Blue Pigment from Engineered Fungi Could Help Turn the Textile Industry Green

By Aliyah Kovner

Often, the findings of fundamental scientific research are many steps away from a product that can be immediately brought to the public. But every once in a while, opportunity makes an early appearance.

Such was the case for a team from the Department of Energy’s Joint BioEnergy Institute (JBEI), whose outside-the-box thinking when investigating microbe-based biomanufacturing led straight to an eco-friendly production platform for a blue pigment called indigoidine. With a similar vividly saturated hue as synthetic indigo, a dye used around the world to color denim and many other items, the team’s fungi-produced indigoidine could provide an alternative to a largely environmentally unfriendly process.

Lead researcher Aindrila Mukhopadhyay holds a vial of purified indigoidine powder. (Credit: Marilyn Chung/Berkeley Lab)

“Originally extracted from plants, most indigo used today is synthesized,” said lead researcher Aindrila Mukhopadhyay, who directs the Host Engineering team at JBEI. “These processes are efficient and inexpensive, but they often require toxic chemicals and generate a lot of dangerous waste. With our work we now have a way to efficiently produce a blue pigment that uses inexpensive, sustainable carbon sources instead of harsh precursors. And so far, the platform checks many of the boxes in its promise to be scaled-up for commercial markets.”

Importantly, these commercial markets already have considerable demand for what the scientists hope to supply. After meeting with many key stakeholders in the textile industry, the team found that many companies are eager for more sustainably sourced pigments because customers are increasingly aware of the impacts of conventional dyes. “There seems to be a shift in society toward wanting better processes for creating everyday products,” said Maren Wehrs, a graduate student at JBEI and first author of the paper describing the discovery, now published in Green Chemistry. “That’s exactly what JBEI is trying to do, using tools derived from biological systems – it just so happens that our engineered biological platform worked very well.”

Droplets of purified indigoidine, produced by bioengineered fungi, are added to water to showcase the pigment’s rich, saturated hue. (Marilyn Chung/Berkeley Lab)

The story began when the team set out to test how well a hardy fungi species called Rhodosporidium toruloides could express nonribosomal peptide synthetases (NRPSs) – large enzymes that bacteria and fungi use to assemble important compounds. The scientists examined this fungi’s NRPS expression capability by inserting a bacterial NRPS into its genome. They chose an NRPS that converts two amino acid molecules into indigoidine – a blue pigment – to make it easy to tell if the strain engineering had worked. Quite simply, when it did, the culture would turn blue.

Going into this experiment, indigoidine itself was not the main interest for the team. Instead, they were focused on the larger picture: exploring how the assembly line functionality of these enzymes could be harnessed to create biosynthetic manufacturing pathways for valuable organic compounds, such as biofuels, and assessing whether or not the fungi represented a good host species for the production of these compounds. But when they cultivated their engineered strain, and saw just how blue the culture was, they knew something incredible had happened.

Aindrila Mukhopadhyay and Maren Wehrs inspect a bioreactor full of their Bluebelle strain at JBEI. (Credit: Marilyn Chung/Berkeley Lab)

With an average titer of 86 grams of indigoidine per liter of bioreactor culture, the yield of the strain – which they named Bluebelle – is by far the highest that has ever been reported. (Other research groups, including the JBEI team, have synthesized indigoidine using different host  microbes.) Adding to the weight of the achievement, the record-breaking yield was obtained from a culture process that uses nutrient and precursor inputs sourced from sustainable plant material. Previous pathways required considerably more expensive inputs yet made about one-tenth the amount of indigoidine.

Beyond the potential applications of indigoidine, the study succeeded in its original goal of providing a potential production pathway for other NRPSs – something that is much more valuable than any single product. These complex enzymes have multiple subunits that each perform a distinct and predictable action in assembling a compound out of smaller molecules. Scientists at JBEI and beyond are keen to engineer enzymes that use NRPSs’ Lego block-like features to produce advanced bioproducts that are currently hard to make.

“A big challenge is to get a microbe to efficiently express such enzymes. This host has huge potential to fulfill that need,” said Mukhopadhyay.

The team’s next steps will be to characterize how indigoidine could be used as a dye and to dig deeper into the capabilities of R. toruloides.

This work was made possible by the expertise of multiple JBEI groups including John Gladden from Sandia National Laboratories, who leads JBEI’s Fungal Biotechnology team, as well as Berkeley Lab’s Advanced Biofuels and Bioproducts Process Development Unit (ABPDU). The other researchers included Yuzhong Liu, Lukas Platz, Jan-Philip Prahl, Jadie Moon, Gabriella Papa, Eric Sundstrom, Gina Geiselman, Deepti Tanjore, Todd Pray, Jay Keasling, and Blake Simmons. JBEI is funded by the Department of Energy Office of Science. ABPDU is funded by the DOE Office of Energy Efficiency and Renewable Energy.

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.

JBEI participates at 2019 East Bay STEM Career Awareness Day

East Bay STEM Career Awareness Day took place on May 16 in Emeryville. At this annual event, local businesses and organizations collaborated to provide activities that provided insight into potential careers in STEM to 200 students from schools in Berkeley, Emeryville, Richmond, and Oakland. The event was hosted by the Institute for STEM Education at California State University, East Bay in partnership with Bayer, Wareham Development, the East Bay EDA, the City of Emeryville, and the City of Berkeley. We thank the JBEI volunteers  that participated at this event: Deepika Awasthi, Irina Silva, Nicole Ing, Peter Otoupal, and Tina Wang.

2019 Undergraduate Poster Presentation

Undergraduates who have interned at Emery Station East (ESE), where JBEI is located, participated at an on-site poster presentation and celebration on May 10. The students who interned at JBEI and other research programs hosted at ESE presented results of their research. Twelve posters and two talks were presented to the JBEI community.

The undergraduates were judged for their presentation and poster making skills. This year, there was a tie in both categories. Julie Lake and Allison Pearson won for “Best Poster Design” and Kenneth Workman and Isabel Honda won for “Best Poster Presentation”.

The following students presented seminar talks:

  • Marian-Joy Baluyot (Mentor: Sam Curran)
  • Will Sharpless (Mentor: Mitch Thompson)

And the students below presented posters:

  • Alex Cardia (Mentor: Luis Valencia)
  • Allison Pearson (Mentor: Mitch Thompson)
  • Amanda Hernandez, Samantha Chang (Mentor: Amin Zargar)
  • Isabel Honda (Mentor: Laure Leynaud-Kieffer)
  • Jasmine Cisneros, Jennifer Zhang (Mentor: Paul Opgenorth)
  • Julie Lake (Mentor: Sam Curran)
  • Kenneth Workman (Mentor: Henrique De Paoli)
  • Marty Ng (Mentor: Ankita Kothari)
  • Maya Ramamurthy (Mentors: Connie Bailey, Luis Valencia)
  • Shania Tedja (Mentor: Deepika Awasthi)
  • Silver Alkhafaji (Mentor: Laure Leynaud-Kieffer)
  • Suneil Acharya (Mentor: Pablo Cruz-Morales)
Allison Pearson, one of the “Best Poster Design” winners seen here presenting her poster.
Kenneth Workman (on the right), one of the “Best Poster Presentation” winners, with his mentor Henrique De Paoli.

Making fuel out of algae could clean up dirty planes

Corinne Scown, JBEI’s VP of Life-cycle, Economics and Agronomy, recently spoke to Wired UK about how biofuels have the potential to play an important role in the reduction of greenhouse gas emissions in aviation.

Read more

Pam Ronald Elected to National Academy of Sciences

Pam Ronald, scientific lead of plant pathology at the Joint BioEnergy Institute (JBEI), was elected to the National Academy of Sciences in recognition of her distinguished and continuing achievements in original research. She joins 100 scientists and engineers from the U.S. and 25 from across the world as new lifelong members and foreign associates.

All new NAS members and foreign associates are nominated by existing NAS members for outstanding contributions to their field. Only 100 or fewer researchers make it through the selection process each year. The four new members bring the number of Berkeley Lab scientists elected as NAS members to 84.

On top of being one of the highest honors a scientist or engineer can receive, membership to NAS also provides a platform for advocacy and leadership. Since its creation in 1863, NAS has served as a nonpartisan, nonprofit institution that offers science, engineering, and health policy advice to the federal government and other organizations.

Pam Ronald is also a distinguished professor in the College of Agricultural and Environmental Sciences at UC Davis. Ronald’s research focuses on plant genes that control resistance to disease and tolerance to environmental stressors, with the goal of using genetic engineering to improve food security for the world’s poorest farmers. Read more.

New Study Sees Competitive SAFJ Prices in Future, AIN Online

Aviation International News covered JBEI’s study “Techno-economic analysis and life-cycle greenhouse gas mitigation cost of five routes to bio-jet fuel blendstocks,” published in the journal Energy & Environmental Science which provided evidence that optimizing the biofuel production pipeline is well worth the effort.

Read more

Bright Skies for Plant-Based Jet Fuels

Joint BioEnergy Institute researchers demonstrate that jet fuels made from plants could be cost competitive with conventional fossil fuels

With an estimated daily fuel demand of more than 5 million barrels per day, the global aviation sector is incredibly energy-intensive and almost entirely reliant on petroleum-based fuels. Unlike other energy sectors such as ground transportation or residential and commercial buildings, the aviation industry can’t easily shift to renewable energy sources using existing technologies.

However, a new analysis by scientists at the Department of Energy’s Lawrence Berkeley National Laboratory (Berkeley Lab) shows that sustainable plant-based bio-jet fuels could provide a competitive alternative to conventional petroleum fuels if current development and scale-up initiatives continue to push ahead successfully.

“Techno-economic analysis and life-cycle greenhouse gas mitigation cost of five routes to bio-jet fuel blendstocks” published recently in the journal Energy & Environmental Science, provides promising evidence that optimizing the biofuel production pipeline – taking carbohydrate-rich plant material and using genetically modified bacteria to digest the isolated sugars into energy-dense molecules that are then chemically converted into a fuel product – is well worth the effort.

From left: Nawa Baral, Daniel Mendez-Perez Aindrila Mukhopadhyay, Blake Simmons, Corinne Scown and Taek Soon Lee.

“It’s challenging to electrify aviation using batteries or fuel cells in part because of the weight restrictions on aircraft, so liquid biofuels have the potential to play a big role in greenhouse gas emissions reductions,” said lead author Corinne Scown, a researcher in Berkeley Lab’s Energy Technologies Area as well as DOE’s Joint BioEnergy Institute (JBEI). “The team at JBEI has been working on biological routes to advanced bio-jet fuel blends that are not only derived from plant-based sugars but also have attractive properties that could actually provide an advantage over conventional jet fuels.”

How to get fuel from plant material

Currently, multidisciplinary teams based at JBEI are focused on optimizing each stage of the bio-jet fuel production process. Some researchers specialize in engineering ideal source plants – referred to as biomass – that create a high proportion of carbohydrates and a low proportion of lignin, a type of material that, as of now, is more challenging to make useful. Meanwhile, others are developing methods for efficiently isolating the carbohydrates in non-food biomass and breaking them into sugar molecules that bacteria can digest, or “bioconvert,” into a fuel molecule. To obtain the highest possible yield from bioconversion, yet other JBEI researchers are examining what genetic and environmental factors make the modified bacteria more efficient.

Once these stages are optimized, JBEI scientists can transition the technologies to commercial partners who may then modify and blend the fuels into ready-to-use products and devise strategies to industrialize the scale of production. Given the vast amount of experimentation and innovation needed to accomplish all this, Scown and her co-authors used innovative analysis methods to assess whether the undertaking could actually reach the end game of a jet fuel alternative that airlines will want to use.

“Our hope is that early in the research stages, we can at least simulate what we think it would look like if you develop these fuel production routes to the point of maturity,” Scown said. “If you were to push them to the ethanol benchmark – the technology to create ethanol from plant material like corn stalks, leaves, and cobs has been around a long time, and we can ferment sugars with a 90 percent efficiency – how close would this get us to the market price of petroleum fuels? That is important to know now.

“Thankfully, the answer is they can be viable. And we’ve identified improvements that need to happen all along the conversion process to make that happen.”

Imagining the production process at scale

From left: Co-authors Nawa Baral and Daniel Mendez-Perez

Due to the biomass deconstruction and fuel synthesis technologies developed at JBEI, the theoretical cost of bio-jet fuel has declined steadily in recent years and is currently as low as $16 per gallon, as compared to $300,000 per gallon when JBEI was established, according to co-author and JBEI postdoctoral fellow Nawa Baral. The cost of standard jet fuel is about $2.50 per gallon.

To explore how bio-jet fuel could bridge the remaining price gap, the research team used complex computer simulations that modeled the necessary technology and subsequent costs of complete, scaled-up production pathways at different efficiency levels and with a range of biomass and chemical inputs. The authors simulated a total of five different production pathways to four distinct fuel molecules.

The results showed that all five pathways could indeed create fuel products at the target price of $2.50 per gallon if manufacturers are able to convert the leftover lignin into a valuable chemical – something JBEI researchers are currently working toward – that could be sold to offset the cost of biofuels. The net price of a gallon of biofuel could be lowered further if airlines were offered even a modest financial credit for emissions reduction.

Following some industry research, the team also found that airlines may be willing to pay a premium of as much as fifty cents per gallon because all four biofuels deliver more energy per unit volume, meaning a plane could fly farther on a tank of the same size.

“The development of plant-based compounds that have a performance advantage over their petroleum-based counterparts is an important factor in determining their marketplace viability,” said Blake Simmons, a co-author and the Chief Science and Technology Officer at JBEI.

However, as promising as these findings are, getting the biofuel production technology to the gold-standard yields assumed in these simulations will require further advances.

“It’s clear that, to get these fuels to commercial viability, we need all hands on deck,” Scown noted. “But this analysis highlights the importance of multi-institutional, integrative research centers like JBEI because no group working on one phase of the process alone can make it happen.”

The other co-authors on the paper are JBEI scientists Olga Kavvada, Daniel Mendez-Perez, Aindrila Mukhopadhyay, and Taek Soon Lee.

Funded by the DOE’s Office of Science, JBEI was created with a mission to develop economically-viable, carbon-neutral biofuels and bioproducts that utilize the sunlight energy stored in biomass.

Sloan Fellowship Will Help Patrick Shih Investigate Ancient Origins of Photosynthesis

Patrick Shih, JBEI’s Director of Plant Biosystems Design who also serves as an Assistant Professor at the Department of Plant Biology at UC Davis, was recently selected as a 2019 Alfred P. Sloan Research Fellow in Computational and Evolutionary Molecular Biology.

Shih, will use this fellowship to help fund his research to reconstruct the evolution of photosynthesis, a process that originated billions of years ago.

Read more