(326b) Comparative Functional Analysis of Different ?-Carotene Ketolase (bkt) Variants for Enhanced Biosynthesis of Natural Astaxanthin in Haematococcus algae.

Astaxanthin is a valuable ketocarotenoid with diverse applications in the food, pharmaceutical, and cosmetic industries. While naturally produced by certain microorganisms and flowering plants, limitations such as slow growth rates and extraction difficulties hinder their utility. Studies suggest that astaxanthin from petrochemical precursors lacks controllable stereochemistry, rendering it unsuitable for human consumption. Therefore, efficient production methods for natural astaxanthin are in demand. The green alga Haematococcus accumulates significant astaxanthin levels in its immobile (akinete) stage. While numerous studies have investigated factors influencing astaxanthin yield in Haematococcus cells, the role of the alga itself is often overlooked. Astaxanthin biosynthesis in Haematococcus involves at least seven enzymatic steps, with the β-carotene ketolase (bkt) enzyme being pivotal in ketocarotenoid production. Recent genome sequencing of Haematococcus pluvialis has unveiled up to six distinct copies of the bkt gene in the nuclear genome. It is hypothesized that these variant copies may possess unique functional roles in astaxanthin production. Preliminary analyses of various Haematococcus isolates have revealed up to a ten-fold variation in astaxanthin accumulation among four different isolates. We selected these isolates and cloned the bkt variants within each to ascertain their contribution to astaxanthin production. Bkt activity was assayed in E. coli through co-expression with a heterologous zeaxanthin biosynthesis pathway, and the resulting astaxanthin products were measured using HPLC. Our study provides insights into the biosynthetic capability of each bkt variant in different isolates for natural astaxanthin production. Additionally, it lays the groundwork for engineering a Haematococcus strain with high natural astaxanthin biosynthetic activity, utilizing the CRISPR-Cas gene-editing technique.