Optimization of transconjugation and characterization of attB integration site for Streptomyces cinnamoneus producing transglutaminase

Abstract

An effective transformation method for molecular genetic studies of Streptomyces cinnamoneus producing transglutaminase was established using intergeneric conjugal transfer based on the bacteriophage φ{symbol}C31 att/int system. The high efficiency of S. cinnamoneus transconjugation was obtained on mannitol soya flour medium containing 50 mM MgCl2, using 2.5 × 108 Escherichia coli as donor and spores treated with heat treatment at 35°C for 10 min as host. By cloning and sequencing the attB integration site, a single attB site within an open reading frame coding for a pirin homologue was located in S. cinnamoneus genome. Its sequence exhibited the highest degree of sequence similarity with that of Streptomyces clavuligerus. These results provide sufficient efficiency to enable conjugal transfer of genetic elements for S. cinnamoneus, and also should facilitate molecular genetic studies for improvement in production ability of transglutaminase used in food industry. © 2014 Versita Warsaw and Springer-Verlag Wien.