Branch-point engineering of carotenoid pathways reshapes ketocarotenoid profiles and photosynthetic performance in Chlamydomonas reinhardtii

Abstract

Carotenoid biosynthesis in Chlamydomonas reinhardtii is tightly partitioned into α- and β-branches, yielding lutein and violaxanthin but lacking ketocarotenoids under standard conditions. Previous engineering efforts have focused mainly on β-carotene ketolase (BKT) overexpression to enhance astaxanthin accumulation, but the interplay between pathway branch points and photosynthetic performance remains poorly understood. Here we combined targeted knockouts of β-carotenoid hydroxylase (CHYB) and lycopene ε-cyclase (LCYE) with BKT overexpression to reprogram metabolic flux toward non-hydroxylated ketocarotenoids. BKT expression in the wild-type background (B7) resulted in mixed profiles of canthaxanthin (1.28 ± 0.16 mg L−1), adonirubin (2.19 ± 0.09 mg L−1), astaxanthin (2.39 ± 0.15 mg L−1), whereas CHYB disruption (dc_B10) abolished astaxanthin production and redirected flux predominantly to canthaxanthin (4.44 ± 0.01 mg L−1; 3.47-fold vs. B7). Additional LCYE removal (dcl_B8) eliminated α-branch products and yielded simplified pigment profiles enriched in non-hydroxylated ketocarotenoids. Despite the lack of astaxanthin, dcl_B8 exhibited partial recovery of the chlorophyll content (2.87 ± 0.15 vs. 1.29 ± 0.24 pg cell−1 in dc_B10), PSII efficiency (Fv/Fm = 0.49 vs. 0.19 in dc_B10), and non-photochemical quenching (NPQ, 0.15 vs. 0.016 in dc_B10), while achieving the highest oxygen evolution under saturating light and elevated half-saturation irradiance conditions. These results demonstrate that photosynthetic impairment in ketocarotenoid-accumulating strains is not dictated by the total pigment content but by the specific configuration of carotenoids supporting PSII stability and photoprotection.