• Food Science and Biotechnology
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• v.15 no.4
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• pp.485-493
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• 2006

Lactococcus species are noninvasive and nonpathogenic microorganisms that are widely used in industrial food fermentation and as well-known probiotics. They have been modified by traditional methods and genetic engineering to produce useful food-grade materials. The application of genetically modified lactococci in the food industry requires their genetic elements to be safe and stable from integration with endogenous food microorganisms. In addition, selection for antibiotic-resistance genes should be avoided. Several expression and secretion signals have been developed for the production and secretion of useful proteins in lactococci. Food-grade systems composed of genetic elements from lactic acid bacteria have been developed. Recent developments in this area have focused on food-grade selection markers, stabilization, and integration strategies, as well as approaches for controlled gene expression and secretion of foreign proteins. This paper reviews the expression and secretion signals available in lactococci and the development of food-grade markers, food-grade cloning vectors, and integrative food-grade systems.

• Food Science and Biotechnology
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• v.17 no.6
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• pp.1221-1227
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• 2008

The application of the cellulase gene (celA) as a selection marker of food-grade integration system was investigated in Lactobacillus (Lb.) casei, Lactococcus lactis, and Leuconostoc (Leu.) mesenteroides. The 6.0-kb vector pOC13 containing celA from Clostridium thermocellum with an integrase gene and a phage attachment site originating from bacteriophage A2 was used for site-specific recombination into chromosomal DNA of lactic acid bacteria (LAB). pOC13 was also equipped with a broad host range plus replication origin from the lactococcal plasmid pWV01, and a controllable promoter of nisA ($P_{nisA}$) for the production of foreign proteins. pOC13 was integrated successfully into Lb. casei EM116, and pOC13 integrants were easily detectable by the formation of halo zone on plates containing cellulose. Recombinant Lb. casei EM 116::pOC13 maintained these traits in the absence of selection pressure during 100 generations. pOC13 was integrated into the chromosome of L. lactis and Leu. mesenteroides, and celA acted as an efficient selection marker. These results show that celA can be used as a food-grade selection marker, and that the new integrative vector could be used for the production of foreign proteins in LAB.