One amino acid substitution in phytoene desaturase makes Chlorella zofingiensis resistant to norflurazon and enhances the biosynthesis of astaxanthin.

A stable Chlorella zofingiensis mutant (E17) produced by chemical mutagen was characterized with respect to growth, astaxanthin biosynthesis, and phytoene desaturation. The mutant E17 could grow well and produce normal levels of colored carotenoids in the presence of 0.25 microM norflurazon, in which the growth of wild type (WT) cells was greatly limited due to inhibited carotenoid formation. Induced by high-light irradiation or glucose, E17 produced 44 or 36% more astaxanthin than WT when cultured in media without norflurazon. A point mutation (C-T) was revealed to occur in the PDS gene of E17, leading to an amino acid change (L516F) in its coding region. The mutated PDS exhibited 31-fold resistance to norflurazon when compared to WT as determined by an in vitro assay. Surprisingly, the mutated PDS exhibited higher efficiency in converting phytoene to zeta-carotene. No difference in PDS transcripts was found between E17 and WT cells cultured either in normal or induced conditions. In contrast, higher transcript levels of beta-carotene ketolase and hydroxylase were found in the E17 cells. Taken together, we conclude that a point mutation in Chlorella PDS gene makes E17 resistant to norflurazon and synthesize higher amounts of carotenoids including astaxanthin.