, 2001; Sabathe, Belaich, et al., 2002). Although overexpression of single genes led to functional proteins (Lopez — Contreras et al., 2003; Perret et al., 2004) and also a minicellulosome could be produced in vivo (Sabathe and Soucaille, 2003), C. acetobutylicum is unable to ferment cellulose (Lee et al., 1985a; Lopez-Contreras et al., 2003).

However, xylan, another major component of lignocellulose besides cellulose, can be degraded by C. acetobutylicum as well as C. beijerinckii (Lemmel et al., 1986; Qureshi et al., 2006), and respective enzymes have been isolated and characterized (Ali et al., 2004; Ali et al., 2005; Lee and Forsberg, 1987; Lee et al., 1985b; Lee et al., 1987).

Several a-amylases for degradation of starch were found in C. acetobutylicum. Two of them were purified and analyzed in detail (Annous and Blaschek, 1994; Paquet et al., 1991), but only one of the respective genes (amyP = CAP0168) has been confidently identified from the genome sequence (Sabathe, Croux, et al., 2002).

Mono — and disaccharides are then taken up by phosphoenolpyruvate-dependent phosphotransferase systems, which are already well described in C. acetobutylicum and C. beijerinckii (see Table 10.3). Only galactose is transported by a non­phosphotransferase mechanism (Mitchell and Tangney, 2005).

Inside the cell, sugars are metabolized to pyruvate, hexoses via glycolysis and pentoses via the pentose phosphate pathway. Respective genes were found in the genome sequence of C. acetobutylicum (Nolling et al., 2001) and C. beijerinckii (JGI, 2005). Pyruvate is then converted to acetyl-CoA by a pyruvate:ferredoxin — oxidoreductase, one of the most oxygen-sensitive enzymes known (Meinecke et al., 1989). Lactate and acetoin are also produced from pyruvate (Fig. 10.2), catalyzed by lactate dehydrogenase (Freier and Gottschalk, 1987) and acetolactate synthase plus acetolactate decarboxylase, respectively, while the formation of acetate, butyrate, ethanol, acetone, isopropanol and butanol starts from acetyl-CoA (Fig. 10.2).

Acetate is produced via acetyl phosphate by successive action of phosphotransacetylase (Pta) and acetate kinase (Ack) (Fig. 10.2). The latter was purified from C. acetobutylicum and was shown to be a highly specific enzyme (Winzer et al, 1997). The respective genes pta and ack are located in a common operon (CAC1742-CAC1743; Fig. 10.3) on the genome of C. acetobutylicum (Boynton et al., 1996) and are also present in C. beijerinckii, arranged in exactly the same order. Butyrate is formed in analogous reactions from butyryl-CoA (Fig. 10.2). The respective enzymes phosphotransbutyrylase (Ptb) and butyrate kinase (Buk) are already characterized in detail (Hartmanis, 1987; Thompson and Chen, 1990; Wiesenborn et al., 1989b). The corresponding genes ptb and buk are clustered in a common operon on the genome of C. acetobutylicum (CAC3075-CAC3076; Fig. 10.3; Cary et al, 1988; Walter et al., 1993) and C. beijerinckii, respectively. Gene expression is relatively stable over the whole growth, similar to pta and ack (Fig. 10.3; Alsaker and Papoutsakis, 2005). However, in contrast to the acetate-producing enzymes, butyrate kinase activity can also be detected during solventogenesis (Andersch et al, 1983; Hartmanis and Gatenbeck, 1984). This might be attributed to a second butyrate kinase (BKII) found in C. acetobutylicum, whose physiological function is yet unknown (Huang et al., 2000).

Butyryl-CoA itself is produced from two molecules of acetyl-CoA by successive action of thiolase (ThlA), 3-hydroxybutyryl-CoA dehydrogenase (Hbd), crotonase (Crt) and butyryl-CoA dehydrogenase (Bcd) (Fig. 10.2). The respective genes are clustered on the genome of C. acetobutylicum and C. beijerinckii in the bcs operon (CAC2708-CAC2712; Fig. 10.3; Boynton et al., 1996), except the thiolase gene thlA (CAC2873) that is organized monocistronically (Stim-Herndon et al., 1995). Furthermore, a second thiolase operon was found on the megaplasmid of C. acetobutylicum (Winzer et al., 2000). While its physiological role is still unknown, ThlA has already been studied in detail (Wiesenborn et al., 1988). Hbd has been purified and characterized from C. beijerinckii (Colby and Chen, 1992) and Crt from a non-specified C. acetobutylicum strain (Waterson et al., 1972). Data on Bcd are scarce, but Inui et al. (2008) demonstrated that a pair of electron­transferring flavoproteins (EtfA/B), whose genes are also part of the bcs operon,

1 2000 4000 6000 8000 10000

10.3 Arrangement of the genes associated with solvent formation in C. acetobutylicum and their expression profile (according to Alsaker et al., 2004).

is essential for the activity of this enzyme. In Clostridium kluyveri, Bcd was shown to form a stable complex with EtfA/B (Herrmann et al, 2008; Seedorf et al., 2008), which is also involved in energy conservation via an Rnf complex (Herrmann et al., 2008; Seedorf et al., 2008). However, no Rnf complex is present in C. acetobutylicum, while respective genes were found in C. beijerinckii.

Formation of solvents starts with an acetoacetyl-CoA:acetate/butyrate-CoA transferase CtfA/B, which converts the previously produced acids acetate and butyrate into the respective acyl-CoA derivatives and acetoacetate (Fig. 10.2). While the recycled acetyl-CoA and butyryl-CoA are used for the production of alcohols such as ethanol and butanol via a number of aldehyde and alcohol dehydrogenases (see below), an acetoacetate decarboxylase Adc splits acetoacetate into CO2 and acetone, which is in some strains of C. beijerinckii further reduced to isopropanol by action of a primary/secondary alcohol dehydrogenase (Ismaiel et al., 1993).

CoA transferase and acetoacetate decarboxylase have already been studied in detail (Chen, 1993; Gerischer and Durre, 1990; Petersen and Bennett, 1990; Schaffer et al., 2002; Wiesenborn et al., 1989a). In C. acetobutylicum, the respective genes are located on the megaplasmid directly next to each other in a convergent direction (Fig. 10.3). While adc forms a monocistronic operon, ctfA/B are arranged in the sol operon together with the genes orfL (encoding a small peptide of still unknown function) and adhE (coding for a bifunctional butyraldehyde/butanol dehydrogenase) (Fischer et al., 1993; Zickner et al., 1993). This distinguishes C. acetobutylicum from all other solventogenic clostridia, where adc is part of the sol operon and adhE is replaced by an ald gene encoding an aldehyde dehydrogenase (Berezina et al., 2009; Toth et al., 1999). A typical oA-dependent promoter was found upstream of adc (Gerischer and Durre, 1992), whereas two promoter sequences were deduced for the sol operon by primer extension experiments (Fischer et al., 1993; Nair et al., 1994b). However, reporter gene studies revealed that only the distal sequence P1 represents a promoter and the proximal P2 is obviously an mRNA processing site (see below; Thormann et al., 2002). Both operons show a similar expression profile with a massive upregulation at transition from acidogenesis to solventogenesis (Fig. 10.3; Alsaker and Papoutsakis, 2005). The signal leading to this induction is still unknown, but it is proposed that various extra — and intracellular parameters such as temperature, low pH and high concentration of (undissociated) acetic and butyric acid (Ballongue et al., 1985; Gottwald and Gottschalk, 1985; Huesemann and Papoutsakis, 1986; Monot et al., 1983; Terracciano and Kashket, 1986), limiting phosphate or sulphate concentrations (Bahl, Andersch, and Gottschalk, 1982; Kanchanatawee and Maddox, 1990), levels of butyryl phosphate and butyryl-CoA (Boynton et al., 1994; Gottwald and Gottschalk, 1985; Zhao et al., 2005), ATP/ ADP ratio and NAD(P)H level (Grupe and Gottschalk, 1992) are involved. All these factors result in less negative supercoiling and thus relaxation of DNA. This change in topology could serve as a transcriptional sensor by allowing or restricting regulatory proteins to bind (Wang and Syvanen, 1992; Wong and Bennett, 1996; Ullmann and Durre, 1998; Ullmann et al., 1996). Several binding motifs have been identified in intergenic regions of sol and adc; three binding sites for the master regulator of sporulation Spo0A were found upstream of the adc promoter and another 0A box is located upstream of the sol promoter (Ravagnani et al., 2000). Gel retardation and targeted mutation experiments confirmed binding of phosphorylated Spo0A to these sites (Hollergschwandner, 2003; Ravagnani et al., 2000), thus proving that the regulatory networks of solventogenesis and sporulation are linked. Furthermore, a potential binding site for the catabolite control protein CcpA (cre sequence; Feustel, 2004; Nold, 2008) and three imperfect repeats (R1, R2 and R3; Thormann et al., 2002; Scotcher et al., 2003) were found upstream of the sol promoter. The region downstream of the sol promoter forms a very complex secondary structure with several predicted stem loops. This structure seems to be important for processing of the adhE mRNA (Thormann et al., 2002), but might also affect the adhE expression negatively (Scotcher et al, 2003).

The adhE transcript finally yields two different products, the mature bifunctional enzyme and the C-terminal alcohol dehydrogenase, probably due to a second translation start within the same mRNA (Thormann et al., 2002). While overproduction of AdhE in C. acetobutylicum leads to increased NAD+-dependent butyraldehyde/acetaldehyde dehydrogenase activity and NADH-dependent butanol/ethanol dehydrogenase activity (Nair et al, 1994a), only minor aldehyde dehydrogenase activity could be detected after purification (Thormann, 2001). Heterologous overexpression in Escherichia coli resulted in no enzyme activity at all (Lorenz, 1997; Nair et al., 1994a). In addition to AdhE, two butanol dehydrogenases BdhA and BdhB (sometimes also referred to as BdhI and BdhII) are present in C. acetobutylicum. Although these isoenzymes have a high identity, BdhB has a significantly better affinity to butyraldehyde than BdhA (Petersen et al., 1991; Walter et al., 1992). The respective genes are organized in monocistronic operons directly next to each other on the genome of C. acetobutylicum (Fig. 10.3). In C. beijerinckii, three different butanol dehydrogenases could be identified (Chen, 1995) in addition to the aldehyde dehydrogenase Ald (Toth et al., 1999; Yan and Chen, 1990).

Under special conditions of high NAD(P)H availability, a second bifunctional alcohol/aldehyde dehdrogenase AdhE2 is formed in C. acetobutylicum, representing the first case of an organism that possesses two such enzymes (Fontaine et al., 2002). Such conditions could be induced by addition of artificial electron carriers such as methyl viologen dyes (Rao and Mutharasan, 1986, 1987) or growth on reduced substrates such as glycerol (Fontaine et al., 2002) and lead to alcohologenic fermentations with butanol and increased levels of ethanol, but no acetone as products (Girbal et al., 1995). The gene adhE2 is also located on the megaplasmid of C. acetobutylicum and organized as a monocistronic operon approximately 47 kbps upstream of the sol operon. A homologous gene is present in the genome of C. beijerinckii as well.