Alteration of Product Specificity of Rhodobacter sphaeroides Phytoene Desaturase by Directed Evolution

Abstract

Phytoene desaturases occurring in nature convert phytoene to either neurosporene or lycopene in most eubacteria. Approximately 10% of known phytoene desaturases, as in Rhodobacter, produce neurosporene, whereas the rest produce lycopene. These two types of enzymes, although similar in function, have relatively low similarity (below 60%) in terms of nucleotide or amino acid sequence. The mechanism controlling the product specificity of these enzymes is unclear. Here we used directed evolution to change the product of Rhodobacter sphaeroides phytoene desaturase (crtI gene product), a neurosporene-producing enzyme, to lycopene. Two generations of random mutagenesis were performed, from which three positive mutants were isolated and sequenced. We then used site-directed mutagenesis to determine the effect of each amino acid change. Gathering information from random mutagenesis, we further recombined the beneficial mutations by site-directed mutagenesis and increased the percent of lycopene production to 90%.