• 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.

• 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.

• Journal of Microbiology and Biotechnology
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• v.16 no.11
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• pp.1799-1808
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• 2006

A food-grade integration vector based on site-specific recombination was constructed. The 5.7-kb vector, pIMA20, contained an integrase gene and a phage attachment site originating from bacteriophage A2, with the ${\alpha}$-galactosidase gene from Lactobacillus plantarum KCTC 3104 as a selection marker. pIMA20 was also equipped with a controllable promoter of nisA ($P_{nisA}$) and a signal peptide-encoding sequence of usp45 ($SP_{usp45}$) for the production and secretion of foreign proteins. pIMA20 and its derivatives mediated site-specific integration into the attB-like site on the Lactococcus lactis NZ9800 chromosome. The vector-integrated recombinant lactococci were easily detected by the appearance of blue colonies on a medium containing $X-{\alpha}-gal$ and also by their ability to grow on a medium containing melibiose as the sole carbon source. Recombinant lactococci maintained these traits in the absence of selection pressure during 100 generations. The ${\alpha}-amylase$ gene from Bacillus licheniformis, lacking a signal peptide-encoding. sequence, was inserted downstream of $P_{nisA}\;and\;SP_{usp45}$ in pIMA20, and the plasmid was integrated into the L. lactis chromosome. ${\alpha}-Amylase$ was successfully produced and secreted by the recombinant L. lactis, controlled by the addition and concentration of nisin.

• Proceedings of the Korean Society for Applied Microbiology Conference
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• 2001.06a
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• pp.155-157
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• 2001

Bifidobacterium spp. is nonpathogenic, gram-positive and anaerobic bacteria, which inhabit the intestinal tract of humans and animals. In breast-fed infants, bifidobacteria comprise morethan 90％ of the gut bacterial population. Bifidobacteria spp. are used in commericial fermented dairy products and have been suggested to exert health promoting effects on the host by maintaining intestinal microflora balances, improving lactose tolerance, reducing serum cholesterol levels, increasing synthesis of vitamins, and aiding the immune enchancement and anticarcinogenic activity for the host. These beneficial effects of Bifidobacterium are strain-related. Therefore continued efforts to improve strain characteristics are warranted. in these respect, development of vector system for Bifidobacterium is very important not only for the strain improvement but also because Bifidobacterium is most promising in serving as a delivery system for the useful gene products, such as vaccine or anticarcinogenic polypeptides, into human intestinal tract. For developing vector system, we have characterized several bifidobacterial plasmids at genetic level and developed several shuttle vectors between E. coli and Bifidobacterium using them. Also, we have cloned and sequenced several metabolic genes and food grade selection marker. Also we have obtained bifidobacterial surface protein, which will be used as the mediator for surface display of foreign genes. Recently we have succeeded in expressing amylase and GFP in Bifidobacterium using our own expression vector system. Now we are in a very exciting stage for the molecular breeding and safe delivery system using probiotic Bifidobacterium strains.

• Journal of Microbiology and Biotechnology
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• v.6 no.6
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• pp.386-390
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• 1996

The ${\beta}$-galactosidase gene from Lactococcus lactis subsp. lactis ATCC 7962 was cloned and its enzymatic properties were characterized, with a view to assessing its potential use as a selection marker in the food-grade cloning vector. Chromosomal DNA from L. lactis subsp. lactis 7962 was cleaved with PstI and ligated into pBR322 for transformation into Escherichia coli TGl. Transformants showing ${\beta}$-galactosidase activity possessed the pBR322 plasmid containing a 10 kilobase (kb) PstI fragment and this plasmid was named pCKL11. The cloned ${\beta}$-galactosidase gene came from the chromosomal DNA of L. lactis subsp. lactis 7962 was confirmed by Southern hybridization. A restriction map of pCKL11 was constructed from the cleavage of both pCKL11 and the purified 10kb insert fraqment. The. optimum pH of the ${\beta}$-galactosidase determined with the E. coli harboring the pCKL11 was 7.0. The optimum temperature was $50^{\circ}C$, while the pI of the enzyme was 7.4. These values were the same as those of the enzyme from the parent strain.