While trying to unravel how seaweed creates its chemically complex toxins, scientists at the Scripps Institution of Oceanography at the University of California, San Diego, discovered the largest protein yet identified in biology. The discovery of the biological machinery that the algae developed to produce its intricate toxin also revealed previously unknown strategies for assembling chemicals, potentially opening the way to the development of new drugs and materials.
The researchers found the protein, which they named PKZILLA-1, while studying how a type of algae called Prymnesium parvum produces its toxin, which is responsible for mass fish deaths.
“This is the Mount Everest of proteins,” said Bradley Moore, a marine chemist with joint appointments at Scripps Oceanography and Skaggs School of Pharmacy and Pharmaceutical Sciences and senior author of a new study detailing the findings. “This expands our notion of what biology is capable of.”
PKZILLA-1 is 25% larger than the previous record holder, titin, which is found in human muscles and can reach 1 micron in length (0.0001 centimeter or 0.00004 inches).
Published today in Science The study, funded by the National Institutes of Health and the National Science Foundation, shows that this giant protein and another large but non-record-breaking protein, PKZILLA-2, are key to producing primnesin, the large, complex molecule that is the algae toxin. In addition to identifying the massive proteins behind primnesin, the study also discovered unusually large genes that provide Prymnesium parvum with the blueprint for making proteins.
Finding the genes that underpin production of the toxin primnesin could improve efforts to monitor harmful algal blooms in this species by facilitating water testing that looks for the genes rather than the toxins themselves.
“If we monitor genes rather than toxins, we may be able to detect blooms before they start, rather than only being able to identify them when toxins are circulating,” said Timothy Fallon, a postdoctoral researcher in Moore’s lab at Scripps and co-lead author on the paper.
The discovery of the PKZILLA-1 and PKZILLA-2 proteins also reveals the algae’s complex cellular assembly line for producing toxins, which have unique and complex chemical structures. This better understanding of how these toxins are produced could prove useful for scientists trying to synthesize new compounds for medical or industrial applications.
“Understanding how nature has worked its chemical magic gives us, as scientific professionals, the ability to apply that knowledge to create useful products, whether it’s a new cancer drug or a new fabric,” Moore said.
Prymnesium parvumGolden algae, commonly known as golden algae, is a single-celled aquatic organism found worldwide in both fresh and salt water. Golden algae blooms are associated with fish kills due to its toxin primnesin, which damages the gills of fish and other water-breathing animals. In 2022, a golden algae bloom killed between 500 and 1,000 tons of fish in the Oder River, which borders Poland and Germany. The microorganism can wreak havoc on aquaculture systems in places ranging from Texas to Scandinavia.
Primnesin belongs to a group of toxins called polyethers, which include brevetoxin B, a major red tide toxin that regularly plagues Florida, and ciguatoxin, which contaminates reef fish throughout the South Pacific and Caribbean. These toxins are among the largest and most complex chemicals in all of biology, and researchers have struggled for decades to figure out exactly how microorganisms produce such large and complex molecules.
Beginning in 2019, Moore, Fallon and Vikram Shende, a postdoctoral researcher in Moore’s lab at Scripps and co-lead author on the paper, began trying to figure out how golden algae produce their toxin primnesin at the biochemical and genetic level.
The authors of the study began by sequencing the genome of the golden algae and searching for the genes involved in the production of primnesin. Traditional methods of searching the genome did not yield results, so the team turned to alternative methods of genetic research that were better suited to finding super-long genes.
“We were able to locate the genes and it turned out that to make giant toxic molecules this algae uses giant genes,” Shende said.
Once the PKZILLA-1 and PKZILLA-2 genes were located, the team needed to investigate what those genes produced in order to link them to the production of the toxin. Fallon said the team was able to read the coding regions of the genes like sheet music and translate them into the sequence of amino acids that made up the protein.
When the researchers completed the assembly of the PKZILLA proteins, they were stunned by their size. The PKZILLA-1 protein reached a record mass of 4.7 megadaltons, while PKZILLA-2 was also extremely large, at 3.2 megadaltons. Titin, the previous record holder, can reach 3.7 megadaltons, about 90 times larger than a typical protein.
After further testing showed that golden algae actually produce these giant proteins in life, the team set out to find out if the proteins were involved in producing the toxin primnesin. PKZILLA proteins are technically enzymes, meaning they initiate chemical reactions, and the team depicted the long sequence of 239 chemical reactions involved by the two enzymes using pens and notebooks.
“The final result perfectly matched the structure of primnesin,” Shende said.
Moore said studying the cascade of reactions that golden algae use to make their toxin revealed previously unknown strategies for manufacturing chemicals in nature. “The hope is that we can use this knowledge of how nature makes these complex chemicals to open up new chemical possibilities in the laboratory for the drugs and materials of the future,” he added.
The discovery of the genes that trigger the toxin primnesin could allow for more cost-effective monitoring of golden algae blooms. This monitoring could use tests to detect PKZILLA genes in the environment, similar to the PCR tests that became common during the COVID-19 pandemic. Better monitoring could increase preparedness and allow for more detailed study of the conditions that increase the likelihood of blooms occurring.
Fallon said the PKZILLA genes the team discovered are the first genes causally linked to the production of any marine toxin in the polyether group of which primnesin is a member.
Next, the researchers hope to apply the unconventional screening techniques they used to find the PKZILLA genes to other species that produce polyether toxins. If they can find the genes responsible for other polyether toxins, such as ciguatoxin, which can affect up to 500,000 people a year, the same genetic monitoring possibilities would open up for a range of other toxic algal blooms with significant global implications.
In addition to Fallon, Moore and Shende of Scripps, David Gonzalez and Igor Wierzbikci of UC San Diego along with Amanda Pendleton, Nathan Watervoort, Robert Auber and Jennifer Wisecaver of Purdue University were co-authors of the study.