JBEI Researchers Improve Membrane Protein Expression And Function Using Genomic Edits

Development of robust microbial platforms for bioproduction requires strains that have been engineered to have efficient carbon uptake, energy generation, tolerance to biomass pretreatment byproducts and the export of final product. Many of these optimizations require expression and overexpression of native and heterologous membrane proteins. Over the years, scientists at the U.S. Department of Energy’s Joint BioEnergy Institute (JBEI), and others have successfully found many such engineering targets. However, JBEI has also found that expression of membrane proteins is challenging and can also impact the microbial growth, thus negatively limiting the use of these discoveries.

Genome-wide view of single gene disruptions enriched in high GFP populations. Gene disruptions enriched in at least two biological replicates (RHigh/Low, n ≥ 2) are shown for TnLib/gstA-GFP (grey inner band) and all TnLib/IMP tested (green inner bands). The consensus band (blue outer band) represents gene disruptions enriched in at least three replicates across all TnLib/IMP, for which color density increases with instances of enrichment.

In a new paper published in Nature Scientific Reports, Biofuels and Bioproducts Division scientists at JBEI, hypothesized that they could find loci in the genome that could be edited to enhance expression of any given membrane protein of interest.

The hypothesis was tested by using a bar coded gene knockout library in E. coli in conjunction with expression of fluorescent protein tagged membrane proteins. Numerous loci in the genome were identified that when disrupted allowed improved expression, and consequently function, of the proteins of interest. These are host backgrounds that are better suited to serve as the conversion platforms. This study also represents a systematic evaluation of a microbial host genome to improve membrane protein expression.

Published in Nature Scientific Reports, “Improving membrane protein expression and function using genomic edits” is authored by Heather M. Jensen, Thomas Eng, Victor Chubukov, Robin A. Herbert and Aindrila Mukhopadhyay.

Women’s History Month: Breaking Away from the Mold to Establish Your Own Success Path

Women’s History Month is an annual declared month, celebrated in March that highlights the contributions of women to events in history and contemporary society. This year we celebrate the work and achievements of JBEI research scientist, Ee-Been Goh. A talented molecular biologist, Ee-Been has made significant contributions to the development of new biofuels at JBEI in the area of metabolic pathway for diesel-range methyl ketones. In addition to her impressive technical achievements, Ee-Been is widely recognized at JBEI as being an exemplary mentor and contributor to JBEI’s education and outreach efforts, namely to the iCLEM program.

Who has inspired you? And why?
Scientifically, I will have to say my undergraduate advisor, Prof. (Emeritus) Julian E. Davies. Even though he was a highly renowned professor in the field of antibiotic resistance research, he took a chance on me – someone who did not have the best grades or have any research experience and gave me my first opportunity at independent research. It was Julian’s mentorship that really inspired me to pursue a career in scientific research. Julian’s enthusiasm and passion in science was evident because regardless of whether I presented him with negative or positive results, it was always interesting to him. He taught me that if we process the information properly, you could always learn something from your experiments regardless of the outcome – an outlook that we can always use in science or life!

At a personal level, it would be my grandmother. From sunrise to sunset, she would constantly be working, doing house chores, baking something delicious for us, or sewing new clothes for us and never spending much time idle. What amazes me the most is that she would often put together an ingenious device or apparatus to expedite her work. She never had an education but it never stopped her from being creative or industrious. She instilled a strong working habit in me and taught me to always look to improve things in our lives.

Ee-Been Goh works with summer intern Joshua Borrajo (Credit: Roy Kaltschmidt/Berkeley Lab)

What was your most proud moment? And why?
My most proud moment would be getting accepted into the Ph.D. program at UC Davis because I was not the best student in primary and secondary school (i.e. elementary and junior high). I was doing so badly that teachers had to call my parents to let them know of my struggles in school and even had tutors to help me pass my classes. Many people did not expect that I would go far with my education, so graduating from college was considered a miracle by my family. I surprised them when I decided to seek more education after college. Sometimes we can get pigeon-holed by who we are and where we came from and it felt like a major accomplishment on my part to be able to break away from that mold and establish my own path to success.

What do you do to mentor others?
I like to have students intern with me in the lab and give them an opportunity to experience lab research. My main goal is to try to have them learn as much as they can during their internship and not just assist me with my own research. That way they can find out for themselves if they are truly passionate about science and not be influenced by any other factors. More importantly, I try to set a good example by making sure that my positive work habits and attitude inspires the people (and especially women) around me.

Brewing up Breakthroughs, Create Magazine

JBEI’s CEO Jay Keasling was interviewed by Create, Engineers Australia’s Magazine. In this article Keasling discusses how synthetic biology is being used to tackle major global challenges.

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New Cas9-based Toolkit Eases Obstacles in Genetic Engineering

JBEI develops new synthetic biology tools for engineering Saccharomyces cerevisiae

Pioneering work has been led by the U.S. Department of Energy’s Joint BioEnergy Institute (JBEI) to engineer microbes to transform plant derived starting materials into energy-rich biofuels. But despite the progress in genomics and synthetic biology for the optimization of biofuel production in engineered microbes, microbial engineering methods remain slow and laborious. Such is the case of the fungal host, Saccharomyces cerevisiae. The yeast S. cerevisiae has proven to be an excellent organism for commercial-scale production of biological molecules, though its strain development remains painstakingly slow due to difficulties related to the combined effect of different expression parts and host conditions. Now, researchers at JBEI have developed the largest, most comprehensive Cas9-based toolkit to quickly institute genetic changes in S. cerevisiae to optimize heterologous gene expression.

Cas9-based toolkit for programming gene expression in S. cerevisiae.

Cas9-based toolkit for programming gene expression in S. cerevisiae.

S. cerevisiae is a promising microbial host for biofuel production. It is generally regarded as safe (GRAS), and has capacity for high-density fermentation. To build on our findings at JBEI we have developed new tools to enable more sophisticated strain engineering,” said Aindrila Mukhopadhyay, vice president for fuels synthesis at JBEI and the senior author of the study. “Our toolkit is available online to the scientific community and can be applied to other applications beyond biofuel privation in yeast.”

Subcellular localization of GFP can be modified using protein tags.

Subcellular localization of GFP can be modified using protein tags. The CadDesigner tool is available here.

“CRISPR/Cas9 technology was employed to build a cloning-free toolkit that addresses commonly encountered obstacles in metabolic engineering, including chromosomal integration locus and promoter selection, as well as protein localization and solubility,” explained Amanda Reider Apel, Post-Doctoral Researcher at JBEI and co-first author of the study along with Leo d’Espaux and Maren Wehrs. “We constructed high-efficiency, Cas9-sgRNA plasmids targeting 23 characterized integration loci, characterized 37 standardized promoters in different growth phases and media, and validated the use of 10 protein tags conferring specific protein localization, turnover or solubility.”

The applicability of the toolkit was demonstrated by optimizing the expression of a challenging but industrially important enzyme, taxadiene synthase (TXS). This approach enabled the team to diagnose an issue with TXS solubility, the resolution of which yielded a 25-fold improvement in taxadiene production, the highest levels reported to date.

The strains and DNA sequences reported in the study published by Nucleic Acids Research have been deposited in the JBEI registry. The manuscript describing the toolkit and its application entitled, “A Cas9-based toolkit to program gene expression in Saccharomyces cerevisiae” was authored by Amanda Reider Apel, Leo d’Espaux, Maren Wehrs, Daniel Sachs, Rachel A. Li, Gary J. Tong, Megan Garber, Oge Nnadi, William Zhuang, Nathan J. Hillson, Jay D. Keasling and Aindrila Mukhopadhyay.


Navigating an Ocean of Biological Data in the Modern Era

JBEI’s -omics data visualization tool, Arrowland, facilitates new scientific discovery

Scientists and software engineers at the U.S. Department of Energy (DOE)’s Joint BioEnergy Institute (JBEI) have developed a new -omics visualization tool, Arrowland, which combines different realms of functional genomics data in a single intuitive interface. The aim of this system is to provide scientists an easier way to navigate the ever-growing amounts of biological data generated by the postgenomic revolution. JBEI researchers hope that Arrowland will make it far easier for scientists to reach their next “a-ha!” moment in scientific discovery.

Modern experiment methods in biology can generate overwhelming amounts of raw data.  To manage this, scientists must create entirely new workflows and systems capable of merging large, disparate data sets and presenting them intuitively.  Arrowland is a combined -omics visualization tool that runs on a web browser, tablet, or cellphone.  It shows functional genomics data (fluxomics, metabolomics, proteomics and transcriptomics) together on a zoomable, searchable map, similar to the street maps used for navigation. In addition to providing a coherent layout for -omics measurements, the maps in Arrowland are also an easy-to-use reference for the relationships between the reactions, genes, and proteins involved in metabolism.

“At JBEI we have leveraged the insights provided by metabolic fluxes measured through 13C Metabolic Flux Analysis to make quantitative predictions for E. coli, and we shared these fluxes on Arrowland” said Hector Garcia Martin, Director of Quantitative Metabolic Modeling at JBEI. “By making this cloud-based interactive tool publicly available, our hope is to enable application of this tool in a wide range of fields, not only bioenergy. We are open to collaborations with the broader scientific community and industry.”

Setting itself apart from other available tools, Arrowland is beneficial for its clarity and ease of use. With its unique interface and presentation method, Arrowland makes the exploration of -omics data as intuitive as possible, on a platform that does not require special hardware or configuration.  Four different types of -omics data are integrated into the same map – an ability that no competing visualization platforms share – making important correlations and progressions easy to recognize and examine.

Arrowland developers team. From left: Entrepreneurial Lead, Ling Liang; Principal Investigator, Hector Garcia Martin; and Technical Lead, Garrett Birkel.

Arrowland developers team. From left: Entrepreneurial Lead, Ling Liang; Principal Investigator, Hector Garcia Martin; and Technical Lead, Garrett Birkel.

Arrowland is open-source and currently under active development, with plans to add more maps, editing and curation features, and is integrated with an open-source modeling package that JBEI plans to release separately, called the JBEI Quantitative Metabolic Modeling Library. For the time being, a sample data set from the flux profiles published in “A Method to Constrain Genome-Scale Models with 13C Labeling Data”, PLOS Computational Biology (Garcia Martin et al, 2015) can be viewed at http://public-arrowland.jbei.org.

JBEI scientists explore novel enzyme for aromatic biofuel synthesis

In a Scientific Reports (Nature) paper entitled In vitro characterization of phenylacetate decarboxylase, a novel enzyme catalyzing toluene biosynthesis in an anaerobic microbial community”, researchers at JBEI investigated an enzyme that could enable first-time biochemical production of the widely used octane booster, toluene.

Twenty years ago, it was reported that bacteria in anoxic environments could produce the important industrial chemical, toluene, a widely used octane booster with a global market of 29 million tons per year.  Since that time, the enzyme responsible for catalyzing the intriguing and potentially useful reaction of toluene biosynthesis has remained a mystery.  If biotechnology could harness this enzyme, it could promote sustainability by offsetting the enormous volume of petroleum-derived toluene with biochemically produced toluene made from a renewable resource, such as lignocellulosic biomass.

Researchers at JBEI, led by Harry Beller (Director of Biofuel Pathways at JBEI), and colleagues at DOE’s Joint Genome Institute (JGI), have made significant progress in elucidating the nature of the toluene synthase enzyme.  After establishing an anaerobic toluene-producing microbial community that was inoculated with sewage sludge, they investigated the proteins being expressed in this community using a combination of protein separation technologies, custom mass spectrometric techniques, and metagenomic and metaproteomic analysis.  Although the exact identity (i.e., gene/protein sequence) of the novel enzyme was not yet obtained, the detailed characteristics of the enzymatic reaction that were reported will facilitate its identification.  The researchers are confident that they will soon be completing the discovery of the toluene synthase enzyme, i.e., determining its sequence, thus enabling first-time production of an aromatic hydrocarbon for biofuels.

Using hydrogen sulfide and CO2 to drive production of renewable fuels and chemicals

Berkeley Lab scientists at JBEI have demonstrated a promising biological approach to convert nuisance chemicals in municipal wastewater (sewage) treatment plants into renewable fuels or chemicals. Hydrogen sulfide (which is responsible for the odor of rotten eggs) is a malodorous and corrosive chemical that is problematic in municipal wastewater treatment plants and is typically removed by relatively expensive chemical treatment. JBEI scientists have engineered a common soil bacterium, Thiobacillus denitrificans, which naturally consumes hydrogen sulfide and nitrate (another problem chemical), and fixes CO2 (a greenhouse gas), to simultaneously overproduce fatty acids; these can be further converted to biofuels or value-added chemicals.

fig for summary textIn recently published research, Harry Beller (Senior Scientist at Berkeley Lab) and LBNL colleagues (Peng Zhou, Talia Jewell, Ee-Been Goh, and Jay Keasling) showed that engineered Thiobacillus denitrificans strains could produce more than 50 times the amount of fatty acids compared to the unmodified (wild-type) strain when growing on reduced sulfur compounds, CO2, and nitrate. This is the first demonstration of engineering autotrophic (CO2-fixing) bacteria to make renewable fuels/chemicals using reduced sulfur compounds as a source of energy. In other studies, Beller’s group has engineered different bacteria to produce diesel fuel replacements and valuable fragrance chemicals (medium-chain methyl ketones) from fatty acids, and in the future, this capability could be added to the engineered Thiobacillus denitrificans strains. This proof-of-principle study suggests that engineering sulfide-oxidizing bacteria to overproduce fatty acid-derived products merits consideration as a technology that could simultaneously produce renewable (CO2-based) fuels/chemicals as well as cost-effectively remediate sulfide-contaminated wastewater.

Top image: Thiobacillus denitrificans, credit: Joseph Tringe

JBEI Scientists Unravel Omics Data Using Systems Biology-Based Workflow To Improve Biofuels Productivity

JBEI team developed a workflow to assess and interpret multi-omics data and use it to characterize strain variation in biofuel-producing E. coli.

Researchers at the U.S. Department of Energy (DOE)’s Joint BioEnergy Institute (JBEI) in collaboration with researchers at the University of California, San Diego, have developed a workflow that integrates various omics data and genome-scale models to study the effects of biofuel production in a microbial host.

The development of omics technologies, such as metabolomics and proteomics, and systems biology have dramatically enhanced our ability to understand biological phenomena. Nevertheless the interpretation of large omics data into meaningful ‘knowledge’ as well as the understanding of complex metabolic interactions in engineered microbes remains challenging. This new open-source workflow which integrates various omics data and genome-scale models drives the transition from vision to conception of a designed working phenotype.

The findings were reported in a paper entitled “Characterizing strain variation in engineered E. coli using a multi-omics based workflow” published in Cell Systems (published online on Thursday, May 19). Taek Soon Lee, JBEI’s Director of Metabolic Engineering and Deputy Vice President of the Fuels Synthesis Division is the study’s co-corresponding author along with Bernhard Palsson at University of California, San Diego. Elizabeth Brunk (formerly at JBEI, and currently at UC San Diego) is the study’s co-first author along with Kevin George at JBEI (currently at Amyris).

Metabolic engineering, Proteomics, and Metabolomics teams at the Joint BioEnergy Institute. Taek Soon Lee (left, corresponding author), Christopher Petzold, Jorge Alonso-Gutierrez, Edward Baidoo, George Wang, and Kevin George (co-1st author) (Credit: Irina Silva/JBEI, Berkeley Lab)

Lee’s team analyzed a large omics data set from eight engineered strains producing three different biofuels, applied the workflow to identify the roles of candidate genes, pathways, and biochemical reactions in observed experimental phenomena, and finally used this approach to facilitate the construction of a mutant strain with improved productivity.

“Synthetic biology and systems biology have been considered as two distinct technical frameworks. We hope the confluence of these two fields will benefit biofuels research and its scientific community,” says Lee. “Our team is sharing this workflow as an open-source tool in the form of iPython notebooks, which allows anyone in the microbial engineering field to easily apply this workflow to their system”.

From Near-Dropout to PhD, JBEI Scientist Now at Forefront of Biofuels Revolution

To see biochemist Ee-Been Goh in the lab today, figuring out how to rewire bacteria to produce biofuels, one would never guess she was once so uninterested in school that she barely made it through junior high. Today she is a project scientist at the Joint BioEnergy Institute (JBEI), a Department of Energy Bioenergy Research Center led by Lawrence Berkeley National Laboratory.

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