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 ΦC31 att/int system. The high efficiency of S. cinnamoneus transconjugation was obtained on mannitol soya flour medium containing 50 mM MgCl2, using 2.5 × 10 8 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.

Thailandins A and B, New Polyene Macrolactone Compounds Isolated from Actinokineospora bangkokensis Strain 44EHWT, Possessing Antifungal Activity against Anthracnose Fungi and Pathogenic Yeasts

“…To investigate a suitable medium for the production of active secondary metabolites, strain 44EHW T was cultured in 100 mL of four different media. Medium utilized were HA (0.4% yeast extract, 1% malt extract, and 0.4% glucose), NL5 (0.1% NaCl, 0.1% KH 2 PO 4 , 0.05% MgSO 4 ·7H 2 O, 2.5% glycerol, 0.584% l -glutamine, and 0.2% trace elements), MS (2% soya flour and 2% d -mannitol), and SG (2% glucose, 1% soya flour, 0.2% CaCO 3, 0.1% CoCl 2 , and 0.2% l -valine). , Strain 44EHW T was precultured in TSB medium (Difco, Sparks, MD) for 2 d at 28 °C and then further cultivated in the four media at 28 °C for 10 d. Crude extract samples were prepared separately from supernatants and pellets. Supernatants obtained from strain 44EHW T cultivation were adjusted to pH 4 and pH 7, followed by two extractions with an equal volume of ethyl acetate.…”