Biology Reference
In-Depth Information
sunlight would be needed for growth of
R. sphaeroides
, and given the large membrane space
generated during photosynthetic growth, it could be an ideal host for production of
hydrophobic compounds or membrane proteins.
COMBINING LIGHT-ENERGY CONVERSION AND CO
2
FIXATION
Carbon fixation is an integral part of photosynthesis, and something that must be taken
into account when engineering photosynthesis into a new host. The addition of carbon
fixation into a nonnative host can present numerous advantages for an engineered system.
Carbon fixation can be used to reduce the host
s dependence on organic material as a
carbon source and allow for a wider range of growth conditions. The prospect of utilizing
atmospheric CO
2
during growth is especially desirable for biofuel production processes, as
this directly generates carbon-neutral fuels. Recent advances in our understanding and
engineering of protein-based bacterial microcompartments, which serve to increase the
efficiency of metabolic pathways
'
enable new
approaches for the introduction of efficient carbon fixation in heterologous hosts.
39
42
In
fact, the functional expression of
H. neapolitanus
RuBisCO, inside of carboxysome, has very
recently been demonstrated in
E. coli
.
43
Efforts to increase the efficiency of recombinant
RuBisCO, coupled to optimized carboxysome expression in
E. coli
, could produce cells
capable of efficiently coupling carbon fixation to a biosynthetic process.
44
Reconstructing
the carboxysome along with carbon fixation using RuBisCO is appealing, because its
catalytic activity is inherently poor (reviewed in
45
). Native RuBisCo has turnover rates on
the order of several reactions per second, depending on environmental conditions; RuBisCO
can also incorporate O
2
instead of CO
2
during the reaction cycle to generate a molecule of
2-phosphoglycolate. 2-Phosphoglycolate is of little use to the host and must be
enzymatically converted to a useful metabolite.
45
However, by localizing RuBisCo together
with a carbonic anhydrase to carboxysomes, cells are able to substantially increase local CO
2
concentrations by transporting inorganic bicarbonate from the cytoplasm into
compartments where it is converted into CO
2
, which helps to overcome some of RuBisCo
including CO
2
fixation in carboxysomes
311
s
inefficiencies. Any attempt to engineer efficient carbon fixation into a novel host must take
into account these limitations.
'
Not all organisms use RuBisCo to fix CO
2
, other strategies for carbon fixation include the
reductive TCA cycle or the 3-hydroxypropionate cycle.
46,47
Both of those cycles have
interesting biotechnology implications if they were engineered into a heterologous host.
Driving the regular TCA cycle in reverse yields the reductive TCA cycle, and the majority of
reactions in the TCA cycle are reversible. Only three key enzymes need to be replaced to
operate the TCA cycle in reverse and yield the reductive TCA cycle.
46
Carbon fixed in this
manner would enter cell metabolism as acetyl-CoA, or with the addition of another CO
2
molecule, pyruvate, and would be easily integrated into cellular metabolism. The 3-
hydroxypropionate cycle fixes CO
2
by combining it with an acetyl-CoA molecule, making
malonyl-CoA, followed by reduction and elongation using a second molecule of CO
2
.
46
This form of carbon fixation is especially useful in hosts engineered to produce biofuels
or biopolymers derived from fatty acids, because malonyl-CoA is a basic building block
for those compounds. The first step, addition of CO
2
to acetyl-CoA, is catalyzed by
acetyl-CoA carboxylase in a reaction that requires hydrolysis of ATP.
46
As we have
previously discussed, ATP can be regenerated using ATP-synthase and the proton gradient
established by either proteorhodopsin or a bacterial RC. If the final goal is to simply
boost the availability of malonyl-CoA, while consuming CO
2
, then the addition of
acetyl-CoA carboxylase to the host should be sufficient. Subsequent enzymes of the
3-hydroxypropionate cycle transform malonyl-CoA into an assortment of small, branched
biosynthetic precursors including 4-hydroxybutyrate, a feedstock for biopolymer
production.
47
