Heterologous Gene Expression in Lactococcus lactis subsp. lactis : Synthesis, Secretion, and Processing of the Bacillus subtilis Neutral Protease

TheBacillus subtilis nprEgenelacking its own promoter sequencewas inserted inthelactococcal expression vector pMG36e.Uponintroduction oftherecombinant plasmid intoLactococcus lactis subsp. lactis strain MG1363,neutral protease activity couldbevisualized bytheappearanceoflargeclearing zonesaround colonies grown on milkagarplates. Bymeasuring theactivities oftheneutral protease andtheintracellular enzyme lactate dehydrogenase inculture supernatants andcell fractions, itwas demonstrated thattheneutral protease was actively secreted intothegrowthmedium.Thiswas corroborated byusingtheWesternblot (immunoblot) technique, whichshowedthepresenceofthematureformoftheneutral protease intheculture supernatant. On thebasis oftheseresults, itisconcluded thattheB.subtilis neutral protease gene was expressed inL.lactis andthatthegeneproduct was secreted intothegrowthmediumandwas apparently correctly processed toproduce a biologically active protein. Thesecretion ofthisparticular enzyme may be helpful inachieving accelerated cheese ripening. Recentyearshaveseenanincreasing interest inthe genetics andphysiology oflactic acidbacteria because of their importance formajoreconomic activities, primarily their useinavariety ofdairy fermentations. Mucheffort has beenputinto thedevelopment ofmethods tomodify these microorganisms genetically. Transformation systems were developed, general-purpose cloning vectors becameavailable, andgeneexpression signals originating fromLactococcuslactis wereanalyzed inourandother laboratories (for a review, seereference 6).