Cloning and expression of three new Azotobacter vinelandii genes closely related to a previously described gene family encoding mannuronan C-5-epimerases

The cloning and expression of a family of five modular-type mannuronan C-5- epimerase genes from Azotobacter vinelandii (algE1 to -5) has previously be en reported. The corresponding proteins catalyze the Ca2+-dependent polymer -level epimerization of beta-D-mannuronic acid to alpha-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 Esc herichia call. 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 s ingle 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 corresp onding region in algE4 expressed an active epimerase. Southern blot analysi s of genomic A. vinelandii DNA, using the 5' part of algE2 as a probe, indi cated that all hybridization signals originated from algE1 to -5 or the thr ee new genes reported here.