Cloning and expression of the bacillus licheniformis a-amylase gene in saccharomyces cerevisiae

摘要

The a-amylase gene from Bacillus licheniformis together withudits signal peptide seguence was cloned into a yeast expression vector under the control of the yeast ADH1 promoter and terminator. The resulting construct, pAAMY, when introduced into Saccharomyces cerevisiae cells which when subsequently grown under pH-controlled conditions, produced active a-amylase enzyme. At least 95% of the recombinant amylase is located extracellurarly. Temperature sensitive yeast mutants defective in the specific steps in the secretory pathway were used to show that the native Bacillus signal peptide is capable of effeciently directing the a-amylase through the yeast secretory pathway.ududThe extracellular amylase produced by the yeast was found toudhave similar pH and temperature profiles to the nativeudBacillus enzyme. Using Western blotting and activity gels,udthis extracellular a-amylase fraction was found to beudheterogeneous with respect to molecular weight, varying fromudapproximately 55 to 71 kilodaltons. After treatment withudendoglycosidaseHf the amylase specific bands were resolved to two bands, one major and one minor. The major band corresponded in size to the the control native bacterial enzyme, indicating that most of the heterogeniety in the band size originally observed was due to glycosylation. The second faint amylase band was approximately 3KD larger. This form of the enzyme is likely to represent the unprocessed form of the enzyme with its signal peptide still attached.ududSeveral approaches were taken in an attempt to improve bothudthe plasmid stability and the quantity of the active enzymeudproduced by the yeast cells. These included the isolation ofudyeast mutants using ethyl methane sulphonic acid(EMS), theudtargeted integration of the a-amylase gene into single andudreiterated regions of the yeast chromosomal DNA, and finallyudthe removal of part of the 3' untranslated region of the DNAudfragment carrying the Bacillus a-amylase gene.