Scientists at the Department of Energy's Oak Ridge National Laboratory were part of a team that identified the existence of a unique genetic code in microbes that can expand cellular building blocks in living organisms.
The discovery, detailed in Science , gives researchers a new understanding of microbial genetics and opens a novel bioengineering pathway for innovations such as the development of custom microbes that produce new fuels, chemicals and materials. The findings could also accelerate the development of better drug therapies - advancing U.S. competitiveness in the biotechnology sector.
In most organisms, a DNA sequence of TAG, called a stop codon, signals where amino acid sequences should end as cells construct proteins - the workhorses inside cells that perform important tasks like building and repairing tissue or fighting disease. While proteins are usually made of 20 common amino acids, there are examples in nature where other amino acids can be beneficial for making specialized proteins.
Researchers on a team led by the Innovative Genomics Institute (IGI) at the University of California, Berkeley, found that some microbes called Archaea have developed the capability to reinterpret this stop codon signal. The microbes use it to instead add another building block - a rare amino acid called pyrrolysine (Pyl) that helps cells customize protein functions and may play a role in their tolerance of extreme environments. Such alternate genetic codes are known for bacteria and eukaryotes, but no alternative genetic codes were known for Archaea.
ORNL's capabilities and expertise in ultra-sensitive biological mass spectrometry experimentally confirmed that certain Archaea consistently incorporate Pyl into their protein where their genome uses TAG codons. Scientists confirmed, for the first time ever, what had been suspected about the code's presence and function, said Robert Hettich, lead for ORNL's Bioanalytical Mass Spectrometry Group.
The lab analyzed hundreds of proteins simultaneously, confirming that the presence of Pyl was not an isolated event, but was incorporated throughout the cells' proteomes on a much wider scale than previously detected.
Researchers further confirmed that Pyl is incorporated into proteins of two Archaea hypothesized to use Pyl based on the presence of metabolic genes. Experiments confirmed the genetic machinery functioned in the same way, by adding Pyl, after it was transplanted into the proteins of a different organism, E. coli, a well-studied bacterium used as a common platform for biotechnology applications.
"The findings show that the genetic code is not fixed; it can change naturally over time," Hettich said. "The project also demonstrated the utility of this knowledge for engineering proteins for innovative biological systems."
Researchers could use the new protein building blocks to develop custom microbes tolerant of industrial processes for new fuels, chemicals and materials or to improve plant microbiomes to boost bioenergy crop performance. The knowledge might also help engineer proteins for medicines that bind more precisely to cancer cells and last longer in the body with fewer side effects.
ORNL mass spectrometry specialists confirm new code
Hettich and colleague Samantha Peters used mass spectrometry to measure the precise molecular masses and amino acid sequences of protein fragments called peptides to confirm that Pyl was present from translation of a TAG sequence, providing direct proof that TAG was being reinterpreted as a building block rather than a stop signal.
"There aren't many other groups in the world that can do this level of mass spectrometry with very complex mixtures," Hettich said, referencing ORNL's expertise in characterizing proteins in microbial communities. "The phenomenon we confirmed is in fairly low abundance in this complex system, so it was rather like looking for a needle in a needle haystack."
"Our team excelled in optimizing sample preparation conditions to achieve sensitive detection and then performing the complicated pattern-matching of our measured peptides to the predicted genomic pattern," Hettich added. They examined the datasets against predicted genomes, and interpreted peptide sequences directly from raw mass spectrometric data. "These two bioinformatics approaches cross-validated the results, a critical step in the project."
For ORNL in particular, the findings provide a valuable step forward in the lab's research to understand and engineer better bioenergy plants and the microbiomes essential to plant health. Insight into how plants and microbes, including Archaea, communicate and interact at the molecular level is key to that mission, Hettich noted.
"This expands our scientific toolbox in many respects, including a new understanding of Archaea, their durability and their potential function in environmental processes such as methane cycling," Hettich said. "We have not previously had the ability to modify Archaea for specific tasks or purposes, and it is exciting to know that we now have a potential mechanism to do so."
ORNL's contributions were supported by the DOE Office of Science Biological and Environmental Research program. Peters, a postdoctoral researcher at ORNL during the project, is now an Innovation Fellow at the Defense Advanced Research Projects Agency. The research was led by Jillian Banfield and Veronika Kivenson at UC Berkeley's Innovative Genomics Institute , along with colleagues at other institutions in the U.S. and France.
UT-Battelle manages ORNL for the Department of Energy's Office of Science, the single largest supporter of basic research in the physical sciences in the United States. The Office of Science is working to address some of the most pressing challenges of our time. For more information, please visit energy.gov/science . - Stephanie Seay
