Engineering of ketocarotenoid biosynthetic pathway in Chlamydomonas reinhardtii through exogenous gene expression

Microalgal biorefineries have emerged as significant reservoirs of therapeutic compounds, including pigments and proteins. Facilitating a robust circular bioeconomy necessitates the augmentation of pigment synthesis alongside algae biofuel production. Nevertheless, inherent constraints in ketocarotenoid synthesis exist in naturally fast-growing microalgae strains, such as Chlamydomonas reinhardtii. To address this limitation, we overexpressed two pivotal enzymes in the carotenoid biosynthetic pathway, namely β-carotene hydroxylase (crt) and β-carotene ketolase (bkt), in C. reinhardtii utilizing strong promoters to amplify carotenoid production. The genetically modified (GM) microalgae were validated through PCR, Southern hybridization, and Western blot assays, confirming the presence and expression of both genes in the C. reinhardtii strains. These GM lines exhibited a substantial enhancement over wild-type (WT) algae, showcasing a remarkable 5.39-fold increase in β-carotene concentration and twofold increase in total carotenoids compared to the WT microalgae. Notably, the GM microalgae achieved astaxanthin production up to 1.47 ± 0.063 mg/g DCW, a compound absent in WT C. reinhardtii. These findings indicate the successful functionalization of Hematococcus pluvialis genes through nuclear expression in C. reinhardtii, facilitating ketocarotenoid production. This study presents a valuable strategy to boost carotenoid production in microalgae by stable overexpression of two heterologous genes within the nuclear genome of C. reinhardtii. Graphical abstract for the study carried out which represents the in silico plasmid vector designing, algae transformation by electroporation, selection on antibiotic plates, PCR amplification for GM confirmation, Southern hybridization to confirm gene integration, Western blotting to check protein expression, pigment quantification, and algae growth determination.