Expression and secretion of Bacillus polymyxa neopullulanase in Saccharomyces cerevisiae

We have isolated the gene encoding the neopullulanase enzyme from Bacillus polymyxa CECT 155. It consists of an open reading frame of 1545 bp that cou ld code for a protein of 515 amino acids. This open reading frame was expre ssed in Bacillus subtilis and the corresponding transformants produced extr acellular neopullulanase. The neopullulanase gene was also expressed in Sac charomyces cerevisiae placing it under the control of the yeast actin gene (ACT1) promoter. Clones containing the intact neopullulanase gene, includin g its own bacterial signal sequence, gave rise to the synthesis of active, but intracellular, enzyme by S. cerevisiae transformants. When sequences sp ecifying the signal sequence and leader region of the yeast mating pheromon e alpha-factor (MF alpha 1) were fused upstream of the gene encoding the ne opullulanase enzyme, the enzyme was secreted by S. cerevisiae. The secreted protein presented the same biochemical properties and the same apparent mo lecular mass as the Bacillus polymyxa original enzyme. The predicted amino acid sequence of the neopullulanase protein contained sequence motifs conse rved among amylolytic enzymes. Northern blot analysis indicated that the tr anscription of the neopullulanase gene in B. polymyxa was induced by the pr esence of the substrate, pullulan, in the culture, and was repressed by glu cose. (C) 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. Ail rights reserved.