Bio diesel compared to ethanol is way behind in displacing petroleum products.  Add in the other middle distillates such as jet fuel and kerosene and the very small percentage looks even smaller.  Ethanol has been used for centuries and has a huge head start, but a new breakthrough at the U.S Department of Energy (DOE)’s Joint BioEnergy Institute (JBEI) may just crack open the barrier.  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, CA.

Earlier a team discovered that bisabolane, a member of the terpene class of chemical compounds holds high promise as a biosynthetic alternative to No.2 diesel fuel that generated keen interest in the green energy community and the trucking industry. This team identified bisabolane as a potential new advanced biofuel that could replace No.2 diesel, today’s standard fuel for diesel engines, with a 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.

Now a second team led by bioengineers Paul Adams and Jay Keasling, solved the protein crystal structure of an enzyme in the Grand fir (Abies grandis) called “Abies grandis α-bisabolene synthase” (AgBIS) that synthesizes bisabolene, the immediate terpene precursor to bisabolane.  But when AgBIS is engineered into microbes, the enzyme results in a bottleneck that hampers the conversion by the microbes of simple sugars into bisabolene.  The opportunity suddenly became a problem.

AgBIZ Enzyme. Click image for more info.

Adams, a leading authority on x-ray crystallography, explains what the team has figured out saying, “Our high resolution structure of AgBIS should make it possible to design changes in the enzyme that will enable microbes to make bisabolene faster. 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.”

The paper “Structure of a Three-Domain Sesquiterpene Synthase: A Prospective Target for Advanced Biofuels Production.” About AgBIS has been published at the Cell Press journal Structure.

Pamela Peralta-Yahya, a lead member of the earlier JBEI team as well as the current team discusses the situation saying, “The inefficient terpene synthase enzyme is one of the bottlenecks in the metabolic pathway used by the engineered microbes. 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.”

Ryan McAndrew a co-lead author explains 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.

McAndrew continues, “That we found the structure of AgBIS to be more similar to diterpene (20 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.”

The look into AgBIS was made possible by the protein crystallography capabilities of Berkeley Lab’s Advanced Light Source (ALS) for synchrotron radiation, and the first of the world’s third generation light sources.  The JBEI team used three of the five protein crystallography beamlines operated by the Berkeley Center for Structural Biology (BCSB).  Adams, who headed the BCSB from 2004 to 2011, tells us, “We needed to use multiple beamlines because we collected data on several crystals – the protein by itself, and the protein with different inhibitors/cofactors.”

That’s a lot of unfamiliar chemistry terms in one post.  To summarize, the JBEI teams have found AgBIS will convert sugars to the heavier chemicals in the 15-carbon atom range – which is where development needs to be for diesel and other products of that molecular size.  But when AgBIS was engineered in the activity came up short, and the question ‘why?”, now has a visual means of understanding and that offers a much clearer means to manipulate the structure and likely, redevelop a new enzyme that could be very productive indeed.

It’s still way, way early in the research.  Having a leading candidate for engineering bio organisms to join in making the products in the 15 carbon atom and up range is very enticing news.  The ability to make these fuels over just extracting them from plants offers massive growth potential.


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