title: Gene knockout and overexpression analysis revealed the role of N-acetylmuramidase in autolysis of Lactobacillus delbrueckii subsp. bulgaricus ljj-6.
abstract: Autolysis of lactic acid bacteria (LAB) plays a vital role in dairy processing. During cheese making, autolysis of LAB affects cheese flavor development through release of intracellular enzymes and restricts the proliferation of cells in yogurt fermentation and probiotics production. In order to explore the mechanism of autolysis, the gene for the autolytic enzymes of L. bulgaricus, N-acetylmuramidase (mur), was cloned and sequenced (GenBank accession number: KF157911). Mur gene overexpression and gene knockout vectors were constructed based on pMG76e and pUC19 vectors. Recombinant plasmids were transformed into L. bulgaricus ljj-6 by electroporation, then three engineered strains with pMG76e-mur vector and fifteen engineered strains with pUC19-mur::EryBII were screened. The autolysis of the mur knockout strain was significantly lower and autolysis of the mur overexpressed strain was significantly higher compared with that of the wild type strain ljj-6. This result suggested that the mur gene played an important role in autolysis of L. bulgaricus. On the other hand, autolytic activity in a low degree was still observed in the mur knockout strain, which implied that other enzymes but autolysin encoded by mur were also involved in autolysis of L. bulgaricus.
authors.affiliation: Key Laboratory of Agro-Food Processing and Quality Control, Institute of Agro-Food Science and Technology, Chinese Academy of Agricultural Science, Beijing, P.R. China.
authors.forename: Xiao-Yang
authors.initials: XY
authors.lastname: Pang
authors.affiliation: Key Laboratory of Agro-Food Processing and Quality Control, Institute of Agro-Food Science and Technology, Chinese Academy of Agricultural Science, Beijing, P.R. China.
authors.forename: Wen-Ming
authors.initials: WM
authors.lastname: Cui
authors.affiliation: Key Laboratory of Agro-Food Processing and Quality Control, Institute of Agro-Food Science and Technology, Chinese Academy of Agricultural Science, Beijing, P.R. China.
authors.forename: Lu
authors.initials: L
authors.lastname: Liu
authors.affiliation: Key Laboratory of Agro-Food Processing and Quality Control, Institute of Agro-Food Science and Technology, Chinese Academy of Agricultural Science, Beijing, P.R. China.
authors.forename: Shu-Wen
authors.initials: SW
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Agro-Food Processing and Quality Control, Institute of Agro-Food Science and Technology, Chinese Academy of Agricultural Science, Beijing, P.R. China.
authors.forename: Jia-Ping
authors.initials: JP
authors.lastname: Lv
mesh_terms: D001329:Autolysis / Q000235:genetics*; D001483:Base Sequence; D002611:Cheese / Q000382:microbiology*; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D005285:Fermentation / Q000235:genetics*; D055786:Gene Knockout Techniques; D006026:Glycoside Hydrolases / Q000096:biosynthesis / Q000235:genetics*; D048668:Lactobacillus delbrueckii / Q000201:enzymology / Q000235:genetics*; D008969:Molecular Sequence Data; D017422:Sequence Analysis, DNA; D015014:Yogurt / Q000382:microbiology
chemical_list: D004269:DNA, Bacterial; D006026:Glycoside Hydrolases; C026255:endo-N-acetylmuramidase
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