Cloning And Expression Of Three NewAzotobacter Vinelandii Genes Closely Related To A Previously Described Gene Family Encoding Mannuronan C-5-Epimerases

Britt Iren Glærum Svanem, Gudmund Skjåk-Bræk, Helga Ertesvåg, Svein Valla

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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 Ca2+-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 ofalgE2 as a probe, indicated that all hybridization signals originated from algE1 to -5 or the three new genes reported here.