For the production of butanol, two molecules of acetyl-CoA are condensed to
acetoacetyl-CoA by a thiolase (ThlA) in an initial step. Acetoacetyl-CoA is then
reduced to 3-hydroxypropionyl-CoA with NADH. After water is split off by a
crotonase (Crt), the formed crotonyl-CoA is then reduced to butyryl-CoA by an
electron-bifurcating butyryl-CoA dehydrogenase (Bcd). The Bcd of Clostridium
kluyveri is the prototype of a flavin-dependent, electron-bifurcating enzyme [95]. It
uses the exergonic reaction of the NADH-dependent reduction of crotonyl-CoA to
drive reduction of ferredoxin with NADH. Recently, we have demonstrated the
presence of an electron-bifurcating Bcd in E. limosum KIST612, an acetogen which
produces butyrate from CO [96]. Butyryl-CoA can be either reduced via
butyraldehyde directly to butanol by NADH-dependent aldehyde/alcohol dehydrogenases or first converted to butyrate by a phosphotransbuturylase (Ptb) and a
butyrate kinase (Buk), which is then reduced to butaraldehyde by an AOR again.
As mentioned above, C. carboxidivorans can also produce hexanol from syngas
which makes it also a promising alternative for hexanol production. Hexanol is an
interesting alternative to ethanol and is already used in the pharmaceutical and
cosmetic industry as well as in the texile industry [97]. In C. carboxidivorans, the
enzymes involved in hexanol formation have not been characterized yet but are
assumed to run in a similar fashion as the enzymes involved in the formation of the
intermediate butyryl-CoA. The formation of hexanol takes place via a reverse
b-oxidation. In a first step, acetyl-CoA is elongated with butyryl-CoA to
3-ketohexanoyl-CoA by a thiolase (Thl2), which is then reduced to
3-hydroxyhexanoyl-CoA and subsequently converted to hexanoyl-CoA.
Hexanoyl-CoA is then reduced by NADH-dependent aldehyde/alcohol dehydrogenases to hexanol, similar to the formation of butanol.
So far, butanol as well as hexanol titers from syngas are rather low due to a
higher demand of reducing equivalents such as NADH or NADPH in comparison to
other primary metabolites such as ethanol and a rather low solubility of H 2 , CO, and
CO 2 at moderate temperatures. Since C. carboxidivorans is not genetically accessible up to date, the main focus of research was on process optimization including
parameters such as medium composition, pH and temperature. The highest concentrations of hexanol (0.94 g/L) were achieved by increasing molybdenum (to
2 mg/L) and omitting copper in the defined medium at 37 °C [89]. One important
parameter for the production of these alcohols is the temperature. Generally, C.
carboxidivorans can produce alcohols within a range of 24 − 42 °C. Even though
the optimal growth temperature of C. carboxidivorans is at 37 °C, it is not as
beneficial to produce these alcohols at 37 °C. Optimal growth temperatures come
along with higher metabolic rates and thus higher production rates of acetate.
However, this leads to a fast accumulation of acetate in high concentrations
(>60 mmol/l) which inhibits solventogenesis. This inhibitory effect of solventogenesis, also known as “acid crash”, can be prevented by lowering the growth
temperature to 25 °C which leads to lower acetate concentration and therefore to an
increased yield of the produced alcohols [98].
Acetogenic Bacteria for Biotechnological Applications
121
acetoacetyl-CoA by a thiolase (ThlA) in an initial step. Acetoacetyl-CoA is then
reduced to 3-hydroxypropionyl-CoA with NADH. After water is split off by a
crotonase (Crt), the formed crotonyl-CoA is then reduced to butyryl-CoA by an
electron-bifurcating butyryl-CoA dehydrogenase (Bcd). The Bcd of Clostridium
kluyveri is the prototype of a flavin-dependent, electron-bifurcating enzyme [95]. It
uses the exergonic reaction of the NADH-dependent reduction of crotonyl-CoA to
drive reduction of ferredoxin with NADH. Recently, we have demonstrated the
presence of an electron-bifurcating Bcd in E. limosum KIST612, an acetogen which
produces butyrate from CO [96]. Butyryl-CoA can be either reduced via
butyraldehyde directly to butanol by NADH-dependent aldehyde/alcohol dehydrogenases or first converted to butyrate by a phosphotransbuturylase (Ptb) and a
butyrate kinase (Buk), which is then reduced to butaraldehyde by an AOR again.
As mentioned above, C. carboxidivorans can also produce hexanol from syngas
which makes it also a promising alternative for hexanol production. Hexanol is an
interesting alternative to ethanol and is already used in the pharmaceutical and
cosmetic industry as well as in the texile industry [97]. In C. carboxidivorans, the
enzymes involved in hexanol formation have not been characterized yet but are
assumed to run in a similar fashion as the enzymes involved in the formation of the
intermediate butyryl-CoA. The formation of hexanol takes place via a reverse
b-oxidation. In a first step, acetyl-CoA is elongated with butyryl-CoA to
3-ketohexanoyl-CoA by a thiolase (Thl2), which is then reduced to
3-hydroxyhexanoyl-CoA and subsequently converted to hexanoyl-CoA.
Hexanoyl-CoA is then reduced by NADH-dependent aldehyde/alcohol dehydrogenases to hexanol, similar to the formation of butanol.
So far, butanol as well as hexanol titers from syngas are rather low due to a
higher demand of reducing equivalents such as NADH or NADPH in comparison to
other primary metabolites such as ethanol and a rather low solubility of H 2 , CO, and
CO 2 at moderate temperatures. Since C. carboxidivorans is not genetically accessible up to date, the main focus of research was on process optimization including
parameters such as medium composition, pH and temperature. The highest concentrations of hexanol (0.94 g/L) were achieved by increasing molybdenum (to
2 mg/L) and omitting copper in the defined medium at 37 °C [89]. One important
parameter for the production of these alcohols is the temperature. Generally, C.
carboxidivorans can produce alcohols within a range of 24 − 42 °C. Even though
the optimal growth temperature of C. carboxidivorans is at 37 °C, it is not as
beneficial to produce these alcohols at 37 °C. Optimal growth temperatures come
along with higher metabolic rates and thus higher production rates of acetate.
However, this leads to a fast accumulation of acetate in high concentrations
(>60 mmol/l) which inhibits solventogenesis. This inhibitory effect of solventogenesis, also known as “acid crash”, can be prevented by lowering the growth
temperature to 25 °C which leads to lower acetate concentration and therefore to an
increased yield of the produced alcohols [98].
Acetogenic Bacteria for Biotechnological Applications
121