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J. Biol. Chem., Vol. 276, Issue 44, 41161-41164, November 2, 2001

This Article Full Text Full Text (PDF) All Versions of this Article: 276/44/41161    most recent M105786200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Wang, C.-w. Articles by Liao, J. C. Search for Related Content PubMed PubMed Citation Articles by Wang, C.-w. Articles by Liao, J. C. Social Bookmarking                      What's this?

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

Chia-wei Wang and James C. Liao

From the Department of Chemical Engineering, University of California, Los Angeles, California 90095-1592

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%.

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