Biology Reference
In-Depth Information
In the past several decades major advances have been made in our knowledge about the
diverse metabolic networks, and our ability to manipulate and engineer metabolic pathways
of microorganisms for the production of complex molecules previously impossible or
expensive to synthesize chemically. Metabolically engineered microorganisms are now used
for the synthesis of a large number of chemicals ranging from biofuels to complex polymers
and secondary metabolites such as drugs and anticancer compounds.
4
However, production
of many of these compounds poses a heavy metabolic burden for such engineered producer
organisms by depleting their ATP and NAD(P)H pools. Augmenting their energy pools by
introducing a functional light-energy capture and conversion mechanism has the potential
to reduce the energy strain on production hosts, especially during the synthesis of products
with high energy requirements such as fatty acids. Carbon fixation is another very desirable
process in an engineered microbe, especially during biofuel production, but it is also an
energetically expensive process. With the help of recent developments in synthetic biology,
genome-scale modifications of microbes are now becoming more practical. Consequently,
introduction of light-energy converting machinery into a nonphotosynthetic host does not
seem as impossible as it was even five years ago. In the following sections we will
summarize current progress and challenges in implementing light-energy capture and
conversion into microbial hosts.
INCORPORATION OF SIMPLE LIGHT-DRIVEN PROTON PUMPS
INTO ENGINEERED MICROORGANISMS
The simplest and best characterized of the currently known mechanisms for light-energy
conversion involves the rhodopsin protein family which is found in all kingdoms of life.
Rhodopsin is a
trans
-membrane protein containing a single, light-responsive retinal cofactor.
Depending on the host, rhodopsins function as light sensors, proton pumps, or ion pumps
(reviewed in
5
7
). Bacteriorhodopsin from the archaea
Halobacterium salinarium
was the first
light-driven proton pump discovered in the early 1970s.
8
To date, many archaeal
bacteriorhodopsins have been identified in a wide range of environments using
metagenomic sequencing techniques. Bacteriorhodopsin homologues appear to be
especially prevalent in the oceans, where resources are sparse and any boost in energy
generation can give an organism a significant competitive advantage. The first rhodopsin
homologue from a proteobacteria, proteorhodopsin, was isolated from a metagenomic
sample from the Sargasso Sea.
9
Recent work on light-driven proton pumping has centered
around proteorhodopsins as opposed to bacteriorhodopsin, as the proteobacterial
membrane proteins proved to be much easier to express and isolate from a wide range of
heterologous hosts.
9
Both bacteriorhodopsin and proteorhodopsin work by translocating a
proton from the cytoplasm into the periplasm during the light-induced isomerization of a
retinol cofactor.
7
Overall, this creates a very simple mechanism of establishing and
maintaining a proton gradient.
304
Being such a simple system, proteorhodopsin is an obvious candidate to add into a
recombinant host as a first step in engineering a heterologous light-energy conversion
system. Successful addition of proteorhodopsin to a new host requires not only proper
folding and transmembrane localization, but also the availability of retinal as cofactor.
Retinal can either be supplemented in the growth media or alternatively, a heterologous
retinal biosynthetic pathway can be engineered for endogenous cofactor synthesis. For
example, in
E. coli
, four additional gene products are needed to convert the isoprenoid
precursor farnesyl diphosphate into
-carotene, which is then cleaved by another
heterologous enzyme at the central 15,15
β
-double-bond to yield retinal.
10
'
Since the discovery and characterization of proteorhodopsin, several groups have attempted
to use it to supplement a heterologous host
s energy pool, though with somewhat limited
success.
11,12
There is evidence that expression of proteorhodopsin can aid in the
'
