Biology Reference
In-Depth Information
membrane invaginations. A similar tactic could be applied to a recombinant host. Unfortunately,
the mechanism by which
R. sphaeroides
creates membrane invaginations is poorly-understood.
There are
E. coli
strains, such as C43(DE3),
31
that have more membrane space and are used for
theoverexpressionofsomemembraneproteins, but it does not solve toxicity problems
associated with overexpression for all membrane proteins. In addition to the reaction center core
complex, the peripheral light-harvesting complexes should ideally also be added to an
engineered host for maximum light-capture efficiency. In
R. sphaeroides
, these are encoded by at
least four more genes that express the alpha and beta peptides of the LH1 and LH2 complexes. It
is likely that other genes, such as
pufX
, will need to be coexpressed as well to facilitate proper
super complex formation and function.
32
Lastly, bacteriochlorophyll and carotenoid pigments need to be introduced into a
nonphotosynthetic host either via exogenous addition or by engineering of their
biosynthetic pathways. Biosynthesis of porphyrin-derived Bchl a pigments in
E. coli
requires
the assembly of at least 10 different enzymatic steps. In previous research, we have shown
that overexpression of heme biosynthetic genes leads to accumulation of significant
quantities of porphyrins in
E. coli
.
33,34
In subsequent work, we demonstrated that
expression of the three-subunit Mg
chelatase complex in
E. coli
introduces magnesium into
protoporphyrin IX as the first committed step of (bacterio)chlorophyll biosynthesis.
Extension with the next enzyme in the Bchl pathway produced Mg-protoporphyrin IX
monomethylester in
E. coli
.
35
The remaining enzymes required for Bchla synthesis in
Rhodobacter
species have been elucidated using gene deletions; however, not all enzyme
activities have been confirmed via biochemical methods.
3
One key enzyme that has yet to be biochemically characterized is the anaerobic
Mg-protoporphyrin IX monomethylestercyclase (BchE). This enzyme is responsible for the
introduction of the characteristic fifth ring of (bacterio)chlorophylls.
36
The same reaction
has also been proposed to be carried out under aerobic conditions by AcsF. Very little is
known about either of the two enzymes, except that strains with genomic deletions of those
genes accumulate Mg-protoporphyrin IX monomethylester.
36
BchE is predicted to have a
Fe
310
S cluster and is expressed under anaerobic conditions, so any host cell must have the
machinery for proper Fe
S cluster maturation. If the host is not inherently competent at
Fe
S protein maturation, a suitable maturation pathway would have to be engineered into
the host. Even less is known about AcsF, except that it likely requires the presence of at least
one more protein in order to be active.
36
Carotenoid pigments serve as accessory pigments in light-harvesting complexes, and in the
RC complex. Many different carotenoid biosynthetic pathways have been successfully
engineered into
E. coli
, including pathways for the synthesis of the acyclic carotenoid present
in
Rhodobacter
reaction centers.
37,38
Although these pigments are not immediately needed to
show function of an engineered RC complex, they can be easily produced in a recombinant
host to provide photoprotection to the photosystem.
The primary focus has so far been on possibilities and challenges of taking a light-capturing
system and reconstituting it in a nonphotosynthetic host. The primary assumption has been
that the nonphotosynthetic host is well-studied, with a good understanding of its metabolic
processes and, most importantly, is amenable to genetic engineering. A lack of a good set of
molecular biology tools for the majority of photosynthetic organisms prevents the
introduction of complex metabolic features required for the development of industrial
production processes for chemicals or biofuels. Well-studied photosynthetic organisms, such
as
R. sphaeroides
or some cyanobacteria, do have some genetic tools presently available, but
they pale in comparison to what is possible in the well-studied biotechnology workhorse
E. coli
. Once we generate a comprehensive genetic toolkit, as well as thorough
understanding of metabolic flux and signaling pathways in
R. sphaeroides
, it could become a
very attractive host for industrial use. Being naturally photosynthetic, only water, CO
2
,and
