Carotenoids and apocarotenoids play significant roles in various biological processes and have valuable applications in the food, feed, and pharmaceutical industries. However, the production of non-natural carotenoids and apocarotenoids with unique properties remains challenging due to limitations in existing biosynthetic pathways. This seminar will explore the diversification of non-natural carotenoid and apocarotenoid pathways using directed evolution techniques, with a particular focus on long-chain (C50) carotenoids. By subjecting the carotenoid pathway enzymes to iterative rounds of mutagenesis, selection, and screening, we have successfully generated a library of enzymes with altered and expanded substrate specificities. However, merely co-expressing these promiscuous enzymes resulted in the production of numerous byproducts, including unwanted natural carotenoids, which made it difficult to extend the pathway and produce compounds that deviate significantly from natural structures. Instead, by generating a series of enzymes with slightly shifted specificities and strategically combining them, we found combinations that limited byproduct formation, enabling the pathway to extend further. This approach allowed us to create a non-natural C50-lycopene specific pathway, which we then further extended to produce a variety of different C50-carotenoids.

Our findings demonstrate that a combination of directed evolution and strategic enzyme selection is an effective approach for diversifying non-natural carotenoid and apocarotenoid pathways, paving the way for the production of novel compounds with potential applications in various industries. Further research could focus on optimizing the directed evolution process and exploring the functional properties of these novel carotenoids and apocarotenoids.

1. Furubayashi M. et al. (2015) A Highly Selective Biosynthetic Pathway to Non-Natural C50 Carotenoids Assembled from Moderately Selective Enzymes. Nature Communications 6, 7534. https://doi.org/10.1038/ncomms8534.
2. Li L.*, Furubayashi M.* et al.. (2019) Construction of a Nonnatural C60 Carotenoid Biosynthetic Pathway. ACS Synthetic Biology 8, 511–20. https://doi.org/10.1021/acssynbio.8b00385.
3. Furubayashi M. and Umeno D. (2022) Use of Directed Enzyme Evolution to Create Novel Biosynthetic Pathways for Production of Rare or Non-Natural Carotenoids. Methods in Enzymology 671 (2022): 351–82. https://doi.org/10.1016/bs.mie.2022.03.008.