Functional assignment of Erwinia herbicola EholO carotenoid genes expressed in E$cherichia coil

Erwinia herbicola is a nonphotosynthetic bacterium that is yellow pigmented due to the presence of carotenoids. When the Erwinia carotenoid biosynthetic genes are expressed in Escherichia coli, this bacterium also displays a yellow phenotype. The DNA sequence of the plasmid pPL376, carrying the entire Erwinia carotenoid gene cluster, has been found to contain 12 open reading frames (ORFs). Six of the ORFs have been identified as carotenoid biosynthesis genes that code for all the enzymes required for conversion of farnesyl pyrophosphate (FPP) to zeaxanthin diglucoside via geranylgeranyl pyrophosphate, phytoene, lycopene, [3carotene, and zeaxanthin. These enzymatic steps were assigned after disruption of each ORF by a specific mutation and analysis of the accumulated intermediates. Carotenoid intermediates were identified by the absorption spectra of the colored components and by high pressure liquid chromatographic analysis. The six carotenoid genes are arranged in at least two operons. The gene coding for [~-carotene hydroxylase is transcribed in the opposite direction from that of the other carotenoid genes and overlaps with the gene for phytoene synthase. Communicated by A. Kondorosi B. Hundle 1 • M. Alberti. V. Nievelstein 2 • P. Beyer 2 H. Kleinig 2 ' G.A. Armstrong 3 • D.H. Burke 4 • J.E. Hearst (~]) Department of Chemistry, University of California and Division of Structural Biology, Lawerence Berkeley Laboratory, Berkeley, CA 94720, USA Present addresses: Gallo Center, Department of Neurology, University of California San Francisco, San Francisco, CA 94110, USA 2 Institute of Biology II, Albert-Ludwigs-Universit~it, SchS, nzlestr. 1, 79104 Freiburg, Germany 3 Institute for Plant Sciences, Department of Plant Genetics, Swiss Federal Institute of Technology (ETH), CH-8092 Zurich, Switzerland 4 University of Colorado, Department of Chemistry, Boulder, ColoradoTo whom reprint requests should be addressed