Strains of E. coli bacteria were engineered to digest switchgrass biomass and synthesize its sugars into gasoline, diesel, and jet fuel. Image: Lawrence Berkeley National Laboratory |
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.
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“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.”
