Presentation Information
[P04-455]Enhancing Ergot Alkaloid Diversity via Cytochrome P450 Redox Partners
○Alefiya Taher Dohadwala1, Garrett Wong1, Wen Shan Yew1 (1. National University of Singapore (Singapore))
Keywords:
Ergot Alkaloids,Cytochrome P450,Alkaloid Biosynthesis,Elymoclavine Monooxygenase,Lysergic Acid,Lysergol
The ergot alkaloids (EAs) and their derivatives have numerous applications in medicine, including the treatment of various neurological conditions, such as dementia and migraines. Their bioactivity is attributed to their common skeleton, the tetracyclic ergoline, which is molecularly similar to the monoamine neurotransmitters (eg. dopamine and serotonin). Due to their psychoactive properties and importance in medicine, it is important to explore this family of therapeutics and their biosynthetic pathways.
EAs are a structurally diverse family produced collectively by multiple fungal lineages. Their biosynthesis pathways diverge at several key branch points. Such divergence is usually attributed to differences in enzyme classes acting on the branch point substrate, or differences in enzyme homologs that demonstrate different specificities. The branch point involving agroclavine is of particular interest as it leads to the creation of the pharmaceutically important ingredients D-lysergic acid and lysergol. While differences in enzyme homologs can explain some diversity arising from the agroclavine branch point, the nature of the enzyme acting on agroclavine could offer an alternative explanation as well.
Agroclavine’s conversion to D-lysergic acid and lysergol is catalysed by cloA, a cytochrome P450 (CYP450) enzyme. CYP450s need redox partners (CRPs) to function. In fungal systems, they typically consist of a CYP450 partner reductase and a cytochrome b5. Some studies have shown that altering the CRPS of plant CYP450s can lead to different products and yields. These findings suggest that changing the CRPs of cloAs could also lead to changes in product titre and diversity.
To test whether changing the CRPs of cloA changes their output, we heterologously expressed cloA in Saccharomyces cerevisiae alongside different combinations of CPRs derived from closely and distantly related fungi. We then fed the recombinant strains agroclavine and monitored the output of the expected oxidation products. We have found that manipulation of CPRs results in wide variation in product outputs and yield.
These results demonstrate that changes in CPRs can lead to product diversity at the agroclavine branch point. They also shed light on how such manipulation can be used to increase the yield of D-lysergic acid and lysergol during bioproduction.
EAs are a structurally diverse family produced collectively by multiple fungal lineages. Their biosynthesis pathways diverge at several key branch points. Such divergence is usually attributed to differences in enzyme classes acting on the branch point substrate, or differences in enzyme homologs that demonstrate different specificities. The branch point involving agroclavine is of particular interest as it leads to the creation of the pharmaceutically important ingredients D-lysergic acid and lysergol. While differences in enzyme homologs can explain some diversity arising from the agroclavine branch point, the nature of the enzyme acting on agroclavine could offer an alternative explanation as well.
Agroclavine’s conversion to D-lysergic acid and lysergol is catalysed by cloA, a cytochrome P450 (CYP450) enzyme. CYP450s need redox partners (CRPs) to function. In fungal systems, they typically consist of a CYP450 partner reductase and a cytochrome b5. Some studies have shown that altering the CRPS of plant CYP450s can lead to different products and yields. These findings suggest that changing the CRPs of cloAs could also lead to changes in product titre and diversity.
To test whether changing the CRPs of cloA changes their output, we heterologously expressed cloA in Saccharomyces cerevisiae alongside different combinations of CPRs derived from closely and distantly related fungi. We then fed the recombinant strains agroclavine and monitored the output of the expected oxidation products. We have found that manipulation of CPRs results in wide variation in product outputs and yield.
These results demonstrate that changes in CPRs can lead to product diversity at the agroclavine branch point. They also shed light on how such manipulation can be used to increase the yield of D-lysergic acid and lysergol during bioproduction.
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