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 <i>Haematococcus pluvialis</i> is constrained by multi-stage cultivation and a rigid cell wall that hinders downstream extraction. The marine diatom <i>Phaeodactylum tricornutum</i>, 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 <i>P. tricornutum</i> through rational metabolic engineering. To address challenges in protein targeting posed by the host's complex plastid architecture, we performed heterologous expression of the <i>Chlamydomonas reinhardtii</i> 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 <i>P. tricornutum</i> 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.