The WLP enables carbon fixation and is the only CO 2 fixing pathway that allows
for energy conservation [17, 18]. During heterotrophic growth with sugars as
carbon and energy source, glycolysis is coupled to the WLP via a pyruvate:ferredoxin oxidoreductase (PFOR) leading to conversion of pyruvate to acetyl-CoA. The
resulting CO 2 of this reaction is then reduced in the WLP with reducing equivalents
formed during glycolysis to produce additional acetyl-CoA [4]. The WLP is also
well suited for the conversion of other C1 substrates such as CO, formate or
methanol that enter the WLP by different routes. It consists of a methyl branch and
a carbonyl branch serving for the formation of the central intermediate acetyl-CoA,
starting from the reduction of one molecule of CO 2 each (Fig. 1). In the methyl
branch, CO 2 is converted in several reactions to methyl-THF (tetrahydrofolate). In
an initial step, CO 2 is reduced to formate. In most acetogens, this reaction is
catalyzed by a formate dehydrogenase (FDH) with NADPH as electron donor [19–
21], whereas in A. woodii, the reaction is catalyzed by recently discovered
hydrogen-dependent CO 2 reductase (HDCR), where the electrons are derived
directly from molecular hydrogen [15]. Formate is then bound, in a reaction driven
by ATP hydrolysis, to the C1 carrier THF. Water is then split off from formyl-THF,
and methenyl-THF is subsequently reduced to methenyl-THF by a methenyl-THF
cyclohydrolase. The electron donor for this reaction is either NADH or NADPH
[22–24]. In a second reduction step, the THF-bound methenyl group is reduced via
methylene-THF to methyl-THF. The electron donor for the second reduction step,
catalyzed by a methylene-THF reductase, varies among different acetogens. In
some acetogens, NADH serves as electron donor [25], whereas in others, the
electrons are derived via reduced ferredoxin [26]. The final step is transfer of the
methyl group onto a corrinoid/FeS protein (CoFeSP) by a methyl transferase.
In the carbonyl branch of the WLP, another CO 2 is reduced to CO by the
bifunctional CODH/ACS in a ferredoxin-dependent reaction. In a second reaction
step, the methyl group of methyl-THF is condensed with CO of the carbonyl branch
and coenzyme A (CoA) by the CODH/ACS to the central intermediate of the WLP,
acetyl-CoA. The acetyl-CoA formed is then either channeled into the anabolic
metabolism to provide the cell with organic material for biomass production or
converted to acetate by a phosphotransacetylase and an acetate kinase. This last
reaction step leads to the formation of one mol ATP per mol acetate. Since one mol
ATP was also invested in the methyl branch to activate formate to formyl-THF, the
WLP does not lead to a net gain of ATP. Therefore, the WLP is coupled to energy
conservation by a chemiosmotic mechanism [2, 18]. Every acetogen examined to
date uses reduced ferredoxin as the electron donor for an ion-translocating membrane protein complex, and acetogens can have either an Fd:NAD
+ oxidoreductase
(Rnf) [27] or an Fd:H
+ oxidoreductase (Ech) complex [28] for generation of an ion
motive force [18]. In both cases, the ion gradient can be either an H
+ or an Na
+
gradient. The electrochemical ion gradient thus established is then used by a
membrane bound, H
+
- or Na
+ -translocating ATP synthase [29, 30]. For the
reduction of ferredoxin with H 2 as reductant during autotrophic growth, acetogens
may employ different enzymes for redox balancing [18]. One possibility of providing the reducing equivalents is by an electron-bifurcating hydrogenase.
Acetogenic Bacteria for Biotechnological Applications
111
for energy conservation [17, 18]. During heterotrophic growth with sugars as
carbon and energy source, glycolysis is coupled to the WLP via a pyruvate:ferredoxin oxidoreductase (PFOR) leading to conversion of pyruvate to acetyl-CoA. The
resulting CO 2 of this reaction is then reduced in the WLP with reducing equivalents
formed during glycolysis to produce additional acetyl-CoA [4]. The WLP is also
well suited for the conversion of other C1 substrates such as CO, formate or
methanol that enter the WLP by different routes. It consists of a methyl branch and
a carbonyl branch serving for the formation of the central intermediate acetyl-CoA,
starting from the reduction of one molecule of CO 2 each (Fig. 1). In the methyl
branch, CO 2 is converted in several reactions to methyl-THF (tetrahydrofolate). In
an initial step, CO 2 is reduced to formate. In most acetogens, this reaction is
catalyzed by a formate dehydrogenase (FDH) with NADPH as electron donor [19–
21], whereas in A. woodii, the reaction is catalyzed by recently discovered
hydrogen-dependent CO 2 reductase (HDCR), where the electrons are derived
directly from molecular hydrogen [15]. Formate is then bound, in a reaction driven
by ATP hydrolysis, to the C1 carrier THF. Water is then split off from formyl-THF,
and methenyl-THF is subsequently reduced to methenyl-THF by a methenyl-THF
cyclohydrolase. The electron donor for this reaction is either NADH or NADPH
[22–24]. In a second reduction step, the THF-bound methenyl group is reduced via
methylene-THF to methyl-THF. The electron donor for the second reduction step,
catalyzed by a methylene-THF reductase, varies among different acetogens. In
some acetogens, NADH serves as electron donor [25], whereas in others, the
electrons are derived via reduced ferredoxin [26]. The final step is transfer of the
methyl group onto a corrinoid/FeS protein (CoFeSP) by a methyl transferase.
In the carbonyl branch of the WLP, another CO 2 is reduced to CO by the
bifunctional CODH/ACS in a ferredoxin-dependent reaction. In a second reaction
step, the methyl group of methyl-THF is condensed with CO of the carbonyl branch
and coenzyme A (CoA) by the CODH/ACS to the central intermediate of the WLP,
acetyl-CoA. The acetyl-CoA formed is then either channeled into the anabolic
metabolism to provide the cell with organic material for biomass production or
converted to acetate by a phosphotransacetylase and an acetate kinase. This last
reaction step leads to the formation of one mol ATP per mol acetate. Since one mol
ATP was also invested in the methyl branch to activate formate to formyl-THF, the
WLP does not lead to a net gain of ATP. Therefore, the WLP is coupled to energy
conservation by a chemiosmotic mechanism [2, 18]. Every acetogen examined to
date uses reduced ferredoxin as the electron donor for an ion-translocating membrane protein complex, and acetogens can have either an Fd:NAD
+ oxidoreductase
(Rnf) [27] or an Fd:H
+ oxidoreductase (Ech) complex [28] for generation of an ion
motive force [18]. In both cases, the ion gradient can be either an H
+ or an Na
+
gradient. The electrochemical ion gradient thus established is then used by a
membrane bound, H
+
- or Na
+ -translocating ATP synthase [29, 30]. For the
reduction of ferredoxin with H 2 as reductant during autotrophic growth, acetogens
may employ different enzymes for redox balancing [18]. One possibility of providing the reducing equivalents is by an electron-bifurcating hydrogenase.
Acetogenic Bacteria for Biotechnological Applications
111