Installing a Ketocarotenoid Branch in Phaeodactylum tricornutum via Functional Activation of Chlamydomonas reinhardtii β-Carotene Ketolase

Astaxanthin is a high-value ketocarotenoid antioxidant, but its industrial production from Haematococcus pluvialis is constrained by multi-stage cultivation and a rigid cell wall that hinders downstream extraction. The marine diatom Phaeodactylum tricornutum, which lacks these limitations, represents a promising alternative chassis because it grows fast, lacks a recalcitrant wall, and supports efficient pigment accumulation. This study establishes a functional ketocarotenoid biosynthetic branch in P. tricornutum through rational metabolic engineering. To address challenges in protein targeting posed by the host’s complex plastid architecture, we performed heterologous expression of the Chlamydomonas reinhardtii β-carotene ketolase (CrBKT), fused at its N-terminus to bipartite transit peptides derived from two endogenous proteins. Western blotting and UPLC-MS/MS analysis confirmed that only the transit peptide fused constructs produced stable protein and functional activity, whereas the native CrBKT failed. The rationally engineered strain successfully accumulated ~45 µg/g DCW of canthaxanthin and ~15 µg/g DCW of astaxanthin. Metabolomic profiling revealed a 50% reduction in fucoxanthin, indicating a substantial redirection of metabolic flux from the native pathway toward the engineered ketocarotenoid branch. This work establishes P. tricornutum as a viable platform for ketocarotenoid production and highlights the critical role of evolution-aware plastid targeting in heterologous pathway reconstruction within complex algal systems.