A decade ago, the Department of Energy’s Office of Biological and Environmental Research Grand Challenges program was the perfect venue to study the membrane biology of cyanobacteria.
David Koppenaal, Pacific Northwest National Laboratory chief technology officer, and Himadri Pakrasi, professor of biology at Washington University in St. Louis, used resources made available through the program to start developing systems-level models of cyanobacterial processes. They wanted to understand how these photosynthetic microorganisms harness solar energy to transform atmospheric carbon dioxide into biomass.
Then in 2010, Koppenaal and Pakrasi responded to a call for proposals by EMSL, the Environmental Molecular Sciences Laboratory, a DOE Office of Science User Facility at PNNL. That call led to a continuation of their Grand Challenge research through an EMSL Synthetic Biology Research Campaign.
The team’s goal under the campaign was to discover how to genetically optimize cyanobacteria for increased biomass production. As one of the oldest known life forms on Earth, cyanobacteria were responsible for enriching our planet’s atmosphere with oxygen. They have attracted attention as potential catalysts for producing clean energy from sunlight and atmospheric carbon dioxide.
“The Synthetic Biology Research Campaign was a follow up to the first effort, and the whole point of the Grand Challenge project was to show how useful and informative EMSL capabilities could be for environmental science,” says Koppenaal, EMSL lead on the Synthetic Biology Internal Project Team.
BER’s main interest in cyanobacteria is biomass production. However, Koppenaal adds, “fundamental knowledge is really captivating about these organisms.” Now, as the EMSL campaign concludes, it has yielded results both practically useful and fundamentally interesting.
The cost of saving energy
How to increase a photosynthetic organism’s efficiency is a long-standing question in synthetic biology. Genetically modifying the light-harvesting machinery within the organism can elucidate design principles that drive the production of renewable energy sources in a more efficient way.
Pakrasi’s lab has spent years studying how the cyanobacterial strain Synechocystis sp. PCC 6803 behaves under different growth conditions, placing it high on the candidate list for bioenergy production. As the Synthetic Biology External Project Team lead, he wanted to investigate in detail how changes to the bacteria’s light-harvesting machinery affected its overall morphology and physiology.
In cyanobacteria, a light-harvesting antenna complex called the phycobilisome sits at the surface of the bacteria’s photosynthetic membrane. “They represent one of the most complex things we’ve seen. The cell puts great energy into producing these structures,” says Jon Jacobs, a member of the EMSL Internal Project Team and biochemist at PNNL.
Previous studies suggested cyanobacteria could be a more efficient producer if it didn’t have to use energy to grow these huge complexes. To test this hypothesis, the team used a set of cyanobacteria mutants with diminished, and even fully removed, phycobilisomes.
Jacobs applied the suite of mass spectrometry tools in EMSL’s global proteomics facility to reveal changes in the cell’s proteome, or the entire complement of proteins expressed in the modified organisms. This analysis provided a detailed understanding of physiological changes in the organism with a truncated phycobilisome. Pakrasi was surprised to find reducing the antenna size caused productivity to go down.
The proteome helped explain some characteristics identified in previous research. “You have a broad impact on cellular physiology. It’s not just light harvesting; lots of unpredictable things happen in the cell,” says Michelle Liberton of the Washington University team, and lead author on the PLoS ONE paper discussing their results.
Cyanobacteria mutants with severely reduced phycobilisomes struggled to regulate uptake of bicarbonate and iron from their surroundings. Previous work had shown if the pH of their environment got too high, the organism struggled to survive. “You had a massive die-off of cells, but they hadn’t linked this to bicarbonate control,” says Jacobs. The proteomics analysis revealed the bicarbonate transporters in the bacteria’s membrane were severely compromised. Similarly, the same mutants struggled to take up enough iron, even in an iron-rich medium.
“The take-home message was, if you’re going to do a comprehensive synthetic alteration of an organism, chances are you’re going to affect other cellular mechanisms besides what you want to modify,” concludes Jacobs. In the cyanobacteria case, that meant the cell’s overall growth capabilities. While more cellular resources may be available to capture light energy, the organism will suffer, and researchers need to understand and mitigate these results when creating mutants as bioenergy producers.
Faster growing strains
A separate limitation for using photosynthetic microbes as producers in biotechnology is their relatively slow growth rate compared to their non-photosynthetic counterparts such as E. coli or yeast. For a cyanobacterial strain to be useful as a production organism, it must rapidly produce a large amount of biomass. As part of the Synthetic Biology program, the EMSL team took on the challenge of finding a cyanobacterium that grew faster than Synechocystis 6803 — and explaining why.
The cyanobacterial strain Synechococcus elongatus UTEX 2973 has received attention due to its rapid growth and amenability to genetic modification. This strain’s genome is 99.8 percent identical to other slower-growing strains, but it grows more than twice as quickly. Jacobs led the efforts at EMSL to conduct a global proteomics analysis to reveal which protein components the fast-growing UTEX 2973 expresses.
“We can look at the peptides and see if they’re being expressed differently than what would be expected from the genes,” says Jacobs. “That’s a complementary but often different story than what’s happening based on looking at genes alone.”
The analysis revealed how differences between the two genomes led to differences in protein expression, indicating a few single nucleotide differences determine how cells utilize available resources. These differences control the cell’s growth rate. Thus, the presence of certain proteins provides a growth advantage to cyanobacteria under natural conditions. The researchers published their results in Nature Scientific Reports in 2015, arguing this rapidly growing cyanobacterial strain can be genetically manipulated and is an ideal candidate cyanobacterial system for producing valuable products.
EMSL’s role and the future
EMSL’s cutting-edge mass spectrometers made these studies possible. For global proteomics, the ideal mass spectrometer is used to analyse peptides in a very fast, high-turnover mode, giving in-depth coverage. One instrument allowed the team to detect all the proteins synthesized by the cells. Other instruments are specifically designed to quantify the masses of peptides, while others still can identify larger, intact proteins. EMSL offers all of these capabilities, along with the technical expertise, under the same roof.
Further, the Synthetic Biology Research Campaign’s success rode on additional facilities at EMSL, including state-of-the-art nuclear magnetic resonance spectrometers for studying the bacteria’s structural changes and microscopy for observing its morphology.
“The phycobilisome paper is the completion of a number of years of work,” concludes Koppenaal. “It helped to explain some of how cyanobacteria function; there are all kinds of follow-on questions.”
Until the next proposal, the team hopes smaller-scale user projects will continue to make the most of EMSL’s expertise and proteomics facilities for understanding cyanobacterial systems and exploring possibilities for increasing photosynthetic productivity.
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Rachel Berkowitz is a freelance writer.
[Editor’s Note: Himadri Pakrasi is the director of the International Center for Energy, Environmental and Sustainability (InCEES), supported by the Photosynthetic Antenna Research Center (PARC) and the Department of Energy’s Offices of Basic Energy Sciences (BES) and Biological Environmental Research (BER).]