E. coli engineered to turn switchgrass into fuel

Nov. 30, 2011 – A milestone has been reached on the road to developing advanced biofuels
that can replace gasoline, diesel and jet fuels with a
domestically-produced clean, green, renewable alternative.

Researchers with the U.S. Department of Energy (DOE)’s Joint
BioEnergy Institute (JBEI) have engineered the first strains of
Escherichia coli bacteria that can digest switchgrass biomass and
synthesize its sugars into all three of those transportation fuels.
What’s more, the microbes are able to do this without any help from
enzyme additives.

“This work shows that we can reduce one of the most expensive parts
of the biofuel production process, the addition of enzymes to
depolymerize cellulose and hemicellulose into fermentable sugars,” says
Jay Keasling, CEO of JBEI and leader of this research. “This will enable
us to reduce fuel production costs by consolidating two steps –
depolymerizing cellulose and hemicellulose into sugars, and fermenting
the sugars into fuels – into a single step or one pot operation.”

Keasling, who also holds appointments with the Lawrence Berkeley
National Laboratory (Berkeley Lab) and the University of California (UC)
Berkley, is the corresponding author of a paper in the Proceedings of
the National Academy of Sciences (PNAS) that describes this work. The
paper is titled “Synthesis of three advanced biofuels from ionic
liquid-pretreated switchgrass using engineered Escherichia coli.”

Advanced biofuels made from the lignocellulosic biomass of non-food
crops and agricultural waste are widely believed to represent the best
source of renewable liquid transportation fuels. Unlike ethanol, which
in this country is produced from corn starch, these advanced biofuels
can replace gasoline on a gallon-for-gallon basis, and they can be used
in today’s engines and infrastructures. The biggest roadblock to an
advanced biofuels highway is bringing the cost of producing these fuels
down so that they are economically competitive.

Unlike the simple sugars in corn grain, the cellulose and
hemicellulose in plant biomass are difficult to extract in part because
they are embedded in a tough woody material called lignin. Once
extracted, these complex sugars must first be converted or hydrolyzed
into simple sugars and then synthesized into fuels. At JBEI, a DOE
Bioenergy Research Center led by Berkeley Lab, one approach has been to
pre-treat the biomass with an ionic liquid (molten salt) to dissolve it,
then engineer a single microorganism that can both digest the dissolved
biomass and produce hydrocarbons that have the properties of
petrochemical fuels.

“Our goal has been to put as much chemistry as we can into microbes,”
Keasling says. “For advanced biofuels this requires a microbe with
pathways for hydrocarbon production and the biomass-degrading capacity
to secrete enzymes that efficiently hydrolyze cellulose and
hemicellulose. We’ve now been able to engineer strains of Escherichia
coli that can utilize both the cellulose and hemicellulose fractions of
switchgrass that’s been pre-treated with ionic liquids.”

E. coli bacteria normally cannot grow on switchgrass, but JBEI
researchers engineered strains of the bacteria to express several
enzymes that enable them to digest cellulose and hemicellulose and use
one or the other for growth. These cellulolytic and hemicellulolytic
strains of E. coli, which can be combined as co-cultures on a sample of
switchgrass, were further engineered with three metabolic pathways that
enabled the E. coli to produce fuel substitute or precursor molecules
suitable for gasoline, diesel and jet engines. While this is not the
first demonstration of E. coli producing gasoline and diesel from
sugars, it is the first demonstration of E. coli producing all three
forms of transportation fuels. Furthermore, it was done using
switchgrass, which is among the most highly touted of the potential
feedstocks for advanced biofuels.

Gregory Bokinsky, a post-doctoral researcher with JBEI’s synthetic
biology group and lead author of the PNAS paper, explains that the
pre-treatment of the switchgrass with ionic liquids was essential to
this demonstration.

“The magic is in the ionic liquid pre-treatment,” Bokinsky says. “If
properly optimized, I suspect you could use ionic liquid pre-treatment
on any plant biomass and make it readily digestible by microbes. For us
it was the combination of biomass from the ionic liquid pretreatment
with the engineered E. coli that enabled our success.”

The JBEI researchers also attribute the success of this work to the
“unparalleled genetic and metabolic tractability” of E. coli, which over
the years has been engineered to produce a wide range of chemical
products. However, the researchers believe that the techniques used in
this demonstration should also be readily adapted to other microbes.
This would open the door to the production of advanced biofuels from
lignocellulosic feedstocks that are ecologically and economically
appropriate to grow and harvest anywhere in the world. For the JBEI
researchers, however, the next step is to increase the yields of the
fuels they can synthesize from switchgrass.

“We already have hydrocarbon fuel production pathways that give far
better yields than what we obtained with this demonstration,” says
Bokinsky. “And these other pathways are very likely to be compatible
with the biomass-consumption pathways we’ve engineered into our E. coli.
However, we need to find enzymes that can both digest more of the ionic
liquid pre-treated biomass and be secreted by E coli. We also need to
work on optimizing the ionic liquid pre-treatment steps to yield biomass
that is even easier for the microbes to digest.”

Co-authoring the PNAS paper with Keasling and Bokinsky were Pamela
Peralta-Yahya, Anthe George, Bradley Holmes, Eric Steen, Jeffrey
Dietrich, Taek Soon Lee, Danielle Tullman-Ercek, Christopher Voigt and
Blake Simmons.

This research was supported in part by the DOE Office of Science and a UC Discovery Grant.

JBEI is one of three Bioenergy Research Centers established by the
DOE’s Office of Science in 2007. It is a scientific partnership led by
Berkeley Lab and includes the Sandia National Laboratories, the
University of California campuses of Berkeley and Davis, the Carnegie
Institution for Science, and the Lawrence Livermore National Laboratory.
DOE’s Bioenergy Research Centers support multidisciplinary,
multi-institutional research teams pursuing the fundamental scientific
breakthroughs needed to make production of cellulosic biofuels, or
biofuels from nonfood plant fiber, cost-effective on a national scale.

Lawrence Berkeley National Laboratory addresses the world’s most
urgent scientific challenges by advancing sustainable energy, protecting
human health, creating new materials, and revealing the origin and fate
of the universe. Founded in 1931, Berkeley Lab’s scientific expertise
has been recognized with 13 Nobel prizes. The University of California
manages Berkeley Lab for the U.S. Department of Energy’s Office of
Science. For more, visit www.lbl.gov.

DOE’s Office of Science is the single largest supporter of basic
research in the physical sciences in the Unites States, and is working
to address some of the most pressing challenges of our time. For more
information, please visit the Office of Science website at science.energy.gov.

For more about the Joint BioEnergy Institute (JBEI), visit the Website at www.jbei.org