Abstract
Carotenoids are widespread terpenoid pigments with applications in the food and feed industries. Upon illumination, the gibberellin-producing fungus Fusarium fujikuroi (Gibberella fujikuroi mating population C) develops an orange pigmentation caused by an accumulation of the carboxylic apocarotenoid neurosporaxanthin. The synthesis of this xanthophyll includes five desaturation steps presumed to be catalysed by the carB-encoded phytoene desaturase. In this study, we identified a yellow mutant (SF21) by mutagenesis of a carotenoid-overproducing strain. HPLC analyses indicated a specific impairment in the ability of SF21-CarB to perform the fifth desaturation, as implied by the accumulation of gamma-carotene and beta-carotene, which arise through four-step desaturation. Sequencing of the SF21 carB allele revealed a single mutation resulting in an exchange of a residue conserved in other five-step desaturases. Targeted carB allele replacement proved that this single mutation is the cause of the SF21 carotenoid pattern. In support, expression of SF21 CarB in engineered carotene-producing Escherichia coli strains demonstrated its reduced ability to catalyse the fifth desaturation step on both monocyclic and acyclic substrates. Further mutagenesis of SF21 led to the isolation of two mutants, SF73 and SF98, showing low desaturase activities, which mediated only two desaturation steps, resulting in accumulation of the intermediate zeta-carotene at low levels. Both strains contained an additional mutation affecting a CarB domain tentatively associated with carotenoid binding. SF21 exhibited higher carotenoid amounts than its precursor strain or the SF73 and SF98 mutants, although carotenogenic mRNA levels were similar in the four strains.