Hansenula polymorpha maltase gene promoter with sigma 70-like elements is feasible for Escherichia coli-based biotechnological applications: Expression of three genomic levansucrase genes of Pseudomonas syringae pv. tomato

PMAL1, the sigma 70-like sequences possessing promoter of the maltase gene of Hansenula polymorpha, was evaluated for its application in heterologous protein production in Escherichia coli. Levansucrases Lsc1, Lsc2 and Lsc3 of Pseudomonas syringae pv. tomato DC3000 were expressed from PMAL1 in E. coli as biotechnologically relevant model proteins. Production of soluble levansucrases with high specific activity confirmed appropriate strength of PMAL1 in a prokaryotic host. As about 90% of levansucrase activity was present in the cytoplasm of E. coli, no levan-synthesis related sucrose intolerance of bacteria was observed. All three levansucrases hydrolyzed and polymerized both, sucrose and raffinose. The raffinose-related activity of levansucrases has not been previously described in P. syringae. The PMAL1 expression system was used to produce Lsc3 protein in E. coli for purification. The purified levansucrase showed much higher affinity for sucrose cleavage (Km = 21 mM) than the levansucrase from P. syringae pv. phaseolicola described in the literature with Km of 160 mM.