Co-overexpression of prlF increases cell viability and enzyme yields in recombinant Escherichia coli expressing Bacillus stearothermophilus alpha-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 systems: (i) a high copy number plasmid with the Bacillus stearothermophilus 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 pL promoter-controlled gene expression. In addition, protease mutants (lon-) of E. coli C600 were used to evaluate the influence of the Lon protease on net enzyme formation and activity degradation during batch fermentations. Our results show that alpha-amylase synthesis occurred during exponential growth and ceased in the stationary phase. While strong promoters on high copy number plasmids severely impaired cell viability, resulting in culture lysis at mid-log phase, co-overexpression of prlF greatly improved cell viability, as well as the yield and specific production of alpha-amylase for the expression constructs considered. lon deficiency slightly increased amylase stability during the late stationary phase. However, the specific productivity of lon- strains was only about 40-60% that of the isogenic E. coli C600 equivalent.
Minas
W
W
Institut für Biotechnologie, ETH-Hönggerberg, Zürich, Switzerland.
Bailey
J E
JE
D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D005285:Fermentation; D015870:Gene Expression; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D009154:Mutation; D010957:Plasmids; D011994:Recombinant Proteins / Q000096:biosynthesis; D000516:alpha-Amylases / Q000096:biosynthesis*
D011994:Recombinant Proteins; D000516:alpha-Amylases; D010450:Endopeptidases