7 Engineering and Isolation Strategies for Efficient
and Robust N 2 O-Respiring Bacteria
In the light of the complexity of the N 2 O reduction systems described above, it is
quite clear that any attempt to engineer the effectiveness of NosZ activity or N 2 O
respiration needs to consider the respective cellular context and the physiology of
the host organism. In other words, one has to take into account that the activity of
any existing NosZ depends on other functional entities of the cell such as the
electron transport network, the machinery ensuring copper homoeostasis and the
defence systems against oxidative and nitrosative stress, caused by reactive oxygen
and nitrogen species, respectively.
The following properties of a prototypic ‘enhanced NRB’ appear desirable:
• A high specific cellular N 2 O turnover rate in natural and/or man-made
N 2 O-emitting environments and in the presence of N 2 O concentrations in the
lM or low mM range. To facilitate comparison, such activities should be given
in units such as lmol N 2 O min
−1 mg
−1 dry biomass or fmol N 2 O cell
−1 h
−1 .
• A low apparent K M value for N 2 O, for example, in the low lM range.
• Oxygen-resistant N 2 O reduction or fast reactivation of N 2 O reduction after
inhibition due to oxygen exposure. In this context, it is notable that it is unknown
so far whether oxygen-resistant N 2 O reduction is due to an oxygen-insensitive
NosZ enyzme or, more likely, whether the corresponding cells benefit from a
superior defence system against reactive oxygen species that effectively shields
NosZ from oxygen damage (see Sect. 2).
• N 2 O reduction capacity over a wide range of pH values
• Functional maintenance of N 2 O reduction over a wide range of copper concentrations, for example, due to the presence of efficient copper homoeostasis
and/or storage systems, copper chaperones and enzymic copper centre assembly
machineries.
• Efficient and fast electron transport systems delivering electrons to NosZ based
on an adaequate cellular architecture that might involve an N 2 O respirasome.
• Fast (and versatile) growth in cheap (minimal) media with high cell yields.
• Genetic accessibility allowing mutant construction and Metabolic Engineering.
Note that only few NRBs such as P. stutzeri, Bradyrhizobium sp. and W. succinogenes are genetically tractable.
Given this variety of constraints, the question whether a clade I or clade II
organism might be better suited as a chassis organism seems to be of secondary
importance. Furthermore, it appears to be an enormous task to accomplish
heterologous production of a complex metalloenzyme such as NosZ, let alone to
transfer functional N 2 O respiration to a non-N 2 O reducing host cell, especially to
cells of higher organisms such as plants. After all, an enzymatically active and
copper-containing NosZ from P. stutzeri ZoBell has been produced recently in
Escherichia coli upon co-production of the biosynthetic machinery for Cu Z centre
Mitigation of Laughing Gas Emissions …
203
and Robust N 2 O-Respiring Bacteria
In the light of the complexity of the N 2 O reduction systems described above, it is
quite clear that any attempt to engineer the effectiveness of NosZ activity or N 2 O
respiration needs to consider the respective cellular context and the physiology of
the host organism. In other words, one has to take into account that the activity of
any existing NosZ depends on other functional entities of the cell such as the
electron transport network, the machinery ensuring copper homoeostasis and the
defence systems against oxidative and nitrosative stress, caused by reactive oxygen
and nitrogen species, respectively.
The following properties of a prototypic ‘enhanced NRB’ appear desirable:
• A high specific cellular N 2 O turnover rate in natural and/or man-made
N 2 O-emitting environments and in the presence of N 2 O concentrations in the
lM or low mM range. To facilitate comparison, such activities should be given
in units such as lmol N 2 O min
−1 mg
−1 dry biomass or fmol N 2 O cell
−1 h
−1 .
• A low apparent K M value for N 2 O, for example, in the low lM range.
• Oxygen-resistant N 2 O reduction or fast reactivation of N 2 O reduction after
inhibition due to oxygen exposure. In this context, it is notable that it is unknown
so far whether oxygen-resistant N 2 O reduction is due to an oxygen-insensitive
NosZ enyzme or, more likely, whether the corresponding cells benefit from a
superior defence system against reactive oxygen species that effectively shields
NosZ from oxygen damage (see Sect. 2).
• N 2 O reduction capacity over a wide range of pH values
• Functional maintenance of N 2 O reduction over a wide range of copper concentrations, for example, due to the presence of efficient copper homoeostasis
and/or storage systems, copper chaperones and enzymic copper centre assembly
machineries.
• Efficient and fast electron transport systems delivering electrons to NosZ based
on an adaequate cellular architecture that might involve an N 2 O respirasome.
• Fast (and versatile) growth in cheap (minimal) media with high cell yields.
• Genetic accessibility allowing mutant construction and Metabolic Engineering.
Note that only few NRBs such as P. stutzeri, Bradyrhizobium sp. and W. succinogenes are genetically tractable.
Given this variety of constraints, the question whether a clade I or clade II
organism might be better suited as a chassis organism seems to be of secondary
importance. Furthermore, it appears to be an enormous task to accomplish
heterologous production of a complex metalloenzyme such as NosZ, let alone to
transfer functional N 2 O respiration to a non-N 2 O reducing host cell, especially to
cells of higher organisms such as plants. After all, an enzymatically active and
copper-containing NosZ from P. stutzeri ZoBell has been produced recently in
Escherichia coli upon co-production of the biosynthetic machinery for Cu Z centre
Mitigation of Laughing Gas Emissions …
203