CO-OVEREXPRESSION OF PRLF INCREASES CELL VIABILITY AND ENZYME YIELDS IN RECOMBINANT ESCHERICHIA-COLI EXPRESSING BACILLUS-STEAROTHERMOPHILUSALPHA-AMYLASE

The effects on cloned amylase production of co-overexpression of prlF, a gene that appears to interact with the sec protein export machinery in Escherichia coli, was investigated by comparing three expression s ystems: (i) a high copy number plasmid with the Bacillus stearothermop hilus alpha-amylase gene (amyS) cloned with its promoter downstream of the lac promoter; (ii) a pBR322-based vector with amyS under control of the indigenous Bacillus promoter; and (iii) a temperature-inducible vector with runaway replicon and lambda p(L) promoter-controlled gene expression. In addition, protease mutants (lon(-)) of E. coli C600 we re used to evaluate the influence of the Lon protease on net enzyme fo rmation and activity degradation during batch fermentations. Our resul ts show that alpha-amylase synthesis occurred during exponential growt h and ceased in the stationary phase. While strong promoters on high c opy number plasmids severely impaired cell viability, resulting in cul ture lysis at mid-log phase, co-overexpression of prlF greatly improve d cell viability, as well as the yield and specific production of alph a-amylase for the expression constructs considered. ion deficiency sli ghtly increased amylase stability during the late stationary phase. Ho wever, the specific productivity of lon(-) strains was only about 40-6 0% that of the isogenic E. coli C600 equivalent.