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The recent discovery that bisabolane, a member of
the terpene class of chemical compounds used in fragrances and flavorings,
holds high promise as a biosynthetic alternative to D2 diesel fuel has
generated keen interest in the green energy community and the trucking
industry. Now a second team of researchers with the U.S. Department of Energy
(DOE)’s Joint BioEnergy Institute (JBEI) has determined the 3D crystal
structure of a protein that is key to boosting the microbial-based production
of bisabolane as an advanced biofuel.
The JBEI research team, led by bioengineers Paul
Adams and Jay Keasling, solved the protein crystal structure of an enzyme in
the Grand fir (Abies grandis) that synthesizes bisabolene, the
immediate terpene precursor to bisabolane. The performance of this enzyme—the
Abies grandis ?-bisabolene synthase (AgBIS)—when engineered into microbes, has
resulted in a bottleneck that hampers the conversion by the microbes of simple
sugars into bisabolene.
“Our high-resolution structure of AgBIS should make
it possible to design changes in the enzyme that will enable microbes to make
bisabolene faster,” says Adams, a leading
authority on X-ray crystallography. “It should also enable us to engineer out
inhibition effects that slow throughput, and perhaps also engineer the enzyme
to produce other kinds of fuels similar to bisabolane.”
Adams, who heads JBEI’s Technologies Division, is
the corresponding author of a paper describing this work in Structure.
The paper is titled “Structure of a Three-Domain Sesquiterpene Synthase: A
Prospective Target for Advanced Biofuels Production.” Co-authoring it with
Adams and Keasling were Ryan McAndrew, Pamela Peralta-Yahya, Andy DeGiovanni,
Jose Pereira, and Masood Hadi.
JBEI is one of three DOE Bioenergy Research Centers
established by DOE’s Office of Science to advance the technology for the
commercial production of advanced biofuels. It is a multi-institutional
partnership led by the Lawrence Berkeley National Laboratory (Berkeley Lab) and
headquartered in Emeryville,
Calif.
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This past fall, JBEI researchers identified
bisabolane as a potential new advanced biofuel that could replace D2 diesel,
today’s standard fuel for diesel engines, with a clean, green, renewable
alternative that’s produced in the United States. Using the tools of synthetic
biology, the researchers engineered strains of bacteria and yeast to produce
bisabolene from simple sugars, which was then hydrogenated into bisabolane.
While showing much promise, the yields of bisabolene have to be improved for
microbial-based production of bisabolane fuel to be commercially viable.
“The inefficient terpene synthase enzyme is one of
the bottlenecks in the metabolic pathway used by the engineered microbes,” says
Peralta-Yahya, a lead member of the earlier JBEI team as well as the current
team. “Knowing the AgBIS crystal structure will guide us in engineering it for
improved catalytic efficiency and stability, which should bring our bisabolene
yields closer to economic competitiveness.”
Peralta-Yahya and her colleagues determined that
the AgBIS enzyme consists of three helical domains, the first three-domain
structure ever found in a synthase of sesquiterpenes—terpene compounds that
contain 15 carbon atoms. The discovery of this unique structure holds
importance on several fronts, as co-lead author of the Structure paper
McAndrew explains.
“That we found the structure of AgBIS to be more
similar to diterpene (two carbon terpene compounds) synthases not only provides
us with insight into the function of these less well characterized enzymes, it
also provides us with clues to the evolutionary heritage as the archetypal
three-domain terpenoid synthases became two-domain sesquiterpene synthases in
plants.
“Furthering our knowledge of the structures and
functions of terpenoid synthases may prove to have abundant practical
applications aside from advanced biofuels because these enzymes produce a wide
variety of specialized chemicals.”
Solving the 3D crystal structure of AgBIS was made
possible by the protein crystallography capabilities of Berkeley Lab’s Advanced
Light Source (ALS). For this work, the JBEI team used three of the five protein
crystallography beamlines operated by the Berkeley Center
for Structural Biology (BCSB)—beamlines 8.2.1, 8.2.2, and 5.0.3.
“We needed to use multiple beamlines because we
collected data on several crystals—the protein by itself, and the protein with
different inhibitors/cofactors,” says Adams, who headed the BCSB from 2004 to 2011. “Also, the approach
we used to solve the AgBIS structure required high flux tunable X-rays such as
those provided at 8.2.1 and 8.2.2, which are superbend beamlines.”
