Sit Down with Sabin: Henrik Scheller: Customizing plants for biofuels.

Uploaded on Aug 10, 2011

Henrik Scheller from the JBEI appeared on August 3rd, 2011 for this installment of "Sit Down with Sabin," a conversation in which former reporter Sabin Russell chats with Lab staff about innovative science. They will discuss "Customizing plants for biofuels." During this series of conversations, Russell and Lab staff will explore the ups and downs of pioneering science, all without the aid of PowerPoints.

Tags: genetic engineering, feedstock, mutant, mutation, acetic acid,

Clearing a Potential Roadblock to Bisabolane

From the Lawrence Berkeley National Laboratory:

Clearing a Potential Roadblock to Bisabolane

Joint BioEnergy Institute Researchers Identify Key Enzyme Structure

January 09, 2012

Lynn Yarris (510) 486-5375  lcyarris@lbl.gov

JBEI researchers determined the structure of the AgBIS enzyme and found it to consist of three helical domains, the first three-domain structure ever found in a synthase of sesquiterpenes. This discovery holds importance for advanced biofuels and other applications.

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 three-dimensional 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 α-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 the Cell Press journal 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, CA.

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 (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.”

Solving the three-dimensional crystal structure of AgBIS was made possible by the protein crystallography capabilities of Berkeley Lab’s Advanced Light Source (ALS), a DOE Office of Science national user facility for synchrotron radiation, and the first of the world’s third generation light sources. 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.”

This research was supported by the DOE Office of Science.

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Insight into Biochemical Balance Needed for Plant Growth

Study Offers Insight into Delicate Biochemical Balance Required for Plant Growth

Implications for producing sustainable biomass, biofuels, and food-processing agents

January 13, 2012

Compared with control plants (left) transgenic plants with overexpression of a gene for pectin acetylesterase had altered leaf shape as well as deformed anther sacs and pollen grains. These findings imply that pectin acetyl esters are essential for normal plant growth and reproduction.

UPTON, NY — In an ongoing effort to understand how modifying plant cell walls might affect the production of biomass and its breakdown for use in biofuels, scientists at the U.S. Department of Energy’s (DOE) Brookhaven National Laboratory have uncovered a delicate biochemical balance essential for sustainable plant growth and reproduction. Their research on pectin, a sugary component of plant cell walls commonly used as a gelling and stabilizing agent in foods, might also suggest new ways to improve its properties for industrial and food applications.

The research findings appear online in the journal The Plant Cell.

“Pectin is the most structurally complex polysaccharide (sugar) component of plant cell walls, and is mainly associated with cell walls that form in fast-growing tissues that are important for plant growth and development,” said Brookhaven biologist Chang-Jun (C.J.) Liu, lead author of the paper. “Our aim was to understand how small molecules, such as acetyl esters, that commonly bind to the sugar backbone affect pectin’s structure and its biological and biophysical properties.”

By analyzing gene sequences available for poplar, a dedicated bioenergy crop and common experimental plant species, they isolated and characterized a gene encoding what they thought might be an enzyme able to split acetyl esters from the pectin in cell walls. Biochemical experiments revealed that this enzyme, which they named pectin acetylesterase, was indeed able to specifically liberate the acetyl ester from cell wall pectins.
They then inserted the gene into tobacco, another experimental plant, to see what effects “disturbing” the acetyl esters would have on pectin in a growing plant, and examined the consequences for plant growth and biomass digestibility.

They used a laser scanning confocal microscope at Brookhaven’s Center for Functional Nanomaterials (CFN) to identify where the enzyme, fused with a green fluorescent protein, was being expressed within the plant cells. Studies using a form of infrared microspectroscopy at the National Synchrotron Light Source (NSLS), aided by collaborator Lisa A. Miller, allowed them to precisely monitor the changes in chemical composition of the plant cell walls.

The findings were dramatic: Removing acetyl esters from pectin drastically impaired the ability of cell walls to elongate with dire consequences for plant growth.

Chang-Jun (C.J.) Liu

“During plant growth, cell-wall components are constantly changed or remodeled, thus enabling the plant cells to continuously expand, build their biomass, and become bigger and taller,” Liu explained. In many fast-growing plant tissues, the major cell wall component is pectin. So disrupting pectin by expressing the pectin acetylesterase gene severely impeded cell growth.

“The most dramatic case that we observed was that removing the acetyl esters retarded the germination of pollen grains and the growth of pollen tubes. Eventually, the plants were completely sterile, unable to produce seeds,” Liu said.

Equally dramatic — but unexpected — was the effect on biomass digestibility.

“Previously, many in vitro studies had demonstrated that acetylesters on the polysaccharide backbone of cell walls act as a physical barrier, preventing the breakdown of cell-wall polysaccharides,” Liu said. Consequently the scientists thought that removing those acetyl esters might be helpful for enzymatic digestion of cell-wall biomass, therefore facilitating the production of biofuels.

“In contrast, we found that reducing acetyl moieties from pectin actually impairs its digestibility, making it more difficult to break down with digestive enzymes,” Liu said. “This suggests that precise acetylation patterns in cell-wall polysaccharides — at least for pectin — are required for the action of the digestive enzymes in breaking down those cell-wall polymers.”

Understanding the details of this delicate biochemical balance will be essential as attempts are made to manipulate plants to maintain the sustainability of plant biomass and improve cell wall biomass digestibility for applications such as biofuel production.

Though not the direct focus of Liu’s research, the current findings might also offer insight into a more delectable aspect of “digestion” — the application of pectin as a food-processing agent. According to Liu, altering acetyl ester content in pectin can dramatically affect its properties, such as solubility and its ability to form gels (as in jellies and jams). “Therefore, characterization of this pectin-specific deacetylase provides a valuable molecular tool to manipulate pectin properties for improving applications in industry and food processing,” he said.

This research was funded by the DOE Office of Science and the National Science Foundation.