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       kimsg7596@kaist.ac.kr        
       Çѳ²¼ö ±³¼ö        2014.07.29 17:02        21171
´Ù¿î·Îµå : À¯»ê±Õ ¹ßÇö ½Ã½ºÅÛ _Çѳ²¼ö ±³¼ö.pdf(437 Kb)
 

À¯»ê±Õ ¹ßÇö½Ã½ºÅÛ °³¹ß ÇöȲ

 

                                                                                                                                      

                                                                                                                      ÃæºÏ´ëÇб³ ½ÄÇ°»ý¸í°øÇаú Çѳ²¼ö ±³¼ö

 

1. Lactic acid bacteria
À¯»ê±Õ (Á¥»ê±Õ, lactic acid bacteria)Àº ´Ù¾çÇÑ ½ÄÇ°»ê¾÷¿¡ ÀÌ¿ëµÇ°í ÀÖ´Â »ê¾÷ÀûÀ¸·Î Áß¿äÇÑ ¹Ì»ý¹°·Î, ´Ù¾çÇÑ Åº¼Ò¿øÀ» ÀÌ¿ëÇÏ¿© ºü¸¥ ¼Óµµ·Î Á¥»êÀ» »ý¼ºÇÏ°í ÀÌ·Î ÀÎÇØ ½ÄÇ°ÀÇ ÀúÀå±â°£À» ¿¬Àå½ÃÅ°´Â ¿ªÇÒÀ» Çϸç ÀÌ¿Ü¿¡µµ ½ÄÇ°¿¡ ÀÖ¾î Çâ±â, ¹°¼º, ±×¸®°í ¿µ¾çÇÐÀûÀ¸·Î À¯ÀÍÇÑ Ãø¸éÀ» Á¦°øÇÑ´Ù. ƯÈ÷, Ä¡Á À¯»ê±Õ ¹ßÈ¿À¯·Î ´ëÇ¥µÇ´Â À¯°¡°ø »ê¾÷À̳ª ±èÄ¡ µîÀÇ Ã¤¼Ò¹ßÈ¿»ê¾÷¿¡ ÈçÈ÷ ÀÌ¿ëµÇ´Âµ¥, ¾à 20Á¾ÀÇ ´Ù¾çÇÑ generaÀÇ À¯»ê±Õ Áß ½ÄÇ°»ê¾÷¿¡¼­´Â Lactococcus, Lactobacillus, Leuconostoc, Oenococcus,

Pediococcus, ±×¸®°í Streptococcus°¡ ¸¹ÀÌ ÀÌ¿ë µÇ°í ÀÖ´Ù.
À¯»ê±ÕÀÇ ´ë»ç°æ·Î´Â »ý¹°°øÇÐÀûÀ¸·Î »ç¿ëµÇ´Â ´Ù¸¥ ¹Ì»ý¹°¿¡ ºñÇØ ´Ü¼øÇÏ¿© ÇÕ¼º´É·Â°ú »ý»êµÇ´Â ´ë»ç¹°ÁúÀÇ ´Ù¾ç¼ºÀÌ Á¦ÇѵǴ ¹Ý¸é, ¿ÀÈ÷·Á ´Ü¼øÇÑ ´ë»ç°æ·Î´Â º» ¹Ì»ý¹°À» ´ë»ç°øÇÐÀû ¿ëµµ·Î °³¹ßÇÏ´Â µ¥ À־ ÀåÁ¡À¸·Î Æò°¡µÈ´Ù. ´õ¿íÀÌ ¿¡³ÊÁö ´ë»ç°æ·Î¿Í »ýÇÕ¼º °æ·Î°¡ °ÅÀÇ ¿Ïº®ÇÏ°Ô ºÐ¸®µÇ¾î ÀÖ¾î ¼­·Î °£¿¡ ¿µÇâÀ» ¹ÌÄ¡Áö ¾Ê´Â Á¡Àº Å« ÀåÁ¡À¸·Î °£ÁֵȴÙ. À¯»ê±ÕÀ» ÀÌ¿ëÇÑ »ý¹°°øÇÐÀû ¿¬±¸´Â ´ëÇ¥ÀûÀ¸·Î Lactococcus lactis¸¦ ÀÌ¿ëÇÏ¿© ´Ù¾çÇÑ ´ë»ç°øÇÐÀû ½ÃµµµéÀ» ¼º°øÀûÀ¸·Î ¼öÇàÇϸç È°¹ßÈ÷ ÁøÇàµÇ¾î ¿Ô´Ù. º» ¿¬±¸µ¿Çâ¿¡¼­´Â Áö±Ý±îÁö °³¹ßµÈ À¯»ê±ÕÀÇ À¯ÀüÀÚ ÀçÁ¶ÇÕ ±â¼úµéÀ» ¼Ò°³ÇÏ°í À̵éÀ» ¹ÙÅÁÀ¸·Î ¼öÇàµÈ ´Ù¾çÇÑ ´ë»ç°øÇÐÀû ¿¬±¸ »ç·Ê¸¦ Á¤¸®ÇÏ¿´´Ù.

 

2. Food-grade concept: Consideration of regulatory framework
ÀçÁ¶ÇÕ food-grade À¯»ê±ÕÀ» ´Ü¹éÁú, È¿¼Ò¿Í °°Àº ´Ù¾çÇÑ ¹°ÁúµéÀ» »ý»êÇÒ ¼ö ÀÖ´Â ¼¼Æ÷°øÀåÀ¸·Î¼­ »õ·Ó°Ô ÀÌ¿ëÇϱâ À§Çؼ­´Â ¿ì¼± À¯»ê±ÕÀÇ ¾ÈÀüÇÑ »ç¿ëÀ» À§ÇÑ ¹ý·ü ü°è¿¡ ´ëÇÑ ÀÌÇØ°¡ ÇÊ¿äÇÏ´Ù. [de Vos 1999a; Salminen et al. 1998; Sybesma et al. 2006]. ¿¹¸¦ µé¾îLactobacillus±ÕÀº food-grade ¹Ì»ý¹°ÀÌÁö¸¸ GMO(genetically modified organism)·Î½á ½ÄÇ°¿¡ ÀÌ¿ëÇϱâ À§Çؼ­´Â ¾ö°ÝÇÏ°Ô ±ÔÁ¦¸¦ ¹Þ°í ÀÖ´Ù[Gruère and Rao 2007; Renault 2002].
½ÄÇ°¿¡ GMOÀÇ »ç¿ë±ÔÁ¦´Â Àü¼¼°èÀûÀ¸·Î ´Ù¸£°Ô ±ÔÁ¤µÇ¾î Àִµ¥ [Gruère and Rao 2007; Renault 2002], ¹Ì±¹ÀÇ °æ¿ì¿¡´Â ¾à°£ÀÇ ±ÔÁ¦¸¸À» ÇÏ´Â ¹Ý¸é, À¯·´À̳ª ¿À¼¼¾Æ´Ï¾Æ¿¡¼­´Â ½ÄÇ°»ý»ê¿¡ GMOÀÇ ÀÌ¿ë ±ÔÁ¦¸¦ ¾ö°ÝÈ÷ ÁؼöÇÏ°í ÀÖ´Ù. ¶ÇÇÑ, "À¯ÀüÀÚ º¯Çü(Genetic modification) "°ú GMOÀÇ Á¤ÀÇ´Â ±¹°¡¿Í °ü·Ã ±â°ü¿¡ µû¶ó ´Ù¸£°Ô Á¤Àǵǰí ÀÖ´Ù. ¹Ì±¹ÀÇ °æ¿ì "À¯ÀüÀÚ º¯Çü "Àº ¾î¶² ±â¼úÀ» ÀÌ¿ëÇÏ¿© DNA sequence¸¦ º¯Çü½ÃŲ °ÍÀ¸·Î, ±× µ¹¿¬º¯ÀÌ ±ÕÁÖÀÇ Æ¯¼º¿¡ µû¶ó ½ÂÀÎÀÌ µÇ¸ç [FDA 2001], ÀçÁ¶ÇÕ DNA±â¼úÀ» ÀÌ¿ëÇÏ¿© °³·®µÈ ¹Ì»ý¹°·Î Á¦Á¶µÈ ½ÄÇ°Àº ¡°bioengineered foods¡±À̶ó ÇÏ°í GMO¶ó´Â ¿ë¾î´Â »ç¿ëÇÏÁö ¾Ê´Â´Ù [Pedersen et al. 2005]. ¹Ì±¹ÀÇ ±ÔÁ¤¿¡ ÀÇÇϸé GM(genetically modified) À¯»ê±ÕÀº GRAS¶ó ºÐ·ùµÇ¸ç ¸î ³â µ¿¾È ½ÄÇ° »ê¾÷¿¡¼­ ½ÃÀåÀ» Çü¼ºÇÏ°í ÀÖ´Ù. ´ëÁ¶ÀûÀ¸·Î À¯·´ÀÇ À¯»ê±Õ ½ÃÀåÀº ÀÚ¿¬ÀûÀ¸·Î ¹ß»ýÇÏÁö ¾Ê°í in vitro¿¡¼­ °³·®µÈ ¸ðµç ¹Ì»ý¹°µéÀ» GMO·Î °£ÁÖÇÏ°í[EC 2001], ¿À¼¼¾Æ´Ï¾Æ¿¡¼­´Â in vitro ±â¼úÀ» ÀÌ¿ëÇÑ ¸î °¡Áö ±ÔÁ¤µÈ À¯ÀüÀû ±â¼úÀ» ÀÌ¿ëÇÏ¿© ¸¸µé¾îÁø ¹Ì»ý¹°¸¸À» GMO¶ó Á¤ÀÇÇÑ´Ù [Hobbs 2001].
Lactobacilli´Â µ¶¼Ò°¡ ¾ø°í, °Ç°­ÁõÁø È¿°ú¸¦ ÁÖ´Â ÇÁ·Î¹ÙÀÌ¿ÀƽÀ¸·Î½á ½ÄÇ°»ý»ê°øÁ¤¿¡ ¼ö õ³â µ¿¾È ÀÌ¿ëÀÌ µÇ°í ÀÖ´Â GRASÀÌ°í QPS(Qualified Presumption of Safety) ¹Ì»ý¹°·Î Áö³­ 10³â µ¿¾È ÀçÁ¶ÇÕ ¹ßÇö¼÷ÁÖ·Î ²ÙÁØÈ÷ ÀÌ¿ëµÇ¾î ¿Ô´Ù. ¶ÇÇÑ, Lactococcus lactis À¯»ê±Õ ¸ðµ¨Àº 1990³â ÃÊ¿¡ °³¹ßµÈ ÀÌÈÄ·Î Áö³­ 10³â µ¿¾È ´Ü¹éÁú ÀçÁ¶ÇÕ ¹ßÇö½Ã½ºÅÛÀ¸·Î½á ¹ßÀüµÇ¾î ¿Ô´Ù [Kleerebezem et al. 1997; Konings et al. 2000; Mierau and Kleerebezem 2005].
GM-À¯»ê±Õ¸¦ ÀÌ¿ëÇÑ ¹ßÇö ½Ã½ºÅÛÀº ½ÄÇ°ÀÇ ¾ÈÀüÇÑ »ç¿ëÀ» À§ÇØ ¸î °¡Áö ÃæÁ·µÇ°í °í·ÁµÉ »çÇ×ÀÌ Àִµ¥, À̸¦ "Food-grade concept"À̶ó ÇÑ´Ù. ÀÌ ¿ë¾î´Â À¯»ê±Õ ¹ßÇö½Ã½ºÅÛÀÌ Ã³À½À¸·Î °³¹ßµÈ 1990³â´ëºÎÅÍ »ç¿ëµÇ¾ú°í, Johansen [1999]¿¡ ÀÇÇØ food-grade ¹ßÇö ½Ã½ºÅÛÀÇ ¾ö°ÝÇÑ Á¤ÀÇ ¹× ±âÁØÀº ´ÙÀ½°ú °°ÀÌ ¼³¸íµÇ¾ú´Ù. Food-grade ¹ßÇö½Ã½ºÅÛÀÇ °¡Àå Áß¿äÇÑ ±âÁØÀº À¯Àü °øÇÐÀû ¿ëµµ·Î »ç¿ëµÇ´Â ¼÷ÁÖ´Â ¾ÈÀüÇÏ°í, ¾ÈÁ¤ÀûÀΠƯ¡À» °¡Áö°í ÀÖÀ¸¸ç, GRAS ¶Ç´Â QPS À̾î¾ß ÇÑ´Ù. ´õºÒ¾î ½ÄÇ°°ú ȣȯ °¡´ÉÇÑ ¼±¹ß Ç¥Áö(selectable maker)¸¦ »ç¿ëÇØ¾ß µÇ´Âµ¥, Ç×»ýÁ¦ ÀúÇ× À¯ÀüÀÚ Ç¥Áö´Â ¹èÁ¦µÇ¾î¾ß ÇÏ°í ¼÷ÁÖ ³ª ¹ßÇö ½Ã½ºÅÛ¿¡ ÀÇÇؼ­ ÇطӰųª, µ¶¼Ò¸¦ »ý¼ºÇϰųª, ¾Ë·¹¸£±â¸¦ ÀÏÀ¸Å°´Â ¼ººÐÀÌ »ý¼ºµÇÁö ¾Ê¾Æ¾ß µÇ°í, ¸ðµç À¯ÀüÀû º¯ÇüÀº ÀÚ°¡ º¹Á¦·Î ÀÌ·ïÁ®¾ß ÇÑ´Ù. ¶ÇÇÑ, food-grade ½Ã½ºÅÛÀ¸·Î½á ¶Ç ´Ù¸¥ Áß¿äÇÑ Æ¯Â¡Àº ¹ÙÀÌ¿À»ê¾÷À̳ª ½Ä·® »ý»ê »ê¾÷¿¡ À־ ´ë±Ô¸ð·Î »ê¾÷¿¡ ÀÀ¿ë°¡´ÉÇÏ¸ç »ç¶÷ÀÌ food-grade modifiedµÈ À¯»ê±ÕÀ» ¼·ÃëÇÏ¿´À» ¶§ À§Àå°ü¿¡¼­ ¾ÈÁ¤ÀûÀ̾î¾ß ÇÑ´Ù. ¾ö°ÝÇÑ food-grade´Â °°Àº genusÀÇ Á¾¿¡¼­ À¯·¡ÇÑ À¯ÀüÀÚ³ª ½ÄÇ° ¹Ì»ý¹°¿¡¼­ ¾òÀº À¯ÀüÀÚ¸¦ °¡Áø À¯»ê±Õ±ºÀ¸·Î Á¤ÀÇ ÇÒ ¼ö ÀÖ´Ù. ³ÐÀº ¹üÀ§·Î´Â ÃÖÁ¾ÀûÀ¸·Î food -grade ±ÕÁÖÀÇ DNA°¡ ¿ÏÀüÈ÷ Á¦°Å°¡ µÇ¾úÀ» ¶§, non-GRAS ¶Ç´Â QPS ¹Ì»ý¹°¿¡¼­ À¯·¡ÇÑ À¯ÀüÀÚµµ ½ÂÀÎÀÌ µÈ´Ù. ÀÌ°ÍÀº ½ÄÇ° È¿¼Ò¿Í EFSA(European Food Safety Authority )ÀÇ ¾È³»¿¡ °üÇÑ ÇöÀç EC ±ÔÁ¤°ú´Â ´ëÁ¶ÀûÀ¸·Î[EC 2008; EFSA 2009], ½ÄÇ°È¿¼Ò¿¡ ´ëÇÑ EC ±ÔÁ¦´Â ¾ÈÀü¿¡ ´ëÇØ »ó¼¼ Æò°¡ ÈÄ, °¢ È¿¼Ò¿¡ ´ëÇØ ¾ÈÀü¼ºÀÌ È®º¸µÇ¸é ½ÂÀÎ ÈÄ¿¡ ½ÄÇ°¿¡ ÷°¡ µÉ ¼ö ÀÖ´Ù.
 Food-grade concept¸¦ ±âÃÊ·Î ÇÏ¿© ¸¹Àº ½Ã½ºÅÛµéÀÌ Áö³­ 20³â°£ ¹ßÀüÇØ ¿Ô°í, ƯÈ÷ ÀçÁ¶ÇÕ Lactococcus¿Í LactobacilliÀÇ ¹ßÇö ½Ã½ºÅÛÀÇ ÀÌ¿ëÀÌ Áõ°¡µÇ¾î ¿Ô´Ù. ´Ù¾çÇÑ Á¾·ùÀÇ Lactobacilli ¼÷ÁÖ¿Í ÇÁ·Î¸ðÅÍ, food-grade ¼±¹ß Ç¥Áö°¡ °³¹ßµÇ¾ú°í [Diep et al. 2009; Morello et al. 2008], ÀÌ ½Ã½ºÅÛÀ» ÀÌ¿ëÇÑ ÀçÁ¶ÇÕ È¿¼Ò¿Í »ý¹é½Å(live vaccine)ÀÇ »ý»ê ¿¬±¸°¡ °ü½ÉÀ» ¹Þ°í ÀÖ´Ù.

 

  Ç¥1: Guidelines for the use of GMO in food products in several regions[Carter and Gruère 2007]

 

 

3. Lactic acid bacteria for recombinant protein production
¹Ì»ý¹°À» À¯ÀüÀûÀ¸·Î º¯ÇüÇÏ°í ÀçÁ¶ÇÕ ¹Ì»ý¹°·Î ÀÌ¿ëÇϱâ À§ÇÑ Ã¹ ¹ø° °úÁ¤Àº ÇüÁúÀüȯ±â¼úÀ» ÀÌ¿ëÇÏ¿© ¿Ü·¡ À¯ÀüÀÚ¸¦ ¹Ì»ý¹° ³»·Î µµÀÔÇÏ´Â °ÍÀÌ´Ù. 1980³â ¸»¿¡ LactobacilliÀÇ ÇüÁúÀüȯ¿¡ °üÇÑ ¿¬±¸°¡ óÀ½ ¹ßÇ¥µÈ ÀÌ·¡·Î, ´Ù¾çÇÑ Á¾·ùÀÇ Lactibacilli°¡ ÀçÁ¶ÇÕ ´Ü¹éÁú »ý»ê¿¡ ÀÌ¿ëÀÌ µÇ¾ú´Ù. [Aukrust and Blom 1992; Aukrust and Nes 1988; de Vos 1987; Natori et al. 1990]. Electroporation, protoplast formation, conjugation methodsµîÀÇ ÇüÁúÀüȯ±â¼úµéÀÌ ¹ßÀüµÇ¾î Lb. acidophilus, Lb. casei, Lb. helveticus, Lb. plantarum Lb. reuteri, Lb. sakei, ¿Í Lb. pentosusµé À¯»ê±Õ¿¡ ¿Ü·¡ À¯ÀüÀÚ¸¦ µµÀÔÇÏ¿© ÀçÁ¶ÇÕ Lactobacilli·Î ºÎÅÍ ¿Ü·¡ ´Ü¹éÁú (heterologous protein) ¹× ´Ù¾çÇÑ originÀÇ antigenÀ» »ý»êÇÒ ¼ö ÀÖ¾úÀ¸¸ç [Bermudez-Humaran et al. 2011; Tarahomjoo 2012; Wells and Mercenier 2008] [Hashiba et al. 1992; Kok 1996; Pouwels and Leer 1993; Wanker et al. 1995], ÀÌ¿Ü¿¡µµ ´Ù¾çÇÑ Á¾·ùÀÇ À¯»ê±Õ¿¡¼­ ¸¹Àº ´Ü¹éÁúÀÌ ¼º°øÀûÀ¸·Î ¹ßÇö, »ý»êµÇ¾ú´Ù (Ç¥2).

 

Table 2. Lactobacilli which have been used for the recombinant expression of proteins

  

     * denoted in nkat in order to make them comparable, if no conversion possible similar to that presented in the original publications
sppIP: sakacin P inducing peptide; inducing peptide of 19 amino acids


Table 2. Lactobacilli which have been used for the recombinant expression of proteins

* denoted in nkat in order to make them comparable, if no conversion possible similar to that presented in the original publications
sppIP: sakacin P inducing peptide; inducing peptide of 19 amino acids

 

4. Integrationº¤ÅÍ
Integrationº¤ÅÍ´Â À¯ÀüÀÚÀÇ ³Ë¾Æ¿ô (knock-out), ÁõÆø(amplification), ±³Ã¼ (replacement) ±×¸®°í »ðÀÔ (insertion)¿¡ »ç¿ëµÇ°í ÀÖ´Ù. »ðÀÔ¼­¿­ÀÇ ÀüÀ§ (transposition via IS elements) ¹× att/integrase systemsÀ» ÀÌ¿ëÇÏ¿© ƯÁ¤ºÎÀ§¿¡ ÀçÁ¶ÇÕ, suicide ¶Ç´Âtemperature sensitive vectors¸¦ ÀÌ¿ëÇÑ homologous recombination ¹æ¹ýÀÌ ÈçÈ÷ »ç¿ëÇÑ´Ù [2]. À¯»ê±Õ integrationº¤ÅͽýºÅÛÀº one-step Campbell-like integration°ú one-À̳ª two-step homologous recombination µÎ °¡Áö ÇüÅ·Π±¸ºÐµÈ´Ù. Campbell-like integrationÀº integration µ¿¾È¿¡ complete vector DNA°¡ ¿°»öü ³»ºÎ·Î »ðÀԵǰí, ¿°»öü ³»ºÎ¿Í °°Àº À§Ä¡¿¡ plasmid°¡ multi-copy integrationÀÌ ÀϾ°Ô µÈ´Ù. one-À̳ª two-step homologous recombination ±â¼úÀº º¤ÅÍ¿Íchromosome »çÀÌ¿¡ homologous regionÀÌ Á¸ÀçÇÏ¸é ¹ß»ýÇÒ ¼ö Àִµ¥, ¡°one-step gene replacement¡±°¡ ÀϾ ¶§, µ¿½Ã¿¡ crossover (double crossover)°¡ ÀϾ°í, two-stepÀÌ ÀϾ ¶§´Â ¿¬¼ÓÀûÀ¸·Î integrationº¤ÅÍ°¡ loop-out deletion¿¡ ÀÇÇØ integration (single crossover)ÀÌ ÀϾ°Ô µÈ ÈÄ chromosomal deletionÀÌ µÇ°Å³ª integration geneÀÌ ¿°»öü ³»ºÎ·Î »ðÀԵȴÙ[3].

 Campbell-like integrationÀº Leenhouts¿¡ ÀÇÇØ Lc. lactis¿¡¼­ º¹Á¦°¡ µÇÁö ¾Ê´Â originÀ» °¡Áö°í ÀÖ´ÂpBR322 (E.coli)¿Í pE194 (Staphylococcus aureus)À» ÀÌ¿ëÇÏ¿© óÀ½À¸·Î ½ÃµµµÇ¾ú´Ù. ±× ÈÄ, pBluescript SK vectorÀÇ E.coli replicon À¯ÀüÀÚ¸¦ ÀÌ¿ëÇÑ chromosomal integration¿¬±¸°¡ ÁøÇà µÇ¾ú´Ù [4, 5].  Lactococcal originÀ» ÀÌ¿ëÇÑ integrationº¤ÅÍ´Â º¹Á¦°³½Ã´Ü¹éÁú(RepA)ÀÌ ºÒÈ°¼ºÈ­µÈ lactococcal broad host range plasmidÀÎ pWV01¸¦ ÀÌ¿ëÇÏ¿© °³¹ßµÈ pORI/pINT º¤ÅÍ ½Ã¸®Á »ç¿ëÇÑ´Ù. ÀÌ º¤ÅÍ´Â RepAÀ» Á¦°øÇÏ¿© ±×¶÷¾ç¼º±Õ (Lc. lactis LL108)»Ó¸¸ ¾Æ´Ï¶ó ±×¶÷À½¼º±Õ (E. coli EC1000)¿¡¼­ integration º¤ÅÍ·Î ÀÌ¿ëµÈ´Ù.

Chromosome¿¡ integrationµÈ plasmid´Â Ç×»ýÁ¦¸¶Ä¿¸¦ ÀÌ¿ëÇÏ¿© ¼±¹ßÇÑ ÈÄ integrationº¤ÅÍÀÇ lacZ reporter geneÀ» ÀÌ¿ëÇÏ¿© ÈÄ loop-out deletionÀ» ÇÒ ¼ö°¡ ÀÖ´Ù. ÀÌ¿Í °°Àº ¹æ¹ýÀ¸·Î sucrose-specific enzyme IIÀ» ÄÚµåÇÏ´Â scrAÀ¯ÀüÀÚ¿Í sucrose-6-phosphate hydrolaseÀ» ÄÚµåÇÏ´Â scrBÀ¯ÀüÀÚ·Î ±¸¼ºµÈ pINT (pWV01 replication origin) integrationº¤Å͸¦ erythromycin ³»¼º À¯ÀüÀÚ¸¦ ±³Ã¼ÇÏ¿©, chromosome¾È¿¡ targetÀ¯ÀüÀÚ¸¦ Campbell-like integrationÇÏ¿© food-grade systemÀ¸·Î ÀÌ¿ëÇÑ »ç·Ê°¡ ÀÖ´Ù [6].

 

4.1 ¿Âµµ¹Î°¨Çü Çö󽺹̵å (Temperature-sensitive plasmids)
¿°»öü ³»ºÎ·Î À¯ÀüÀÚ¸¦ »ðÀÔÇϱâ À§ÇØ Á¶°Ç/ºñÁ¶°ÇÀûÀ¸·Î º¹Á¦ÇÏ´Â Çö󽺹̵åÀÇ ¿ø¸®¸¦ ÀÌ¿ëÇÑ temperature-sensitive º¤Å͸¦ ÀÌ¿ëÇÑ´Ù. Integrationº¤ÅÍÀÎ pG+ ½Ã¸®Áî´Â Á¶°ÇºÎ Çö󽺹̵å·Î temperature-sensitive repliconÀ» °¡Áö°í ÀÖ¾î, permissive temperaure (¿¹ 28¡É)¿¡¼­ ±×¶÷¾ç¼º±Õ°ú ±×¶÷À½¼º±Õ¿¡¼­ º¹Á¦°¡ µÇÁö¸¸ »ýÀå¿Âµµ¸¦ ³ôÀÌ°Ô µÇ¸é (35¡É ÀÌ»ó) º¹Á¦°³½Ã´Ü¹éÁú (RepA)ÀÌ ºÒÈ°¼ºÈ­µÇ¾î º¤ÅÍÀÇ º¹Á¦¿Í ¼÷ÁÖ ¿°»öü ¾È¿¡ »ðÀԵǴ ´É·ÂÀ» ÀÒ°Ô µÈ´Ù. RepA+ temperature sensitive helper plasmidÀÎ pVE6007 [7] ¿Í RepA- vectorÀÎ pORI280 [8]´Â ¶Ç ´Ù¸¥ homologous recombination ½Ã½ºÅÛÀ¸·Î »ç¿ëµÈ´Ù. ÀÌµé µÎ°³ÀÇ Çö󽺹̵å´Â permissive temperature¿¡¼­´Â À¯ÁöÇÏÁö¸¸ »ýÀå¿Âµµ°¡ ³ô¾ÆÁö¸é pVE6007 helper plasmid¿¡¼­´Â RepA°¡ ºÒÈ°¼ºÈ­µÇ°í, RepA°¡ Á¸ÀçÇÏÁö ¾Ê´Â pORI280¿¡¼­´Â º¹Á¦¿Í chromosome¾ÈÀ¸·Î »ðÀÔÀ» ÇÒ ¼ö ¾ø°Ô µÈ´Ù [2]. Russell and Klaenhammer [9]´Â thermophilic lactobacilliÀÇ pWV01 repliconÀ» ±âÃÊ·Î ÇÑ temperature-sensitive helper plasmidÀÎ pTRK669¸¦ °³¹ßÇÏ¿´°í, Neu and Henrich [10]´Â thermophilic lactobacilli¸¦ À§ÇÑ pTN1 vector¸¦ ±âÃÊ·Î ÇÑ integration º¤Å͸¦ °³¹ßÇÏ¿´´Ù. ÀÌ Çö󽺹̵å´Â Lb. curvatusÀÇ rolling-circle Çö󽺹̵åÀÎ tpLC2¿¡¼­ À¯·¡ÇÏ¿© 42¡ÉÀÌÇÏ¿¡¼­ º¹Á¦ ´É·ÂÀ» ºÒÈ°¼ºÈ­ ½ÃŲ´Ù.

 

5. ¹ßÇöº¤ÅÍ-ON/OFF À¯µµ½Ã½ºÅÛ

 À¯»ê±Õ¿¡¼­ µ¿ÇüÀ¯ÀüÀÚ(homologous protein)¿Í ÀÌÇüÀ¯ÀüÀÚ(heterologous protein)¸¦ ¹ßÇöÇϱâ À§ÇÑ ¹ßÇöº¤ÅÍ¿¡´Â º¹Á¦µÉ À¯ÀüÀÚÀÇ Àü»ç¸¦ Á¶ÀýÇϱâ À§Çؼ­ ÀûÀýÇÑ ÇÁ·Î¸ðÅÍ°¡ Á¸ÀçÇÑ´Ù. ÁÖ·Î transcription terminator´Â º¹Á¦µÉ À¯ÀüÀÚÀÇ downstream¿¡ À§Ä¡ÇÏ°í ÀÖ¾î º¤ÅÍÀÇ À¯ÀüÀÚ Àü»ç¸¦ Á¾°áÇÏ°í ºÒÇÊ¿äÇÑ Àü»ç¸¦ ¸·¾ÆÁÖ°í, Signal sequences´Â »ý¼ºµÈ ´Ü¹éÁúÀ» ºÐºñ½ÃÅ°±â À§ÇØ »ç¿ëÀÌ µÇ¸ç »ý¼ºµÈ ´Ü¹éÁúÀÇ Á¤Á¦¸¦ ¿ëÀÌÇÏ°Ô Çϱâ À§ÇØ ÀϹÝÀûÀ¸·Î His-tag À̳ª ´Ù¸¥ ÀΰøÀûÀΠű׸¦ ºÙ¿© »ç¿ëÇÑ´Ù.

 ¸¸ÀÏ ¹ßÇö º¤ÅÍÀÇ ÇÁ·Î¸ðÅÍ strength°¡ °­ÇÏ°í À¯ÀüÀÚ ¹ßÇöÀÌ ¸Å¿ì ³ôÀ¸¸é, »ý¼ºµÈ ´Ü¹éÁúÀº ¼¼Æ÷Áú (cytoplasm)¾È¿¡ ÃàÀûµÇ°í »ý¹°ÇÐÀû È°¼ºÀÌ ¾ø´Â inclusion bodies¸¦ »ý¼ºÇÏ°Ô µÈ´Ù. ¶ÇÇÑ ¸î¸îÀÇ ´Ü¹éÁúÀÇ °æ¿ì °í³óµµ·Î »ý¼ºÀÌ µÇ¸é ¿ÀÈ÷·Á ¼¼Æ÷¿¡ µ¶¼ºÀ» ³ªÅ¸³½´Ù [1]. ±×·Î ÀÎÇØ ¹ßÇö¼öÁØÀ» ÀûÀýÈ÷ Á¶ÀýÇÒ ¼ö ÀÖ´Â ¿©·¯ °¡Áö À¯µµ ÇÁ·Î¸ðÅÍ (inducible promoters)°¡ °³¹ßµÇ¾ú´Ù. À¯µµ ÇÁ·Î¸ðÅÍ¿¡´Â sugar¿Í NaCl Á¶Àý ÇÁ·Î¸ðÅÍ, pHÀÇ °¨¼Ò, ¿ÂµµÀÇ º¯È­¿Í ÆľÆÁö °¨¿° ÇÁ·Î¸ðÅÍ°¡ ÀÖ´Ù [2]. Áö±Ý±îÁö´Â NICE¶ó ºÒ¸®´Â nisin À¯µµ ÇÁ·Î¸ðÅÍ°¡ À¯»ê±Õ ±ÕÁÖ¿¡ ³Î¸® »ç¿ëÀÌ µÇ°í ÀÖÀ¸¸ç ƯÈ÷ Lc. lactis¿¡ ÀÌ¿ëµÇ°í ÀÖ´Ù. NisinÀº nisin cluster¿¡ ÀÇÇØ Á¶ÀýµÇ¾î À¯»ê±Õ¿¡¼­ »ýÇÕ¼ºµÇ´Â Ç×±Õ¼ºÀ» °¡Áø ÆéŸÀ̵åÀÌ°í, NICE ½Ã½ºÅÛÀº nisA ÇÁ·Î¸ðÅÍ¿¡ ÀÇÇØ À¯ÀüÀÚ¸¦ Á¶ÀýÇÏ´Â Çö󽺹̵å·Î Lc. lactisÀÇ nisin gene cluster¿¡¼­ histidine kinase¸¦ ÄÚµåÇÏ´Â NisKÀ¯ÀüÀÚ¿Í response regulator¿ªÇÒÀ» ÇÏ´Â NisRÀ¯ÀüÀÚ·Î ±¸¼ºÀÌ µÇ¾î µÎ °³ÀÇ À¯ÀüÀÚÀÇ Á¶Àý¿¡ ÀÇÇØ ½ÅÈ£ º¯È­¸¦ ÁØ´Ù [12]. NICE Çö󽺹̵å´Â nisinÀ» »ý¼ºÇÏÁö ¾Ê°í NisR¿Í NisK ´Ü¹éÁúÀ» »ý¼ºÇÏ´Â ±ÕÁÖ¿¡ µµÀÔµÇ¾î ¼¼Æ÷°¡ »ýÀåÇÏ´Â ´ë¼ö±â¶§ ¹èÁö¿¡ ÷°¡µÈ nisin¿¡ ÀÇÇØ nisA ÇÁ·Î¸ðÅÍ°¡ Á¶Àý µÇ´Âµ¥, ¹ßÇö ¼öÁØÀº ÷°¡µÈ nisin¾ç°ú ºñ·ÊÇÏ°í ´Ü¹éÁúÀÇ »ý¼º ¼öÁØÀº ÃÑ »ý»ê soluble ´Ü¹éÁúÀÇ 10%¿¡¼­ 60%±îÁö Áõ°¡À²À» º¸¿´´Ù [12]. Nisin À¯µµ ½Ã½ºÅÛÀº Lc. lactis¿ÜÀÇ ´Ù¸¥ À¯»ê±Õ¿¡¼­ ¼÷ÁÖ¼¼Æ÷¿Í À¯µµ È¿À²¿¡ ÀÇÇÑ ³»¼ºÀ¸·Î ÀÎÇØ nisR¿Í nisKÀÇ ¹ßÇö ¼öÁØ¿¡¼­ ¹®Á¦°¡ µÉ ¼ö ÀÖÀ¸³ª, nisinÀº food-grade¹°Áú·Î °ªÀÌ Àú·ÅÇÏ°í ÀûÀº ¾ç (0.05-5 ng/mL)À¸·Îµµ Á¤È®ÇÏ°Ô ´Ü¹éÁú »ý»êÀ» on/off·Î À¯µµÇÒ ¼ö°¡ ÀÖ¾î »ý»ê½Ã½ºÅÛÀ¸·Î ¸¹Àº ÀåÁ¡À» °¡Áö°í ÀÖ´Ù. ¶Ç ´Ù¸¥ À¯µµ ÇÁ·Î¸ðÅÍ·Î Lb sakei¿Í Lb. plantarum¿¡¼­ class ¥± bacteriocin sakacin A (sap gene cluster)³ª sakacin P (spp gene cluster)ÀÇ ÇÁ·Î¸ðÅÍ¿Í Á¶Àý À¯ÀüÀÚ¸¦ ÀÌ¿ëÇÑ pSIPº¤ÅÍ°¡ °³¹ßµÇ¾ú°í[13, 14], ÀÌ ½Ã½ºÅÛÀº pheromone ÆéŸÀ̵带 ÷°¡ÇÏ¿© ´Ü¹éÁú ¹ßÇöÀ» À¯µµÇÒ ¼ö ÀÖ´Ù.

 

±×¸²1. Schematic overview of the NICE vector and pSIP vector expression system.

PnisA; promoter, NisR and NisK; regulation, NisI; immunity, PsppA or PsppQ; promoter, sppK and sppR; regulation 

 

6. ÇÕ¼º ÇÁ·Î¸ðÅÍ (synthetic promoters)

Jensen and Hammer [15]Àº ¸ñÇ¥À¯ÀüÀÚ (targeted gene)ÀÇ ¹ßÇö¼öÁØÀ» ³ôÀÌ°í ¸Å¿ì ¾ÈÁ¤µÈ ¹ßÇö ½Ã½ºÅÛÀ» ¾ò°íÀÚ ÇÕ¼ºÇÁ·Î¸ðÅÍÀÇ ¶óÀ̺귯¸®¸¦ ±âÃÊ·Î ÇÑ Lc. lactis ¹ßÇö½Ã½ºÅÛÀ» °³¹ßÇÏ¿´´Ù. ÇÁ·Î¸ðÅÍ ºÎºÐÀÇ -35¿Í -10¿¡ ÀÏÄ¡ÇÏ´Â ¿°±â¼­¿­ (consensus sequences)À» ÀÏÁ¤ÇÏ°Ô À¯ÁöÇÏ°í ¿°±â ¼­¿­°£¿¡ ÀÏÄ¡ÇÏÁö ¾Ê´Â spacer sequencesºÎºÐÀº ·£´ýÇÑ ¿°±â¼­¿­·Î ´ëüÇÏ¿´´Ù. ·£´ýÇÏ°Ô Á¸ÀçÇÏ´Â spacer sequencesÀÇ ÇÁ·Î¸ðÅÍ´Â degenerated single-stranded promoter oligonucleotide¿¡ ÀÇÇØ ÇÕ¼ºÇÏ¿´°í À̸¦ ¥â-galactosidase À¯ÀüÀÚ (lacL and lacM) ¹ßÇö¿¡ Àû¿ëÇÏ¿´´Ù. ÇÕ¼ºÇÁ·Î¸ðÅÍ´Â ´Ü¹éÁú ¹ßÇö·®À» 10-1000¹è·Î ´Ù¾çÇÏ°Ô Á¶Àý ÇÒ ¼ö ÀÖÀ¸¸ç À¯»ê±Õ »Ó ¸¸ ¾Æ´Ï¶ó ´Ù¸¥ ¹Ì»ý¹°¿¡¼­µµ À¯¿ëÇÏ°Ô Àû¿ëÀÌ °¡´ÉÇÏ´Ù[16].

 

Ç¥3. Constitutive promoter for the expression in lactic acid bacteria

pep; proteolytic enzyme genes, hsp; small heat-shock protein gene, slp; surface layer protein gene, ldh; lactate dehydrogenase gene, erm; enterococcal rRNA adenine N-6-methyltransferase gene

 

7. Food-grade  ¼±¹ß Ç¥Áö (selectable marker)
 Food-grade½Ã½ºÅÛÀº ¼÷Á־ȿ¡¼­ integration°ú ¹ßÇö º¤ÅÍÀÇ ¾ÈÁ¤¼ºÀ» ±âÃÊ·Î ÇÏ¿©, ½ÄÇ°¿¡ »ç¿ëÀÌ °¡´ÉÇÑ ´Ù¾çÇÑ ¼±¹ßÇ¥Áö °³¹ßÀÌ ÇÊ¿äÇÏ´Ù. ¹«¾ùº¸´Ù Ç×»ýÁ¦ ³»¼º ¼±¹ßÇ¥ÁöÀÇ °æ¿ì Àΰ£Àå³»¹Ì»ý¹°·Î Ç×»ýÁ¦ ³»¼ºÀÌ ÀüÀÌµÉ ¼ö ÀÖÀ¸¹Ç·Î »ç¿ëÇÒ ¼ö ¾øÀ¸¸ç, À̸¦ ´ëüÇϱâ À§ÇØdominant¿Í complementation ¼±¹ß ¹æ¹ýÀ» ÀÌ¿ëÇÏ¿© ¼±¹ß Ç¥Áö¸¦ °³¹ßÇØ ¿Ô´Ù. [17, 18]. ³ôÀº È°¼ºÀ» º¸ÀÌ´Â dominant¼±¹ßÀº ÀçÁ¶ÇÕ À¯»ê±ÕÀ» ¼±¹ßÇÔ¿¡ À־ Ç×»ýÁ¦ ³»¼º ¼±¹ßÇ¥ÁöÀÇ »ç¿ë°ú °°Àº È¿°ú¸¦ º¸À̸ç hostÀÇ Æ¯Á¤ À¯ÀüÀÚ¿¡ ÀÇÁ¸ÇÏÁö ¾ÊÀ¸¹Ç·Î À¯»ê±ÕÀÇ ´Ù¾çÇÑ Á¾¿¡¼­ ÀÌ¿ëµÈ´Ù.
 Nisin¿¡ ³»¼ºÀÌ ÀÖ´Â nsrÀ¯ÀüÀÚ´Â hydrophobic ´Ü¹éÁú·Î½á, dominant¼±¹ß¿¡ ÀÌ¿ëÀÌ µÈ´Ù. pNP40 (60 kb)º¤ÅÍ¿¡ nsrÀ¯ÀüÀÚ¸¦ µµÀÔÇÏ¿© food-grade ¼±¹ßÇ¥Áö·Î »ç¿ëµÇ°í ÀÖÀ¸¸ç [19], Takala and Saris [20]´Â nisin ¸é¿ª À¯ÀüÀÚÀÎ nisI¸¦ ÀÌ¿ëÇÏ¿© pLEB590 º¤Å͸¦ °³¹ßÇÏ¿´°í, class II bacteriocin lactacin F¸¦ ÄÚµùÇÏ´Â Lb. johnsonii lafIÀ¯ÀüÀÚ¸¦ ÀÌ¿ëÇÏ¿© lactacin F¿¡ ¹Î°¨µµ¸¦ º¸ÀÌ´Â ±ÕÁÖ¿¡ »ç¿ë°¡´ÉÇÑ pTRK434/435¹ßÇö½Ã½ºÅÛÀ» °³¹ßÇÏ¿´´Ù[21].
 Two-component food-grade Ŭ·Î´× ½Ã½ºÅÛÀº Lc. lactis MG 1363°ú »ê¾÷¿ë Lc. Lactis ±ÕÁÖ¿¡¼­ »ç¿ëµÇ°í Àִµ¥ [22], ÀÌ ½Ã½ºÅÛÀº theta type¸ÞÄ¿´ÏÁòÀ¸·Î º¹Á¦°¡ ÀÌ·ç¾îÁö¸ç ¼±¹ß Ç¥Áö°¡ ¾ø´Â pVEC1¿Í, repBÀ¯ÀüÀÚ°¡ ºÒÈ°¼ºÈ­µÇ°í erythromycin ³»¼ºÀ¯ÀüÀÚ¸¦ °¡Áö°í ÀÖ´Â pCOM1À¸·Î µÎ °¡Áö º¤ÅÍ·Î ±¸¼ºµÇ¾î ÀÖ´Ù. Erythromycin÷°¡ ¹èÁö¿¡ pVEC1¿Í pCOM1 º¤ÅÍ°¡ °°ÀÌ º¹Á¦°¡ µÇ¾î¾ß¸¸ »ýÁ¸ÇÒ ¼ö ÀÖÀ¸¸ç, pCOM1¿Í °°Àº theta type¸ÞÄ¿´ÏÁòÀ¸·Î º¹Á¦ÇÏ´Â º¤ÅÍ´Â ¼¼Æ÷ºÐ¿­°ú ±¸Á¶¸é¿¡¼­ ¾ÈÁ¤¼ºÀ» º¸À̱⠶§¹®¿¡ ¼±¹ß ÈÄ Ç×»ýÁ¦°¡ ¾ø´Â ¹èÁö¿¡ »ýÀå½Ãų °æ¿ì¿¡ pCOM1¸¸À» º¹Á¦ÇÏ´Â ¹Ì»ý¹°À» ¼±¹ß ÇÒ ¼ö ÀÖ´Ù.
 O¢¥Sullivan [23, 24]´Â natural co-integrate lactococcal plasmid pAH90(26kb)À» ÀÌ¿ëÇÏ¿© cadmium ³»¼ºÀ¸·Î ÀçÁ¶ÇÕÀ¯»ê±ÕÀ» ¼±¹ßÇÏ´Â ¹æ¹ýÀ» °³¹ßÇÏ¿´À¸¸ç, Strep. thermophilus [25, 26]¿Í L. lactis [27] ±ÕÁÖ¿¡ ÀÌ¿ëÇß´Ù. ÃÖ±Ù¿¡´Â Lb. plantarum À¯·¡ÀÇ bile salt hydrolaseÀ» ÀÌ¿ëÇÑ dominant ¼±¹ßÇ¥Áö°¡ °³¹ßµÇ¾î, Lb. casei¿¡¼­ bshÀ¯ÀüÀÚ¸¦ ¹ßÇö½ÃÄ×À» ¶§, ÀçÁ¶ÇÕÀ¯»ê±ÕÀº 0.1% bile salt (glycodeoxycholic acid sodium, GDCA) ¿¡¼­ »ýÀ°ÀÌ µÇÁö¸¸ wild-type Lb. casei´Â 0.05% GDCA¿¡¼­ »ýÀåÀÌ ÀúÇصǾî ÀçÁ¶ÇÕ À¯»ê±ÕÀ» ¼±¹ßÇÒ ¼ö ÀÖ´Ù.
 Posno [28]Àº D-xylose¿Í °°Àº ´ç ´ë»ç´É·ÂÀ» ÀÌ¿ëÇÑ dominant ¼±¹ß¹æ¹ýÀ» óÀ½À¸·Î °³¹ßÇß´Ù. Lb. pentosus¿¡¼­ D-xylose¸¦ ÄÚµåÇÏ´Â xylRAB À¯ÀüÀÚ¸¦ ÀÌ¿ëÇÑ E. coli-Lactobacillus ¼ÅƲº¤ÅÍ´Â erythromycinÀ» ÷°¡ÇÑ ¹èÁö¿¡¼­ D-xylose¸¦ ´ë»ç ÇÒ ¼ö ÀÖ¾î food-grade ¸¶Ä¿·Î½á »ç¿ëÀÌ µÉ ¼ö ÀÖ´Ù. ¶ÇÇÑ,  Lactococcus raffinolactis±ÕÁÖ¿¡¼­b-GalactosidaseÀ¯ÀüÀÚ¸¦ ÀÌ¿ëÇÏ¿© melibiose ´ë»çÇÒ ¼ö ÀÖ´Â ¹ßÇöº¤Å͸¦ °³¹ßÇÏ¿´´Ù[29].

¾ÈÁ¤ÀûÀÌ°í ¼±ÅÃÀûÀÎ host/vector½Ã½ºÅÛÀ» °³¹ßÇϱâ À§ÇÑ ¶Ç ´Ù¸¥ ¹æ¹ýÀ¸·Î´Â ¹Ì»ý¹°ÀÇ ´ë»ç°æ·Î³ª ¾î¶² Ư¼ºÀ» ºÎ¿©ÇÏ´Â ÇʼöÀûÀÎ ´Ü°è¿¡ °ü·ÃµÈ chromosome À¯ÀüÀÚ¸¦ µ¹¿¬º¯ÀÌ ½ÃÅ°´Â complementation¹æ¹ýÀÌ ÀÌ¿ëµÈ´Ù. ComplementationÀ» ±¸ÃàÇϱâ À§Çؼ­ host chromosome¾È¿¡ ¸ñÀûÀ¯ÀüÀÚ¸¦ knock-outÇÑ µ¹¿¬º¯ÀÌ ±ÕÁÖ¸¦ ¼±¹ßÇÑ ÈÄ, ¾ç¸³ÇÒ ¼ö ÀÖ´Â complementationÀ» µµÀÔÇÑ º¤Å͸¦ ±¸ÃàÇÑ´Ù. Dickley[54]´Â Purine »ýÇÕ¼º °æ·Î¿¡¼­ nonsense suppressor (ocher suppressorÀ¯ÀüÀÚ supB)ÀÇ µ¹¿¬º¯À̸¦ ÀÏÀ¸Å² Lc. Lactis¸¦ ÀÌ¿ëÇÏ¿© ¿µ¾ç¿ä±¸¼º food-grade¸¶Ä¿¸¦ µµÀÔÇÑ pFG1¸¦ °³¹ßÇÏ¿´´Ù. Alanine racemase¸¦ ÄÚµåÇÏ´Â alrÀ¯ÀüÀÚ¸¦ ÀÌ¿ëÇÑ complementationÀÌ °³¹ßµÇ¾ú´Ù. Alanine racemase´Â L-alanine°ú D-alanineÀÇ »óÈ£ÀÛ¿ëÀ» Ã˸ÅÇÏ¿© ¼¼Æ÷º® ÇÕ¼º¿¡ °ü¿©ÇÏ¿© Lb. plantarum¿Í Lc. lactis [31] »ýÀå¿¡ ÇʼöÀûÀ¸·Î ÇÊ¿äÇÑ È¿¼Ò·Î½á, Lb. plantarum¿Í Lc. LactisÀÇ chromosomal alrÀ¯ÀüÀÚ ³»ºÎÀÇ 100bp¿Í 30bpºÎºÐÀ» °¢°¢ Á¦°ÅÇÏ¿© D-alanine ¿µ¾ç¿ä±¸¼º ±ÕÁÖ¸¦ ¼±¹ßÇÏ¿´°í, lactococcal/lactobacillal alrÀ¯ÀüÀÚ·Î ±¸¼ºµÈ pGIPº¤Å͸¦ ÀÌ¿ëµÈ´Ù. ±× ¿Ü¿¡ ´Ù¾çÇÑ À¯»ê±Õ¿¡¼­ chromosomal lactose operonÀ» ƯÁ¤ÀûÀ¸·Î µ¹¿¬º¯À̸¦ ÀÏÀ¸ÄѼ­ complementation¸¶Ä¿·Î ÀÌ¿ëµÈ´Ù [32].

 

8. À¯»ê±Õ¿¡¼­ food-grade gene expression »ç·Ê
HernándezÀº [36] strawberry (Fragariax ananassa)À¯·¡ÀÇ alcohol acyltransferase (SAAT)¿Í linalool/nerolidol synthase (FaNES)¸¦ ÄÚµùÇÏ´Â µÎ °¡Áö À¯ÀüÀÚ¸¦ NICE systemÀ» ÀÌ¿ëÇÏ¿© ¹ßÇö½ÃÄÑ fruit flavor metabolites, esters ±×¸®°í terpenoid¸¦ °¢°¢ ÇÕ¼ºÇÒ ¼ö ÀÖ¾ú°í, ¸é¿ª´Ü¹éÁú »ý»ê¿¡ À־ peanut (Arachis hypogaea) allergen Ara h2À» ÄÚµùÇÏ´Â À¯ÀüÀÚ¸¦ signal sequence (SP310mut2) ¿Í lactate-inducible lactococcal P170 promotor¸¦ ÀÌ¿ëÇÏ¿© Lc. Lactis¿¡¼­ ¹ßÇöÀ» ¼öÇàÇÏ¿´´Ù[37]. Ç׽ùßÇö Lactococcal P32 promoter¿Í NICE systemÀ» ÀÌ¿ëÇÏ¿© Bacillus subtilis nattokinase »ý»êÀ» Lc. lactis¿¡¼­ ¿¬±¸ÇÏ¿© Ç÷ÀüÁõ(thrombosis)À» ¿¹¹æÇϰųª Á¶ÀýÇÒ ¼ö ÀÖ´Â ¹°ÁúÀ» »ý»êÇÏ¿´À¸¸ç [38], ±× ¿Ü·Î oral vaccine ¹× ´Ü¹éÁú delivery ¿¬±¸°¡ Lc. lactis¿¡¼­ ÁøÇàÀÌ µÇ¾î murine cytokine IFN-¥ã[39, 40], Helicobacter pyloris urease subunit B [41], a hepatitis B virus surface antigen [42], ¶Ç´Â Listeria monocytogenes antigens P60¿Í LLO [43]À» »ý»êÇÏ¿´´Ù.

 

Ç¥4. Secretion system for the expression in Lactic acid bacteria

 

Ç¥5. Display system for the expression in Lactic acid bacteria




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