Optimising the production of heterologous exported proteins in Lactococcuslactis by inactivation of HtrA, the unique housekeeping surface protease.

The use of bacteria as cell factories to produce heterologous exported prot eins is often limited by extra-cellular proteolysis. We constructed a Lacto coccus lactis mutant strain in which recombinant or heterologous exported p roteins are stable. A previously unknown membrane protease belonging to the HtrA/DegP family (HtrA(LI)) was first identified in L. lactis subsp. lacti s strain IL1403. Inactivation of the chromosomal gene revealed that HtrA(LI ) acts as a surface housekeeping protease by elimination of abnormal and/or misfolded proteins, and that HtrA(LI) is also responsible for the maturati on of natural exported proteins, such as the L. lactis bacteriolysin, AcmA. From an applied point of view, the most important result of our study is t hat in the absence of HtrA(LI), the extra-cellular proteolysis of all teste d proteins, in particular an heterologous one, is completely abolished, and the yield of intact protein is significantly increased. These results sugg est that HtrA(LI) is the sole extra-cellular housekeeping protease in L. la ctis, in agreement with the analysis of the complete IL1403 genome sequence . In the future, the mutant htrA strain should constitute an efficient tool to improve yields of heterologous exported proteins in L. lactis.