Directing biofuel production

November 27, 2013, Richland, Wash. – Scientists have charted
a significant signaling network in a tiny organism that's big in the world of
biofuels research. The findings about how a remarkably fast-growing organism
conducts its metabolic business bolster scientists' ability to create biofuels
using the hardy microbe Synechococcus, which turns sunlight into useful energy.

The team at the Department of Energy's Pacific Northwest
National Laboratory glimpsed key chemical events, known as redox reactions,
inside living cells of the organism. The publication in ACS Chemical Biology
marks the first time that redox activity, a very fast regulatory network
involved in all major aspects of a cell's operation, has been observed in
specific proteins within living cells.

The findings hone scientists' control over a common tool in
the biofuels toolbox. At a more basic level, the work gives researchers the
newfound ability to witness a basic biological process that occurs every moment
in everything from bacteria to people.

"Redox activity tells us where the action is going on
within a cell," said chemist Aaron Wright, the leader of the PNNL team
whose project was funded by DOE's Office of Science. "We've been able to
get a look at the redox system while it's still operating in a living cell,
without destroying the cell first. This allows us to tell who the players are
when the cells are engaged in the activity of our choice, like making
components for biofuels."

Redox activity is one of the most powerful tools an organism
has to sense and adapt to a changing environment; it's particularly active in
plants that must respond constantly to changing conditions, such as light and
dark.

The PNNL study was aimed at ferreting out proteins that are
potential redox players in the cyanobacterium Synechococcus. Cyanobacteria
absorb light energy from the sun and use it to convert carbon dioxide into food
and other molecules, while also giving off oxygen. Redox reactions play a role
in directing where the harvested energy goes.

Scientists believe the organism and its plant-like cousins,
including algae, were responsible for producing the first oxygen on Earth, more
than 2.5 billion years ago. It's a sure bet that you have inhaled oxygen
molecules produced by Synechococcus, which today contributes a significant
proportion of the oxygen available on Earth.

The organism is attractive to scientists for a number of
reasons. It's adept at converting carbon dioxide into other molecules, such as
fatty acids, that are of interest to energy researchers. Synechococcus is easy
for scientists to change and manipulate as they explore new ideas. And it grows
quickly, doubling in approximately two hours. A patch just two feet wide by
seven feet long – roughly the area of a typical dining room table – could
blossom into an area the size of a football field in just one day.

Biofuels makers and other scientists are trying to exploit
this ability to churn out quantities of materials that might serve as biofuel.
Synechococcus is also remarkably hardy, capable of tolerating the stress caused
by intense sunlight, which kills many other cyanobacteria. Redox reactions that
take place throughout the organism are at the core of this ability, and
understanding them gives scientists a treasured global view of how the cell
lives and responds to change.

Some researchers are working to get the bacteria itself to
create biofuel, growing an organism with more fatty acids that could be
converted to diesel fuel. Others, like Wright, are working to understand the
organism more completely, to direct the organism to create fuels using light
and carbon dioxide.

Wright's team found the signals by keeping the bacteria
hungry, then suddenly flooding it with food – a massive, immediate change in
environment. Within 30 seconds, the team detected redox activity, which changes
the way proteins operate by adding or subtracting electrons.

His team uncovered an extensive network of redox activity,
identifying 176 proteins that are sensitive to signaling in this manner. Before
this study, just 75 of those proteins were known to be part of a redox
signaling network. The scientists found that the system is involved in all the
major processes of a cell – which genes are turned on and off, for example, as
well as how the cell maintains its molecular machinery and converts energy into
fuel.

Central to the work are the chemical probes Wright developed
that are able to cross the cell membrane and get into the cytoplasm of the
cell. The probes flag redox events by binding to certain forms of the amino
acid cysteine, which is a known player in many of these interactions. Then the
probes and the interactions they flag are subjected to scrutiny at EMSL, the
DOE's Environmental Molecular Sciences Laboratory on the PNNL campus, where
instruments detect redox activity through various means, such as through
fluorescent imaging and mass spectrometry. The analysis tells scientists about
when and where within the cell redox activity occurred.

"Knowing the proteins that are sensitive to redox
signaling lets us know where to look as we test out new methods for working
with this organism," said Wright. "We can tinker with a specific
protein, for instance, and then watch the effects immediately.

"This is the type of information we really must have if
we want organisms like this to produce substances that make a difference, like
biofuels, chemicals or potential medicines," he added.