Scalable biosynthesis of the seaweed neurochemical kainic acid
Inventors
Moore, Bradley S. • Chekan, Jonathan R. • McKinnie, Shaun M. K.
Assignees
University of California San Diego UCSD
Publication Number
US-12365928-B2
Publication Date
2025-07-22
Expiration Date
2039-12-03
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Abstract
Provided herein are compounds of Formulas (la), (lb), (II) to (VIII), and salts thereof, and methods of making the same. Also provided herein are recombinant proteins useful in the production of compounds disclosed herein, polynucleotides encoding the same, and cells comprising the same.
Core Innovation
Kainic acid is a well-known neuropharmacological agent important for studying ionotropic glutamate receptors, especially the kainate receptor, in the central nervous system. There has been historical worldwide shortage of this seaweed natural product, prompting the development of numerous chemical syntheses. Despite its discovery over 50 years ago and many synthetic routes, little was known about how kainic acid is biosynthesized by seaweeds. Red macroalgae genomes are difficult to sequence due to contamination from marine microbes, limiting biosynthetic gene discovery.
The invention solves the need for improved and scalable methods for the production of kainic acid and kainic acid lactone. The inventors discovered a two-enzyme biosynthetic pathway to kainic acid in red macroalgae such as Digenea simplex and Palmaria palmata. The biosynthetic genes are co-clustered in the genomes of these species. The invention also reports a series of scalable biosynthetic strategies including enzyme total synthesis, fermentation in a heterologous host, chemoenzymatic synthesis, and biotransformation to efficiently generate kainic acid on a gram scale.
The biosynthetic pathway involves the N-prenylation of L-glutamate with dimethylallyl pyrophosphate (DMAPP) by a KabA polypeptide producing prekainic acid, which is cyclized by KabC, an alpha-ketoglutarate-dependent dioxygenase, to yield kainic acid. The enzymatic activities were validated using recombinant proteins, confirming the proposed pathway and showing efficient enzymatic conversion. The invention further enables the development of recombinant cell lines and biotransformation methods for scalable kainic acid production.
Claims Coverage
The claims describe processes for preparing kainic acid compounds and compositions related to the key biosynthetic enzymes KabA and KabC with focus on enzymatic cyclization, recombinant expression, and chemical synthesis steps. There are multiple inventive features covering the enzymatic cyclization, chemical synthesis, recombinant microorganism production, and nucleic acid compositions.
Enzymatic cyclization of a compound of Formula II to produce kainic acid
A process comprising cyclizing a compound of Formula II, where substituents R1-R6 meet defined criteria, by contacting it with a recombinant KabC polypeptide to produce a kainic acid compound of Formula Ia or Ib.
Chemical synthesis of precursor compound Formula II via imine formation and reduction
A process preparing compound of Formula II by contacting a compound of Formula III with a compound of Formula IV to produce an imine, followed by reducing the imine.
Use of recombinant microorganisms expressing KabC for production of kainic acid
Processes that involve contacting compound of Formula II or other precursors with recombinant microorganisms expressing KabC, optionally in combination with KabA, to produce kainic acid.
Processes using recombinant KabA and KabC polypeptides and substrates for biosynthesis
Producing kainic acid by contacting compounds of Formula III and Formula V with recombinant KabA and KabC polypeptides and α-ketoglutarate.
Recombinant nucleic acids encoding KabA and KabC polypeptides
Recombinant polynucleotides and expression vectors encoding KabA and KabC polypeptides with specified sequence identity ranges to disclosed SEQ ID NOs.
Recombinant cells expressing KabA and KabC polypeptides for kainic acid production
Recombinant cells, including bacteria like E. coli, comprising KabA and/or KabC polynucleotides or vectors, capable of producing kainic acid upon culturing and substrate contact.
The patent claims cover inventive methods for enzymatic cyclization of precursors to kainic acid using KabC polypeptides, routes for chemical synthesis of intermediates, recombinant microorganism-based production methods, and compositions encoding and expressing the key biosynthetic enzymes KabA and KabC, enabling efficient and scalable biosynthesis of kainic acid compounds.
Stated Advantages
The biotransformation strategy provides a biocompatible, green chemistry alternative for producing kainic acid with reduced steps and environmentally friendly reagents.
Enzymes KabA and KabC catalyze challenging transformations with high stereo- and regiospecificity, simplifying synthesis and improving yield and purity.
The scalable biosynthetic approaches enable gram-scale production of kainic acid, overcoming previous shortages and synthetic complexity.
Documented Applications
Use of kainic acid as a neuropharmacological agent to study ionotropic glutamate receptors and related neurological research.
Production of kainic acid to generate mouse models for neurological disease studies, including temporal lobe epilepsy.
Development of biotechnological production methods for kainic acid and related kainoids, including domoic acid and kainic acid analogs, for research and environmental toxin studies.
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