A team of scientists at Scripps Institution of Oceanography at the University of California San Diego and the J. Craig Venter Institute (JCVI) has developed a new way to produce kainic acid, a natural seaweed neurochemical and powerful reagent used in brain research.
In a new study published April 17 in the journal Angewandte Chemie International Edition, the scientists were able to sequence the genome of a seaweed known to produce kainic acid, and they identified the enzymes responsible for production of the natural chemical. They also utilized biotechnology to develop a cheaper and more efficient way to produce the seaweed chemical in gram quantities in the lab—a major breakthrough with implications for large-scale production of kainic acid in the future.
“Because kainic acid is an important molecule still used in research today, we wanted to develop a new way to produce it,” said lead author Jonathan Chekan, a postdoctoral fellow at the Center for Marine Biotechnology and Biomedicine at Scripps Oceanography. “This biotransformation approach allows us to quickly produce kainic acid in a cheaper and more environmentally friendly way than traditional chemical synthesis.”
Kainic acid is found in certain types of red algae, or seaweed, around the world. Centuries ago, people in Japan discovered that eating a local seaweed known as Digenea simplex could treat parasitic worm infections. In the 1950s, scientists identified kainic acid as the active compound in D. simplex seaweed, leading to its clinical use as an anti-parasitic drug in Japan. Kainic acid activates excitatory glutamate receptors that control cell-to-cell communication in the brain and are critical for short-term memory. In recent years, kainic acid has been used as a research tool to study human neurological conditions such as epilepsy and Alzheimer’s disease.
Worldwide shortages of the seaweed natural product in 2000 prompted researchers to use chemical synthetic versions of kainic acid. While more than 70 synthetic versions exist, they require a minimum of six steps to produce, have a low production rate, and are very expensive. These factors have limited many scientists from using kainic acid for research.
The new kainic acid production method described by the authors could open the door for large-scale biotechnological production of the chemical. Their research builds upon the team’s recent work on discovering the genetic origin of domoic acid, a potent neurotoxin produced by planktonic microalgae. The domoic acid study helped the scientists develop a hypothesis for how a chemical compound like kainic acid is formed within a living organism—in this case seaweed—in a process known as biosynthesis.
“Our work represents the first example of a validated cluster of genes that code for the production of a seaweed natural product drug. This knowledge has enabled a new and more affordable supply of kainic acid for researchers studying human neurological diseases,” said senior author Bradley Moore, a professor at Scripps Oceanography and the Skaggs School of Pharmacy and Pharmaceutical Sciences at UC San Diego.
The researchers began the study by sequencing the genomes of two kainic acid producing seaweeds, D. simplex and Palmaria palmata, for the first time ever. Very few seaweed genomes have been sequenced due to the high repeat nature of algal genomes, the relative unknown sizes of seaweed genomes, and the technical challenges in obtaining seaweed samples free of contaminating microbes and other algae.
These challenges were addressed during the sequencing of D. simplex through use of the new PromethION sequencer from Oxford Nanopore Technologies. This equipment has long read sequencing technology, enabling the researchers to assemble the genome of the seaweed and allowing them to observe biosynthesis gene clustering for the first time in a seaweed. They used a smaller version of the PromethION called MinION to sequence P. palmata.
“Nanopore sequencing has been touted as the ‘holy grail’ of sequencing and only recently has this become a reality,” said study co-author Todd Michael, a professor and director of informatics at JCVI.
He worked on sequencing and assembling the seaweed genome where the new pathway was discovered.
“This is one of the first published studies using the promethION to sequence and assemble a genome, and the first red algae genome,” said Michael. “The sequencing and assembly with the long reads and the large amount of data enabled us to assemble the genes in the kainic acid pathway.”
The researchers used the newly discovered seaweed enzyme pathway to develop a concise, two-step process to produce kainic acid. They expressed the seaweed genes in the bacterium Escherichia coli and found that the enzymes were able to form kainic acid in the fermentation.
“It was only when we established a method in which we employed a combination of chemical synthesis and bacterial fermentation with the seaweed gene that we produced gram quantities of pure kainic acid,” said Moore. “I was just totally shocked to see so much kainic acid produced, so simply and so elegantly.”
The researchers are now moving forward to commercialize this biotechnological method to produce kainic acid and other neurochemicals in a more affordable and clean way. They said this kainic acid production method could be rapidly implemented on an industrial scale in the near future, which could provide more opportunities for research on human brain diseases.
In addition to Chekan, Moore, and Michael, the study was authored by Shaun McKinnie and Malia Moore of Scripps Oceanography and Shane Poplawski of JCVI.
This study was funded by the U.S. National Institutes of Health (R01-GM085770), the Simons Foundation Fellowship of the Life Science Research Foundation, and the Natural Sciences and Engineering Research Council of Canada.