Scientists Develop Higher-Performance Fuels, Biofuels and Bioproducts

-By Irina Silva

Researchers at Berkeley Lab’s Joint BioEnergy Institute (JBEI) and the Advanced Biofuels & Bioproducts Process Development Unit (ABPDU) have developed a new polyketide synthase-based platform and prototyped efficient production of potential biofuels, gasoline additives, and commodity chemicals.

Microbial production of biofuels and bioproducts is typically carried out using natural or slightly modified enzymes within the metabolic pathway, which can inherently limit the types of molecules that can be produced. Type I modular polyketide synthases (PKSs) are multi-domain enzymes that resemble a modular metabolic assembly line that naturally produces a wide range of unique and diverse molecular structures by combining particular types of catalytic domains in a Lego®-like fashion. This versatile biocatalytic mechanism intrinsically offers a wealth of bioengineering opportunities that scientists can exploit to improve both the rate and yield of the biofuels and bioproducts generated by PKSs.

In “Short-chain ketone production by engineered polyketide synthases in Streptomyces albus published recently in Nature Communications, co-authors Satoshi Yuzawa (JBEI) and Mona Mirsiaghi (ABPDU), present the results of an engineered modular PKS system in the native host Streptomyces venezualae. JBEI and ABPDU researchers were able to demonstrate production of over 1 g/L of C6 and C7 ketones from plant biomass-, a 200-fold improvement over previous efforts.

Final titers of C6 and C7 ethyl ketones with strain ALB188 (left) and C5 and C6 methyl ketones with strain ALB191 (right), in media MM042 with amino acid supplements. Manipulation of cultivation conditions allows tunable production of shorter or longer chain molecules.

Engine tests, performed in the scope of the Co-Optimization of Fuels & Engines (Co-Optima) project, indicate these short-chain ketones can be added to gasoline to increase its octane. This flexible platform could enable biosynthesis of an array of previously inaccessible molecules, allowing fine-tuning of fuel properties, production of highly branched diesel-range biofuels, and a broad range of commodity chemicals.

Other co-authors on the paper are: Renee Jocic, Tatsuya Fujii, Veronica T. Benites, Edward E. K. Baidoo, Anthe George, John M. Gladden, Blake A. Simmons, Leonard Katz and Jay D. Keasling of JBEI, Fabrice Masson, Eric Sundstrom, Deepti Tanjore, and Todd R. Pray of the ABPDU, and Ryan W. Davis of Sandia National Laboratories.

This work was funded by the Joint BioEnergy Institute, a DOE Bioenergy Research Center funded by DOE’s Office of Science, and the Co-Optimization of Fuels & Engines (Co-Optima) project sponsored by the U.S. DOE Office of Energy Efficiency and Renewable Energy’s Bioenergy Technologies Office (BETO). This work was also funded by the National Science Foundation, and leveraged the ABPDU facility which is maintained by BETO and was initiated with funding from the American Recovery and Reinvestment Act.

JBEI Participates at Bay Area Science Festival’s Discovery Day at AT&T Park

JBEI’s volunteers participated at the Bay Area Science Festival’s Discovery Day at AT&T Park this past Saturday, Nov. 3 in collaboration with Berkeley Lab. The event was the culmination of a series of science events for young scientists in the Bay Area.

Our volunteers interacted with about 500 people guiding them through the bioenergy pipeline, and teaching them about concepts such as plant engineering and the use of microbial hosts for the production of biofuels and bioproducts.

Many thanks to our volunteers for their outreach efforts: Irina Silva, Kavitha Satish Kumar, Kevin Lin, Kosuke Iwai, Nurgul Kaplan, Peter Otoupal and Tina Wang.

For more photos, check out this link.

JBEI Organizes Third Annual Software Developer WetLab Bootcamp

-By Irina Silva

The Biosciences Area’s software developers were invited to participate at the third annual Software Developer Wetlab Bootcamp at the Joint BioEnegy Institute (JBEI). Ten participants from Agile BioFoundry, DOE Joint Genome Institute (JGI), JBEI and KBase participated at the training which took place from October 29 to November 2.

Software developers at Berkeley Lab often develop software infrastructure that support, automate, or enhance laboratory operations. “The more hands-on experience the software engineers have with these laboratory operations, the better they can understand them and develop software for them”, said Nathan Hillson, JBEI’s Director of Synthetic Biology Informatics and the organizer of the training. “The WetLab Bootcamp provides just such an opportunity for software engineers to get this hands-on experience, and actually perform the operations that they are supporting.”

Additionally the WetLab Bootcamp is beneficial as it brings together software developers from across the Biosciences Area’s different facilities that may be working in related domains but rarely, if ever, have the opportunity to meet and deepen their professional relationships in person. “The WetLab Bootcamp can be considered a very special job perk that other Bay Area software companies do not offer to their staff. For Berkeley Lab, providing this kind of work experiences helps with staff recruitment and retention.”

Nurgul Kaplan, one of the WetLab Bootcamp instructors, explains how 384 uniquely indexed samples can be pooled and sequenced together in a single lane on an Illumina sequencer.

WetLab Bootcamp instructors Nurgul Kaplan and Tadeusz Ogorzalek from JBEI’s Synthetic Biology Informatics Group, provided training on how to understand the basics of, as well as perform, a Nextera/MiSeq NGS DNA sequence validation workflow, how to use the Echo acoustic and Biomek liquid handling robotics, how machine learning can help optimize wetlab operations, and finally how MiSeq Data are analyzed, and IGV can be used to visualize results.

For KBase’s Dylan Chivian, one of the trainees, the Bootcamp was very helpful, “The WetLab Bootcamp was really useful for me to get a better sense of the power and limitations of multiplexed sequencing with robotics. All my work is downstream of sequencing, and knowing what goes into the data generation will help me build better tools for analysis of that data. Also, it was fun! Nurgul and Tad were great teachers.”

JGI’s Lisa Simirenko learns to denature and dilute prepared libraries for sequencing on the Illumina MiSeq system.

Lisa Simirenko who also undertook the training and works at JGI’s DNA Synthesis group added, “The people on our team that work in the lab routinely validate the sequences of the constructs that they synthesize. Understanding the wet lab sequencing process, and how the operator interacts with different hardware and software tools will help me design better software to help enable this process”. Simirenko finished the training “with a much deeper understanding of the sequencing process, and the interpretation of the resulting data.”

Metabolic Engineering of Lipids Improves the Respiratory Function of Biofuels and Bioproducts Hosts

JBEI researchers define a systems-level model for cellular respiration

-By Irina Silva

Metabolic engineering leads to fundamental discovery about cellular respiration. JBEI scientists engineered lipid metabolism in bacteria and yeast in order to study physiological effects of changes to membrane structure (left). This endeavor led to the development of a mathematical model for cellular respiration, the primary process by which all cells harness energy (right). This model is based on the diffusion of proteins and small molecules on the cell surface, which takes the form of a random motion shown in the trace. This model explains how respiration rates in bacterial cells change depending on the viscosity of the membrane, which sets how fast membrane diffusion can occur. This work uncovers a potentially universal link between lipid biosynthesis in primary metabolism, explaining why organisms regulate lipid synthesis in order to maintain membrane fluidity.

Metabolic engineering leads to fundamental discovery about cellular respiration.

While much is known about how enzymes and molecules are involved in cellular respiration, the understanding of the respiration system as a whole remains limited. Researchers at the Department of Energy (DOE)’s Joint BioEnergy Institute (JBEI) have gained insight into how cellular respiration is affected by the membrane environment in which it occurs. By engineering lipid synthesis to carefully control the membrane composition, researchers found that lipids, which consist of fatty acid molecules and determine membrane viscosity, also tightly control the rate of bacterial and yeast respiration. As lipid synthesis in these hosts is often engineered in order to produce molecules, these findings suggest new ways by which the pathways to produce biofuels and bioproducts could be optimized to maintain proper respiratory function, thereby increasing production.

This work was described in a paper, “Viscous control of cellular respiration by membrane lipid composition” which was published in Science on October 25. The research team was led by JBEI’s Chief Executive Officer Jay Keasling, corresponding author and also senior faculty scientist at Lawrence Berkeley National Laboratory. In this study, the researchers determined the relationship of membrane viscosity to cellular respiration. Viscosity, in the context of this paper, refers to how fluid a membrane is, and can fluctuate depending on which fatty acids are present.

“We were inspired by an old observation that cells have feedback mechanisms that allow their membrane structure to change”, said Itay Budin, JBEI researcher and lead author of the publication. “So we asked ourselves: How will changes in the types of lipids produced by bacterial cells affect their growth and metabolism?” To find an answer, Budin used synthetic biology and metabolic engineering methods to manipulate lipid synthesis and thereby carefully control membrane composition.

Lipids are commonly known as fats and oils. The split into these two categories depends on the physical state of the different types of fatty acids molecules at room temperature. For example, there are small chemical differences (double bonds) in the chemical structures of lipid molecules in solid butter and liquid olive oil, and these are responsible for their characteristic physical properties. Similarly, the physical properties of cell membranes, oily structures only a few nanometers thick, is also dependent on the chemistry of their lipid components. In this paper, the team modulated the viscosity of membranes in cells using metabolic engineering, a process by which JBEI researchers regularly use to control the relative levels of different chemical pathways in cells.

Simulations of ubiquinone distribution on the surface of a bacterial cell. Each square represents the membrane of a cell with different characteristics viscosities, which are experimentally controlled in this study from high (left) to low (right). The dots represent molecules called ubiquinones––small molecules that change that shuttle electrons between enzymes during respiration. Red dots are carrying two electrons, while blue ones are empty. These electrons are finally designated to oxygen, which gets consumed during respiration. Under the viscous membrane conditions (left), the electron carriers form patches of red and blue dots because diffusion is not fast enough to keep up with the speed of respiration enzymes in these locations. This ‘patchiness’ is a hallmark of diffusion in the reaction.

Simulations of ubiquinone distribution on the surface of a bacterial cell.

Budin found that lipids that determine membrane viscosity also tightly control the rate at which bacteria carried out respiration. Cellular respiration occurs through a set of reactions that occur when different enzymes and their substrates collide in the membrane, and viscosity sets the rate at which these collisions occur by random thermal motion (diffusion). Because of this key insight, Budin, working alongside former JBEI graduate student Tristan de Rond, developed a mathematical model for respiration that accounts for the diffusion of its components within the membrane. As inputs, they used quantitative measurements of the abundance and diffusion of the molecular components in the process, which was aided by mass spectrometry work by co-authors Yan Chen, Leanne Jade G. Chan, and Christopher J. Petzold. The team’s model described several aspects of bacterial metabolism, such as how it responds to inhibitors or changes in enzyme concentrations. They then showed that lipids also mediate respiratory rates in mitochondria, dedicated organelles used by all eukaryotic cells for energy production. Thus, lipids could effectively set the ‘speed limit’ by which cells can ‘breathe’ through their effects on membrane diffusion.

“Itay’s research provides us a better understanding of the central metabolism in the two most commonly used hosts used for biotechnology: E. coli and S. cerevisiae,” said Keasling. “This is knowledge with ample application in future metabolic engineering efforts. Furthermore, it demonstrates how tools developed by synthetic biology can also be applied to address fundamental questions in biology.”

JBEI is a DOE Bioenergy Research Center funded by DOE’s Office of Science, and is dedicated to establishing the scientific knowledge and new technologies to transform the maximum amount of carbon available in bioenergy crops into biofuels and bioproducts. This work was also supported by funding from the National Science Foundation.

Researchers build a genetic profile for a section of Aspergillus fungi

There are millions of fungal species, and those few hundred found in the Aspergillus genus play important roles in areas ranging from industrial production to agricultural plant pathogens. Reported October 22, 2018, in Nature Genetics, a team led by scientists at the Technical University of Denmark, the U.S. Department of Energy (DOE) Joint Genome Institute (JGI), a DOE Office of Science User Facility, and the Joint Bioenergy Institute (JBEI), a DOE Bioenergy Research Center, present the first large analysis of an Aspergillus fungal subgroup, section Nigri.

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Congratulations to the 2018 Nobel Prize Winner in Chemistry

Frances Arnold, the Linus Pauling Professor of Chemical Engineering, Bioengineering, and Biotechnology at the California Institute of Technology, is one of the winners of this year’s Nobel Prize in Chemistry. She was a founding member of Berkeley Lab’s Biosciences Expert Advisory Committee, which was set up in 2013 to help develop a strategic plan for the Lab’s Biosciences Area. Arnold received her Ph.D. in chemical engineering from UC Berkeley under the auspices of Harvey Blanch, former Chief Science and Technology Officer of the Joint BioEnergy Institute. Following her thesis work, she was a postdoctoral scholar for about 18 months with the late UC Berkeley chemist and Biosciences affiliate Ignacio Tinoco, who researched the structure of RNA, or ribonucleic acid. Additionally, she has conducted experiments to solve the structure of engineered proteins at the Advanced Light Source and collaborated with researchers at the Molecular Foundry, both DOE Office of Science User Facilities at Berkeley Lab.

Arnold was awarded one half of this year’s prize for her work on directed evolution of enzymes, which are proteins that catalyze chemical reactions. Further, she refined the methods that are now routinely used to develop new catalysts. The uses of Frances Arnold’s enzymes include more environmentally friendly manufacturing of chemical substances, such as pharmaceuticals, and the production of renewable fuels. Read more about her affiliation with UC Berkeley in the Berkeley News Center.

JBEI Hosts Fossil-Based Resources, Refining, Fuels & Petrochemicals Technology Workshop

On September 20-21, JBEI hosted an internal workshop that covered the fundamentals of the production of petroleum and natural gas and their refining and conversion to chemicals.

The workshop leader was Dr. Paul Bryan, former Director of DOE’s Bioenergy Technology Office (BETO), and the former VP of Biofuels Technology and Founding Manager of the Alliance for Advanced Energy Solutions for Chevron.

During this two-full-day course, 21 participants from JBEI and the Biosciences Area gained a strong grounding in the current chemicals and liquid fuels industries. In addition, they delved into the challenges and opportunities in developing renewable alternatives. This latest update included expanded sections on Fuel Properties and “Non-Commodity” Chemicals.

The workshop was first held with DOE / EERE / BETO personnel in Washington, DC and Golden, CO in 2014, and has since been updated and held several times with National Labs and private-sector firms.

What Termites Can Teach Us, The New Yorker

The New Yorker article “What What Termites Can Teach Us” talks about the termites ability to turn grass into energy. The article mentions Jay Keasling, JBEI’s Chief Executive Officer, as one of the synthetic biology leaders, and quotes Héctor García Martín, JBEI’s Deputy Vice President of Biofuels and Bioproducts.

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All Aboard the Jungle Express!

JBEI researchers pave the way for efficient gene expression at any scale

-By Lida Gifford

In the quest to find the key to a rainforest dwelling bacterium’s lignin-degrading ability, researchers at the Department of Energy (DOE)’s Joint BioEnergy Institute (JBEI) have constructed a gene expression system that outperforms conventional systems. Controlling gene expression is crucial to scientists’ ability to perform basic science and biotechnological research to produce enzymes, bio-based products, and biofuels, both at the bench and on industrial scales.

The JBEI team was led by Michael Thelen, a biochemist in the Deconstruction Division, and included researchers from Lawrence Berkeley National Laboratory (Berkeley Lab), Lawrence Livermore National Laboratory (LLNL), and San Francisco State University. Their work, published on September 6 in Nature Communications, describes the bottom-up engineering of Jungle Express, a versatile expression system that enables efficient gene regulation in diverse gram-negative bacteria.

Thomas Ruegg, JBEI researcher and lead author of the publication, said that he began developing this system while studying Enterobacter lignolyticus, a soil bacterium native to a tropical rainforest in Puerto Rico, giving rise the name Jungle Express. Two genes in E. lignolyticus allow the bacterium to withstand exposure to harsh ionic liquids that are used in the deconstruction of biomass, a necessary step in the production of biofuels. Ruegg focused on the regulatory component of the resistance mechanism and tested its response to a range of chemicals that share certain properties with ionic liquids. One of those chemicals was crystal violet, an antifungal agent commonly found in microbiology labs that is also used as a dye for textiles and printing inks. “When I saw extremely high sensitivity to crystal violet,” said Ruegg, “I decided to engineer a gene expression system that can be efficiently activated by this cheap and readily available resource.”

Visual Abstract for Jungle Express Article

Jungle Express is a highly regulated system that enables efficient gene expression in diverse bacteria at negligible costs. The key component of this expression system is a regulatory DNA binding protein (upper right) that originates from a bacterium isolated from the Puerto Rican El Yunque cloud forest (background). The researchers combined a computationally optimized DNA binding site (bound to the protein, upper right) with several bacteriophage promoters, regions of DNA that initiate transcription of a particular gene (upper left). A number of cationic dyes (lower left) have the ability to release the DNA binding protein from the DNA, enabling gene regulation in various bacteria, including the industrially relevant hosts E. coli and Pseudomonas putida. Low concentrations of crystal violet induce gene expression over four orders of magnitude (center), resulting in high product yields (lower right). The potency and low cost of Jungle Express provides a means for highly controllable gene expression that is drastically cheaper than currently available systems (far right). (Credit: Thomas Ruegg/JBEI)

The researchers performed a combination of computational analysis and rational molecular engineering approaches to develop, understand and optimize performance of Jungle Express. This system encompasses several qualities that are very desirable in gene expression applications: tight control, high level and specificity of gene expression, versatility of host bacteria (from E. coli to industrially relevant strains), cost-effectiveness, and flexibility.

To further characterize the system at the molecular level, Jose Henrique Pereira, a research scientist in JBEI’s Technology Division, performed X-ray crystallography at the Advanced Light Source, a DOE Office of Science User Facility. Using these data, they determined the interactions between the regulatory elements and two molecules, including crystal violet, used to turn on the system, which gives insight into its specificity.

“Our findings have the potential to overcome the bottlenecks encountered in earlier systems, and open the way for tightly controlled and efficient gene expression that is not restricted to host organism, substrate, or scale,” explained Thelen, who is also a biochemist at LLNL. “Overall, this has been a fascinating journey that literally started in a jungle of microbial genetic information,” said Ruegg. “We explored this tremendous resource and were able to change the context for the development of a novel game-changing application.”

JBEI is a DOE Bioenergy Research Center funded by DOE’s Office of Science, and is dedicated to developing advanced biofuels. Other co-authors on the paper are: Joseph Chen, Andy DeGiovanni, Giovanni Tomaleri, Steve Singer, Nathan Hillson, Blake Simmons, and Paul Adams of JBEI and Pavel Novichkov and Vivek Mutalik of the Environmental Genomics and Systems Biology Division at Berkeley Lab. Read more about this research in the LLNL press release.