While trying to unravel how marine algae create their chemically complex toxins, scientists at UC San Diego’s Scripps Institution of Oceanography have discovered the largest protein yet identified in biology. Uncovering the biological machinery the algae evolved to make its complex toxin also revealed previously unknown strategies for assembling chemicals, which could unlock the development of new drugs and materials.
Researchers found the protein, which they named PKZILLA-1, while studying the name of a type of algae Prymnesium parvum produces its toxin, which is responsible for massive fish kills.
“This is the Mount Everest of proteins,” says 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 sense of what biology is capable of.”
PKZILLA-1 is 25% larger than titin, the previous record holder, which is found in human muscle and can reach a length of 1 micron (0.0001 centimeters or 0.00004 inches).
Published today in Science and funded by the National Institutes of Health and the National Science Foundation, the research shows that this giant protein and another super-sized but not record-breaking protein – PKZILLA-2 – are key to the production of prymnesin – the large, complex molecule that the toxin of the algae. In addition to identifying the massive proteins behind prymnesin, the study also uncovered unusually large genes that make it happen Prymnesium parvum with the blueprint for making the proteins.
Finding the genes underlying production of the prymnesin toxin could improve monitoring efforts for harmful algal blooms of this species by allowing water tests that look for the genes rather than the toxins themselves.
“Monitoring the genes rather than the toxin could allow us to catch flowers before they start, rather than identifying them once the toxins are circulating,” says Timothy Fallon, a postdoctoral researcher in Moore’s lab at Scripps and co-first author. of the paper.
The discovery of the PKZILLA-1 and PKZILLA-2 proteins also reveals the alga’s extensive cellular assembly line for building the toxins, which have unique and complex chemical structures. This improved understanding of how these toxins are made could be useful to scientists trying to synthesize new compounds for medical or industrial applications.
“Understanding how nature developed its chemical wizardry gives us as scientific practitioners the opportunity to apply those insights to creating useful products, whether it is a new anti-cancer drug or a new compound,” says Moore.
Prymnesium parvumCommonly known as golden algae, is a single-celled aquatic organism found throughout the world in both fresh and salt water. Golden algae blooms have been linked to fish die-offs due to the toxin prymnesin, which damages the gills of fish and other water-breathing animals. In 2022, a golden algae bloom killed 500 to 1,000 tons of fish in the Oder River, bordering Poland and Germany. The microorganism can wreak havoc in aquaculture systems in places ranging from Texas to Scandinavia.
Prymnesin belongs to a group of toxins called polyketide polyethers that include brevetoxin B, a major redwater toxin that regularly affects Florida, and ciguatoxin, which contaminates reef fish in the South Pacific and the 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, complex molecules.
Starting in 2019, Moore, Fallon, and Vikram Shende, a postdoctoral researcher in Moore’s lab at Scripps and co-first author of the paper, began figuring out how golden algae make their toxin prymnesin at the biochemical and genetic level.
The study authors started by sequencing the genome of the golden algae and looking for the genes involved in prymnesin production. Traditional methods of searching the genome yielded no results, so the team turned to alternative methods of genetic sleuthing that were more adept at finding super-long genes.
“We were able to locate the genes and it turned out that this algae uses giant genes to make giant toxic molecules,” says Shende.
Now that the PKZILLA-1 and PKZILLA-2 genes had been located, the team needed to investigate what the genes made 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 this assembly of the PKZILLA proteins, they were amazed at their size. The PKZILLA-1 protein had a record-breaking mass of 4.7 megadaltons, while PKZILLA-2 was also extremely large at 3.2 megadaltons. Titin, the previous record holder, can be up to 3.7 megadaltons in size – about 90 times larger than a typical protein.
After additional tests showed that golden algae actually produce these giant proteins in life, the team tried to find out if the proteins were involved in making the toxin prymnesin. The PKZILLA proteins are technically enzymes, meaning they initiate chemical reactions, and the team played out the long series of 239 chemical reactions involving the two enzymes with pens and notepads.
“The end result matched the structure of prymnesin perfectly,” Shende said.
Following the cascade of reactions that golden algae use to make their toxin, previously unknown strategies for making chemicals in nature were revealed, Moore said. “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 tomorrow,” he added.
Finding the genes behind the prymnesin toxin could enable more cost-effective monitoring of golden algae blooms. Such monitoring could use tests to detect the PKZILLA genes in the environment, similar to the PCR tests that became known during the COVID-19 pandemic. Improved monitoring could increase preparedness and allow more detailed investigation into the conditions that make blooms more likely.
Fallon said the PKZILLA genes the team discovered are the first genes ever causally linked to the production of a marine toxin in the polyether group that includes prymnesin.
Next, the researchers hope to apply the non-standard screening techniques they used to find the PKZILLA genes to other species that produce polyether toxins. If they can find the genes behind other polyether toxins, such as ciguatoxin, which can affect up to 500,000 people annually, it would open up the same genetic monitoring opportunities for a range of other toxic algae blooms with significant global consequences.
In addition to Fallon, Scripps’ Moore and Shende also co-authored the study with David Gonzalez and Igor Wierzbikci of UC San Diego, along with Amanda Pendleton, Nathan Watervoort, Robert Auber and Jennifer Wisecaver of Purdue University.
