Use of Tn5tac1 to clone a pel gene encoding a highly alkaline, asparagine-rich pectate lyase isozyme from an Erwinia chrysanthemi EC16 mutant with deletions affecting the major pectate lyase isozymes

Erwinia chrysanthemi mutant CUCPB5047, delta(pelA pelE) delta(pelB pelC)::28bp delta(pelX) delta 4bp pehX::omega Cmr, was constructed, mutated with Tn5tac1, and screened for isopropyl-beta-D-thiogalactopyranoside-dependent pectate lyase (Pel) production. A Kmr SacI fragment from the hyperexpressing Pel+ mutant CUCPB5066 was cloned into Escherichia coli and sequenced. The gene identified, pelL, encodes a novel, asparagine-rich, highly alkaline enzyme that is similar in primary structure to PelX and in enzymological properties to PelE.