Turning the Switch on Biofuels

-By Anne M. Stark

Plant cell walls contain a renewable, nearly-limitless supply of sugar that can be used in the production of chemicals and biofuels. However, retrieving these sugars isn’t all that easy.

Imidazolium ionic liquid (IIL) solvents are one of the best sources for extracting sugars from plants. But the sugars from IIL-treated biomass are inevitably contaminated with residual IILs that inhibit growth in bacteria and yeast, blocking biochemical production by these organisms.

Lawrence Livermore National Laboratory (LLNL) scientists and collaborators at the Joint BioEnergy Institute have identified a molecular mechanism in bacteria that can be manipulated to promote IIL tolerance, and therefore overcome a key gap in biofuel and biochemical production processes. The research appears in the Journal of Bacteriology.

“Ionic liquid toxicity is a critical roadblock in many industrial biosynthetic pathways,” said LLNL biologist Michael Thelen, lead author of a paper appearing in the June 10 edition of the Journal of Bacteriology. “We were able to find microbes that are resistant to the cytotoxic effects.”

The team used four bacillus strains that were isolated from compost (and a mutant E. coli bacterium) and found that two of the strains and the E. coli mutant can withstand high levels of two widely used IILs.

Douglas Higgins, a postdoc working with Thelen at the time, dived into how exactly the bacteria do this. In each of the bacteria, he identified a membrane transporter, or pump, that is responsible for exporting the toxic IIL. He also found two cases in which the pump gene contained alterations in the RNA sequence of a regulatory guanidine riboswitch. Guanidine is a toxic byproduct of normal biological processes; however, cells need to get rid of it before it accumulates.

The normal, unmodified riboswitch interacts with guanidine and undergoes a conformational change, causing the pump to switch on and make the bacterial cells resistant to IILs.

“Our results demonstrate the critical roles that transporter genes and their genetic controls play in IIL tolerance in their native bacterial hosts,” Thelen said. “This is just another step in engineering IIL tolerance into industrial strains and overcoming this key gap in biofuel production.”

The results could help identify genetic engineering strategies that improve conversion of cellulosic sugars into biofuels and biochemicals in processes where a low concentration of ionic liquids surpass bacterial tolerance.

Scientists from Sandia National Laboratories and Lawrence Berkeley National Laboratory also contributed to this research.

The work is funded by the Department of Energy, Office of Science.

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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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.

JBEI Pretreatment and Process Development Team Honored by Secretary of Energy

The Pretreatment and Process Development Team at JBEI has been awarded the Secretary of Energy’s Achievement Award. The award is designed to recognize the contributions of Department of Energy (DOE) employees to the mission of the Department and to the benefit of the United States. The JBEI team was recognized for pioneering the development of biomass-derived ionic liquids (“bionic liquids”) to enable one-pot conversion technologies that are efficient, feedstock flexible, scalable, and economically viable to support production of biofuels and co-products.  The team members honored by this award are Tanmoy Dutta, N.V.S.N. Murthy Konda, Corinne D. Scown, Blake A. Simmons, Seema Singh, Aaron M. Socha, Jian Sun, and Feng Xu. This team is a model of inter-institutional collaboration that JBEI has enabled, with about half of the team having been affiliated with Sandia (Dutta, Singh, Socha, Sun, and Xu) and the other half with Lawrence Berkeley National Laboratories (Konda, Scown, and Simmons).

The members have worked together to advance cellulosic biofuels research and development by increasing the economic and environmental sustainability of biomass pretreatment. The bionic liquid-enabled integrated one-pot process reduces annual operating cost by 40 percent and water use/waste water generation by approximately 85 percent, and has the potential to reduce greenhouse gas emissions by as much as 50 to 85 percent compared to conventional gasoline. With clear economic and environmental benefits, the one-pot bionic liquid process may represent a breakthrough technology in the cellulosic biofuel development.

Dutta, Konda, Singh, and Socha received the award on behalf of the team from Secretary Perry at the ceremony held on August 29 (pictured above from left to right with Secretary Perry in the center). For more information about the ceremony, please see the Department of Energy announcement.

Scientists discover how to protect yeast from damage in biofuel production

Some chemicals used to speed up the breakdown of plants for production of biofuels like ethanol are poison to the yeasts that turn the plant sugars into fuel. Researchers from the UW-Madison-based Great Lakes Bioenergy Research Center, Joint BioEnergy Institute, and several Department of Energy National Laboratories have identified two changes to a single gene that can make yeast tolerate the pretreatment chemicals. They published their findings recently in the journal Genetics.

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The highly complex sugarcane genome has finally been sequenced

Sugarcane was the last major cultivated plant to have its genome sequenced. This was because of its huge complexity: the genome comprises between 10 and 12 copies of each chromosome, when the human genome has just two. An international team which included JBEI coordinated by CIRAD achieved this milestone, as reported in Nature Communication on July 6. It will now be possible to “modernize” the methods used to breed sugarcane varieties. This will be a real boon to the sugar and biomass industry.

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All in the Family: Focused Genomic Comparisons

In a study published ahead the week of January 8, 2018 in the Proceedings of the National Academy of Sciences, a team led by researchers at the Technical University of Denmark (DTU), the DOE Joint Genome Institute (JGI), a DOE Office of Science User Facility, and the DOE’s Joint BioEnergy Institute (JBEI), led by Lawrence Berkeley National Laboratory (Berkeley Lab), report the first results of a long-term plan to sequence, annotate and analyze the genomes of 300 Aspergillus fungi. These findings are a proof of concept of novel methods to functionally annotate genomes in order to more quickly identify genes of interest.

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To Find New Biofuel Enzymes, It Can Take a Microbial Village

A new study led by researchers at the Department of Energy’s Joint BioEnergy Institute (JBEI), based at Lawrence Berkeley National Laboratory (Berkeley Lab), demonstrates the importance of microbial communities as a source of stable enzymes that could be used to convert plants to biofuels. The study, recently published in the journal Nature Microbiology, reports on the discovery of new types of cellulases, enzymes that help break down plants into ingredients that can be used to make biofuels and bioproducts. The cellulases were cultured from a microbiome. Using a microbial community veers from the approach typically taken of using isolated organisms to obtain enzymes.

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Study speeds transformation of biofuel waste into useful chemicals, Phys.org

JBEI’s Feedstocks Division collaborated with Sandia National Laboratories in a study that looked into efficient ways to turn discarded plant matter into chemicals.

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