In their quest to understand how marine algae produce complex toxins, scientists at UC San Diego’s Scripps Institution of Oceanography have uncovered the largest protein ever found in biology. This discovery of the unique biological system developed by the algae to generate its sophisticated toxin has unveiled new techniques for chemical assembly, which could lead to advancements in medicine and material science.
During their research on how a certain algae species, Prymnesium parvum, generates a toxin responsible for significant fish fatalities, the scientists identified a protein they dubbed PKZILLA-1.
“This is the Mount Everest of proteins,” remarked Bradley Moore, a marine chemist at Scripps and the leading author of a new study showcasing these findings. “This broadens our understanding of biological capabilities.”
PKZILLA-1 exceeds the size of titin, the previous record-holder found in human muscles, by 25%. Titin can reach up to 1 micron in length (0.0001 cm or 0.00004 inches).
The study, published in Science and supported by the National Institutes of Health and the National Science Foundation, highlights that both PKZILLA-1 and another sizeable protein, PKZILLA-2, are crucial for synthesizing prymnesin, the large and complex molecule acting as the algae’s toxin. The research also pinpointed unusually expansive genes enabling Prymnesium parvum to construct these proteins.
Identifying the genes responsible for prymnesin production could enhance surveillance of harmful algal blooms from this species by enabling water quality tests focused on the genes instead of the toxins themselves.
“By detecting the genes rather than the toxin, we could identify blooms before they occur instead of only recognizing them after toxins are released,” explained Timothy Fallon, a postdoctoral researcher in Moore’s lab and co-first author of the paper.
Discovering PKZILLA-1 and PKZILLA-2 has unveiled the algae’s intricate cellular processes for synthesizing these uniquely structured toxins. This enhanced insight into toxin production might be beneficial for scientists aiming to create new compounds for either medical or industrial use.
“By understanding how nature has mastered its chemical processes, we, as scientists, gain the potential to apply these insights to create beneficial products, whether it’s a new anti-cancer medication or a novel fabric,” stated Moore.
Prymnesium parvum, widely known as golden algae, is a unicellular organism found globally in both freshwater and saltwater environments. Blooms of this algae lead to substantial fish deaths due to its toxin, prymnesin, which harms the gills of fish and other aquatic life. In 2022, a golden algae bloom resulted in the death of 500-1,000 tons of fish in the Oder River between Poland and Germany. This microorganism is known to disrupt aquaculture in locations ranging from Texas to Scandinavia.
Prymnesin falls within a category of toxins known as polyketide polyethers, which includes brevetoxin B—responsible for the red tides in Florida—and ciguatoxin, which poses risks to reef fish in the South Pacific and Caribbean. These toxins rank among the largest and most complex molecular structures in biology, and researchers have struggled for decades to comprehend how microorganisms produce such substantial and intricate molecules.
Starting in 2019, Moore, Fallon, and Vikram Shende, another postdoctoral researcher in Moore’s lab and co-first author of the paper, commenced efforts to delineate how golden algae synthesize their toxin, prymnesin, at biochemical and genetic levels.
The study team began by decoding the genome of golden algae and searching for the genes responsible for prymnesin production. Traditional genome analysis did not yield significant results, prompting the research team to switch to alternative methods suited for identifying extraordinarily long genes.
“We successfully located the genes, revealing that this alga relies on massive genes to create sizeable toxic molecules,” said Shende.
After identifying the PKZILLA-1 and PKZILLA-2 genes, the team investigated what these genes encoded to confirm their role in toxin production. Fallon noted that the team could interpret the coding regions of the genes like musical notation, translating them into the sequence of amino acids that formed the proteins.
Upon assembling the PKZILLA proteins, the researchers were astonished by their sizes. PKZILLA-1 boasts a record-breaking mass of 4.7 megadaltons, while PKZILLA-2 is also notably large at 3.2 megadaltons. In comparison, titin, the previous record-holder, reaches up to 3.7 megadaltons—making it roughly 90 times larger than the average protein.
Subsequent experiments confirmed that golden algae indeed produce these enormous proteins in their life cycle, leading the team to examine if these proteins played a role in synthesizing the prymnesin toxin. The PKZILLA proteins serve as enzymes, initiating chemical reactions, and the team meticulously detailed the lengthy chain of 239 chemical reactions executed by the two enzymes using pens and notepads.
“The final product corresponded exactly with the structure of prymnesin,” shared Shende.
An analysis of the reaction series utilized by golden algae to produce their toxin unveiled previously unknown natural strategies for chemical production, according to Moore. “We hope to leverage our understanding of how nature synthesizes these complicated chemicals to explore new chemical possibilities in the lab for future medicines and materials,” he added.
Uncovering the genes linked to prymnesin could facilitate more economical monitoring of golden algae blooms. This monitoring could employ tests to detect PKZILLA genes in the environment, similar to the PCR tests widely used during the COVID-19 pandemic. Improved monitoring may enhance preparedness and offer a deeper insight into the factors contributing to bloom occurrences.
Fallon noted that the PKZILLA genes identified by the research team are the first genes ever directly associated with the production of any marine toxins within the polyether classification, including prymnesin.
The researchers aim to apply the non-traditional screening techniques used to discover the PKZILLA genes to other species that produce polyether toxins. If they can identify the genes associated with additional polyether toxins, like ciguatoxin—which may impact up to 500,000 individuals each year—they could enable the same genetic monitoring strategies for a variety of toxic algal blooms with significant global ramifications.
In addition to Fallon, Moore, and Shende from Scripps, the study was co-authored by David Gonzalez, Igor Wierzbikci from UC San Diego, and Amanda Pendleton, Nathan Watervoort, Robert Auber, and Jennifer Wisecaver from Purdue University.
