Identification of chloroplast transit peptides inDunaliella salina and their application for heterologous astaxanthin production.

Dunaliella salinais a promising green cell factory, but efficient chloroplast targeting of exogenous proteins requires suitable chloroplast transit peptides (cTPs). In this study, six endogenous cTPs fromD. salinawere shown to direct mCherry import into the chloroplast, and downstream extensions beyond the cleavage site enhanced import efficiency and accuracy. To validate their utility, DAT-2 and DAT-3 were engineered to expressHaematococcus lacustrisβ-carotene hydroxylase and ketolase fused to the cTPs derived from light-harvesting complex II chlorophyll a/b binding protein 1 and tocopherol cyclase, respectively, each extended by 23 amino acids, and DAT-1 expressed the enzymes without any cTP. Under high light (500  μmol photons m-2 s-1), DAT-2 produced 85.7  μg/g astaxanthin, 32.9 % higher than DAT-1, while DAT-3 produced only 37.6  μg/g, suggesting that inappropriate cTP impaired import. This study systematically elucidates the function ofD. salinacTPs and the critical role of downstream extensions, demonstrating the value of suitable cTPs in genetic engineering.