Overexpression of peptidases in Lactococcus and evaluation of their release from leaky cells.
Walker and Klaenhammer (2001) developed a novel expression system in Lactococcus lactis that facilitated the release of beta-galactosidase (117 kDa monomer) without the need for secretion or export signals. The system is based on the controlled expression of integrated prophage holin and lysin cassettes via a lactococcal bacteriophage phi31 transcriptional activator (Tac31A) that resides on a high-copy plasmid. Approximately 85% of beta-galactosidase activity was detected in the supernatant of leaky lactococci without evidence of hindered growth, cell lysis, or membrane damage. The objective of this study was to determine if intracellular peptidases were externalized from leaky lactococci. Five L. lactis peptidases (PepA, PepC, PepN, PepO and PepXP) and two Lactobacillus helveticus peptidases (PepN and PepO) were cloned and overexpressed on two high-copy vectors. The lactococcal peptidases were also cloned into the high-copy vector that contained the Tac31A transcriptional activator to determine if they were externalized from the leaky prophage-containing L. lactis subsp. lactis strain NCK203. Two of the lactococcal peptidases (PepA and PepO) required an additional strong promoter (Lactobacillus paracasei P144) and optimized assay conditions to detect enzyme activity. Results showed different levels of enzymatic overexpression associated with the cellular fraction (2 to 250-fold increases in activity) and negligible amounts of activity present within the supernatant fraction (0 to 6% of total peptidase activity). The lactococcal phage-based protein release mechanism did not facilitate the externalization of the lactococcal peptidases investigated in this study.
Tuler
T R
TR
Department of Food Science, Southeast Dairy Foods Research Center, North Carolina State University, Raleigh 27695, USA.
Callanan
M J
MJ
Klaenhammer
T R
TR
D000626:Aminopeptidases / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins; D003001:Cloning, Molecular; D003374:Coumarins / Q000378:metabolism; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D005822:Genetic Vectors; D043384:Glutamyl Aminopeptidase; D017045:Lactococcus / Q000201:enzymology* / Q000235:genetics; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics; D008666:Metalloendopeptidases / Q000235:genetics / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism; D010447:Peptide Hydrolases / Q000235:genetics* / Q000378:metabolism; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D013379:Substrate Specificity; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D003374:Coumarins; D009842:Oligopeptides; D004262:DNA Restriction Enzymes; D010447:Peptide Hydrolases; D000626:Aminopeptidases; D043384:Glutamyl Aminopeptidase; D008666:Metalloendopeptidases; C080385:oligopeptidase PepO; C028743:7-amino-4-methylcoumarin