Acetyl-CoA generated via the Wood-Ljungdahl pathway serves as key intermediate for syn­thesis of cell mass as well as products. All acetogens are described to produce acetate, in or­der to gain energy via SLP to compensate for the energy invested in activating formate in the Western branch of the reductive acetyl-CoA pathway. Acetate and ATP are formed via acetyl-phosphate through the successive actions of Pta and Ack. pta and ack are arranged in the same operon and they were reported to be constitutively expressed [100]. With CO2 and H2 as substrate, only acetate has been observed as major product [44], with minor amounts of ethanol produced in rare cases with C. ljungdahlii [101], C. autoethanogenum [53], or "Moor — ella sp." [102, 103]. Using the more reduced substrate CO, production of a range of other products have been reported, such as ethanol, butanol, butyrate, 2,3-butanediol [104], and lactate (Figure 4.) [105]. From a biofuel perspective, ethanol and butanol are of particular in­terest. Ethanol and butanol have even been described as the main fermentation products over acetate in some acetogens under specific conditions. Ethanol producers include C. ljungdahlii [62, 63], C. autoethanogenum [53], "C. ragsdalei" ("Clostridium strain P11") [106, 107], "Moorella sp." [102, 103], Alkalibaculum bacchii [44], C. carboxidivorans ("Clostridium strain P7") [54, 55], and B. methylotrophicum [49, 108]. The latter two have also been descri­bed to produce butanol.

Due to historical roles in ABE fermentation, organisms like C. acetobutylicum, C. beijerinckii, C. saccharobutylicum, and C. saccharoperbutylacetonicum have been much more extensively characterized than acetogenic Clostridia [95]. Since C. acetobutylicum was the first Clostridium to be fully sequenced [109] and it remains the most commonly used species for industrial production of solvents to date [110], it provides a model for study of solventogenesis. Al­though sugar — and starch-utilizing ABE Clostridia and acetogens exhibit clear distinctions in substrate utilization and thus metabolism, they share some similarities in the biochemical pathway and genetic organization of product synthesis and can be used as model for com­parison. Structure of key genes and operons (except for the absence of acetone biosynthetic genes) have been found to be very similar in sequenced acetogen C. carboxidivorans [54], and in respect of acetate and ethanol genes to some extent also in C. ljungdahlii [62]. For instance, the operon structure of pta-ack, ptb-buk and the bcs cluster of acetogen C. carboxidivorans are highly similar to starch-utilizing C. acetobutylicum and C. beijerinckii [54, 109] (Figure 5). Due to these reasons, solventogenic genes from starch-utilizing Clostridia are ideal targets for heterologous expression in acetogens for improvement of product yield and expansion of product range.

(A) c. carboxidivorans, C. Ijungdahlii, C. autoethanogenum, C. acetobutylicum, C. beijerinckii

(В) C. carboxidivorans, C. acetobutylicum, C. beijerinckii

Similar to sugar — and starch-utilizing ABE Clostridia, acetogens such as C. carboxidivorans [111, 112], C. Ijungdahlii [113], and C. autoethanogenum [27] also typically undergo biphasic fermentation under autotrophic conditions. The first phase involves the production of car­boxylic acids (acidogenic), H2 and CO2 during exponential growth. This is followed by the solventogenic phase in which part of the produced acids are reassimilated or reduced in­to solvents, which usually occurs during stationary growth phase [114]. This shift from acidogenesis to solventogenesis is of industrial importance and several transcriptional analysis on C. acetobutylicum [100, 115], and C. beijerinckii [116] have been performed to shed light on this process. In both organisms, the onset of solventogenesis coincides with an increase in expression of master sporulation/solventogenesis regulator gene spo0A, sol — ventogenic genes such as ald, ctfA-ctfB, and adc, as well as down-regulation of chemotaxis/ motility genes [100, 115, 116]. Physiologically, the signals that induce solventogenesis were hypothesized to involve temperature, low pH, high concentrations of undissociated acetic and butyric acids, limiting concentrations of sulphate or phosphate, ATP/ADP ratio and/or NAD(P)H levels [117].

For Clostridia such as acetogen C. carboxidivorans [54], which harbour the genes thiolase (thlA), 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase (crt) and butyryl-CoA dehy­drogenase (bcd), the two carbon acetyl-CoA can be converted to four carbon butyryl-CoA [95]. ThlA compete with the activities of Pta, Ald (aldehyde dehydrogenase), and PFOR to condense two acetyl-CoA into one acetoacetyl-CoA, and plays a key role in regulating the C2:C4 acid ratio [110, 118]. Since the formation of acetate yields twice as much ATP per mole of acetyl-CoA relative to butyrate formation, thiolase activity indirectly affects ATP yield [118]. Under physiological conditions, Crt catalyzes dehydration of p-hydroxybutyryl-CoA to crotonyl-CoA [119]. Bcd was shown to require a pair of electron transfer flavoproteins (Et­fA and EtfB) to convert crotonyl-CoA to butyryl-CoA [120]. Furthermore, the Bcd was dem­onstrated to form a stable complex with EtfA and EtfB, and they were shown to couple the reduction of crotonyl-CoA to butyryl-CoA with concomitant generation of reduced ferre — doxins, which can be used for energy conservation via Rnf complex [94, 119]. Subsequent actions of phosphotransbutyrylase (ptb) and butyrate kinase (buk) then generate ATP and butyrate from butyryl-CoA [118].

Under low extracellular pH of 4-4.5, the secreted undissociated acetic acid (pKa 4.79) and/or butyric acid (pXa 4.82) diffuse back into cell cytoplasm and then dissociate into the respec­tive salts and protons because of the more alkaline intracellular conditions. Without further interventions, the result of this is abolishment of the proton gradient and inevitable cell death [95]. The conversion of acetate and butyrate into solvents increase the pH, thus pro­vide some time for the organism to sporulate and secure long term survival. However, the solvents produced are toxic because they increase membrane fluidity and disrupt critical membrane-associated functions such as ATP synthesis, glucose uptake and other transport processes [114, 121]. In C. acetobutylicum, it has been demonstrated that the addition of 7-13 g/l of butanol, or up to 40 g/l of acetone and ethanol resulted in 50% growth inhibition [122]. The bacterium is likely to experience a different cytotoxic effect from endogenously pro­duced solvents because the organism has time to adapt to increasing amount of solvents.

The reassimilation of acetate and butyrate into the respective acyl-CoA and acetoacetate is catalyzed by acetoacetyl-CoA:acetate/butyrate CoA transferase (CtfA and CtfB) [110, 117, 118]. Acetoacetate is deconstructed by acetoacetate decarboxylase (Adc) into acetone and CO2. This enzyme is missing in acetogenic C. carboxidivorans compared to the ABE strains [54, 123]. Some ABE strains such as C. beijerinckii NRRL B593 also possess a primary/secon- dary alcohol dehydrogenase that converts acetone to isopropanol [124]. In acetogenic "C. ragsdalei", reduction of acetone to isopropanol was also observed although the mechanism of this reduction is as yet unknown [124, 125]. Again, C. carboxidivorans lacks this activity [125]. The recycled acetyl-CoA and butyryl-CoA can be converted to ethanol and butanol through the actions of coenzyme A-acylating aldehyde dehydrogenase (Ald) and alcohol de­hydrogenase (Adh) [110, 118]. Ald converts acyl-CoA into aldehydes, and the enzyme has been purified from C. beijerinckii NRRL B593 and was shown to be NADH-specific, exhibit higher affinity with butyraldehyde than acetaldehyde, but possess no Adh activity [126]. In C. ljungdahlii, two variants of aldehyde:ferredoxin oxidoreductases (AOR) are present in the genome, and they are hypothesized to couple reduced ferredoxin from CO oxidation via the CODH (see above) to perform the reversible reduction of acetate into acetaldehyde, which can be further reduced into ethanol [62].

The final step of solventogenesis utilizes Adh to reduce acetaldehyde and butyraldehyde in­to ethanol and butanol, respectively. For ethanol synthesis, transposon mutagenesis and en­zymatic assay in C. acetobutylicum showed the involvement of a specific Ald that does not interact with butyryl-CoA, and a NAD(P)H-dependent Adh [127, 128]. The production of butanol by C. acetobutylicum is mainly due to the action of butanol dehydrogenase A and B (BdhA and BdhB), and bifunctional butyraldehyde/butanol dehydrogenase 1 and 2 (AdhE1 and AdhE2) [95]. In C. carboxidivorans [54] and C. ljungdahlii [62] both adhE1 and adhE2 are arranged in tandem and separated by a 200bp gap which contains a putative terminator [62,

111]. This is likely the result of gene duplication [62]. qRT-PCR analysis from C. carboxidivor — ans fed with syngas showed that the two adhE showed differential expression, and the more abundant adhE2 was significantly upregulated over 1000 fold in a time span that coincided with the greatest rate of butanol production [111].

Pyruvate is a central molecule for anabolism and it is predominantly generated from glycol­ysis during heterotrophic growth. But under autotrophic growth, this four carbon molecule can be synthesized by PFOR and potentially also the pyruvate-formate lyase (PFL). Two variants of PFOR were reported in C. autoethanogenum, and transcriptional analysis showed that they were differentially expressed when grown using industrial waste gases (containing CO, CO2 and H2) [104]. Unlike PFL from most other microorganisms that only catalyze the lysis of pyruvate into formate and acetyl-CoA, clostridial PFL (C. kluyveri, C. butylicum, and C. butyricum) were reported to readily catalyze the reverse reaction (i. e. pyruvate formation) [129]. Apart from roles in anabolism, pyruvate is also a precursor to other products such as lactic acid and 2,3-butanediol. Small amounts of lactic acid are converted from pyruvate in acetogens, a reaction which is catalyzed by lactate dehydrogenase (Ldh) [104, 118]. Recently, Kopke et al. (2011) reported the production of 2mM 2,3-butanediol from acetogenic bacteria (C. autoethanogenum, C. ljungdahlii, and C. ragsdalei) using industrial waste gases (containing CO, CO2 and H2) as feedstock [104]. Pyruvate is first converted into a-acetolactate by the en­zyme acetolactate synthase, followed by acetolactate decarboxylase which split acetolactate into acetoin and CO2, before a final reduction of acetoin into 2,3-butanediol by 2,3-butane — diol dehydrogenase [104] (Figure 4).