Biology Reference
In-Depth Information
introduction into a recombinant host.
26
The core RCs, as well as the surrounding light-
harvesting complexes, have been thoroughly characterized. The RC in
R. sphaeroides
consists
of three core proteins, referred to as the M, L, and H subunits, which are encoded by
pufM
,
pufL
, and
puhH
, respectively. This core complex, with its bound Bchl and carotenoid
pigments, is surrounded by a light-harvesting (LH) superstructure formed by the LH1 and
LH2 protein complexes that contain accessory Bchl pigments (
Fig. 16.1B,C
).
27
After light strikes the reaction center, it excites a low-energy electron from one of the two
Bchla molecules (referred to as the
) located at the center of the type II RC
complex, the excited electron is transferred in the RC via two additional chlorophyll
pigments, and a quinone to a final quinone electron acceptor that exits the RC core upon
reduction by two electrons. Thus, two excitation reactions are necessary before a quinone
molecule is released from the RC complex (reviewed in
28
). Proton translocation across the
inner membrane is coupled to the decrease in the potential energy of the electrons during
each step of the cyclic electron. The established proton gradient can then be utilized by
proton-gradient coupled enzymes such as ATP synthase to regenerate the metabolic needs of
the cell.
12
Alternatively, reduced quinones can be used directly by enzymes such as NADH:
quinoneoxidoreductase to regenerate NADH reducing equivalents needed for CO
2
fixation
or other metabolically taxing reactions (
Fig. 16.2
).
2
In the noncyclic, type I RC mentioned
above, the high-energy electrons are placed on a ferrodoxin molecule instead of a quinone.
The ferrodoxin is subsequently released, and is used as an energy source to convert NAD
(P)
1
to NAD(P)H by the ferrodoxin-NAD reductase (
Fig. 16.2
).
2
Similarly, the electrons in
the special pair are regenerated from a c-type cytochrome, though this time the electrons
originate from a different donor, frequently a sulfur compound.
2
'
special pair
'
The extra energy boost generated by either the type I or type II RC would be ideally coupled
to an energy-expensive metabolic reaction such as the synthesis of long-chain fatty acids as
liquid transportation biofuels. Fatty acid synthesis utilizes a malonyl-CoA molecule as a
basic building block, and requires hydrolysis of ATP to create C
309
C bonds (reviewed in
4,29
).
If a host is engineered for overproduction of fatty acids, any potential boost to its total ATP
pool should prove beneficial to the final product yields. Not only are ATP molecules needed
during the production of long-chain fatty acids, but a number of NADPH molecules are also
used up in the process of making a saturated fatty acid. As discussed previously, NADPH
could be regenerated by the use of reduced quinones, or from ferredoxin molecules
generated during electron transfer from the reaction center (
Fig. 16.2
).
2,25
The addition of a more efficient system for light-energy conversion in an electricity-generating
bacterium like
S. oneidensis
should translate into a greatly increased current production.
Because the RC complex is able to directly generate reduced quinones, it should provide
S.
oneidensis
with the ability to send more electrons to the electrode. One issue that would need
to be circumvented in recombinant
S. oneidensis
is the cyclic transfer of electrons generated by
the RCs from purple bacteria. If electrons are transferred from the quinone pool to the
electrode, a system will need to be engineered to reduce the special pair in the RC. A potential
solution to compensate for the loss of the special pair electrons would be to engineer cells
with a noncyclic, type I, RC complex, and the protein machinery needed to regenerate special
pair electrons through the oxidation of inorganic sulfur or nitrogen.
Functional expression of bacterial RCs in a nonnative host presents a number of challenges. First,
while the core RC from
R. sphaeroides
consists of only three proteins, two of the core subunits, L
and M, are transmembrane proteins. It has been shown that in
R. sphaeroides
both of those
subunits are needed for the stability of the RC complex.
30
Furthermore, overexpression of
heterologous membrane proteins is problematic, often leading to toxicity issues. Successful
overexpression of just the core RC complex in a heterologous host such as
E. coli
will likely
require engineering of a strain that is able to better tolerate high levels of membrane proteins.
R. sphaeroides
solves this problem by creating a large amount of membrane space by using
