A Lactococcus lactis strain deficient in both its major proteases, intracellular (ClpP) and extracellular (HtrA), was constructed and characterized. This strain, hereafter called clpP-htrA, could be obtained only by conjugation between a clpP donor strain and an htrA recipient strain in the NZ9000 context, allowing heterologous gene expression under the control of the NICE (nisin-controlled expression) system. The clpP-htrA double mutant showed both higher stress tolerance (e.g. high temperature and ethanol resistance) and higher viability than single clpP or htrA mutant strains. In addition, the secretion rate of two heterologous proteins (staphylococcal nuclease Nuc and Nuc-E7) was also higher in clpP-htrA than in the wild-type strain. This strain should be a useful host for high-level production and quality of stable heterologous proteins.
BairdL, GeorgopoulosC.
1990; Identification, cloning, and characterization of the Escherichia coli sohA gene, a suppressor of the HtrA (DegP) null phenotype. J Bacteriol 172:1587–1594
BairdL, LipinskaB, RainaS, GeorgopoulosC.
1991; Identification of the Escherichia coli sohB gene, a multicopy suppressor of the HtrA (DegP) null phenotype. J Bacteriol 173:5763–5770
Bermúdez-HumaránL. G,
LangellaP, MiyoshiA, GrussA, GuerraR. T, Le LoirY,
Montes de Oca-LunaR. 2002; Production of human papillomavirus type 16 E7 protein in Lactococcus lactis . Appl Environ Microbiol 68:917–922[CrossRef]
Bermúdez-HumaránL. G,
LangellaP, CommissaireJ, GilbertS, Le LoirY, L'HaridonR, CorthierG.
2003a; Controlled intra or extracellular production of staphylococcal nuclease and ovine omega interferon in Lactococcus lactis . FEMS Microbiol Lett 224:307–313[CrossRef]
Bermúdez-HumaránL. G,
Cortes-PerezN. G, Le LoirY, GrussA, Rodriguez-PadillaC, Saucedo-CardenasO, LangellaP, Montes de Oca-LunaR. 2003b; Fusion to a carrier protein and a synthetic propeptide enhances E7 HPV-16 production and secretion in Lactococcus lactis . Biotechnol Prog 19:1101–1104[CrossRef]
Foucaud-ScheunemannC, PoquetI.
2003; HtrA is a key factor in the response to specific stress conditions in Lactococcus lactis . FEMS Microbiol Lett 224:53–59[CrossRef]
FreesD, VarmanenP, IngmerH.
2001; Inactivation of a gene that is highly conserved in Gram-positive bacteria stimulates degradation of non-native proteins and concomitantly increases stress tolerance in Lactococcus lactis . Mol Microbiol 41:93–103[CrossRef]
GassonM. J.
1983; Plasmid complements of Streptococcus lactis NCDO 712 and other lactic acid streptococci after protoplast-induced curing. J Bacteriol 154:1–9
KuipersO. P, KleerebezemM,
de RuyterP. J,
de VosW. M. 1998; Quorum sensing-controlled gene expression in lactic acid bacteria. J Biotechnol 64:15–21[CrossRef]
LangellaP, ChopinA.
1989; Conjugal transfer of plasmid pIP501 from Lactococcus lactis to Lactobacillus delbruckii subsp. bulgaricus and Lactobacillus helveticus . FEMS Microbiol Lett 51:149–152
Le LoirY, AzevedoV, OliveiraS. C. 12 other authors2005; Protein secretion in Lactococcus lactis : an efficient way to increase the overall heterologous protein production. Microb Cell Fact 4:2[CrossRef]
MadsenS. M, AlbrechtsenB, HansenE. B, IsraelsenH.
1996; Cloning and transcriptional analysis of two threonine biosynthetic genes from Lactococcus lactis MG1614. J Bacteriol 178:3689–3694
MiyoshiA, PoquetI, AzevedoV. 7 other authors2002; Controlled production of stable heterologous proteins in Lactococcus lactis . Appl Environ Microbiol 68:3141–3146[CrossRef]
PoquetI, SaintV, SeznecE, SimoesN, BolotinA, GrussA.
2000; HtrA is the unique surface housekeeping protease in Lactococcus lactis and is required for natural protein processing. Mol Microbiol 35:1042–1051[CrossRef]
RigoulayC, PoquetI, MadsenS. M, GrussA.
2004; Expression of the Staphylococcus aureus surface proteins HtrA1 and HtrA2 in Lactococcus lactis . FEMS Microbiol Lett 237:279–288
van AsseldonkM,
RuttenG, OtemanM, SiezenR. J, SimonsG,
de VosW. M. 1990; Cloning of usp45 , a gene encoding a secreted protein from Lactococcus lactis subsp. lactis MG1363. Gene 95:155–160[CrossRef]