Growth Defects Of Escherichia Coli Cells Which Contain The Gene Of An -Amylase from Bacillus coagulans on a Multicopy Plasmid

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Abstract
An .alpha.-amylase gene from B. coagulans was previously cloned in E. coli and directs the synthesis of an enzymically active protein of 60,000 Dal [daltons]. In 1 particular E. coli host, strain HB101, amylase accumulates in the periplasmic space. To study the processing and the location of the amylase, plasmid pAMY2 was introduced into E. coli 188, which is a strain constitutive for alkaline phosphatase, a periplasmic marker, and for .beta.-galactosidase, a cytoplasmic marker. Abnormally large amounts of .alpha.-amylase and .beta.-galactosidase was found in the culture fluid of cells grown in rich medium. A severe growth defect was found when cells containing pAMY2 were grown in maltose and glycerol media, while the ability to grow on glucose remained normal. This defect could be reversed by 2 types of spontaneous mutations. Mutations in the first class are located on the plasmid and correspond to the insertional inactivation of the amylase gene by IS1. Mutations in the second class are located on the host chromosome. The synthesis and export of B. coagulans .alpha.-amylase may be deleterious to E. coli, especially in media containig maltose or glycerol as sole C source.

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