High-level expression of a recombinant protein in Klebsiella planticola owing to induced secretion into the culture medium.
The Tn5-based transposon Tn5-KIL3 (Miksch et al. 1997c) bearing the kil gene of the ColE1 plasmid of Escherichia coli, which mediates controlled export of periplasmic proteins into the culture medium, was stably integrated into the chromosome of Klebsiella planticola with high transposition frequency. A Bacillus hybrid beta-glucanase located on an RSF1010-derived plasmid was mobilized from E. coli to K. planticola and used as a reporter protein to select strains with high expression and secretion competence. During fermentation experiments it was shown that the production of beta-glucanase in K. planticola was improved to an unexpectedly high level when the enzyme was secreted into the medium. Due to the stationary-phase promoter used for the expression of the kil gene the secretion of beta-glucanase into the medium started at the transition from the exponential to the stationary phase, as in E. coli, and the fraction of secreted protein reached 90%. The results showed that K. planticola may represent an interesting organism for the production of heterologous proteins.
Miksch
G
G
Universität Bielefeld, Technische Fakultät, Germany. gmi@fermtech.techfak.uni-bielefeld.de
Neitzel
R
R
Friehs
K
K
Flaschel
E
E
D001407:Bacillus / Q000201:enzymology; D018533:Biomass; D003470:Culture Media; D004251:DNA Transposable Elements / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005285:Fermentation; D043326:Glucan 1,3-beta-Glucosidase; D007709:Klebsiella / Q000201:enzymology* / Q000235:genetics; D016254:Mutagenesis, Insertional; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013997:Time Factors; D001617:beta-Glucosidase / Q000378:metabolism*
D003470:Culture Media; D004251:DNA Transposable Elements; D011994:Recombinant Proteins; D001617:beta-Glucosidase; D043326:Glucan 1,3-beta-Glucosidase