Cloning and Expression of Three New Azotobacter vinelandii Genes Closely Related to a Previously Described Gene Family Encoding Mannuronan C-5-Epimerases

Abstract

ABSTRACT The cloning and expression of a family of five modular-type mannuronan C-5-epimerase genes from Azotobacter vinelandii ( algE1 to - 5 ) has previously been reported. The corresponding proteins catalyze the Ca 2+ -dependent polymer-level epimerization of β- d -mannuronic acid to α- l -guluronic acid (G) in the commercially important polysaccharide alginate. Here we report the identification of three additional structurally similar genes, designated algE6 , algE7 , and algY . All three genes were sequenced and expressed in Escherichia coli . AlgE6 introduced contiguous stretches of G residues into its substrate (G blocks), while AlgE7 acted as both an epimerase and a lyase. The epimerase activity of AlgE7 leads to formation of alginates with both single G residues and G blocks. AlgY did not display epimerase activity, but a hybrid gene in which the 5′-terminal part was exchanged with the corresponding region in algE4 expressed an active epimerase. Southern blot analysis of genomic A. vinelandii DNA, using the 5′ part of algE2 as a probe, indicated that all hybridization signals originated from algE1 to - 5 or the three new genes reported here.