Presentation Information
[4ASBA-08]Engineering carotenoid cleavage enzymes for apocarotenoid production in Escherichia coli
○Maiko Furubayashi1 (1. National Institute of Advanced Industrial Science and Technology (AIST) (Japan))
Keywords:
apocarotenoid,carotenoid,enzyme engineering,microbial production,isoprenoid
[Purpose]
Apocarotenoids are a structurally and functionally diverse class of compounds that include aroma molecules, pigments, and retinoid analogues, many of which have important biological and industrial value. Growing interest in these molecules has highlighted the need for microbial platforms that can produce not only known apocarotenoids but also structurally diverse derivatives. Apocarotenoids are generated through the oxidative cleavage of carotenoids by carotenoid cleavage dioxygenases (CCDs). However, the establishment of efficient production systems remains challenging because CCDs differ widely in substrate specificity and catalytic behavior. In this study, we aimed to develop and evaluate an Escherichia coli-based platform for apocarotenoid production through carotenoid cleavage enzyme engineering.
[Method]
CCD genes from diverse organisms, representing a range of substrate specificities, were identified through database searches and synthesized. Expression systems were then constructed in E. coli by combining carotenoid biosynthetic pathways with individual CCDs. In addition, because functional characterization of CCDs can be difficult in whole-cell E. coli systems, we developed a simple lysate-based assay as an alternative method for activity evaluation.
[Results]
The engineered systems enabled the production of corresponding apocarotenoids from multiple carotenoid substrates. In addition to well-known apocarotenoids such as β-ionone, α-ionone, β-cyclocitral, and retinal, we also detected terminally modified apocarotenoid derivatives. The lysate-based assay further facilitated rapid comparison of enzyme activity and substrate utilization. By applying this assay to site-saturation mutagenesis of CCDs, we identified key amino acid residues involved in substrate selectivity.
[Consideration]
Our results suggest that apocarotenoid production is influenced not only by the intrinsic substrate specificity of the cleavage enzyme, but also by the design of the evaluation system and the expression conditions used. These findings highlight the importance of combining enzyme discovery with simple and reproducible assay platforms when engineering carotenoid cleavage enzymes for the production of valuable compounds.
[Conclusion]
Taken together, this study demonstrates that carotenoid cleavage enzyme engineering is an effective strategy for apocarotenoid production in E. coli. The results provide useful insights for the future development of microbial production platforms for structurally diverse apocarotenoids.
Apocarotenoids are a structurally and functionally diverse class of compounds that include aroma molecules, pigments, and retinoid analogues, many of which have important biological and industrial value. Growing interest in these molecules has highlighted the need for microbial platforms that can produce not only known apocarotenoids but also structurally diverse derivatives. Apocarotenoids are generated through the oxidative cleavage of carotenoids by carotenoid cleavage dioxygenases (CCDs). However, the establishment of efficient production systems remains challenging because CCDs differ widely in substrate specificity and catalytic behavior. In this study, we aimed to develop and evaluate an Escherichia coli-based platform for apocarotenoid production through carotenoid cleavage enzyme engineering.
[Method]
CCD genes from diverse organisms, representing a range of substrate specificities, were identified through database searches and synthesized. Expression systems were then constructed in E. coli by combining carotenoid biosynthetic pathways with individual CCDs. In addition, because functional characterization of CCDs can be difficult in whole-cell E. coli systems, we developed a simple lysate-based assay as an alternative method for activity evaluation.
[Results]
The engineered systems enabled the production of corresponding apocarotenoids from multiple carotenoid substrates. In addition to well-known apocarotenoids such as β-ionone, α-ionone, β-cyclocitral, and retinal, we also detected terminally modified apocarotenoid derivatives. The lysate-based assay further facilitated rapid comparison of enzyme activity and substrate utilization. By applying this assay to site-saturation mutagenesis of CCDs, we identified key amino acid residues involved in substrate selectivity.
[Consideration]
Our results suggest that apocarotenoid production is influenced not only by the intrinsic substrate specificity of the cleavage enzyme, but also by the design of the evaluation system and the expression conditions used. These findings highlight the importance of combining enzyme discovery with simple and reproducible assay platforms when engineering carotenoid cleavage enzymes for the production of valuable compounds.
[Conclusion]
Taken together, this study demonstrates that carotenoid cleavage enzyme engineering is an effective strategy for apocarotenoid production in E. coli. The results provide useful insights for the future development of microbial production platforms for structurally diverse apocarotenoids.
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