Alteration of Product Specificity ofRhodobacter sphaeroidesPhytoene Desaturase by Directed Evolution

Chia-wei Wang, James C. Liao
2001 Journal of Biological Chemistry  
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
more » ... 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%. PCR, polymerase chain reaction; kb, kilobase pair; HPLC, high performance liquid chromatography; LB, Luria-Bertani.
doi:10.1074/jbc.m105786200 pmid:11526111 fatcat:nuaehmvunzgjngssfi3mpj4ase