title: Food-grade host/vector expression system for Lactobacillus casei based on complementation of plasmid-associated phospho-beta-galactosidase gene lacG.
abstract: A new food-grade host/vector system for Lactobacillus casei based on lactose selection was constructed. The wild-type non-starter host Lb. casei strain E utilizes lactose via a plasmid-encoded phosphotransferase system. For food-grade cloning, a stable lactose-deficient mutant was constructed by deleting a 141-bp fragment from the phospho-beta-galactosidase gene lacG via gene replacement. The deletion resulted in an inactive phospho-beta-galactosidase enzyme with an internal in-frame deletion of 47 amino acids. A complementation plasmid was constructed containing a replicon from Lactococcus lactis, the lacG gene from Lb. casei, and the constitutive promoter of pepR for lacG expression from Lb. rhamnosus. The expression of the lacG gene from the resulting food-grade plasmid pLEB600 restored the ability of the lactose-negative mutant strain to grow on lactose to the wild-type level. The vector pLEB600 was used for expression of the proline iminopeptidase gene pepI from Lb. helveticus in Lb. casei. The results show that the food-grade expression system reported in this paper can be used for expression of foreign genes in Lb. casei.
authors.affiliation: Department of Applied Chemistry and Microbiology, Division of Microbiology, University of Helsinki, Viikinkaari 9, P.O. Box 56, 00014 Helsinki, Finland.
authors.forename: T M
authors.initials: TM
authors.lastname: Takala
authors.forename: P E J
authors.initials: PE
authors.lastname: Saris
authors.forename: S S H
authors.initials: SS
authors.lastname: Tynkkynen
mesh_terms: D000626:Aminopeptidases / Q000235:genetics; D019649:Food Industry; D015964:Gene Expression Regulation, Bacterial; D018014:Gene Transfer Techniques; D005816:Genetic Complementation Test; D005822:Genetic Vectors / Q000235:genetics*; D006026:Glycoside Hydrolases; D007763:Lac Operon; D007780:Lacticaseibacillus casei / Q000201:enzymology* / Q000235:genetics* / Q000378:metabolism; D007785:Lactose / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D008957:Models, Genetic; D009154:Mutation; D010957:Plasmids / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics* / Q000378:metabolism
chemical_list: D006026:Glycoside Hydrolases; D001616:beta-Galactosidase; C022361:6-phospho-beta-galactosidase; D000626:Aminopeptidases; C022450:prolyl aminopeptidase; D007785:Lactose