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

Abstract

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.

Secondary ketocarotenoid astaxanthin biosynthesis in algae: a multifunctional response to stress

“…This mutant harbors a C-T point mutation in the pds gene, leading to the replacement of the leucine residue by a phenylalanine one in position 516. As this mutation confers a higher specific enzyme activity whereas the replacement by an arginine residue reduces this activity, Sandmann et al (1993) and Liu et al (2010) concluded that the leucine residue at the position 516 plays a decisive role in PQ binding. In the E17 mutant, the pds gene expression is not up-regulated while those of bkt and chy-b are enhanced (Lu et al 2010), probably to cope with the higher amount of intermediates produced.…”

“…The astaxanthin production by heterotrophically growing algae can be enhanced by ROS generation system in the medium (Ip and Chen 2005a). Recently, Liu et al (2010) characterized the NF-resistant mutant E17 of C. zofingiensis. This mutant harbors a C-T point mutation in the pds gene, leading to the replacement of the leucine residue by a phenylalanine one in position 516.…”

Chlorella zofingiensis as an Alternative Microalgal Producer of Astaxanthin: Biology and Industrial Potential

“…Mutagenesis has been demonstrated as an effective approach to enhancing the biosynthesis of carotenoids for elevated astaxanthin accumulation in H. pluvialis [68,132]. Recently, enhanced astaxanthin content has also been achieved in C. zofingiensis by mutagenesis [133]. Comprehensive screening with further mutagenesis may produce mutants with even higher astaxanthin content.…”

A New F131V Mutation in Chlamydomonas Phytoene Desaturase Locates a Cluster of Norflurazon Resistance Mutations near the FAD-Binding Site in 3D Protein Models

“…Substitutions at arginine 304 (R304S, R304T, R304H, R304C) of the 580 amino acid PDS protein from the aquatic flowering plant Hydrilla verticillata conferred resistance to norflurazon and the related bleaching herbicide fluridone [17], [18]. A L516F substitution in the 558 amino acid PDS protein of the green alga Chlorella zofingiensis prevented bleaching in media containing 0.25 µM norflurazon; wild type (WT) cells were bleached by 0.05 µM norflurazon [19]. In the cyanobacterium Synechococcus PCC 7942, mutant PDS proteins conferring resistance to norflurazon contain R195P, L320P, V403G and L436R substitutions [11].…”