¿ÞÂÊ ¸Þ´º ŸÀÌƲ À̹ÌÁö
Áֿ伺°ú

Home < Áֿ伺°ú < Áֿ伺°ú

       ÇÕ¼º »ý¹°ÇÐÀ» ÀÌ¿ëÇÑ Ãµ¿¬¹° Á¶ÇÕ »ýÇÕ¼º ±â¼úÀÇ È°¼ºÈ­
               
       ISBC        2018.08.28 17:33        4154
 

ÇÕ¼º »ý¹°ÇÐÀ» ÀÌ¿ëÇÑ Ãµ¿¬¹° Á¶ÇÕ »ýÇÕ¼º ±â¼úÀÇ È°¼ºÈ­


ÀÌÈ­¿©ÀÚ´ëÇб³ À±¿©ÁØ ±³¼ö


Reinvigorating natural product combinatorial biosynthesis with synthetic biology. Nature Chemical Biology, 2015, 11(9), 649-659.


1. ¿¬±¸¹è°æ


ÀÚ¿¬°è¿¡ Á¸ÀçÇÏ´Â ¹Ì»ý¹°À̳ª ½Ä¹°¿¡¼­ À¯·¡ÇÑ Ãµ¿¬ È­ÇÕ¹°Àº À¯¿ëÇÑ »ý¸®È°¼ºÀ» °¡Áö¸ç ÀǾàÇ° °³¹ßÀÇ ÀÚ¿øÀ¸·Î¼­ Áß¿äÇÑ ¿ªÇÒÀ» ÇÑ´Ù. ±×·¯³ª ÀÚ¿¬°è Ž»öÀ» ÅëÇØ »õ ¹°ÁúÀ» ãÀ¸·Á´Â °íÀüÀûÀÎ ¹æ¹ý¿¡´Â ¸¹Àº ½Ã°£°ú ³ë·Â, ºñ¿ëÀÌ ¿ä±¸µÇ¾î È¿¿ë¼ºÀÌ ¶³¾îÁø´Ù. ÀÌ¿¡ õ¿¬ È­ÇÕ¹°ÀÇ ±¸Á¶¸¦ º¯ÇüÇØ À¯¿ëÇÑ ºñõ¿¬ È­ÇÕ¹°À» ¹ß±¼Çϱâ À§ÇÑ ´Ù¾çÇÑ ¹æ¹ýÀÌ ¸¹ÀÌ ¿¬±¸µÇ°í ÀÖ´Ù.


´ëÇ¥ÀûÀÎ ¿¹·Î, õ¿¬ È­ÇÕ¹°ÀÇ »ýÇÕ¼º¿¡ °ü·ÃµÈ À¯ÀüÀÚÀÇ Á¶ÇÕÀ» ÅëÇØ »õ·Î¿î »ýÇÕ¼º °æ·Î¸¦ À籸¼ºÇÏ´Â Á¶ÇÕ»ýÇÕ¼º(combinatorial biosynthesis) ±â¼úÀ» ÀÌ¿ëÇÏ¿© È­ÇÕ¹° ±âº»°ñ°ÝÀÇ ¸ð¾çÀ̳ª ÀÛ¿ë±â µîÀ» º¯È­½ÃÄÑ »õ·Î¿î È­ÇÕ¹°À» ¸¸µé ¼ö ÀÖ´Ù. ¶ÇÇÑ »ýÇÕ¼º °ü·Ã È¿¼ÒÀÇ °íÀ¯¼ºÁú º¯È­¸¦ ÅëÇÑ ÀÎÀ§Àû ÀçÁ¶ÇÕ »ýÇÕ¼º °æ·Î¸¦ ±¸ÃàÇÏ´Â ¹æ¹ý, ±×¸®°í ÀÌÁ¾¼÷ÁÖ¿¡¼­ õ¿¬¹°ÀÇ »ýÇÕ¼º À¯ÀüÀÚµéÀ» È¿À²ÀûÀ¸·Î ¹ßÇö½ÃÅ°´Â ¹æ¹ý µîÀÌ ÀÖ´Ù. À̸¦ È¿À²ÀûÀ¸·Î ¼öÇàÇϱâ À§ÇØ »ý¹°ÇÐÀû ÀÌÇØ¿Í °øÇÐÀû °³³äÀ» Àû¿ëÇÑ ÇÕ¼º »ý¹°ÇÐ (synthetic biology)ÀÌ µµÀԵǰí ÀÖ´Ù.


º» ³í¹®¿¡¼­´Â ÀǾàÇ° °³¹ßÀÇ À¯¿ëÇÑ ÀÚ¿øÀÌ µÇ´Â ºñõ¿¬ È­ÇÕ¹° ¹ß±¼À» À§ÇÑ Á¶ÇÕ»ýÇÕ¼º ±â¼úÀÇ Àû¿ë°ú ±× ÇÑ°èÁ¡À» ³íÀÇÇÏ°í, À̸¦ ±Øº¹Çϱâ À§ÇÑ ÇÕ¼º »ý¹°ÇÐÀÇ µµÀÔÀ» ÅëÇØ ½Å¾à ¹°Áú °³¹ßÀ» È°¼ºÈ­Çϱâ À§ÇÑ Çõ½ÅÀû °üÁ¡À» Á¦½ÃÇÏ°í ÀÖ´Ù.


2. ¿¬±¸³»¿ë


´ëÇ¥ÀûÀΠõ¿¬ È­ÇÕ¹°ÀÇ ¿¹·Î Ç×±Õ, Ç×¾Ï, ¸é¿ª¾ïÁ¦ È°¼ºÀ» °¡Áö´Â polyketide °è¿­À̳ª nonribosomal peptide °è¿­À» µé ¼ö ÀÖÀ¸¸ç, À̵éÀº polyketide synthase (PKS) ¶Ç´Â nonribosomal peptide synthetase (NRPS)¶ó°í ÇÏ´Â ´Ù±â´É ´Ü¹éÁú º¹ÇÕü (multifunctional protein complex)¿¡ ÀÇÇØ »ýÇÕ¼º µÈ´Ù1,2. ÀÌ ´Ü¹éÁú º¹ÇÕü´Â ¿¬¼ÓÀûÀÎ ÃàÇÕ ¹ÝÀÀ¿¡ °ü¿©ÇÏ´Â ¿©·¯ °³ÀÇ module·Î ±¸¼ºµÇ¾î ÀÖÀ¸¸ç, °¢ moduleÀº »ýÇÕ¼º ±âÁú·Î »ç¿ëµÇ´Â carboxylic acid³ª amino acidÀÇ ÃàÇÕ ¹× ȯ¿ø¿¡ °ü¿©ÇÏ´Â ¿©·¯ Á¾·ùÀÇ domain Á¶ÇÕÀ¸·Î ±¸¼ºµÇ¾î ÀÖ´Ù. Àüü moduleÀÇ ¼ö¿¡ µû¶ó È­ÇÕ¹° »ç½½ÀÇ ±æÀÌ°¡ °áÁ¤ µÉ ¼ö ÀÖ°í, domainÀÇ Á¶ÇÕ¿¡ µû¶ó ±âÁúÀÇ Á¾·ù¿Í ÀÛ¿ë±âÀÇ È¯¿ø Á¤µµ°¡ ´Ù¸£°Ô ³ªÅ¸³ª¹Ç·Î, °¢°¢ÀÇ module°ú domainÀÇ Á¶ÇÕ¿¡ µû¶ó ¹«ÇÑÇÑ ±¸Á¶ÀÇ À¯µµÃ¼°¡ »ýÇÕ¼º µÉ ¼ö ÀÖ´Ù. ¶ÇÇÑ polyketideÀÇ °æ¿ì, PKS¿¡ ÀÇÇØ »ýÇÕ¼º µÈ ±âº»°ñ°Ý ¿Ü¿¡ glycosylation, methylation, hydroxylation µîÀÇ post-PKS tailoring modification ´Ü°è¿¡ ÀÇÇØ ±¸Á¶°¡ º¯ÇüµÇ¾î È°¼ºÀ» °¡Áö°Ô µÈ´Ù.


ÀϹÝÀûÀÎ Á¶ÇÕ»ýÇÕ¼ºÀ» ÀÌ¿ëÇÏ¿© gene fusion, gene inactivation, gene replacement, domain substitution, module exchange µîÀ» ÅëÇØ ´Ù¾çÇÑ Ãµ¿¬ È­ÇÕ¹°ÀÇ À¯µµÃ¼¸¦ ¸¸µé¾úÀ¸¸ç (±×¸² 1), ±× Áß ÀϺδ Áõ°¡µÈ »ý¸®È°¼ºÀ» ¶ç´Â ¹°Áúµµ ÀÖ¾ú´Ù3-6 (È­ÇÕ¹° 3).

±×¸² 1. Polyketide¿Í nonribosomal peptideÀÇ ÀϹÝÀûÀÎ Á¶ÇÕ»ýÇÕ¼º ¹æ¹ý. (a) Aureothin PKSÀÇ domain truncation°ú AT domain exchange¸¦ ÅëÇÑ À¯µµÃ¼ luteoreticulin (1)ÀÇ »ý»ê. (b) MethyltransferaseÀÇ inactivationÀ» ÅëÇÑ SF2575 ±¸Á¶ º¯Çü (2). (c) Daptomycin NRPSÀÇ module exchange¸¦ ÅëÇÑ À¯µµÃ¼ (3) »ý»ê.


Á¶ÇÕ»ýÇÕ¼ºÀ» ÀÌ¿ëÇÑ ½Å±Ô ±¸Á¶ÀÇ °³¹ß¿¡ ÀÖ¾î Ãʱ⿡´Â »ýÇÕ¼º °ü·Ã È¿¼ÒÀÇ ÇÑÁ¤µÈ ±âÁú ƯÀ̼ºÀÌ ´Ù¾çÇÑ È­ÇÕ¹°ÀÇ ±¸Á¶ Á¦ÀÛ¿¡ À־ Á¦ÇÑ ¿ä¼Ò·Î ÀÛ¿ëÇßÁö¸¸, ÃÖ±Ù¿¡ µé¾î¼­ È¿¼ÒµéÀÇ °íÀ¯ Ư¼º ±Ô¸í ¹× È°¼º º¯°æÀ» ÅëÇØ À¯¿¬ÇÑ ±âÁú ƯÀ̼ºÀ» °¡Áö°Ô µÇ¾úÀ¸¸ç À̸¦ ÀÌ¿ëÇØ ´Ù¾çÇÑ ±¸Á¶ÀÇ È­ÇÕ¹° °³¹ßÀÌ À¯¿ëÇØÁö°í ÀÖ´Ù. Polyketide¿Í nonribosomal peptideÀÇ »ýÇÕ¼ºÀÇ °æ¿ì, »õ·Î¿î ±âÁúÀÇ »ýÇÕ¼º °æ·Î À籸¼º°ú °ø±Þ, »õ ±âÁúÀÇ È­ÇÕ¹° °ñ°ÝÀ¸·ÎÀÇ °áÇÕ, È­ÇÕ¹° °ñ°ÝÀÇ ±¸Á¶ º¯Çü µî¿¡ ±âÁú ƯÀ̼ºÀÌ À¯¿¬Çϰųª »õ·Î¿î È°¼ºÀÌ ºÎ¿©µÈ È¿¼ÒÀÇ »ç¿ëÀ¸·Î ½Å±Ô ±¸Á¶ÀÇ »ýÇÕ¼º °³¹ßÀÇ °¡´É¼ºÀ» ³ôÀÌ°í ÀÖ´Ù.


¿¹¸¦ µé¾î, ´Ù¾çÇÑ acyl-CoA extender unitÀ» ¸¸µé ¼ö ÀÖ´Â crotonyl-CoA carboxylase/reductase (CCR)ÀÇ ¹ß°ß°ú7 ±×¿Í ¿¬°üµÈ ±âÁú ƯÀ̼ºÀÌ À¯¿¬ÇÑ AT domainÀÇ Á¶ÇÕÀ¸·Î ´Ù¾çÇÑ acyl-CoA extender unitÀ» polyketideÀÇ »ýÇÕ¼º¿¡ »ç¿ëÇÒ ¼ö ÀÖ°Ô µÇ¾ú´Ù. FK506 PKS³ª amtimycin PKS-NRPS hybridÀÇ °æ¿ì ±âÁú ƯÀ̼ºÀÌ À¯¿¬ÇÑ AT domainÀ» °¡Áö°í ÀÖÀ¸¸ç ¿ø·¡ »ç¿ëµÇ´Â ±âÁú ÀÌ¿Ü¿¡ ºñõ¿¬ acyl-CoA¸¦ ¹Þ¾Æµé¿© ±¸Á¶°¡ º¯ÇüµÈ À¯µµÃ¼ (FK506 À¯µµÃ¼ 4-6, antimycin À¯µµÃ¼ 7-10)¸¦ ¸¸µé ¼ö ÀÖ¾úÀ¸¸ç (±×¸² 2), ±× Áß ÀϺΠÀ¯µµÃ¼´Â »ý¸® È°¼ºÀÌ Áõ°¡µÇ°Å³ª º¯ÇüµÇ´Â °ÍÀ» ¾Ë ¼ö ÀÖ¾ú´Ù8,9.


À§¿Í ´Ù¸£°Ô ´ëºÎºÐÀÇ PKS AT domain°ú NRPS¿¡¼­ ±âÁúÀ» ¼±ÅÃÇÏ¿© ¹Þ¾ÆµéÀÌ´Â adenylation (A) domainÀÇ °æ¿ì¿¡´Â ƯÁ¤ ±âÁú¿¡ ´ëÇؼ­¸¸ È°¼ºÀ» °¡Áö°í ÀÖÀ¸¹Ç·Î, active siteÀÇ ÀϺΠamino acid¸¦ ġȯ½ÃÅ°°Å³ª directed evolution ±â¼úÀ» ÅëÇØ È°¼ºÀ̳ª ±âÁúƯÀ̼ºÀÌ °³·®µÈ È¿¼Ò¸¦ »ç¿ëÇÏ¿© À¯µµÃ¼¸¦ »ý»êÇØ ³½´Ù. Erythromycin PKS module 6ÀÇ AT domainÀÇ mutation (Val295Ala)10 (È­ÇÕ¹° 11), calcium-dependent antibiotic (CDA) NRPS module 10ÀÇ A domainÀÇ mutation (Lys278Gln)11 (È­ÇÕ¹° 12-13), andrimid NRPS-PKS Val-activating A domainÀÇ saturation mutagenesis12 (È­ÇÕ¹° 14-16)¸¦ ÅëÇØ ´Ù¾çÇÑ ±âÁúÀ» »ç¿ëÇÏ´Â À¯µµÃ¼¸¦ ¸¸µé ¼ö ÀÖ¾ú´Ù (±×¸² 2).


¶ÇÇÑ, ±âÁúÀÇ ¼±ÅüºÀÌ ¿¹»óµÇ´Â AT domainÀÇ Ä¡È¯À» ÅëÇØ ±¸Á¶¸¦ º¯Çü½Ãų ¼ö Àִµ¥, fluoromalonyl-CoA¸¦ ±âÁú·Î ¼±ÅÃÇÏ´Â disorazole PKSÀÇ trans-AT domainÀ» erythromycin PKSÀÇ ÀϺΠmodule°ú Á¶ÇÕÇÏ¿© fluorinated PK À¯µµÃ¼ (È­ÇÕ¹° 17-18)¸¦ ¸¸µé ¼ö ÀÖ¾ú´Ù13 (±×¸² 2).


ÀÌ¿Ü¿¡, acyl carrier protein (ACP) domain14À̳ª PKSÀÇ docking domain15,16, NRPSÀÇ communication-mediating domain17ÀÇ engineeringÀ» ÅëÇØ ´Ù¾çÇÑ ½Å±Ô À¯µµÃ¼¸¦ »ý»êÇϱâ À§ÇÑ ½Ãµµ¸¦ À̾î¿À°í ÀÖ´Ù.

±×¸² 2. AT ¶Ç´Â A domainÀÇ ±âÁúƯÀ̼º engineering¿¡ ÀÇÇØ »ýÇÕ¼º µÈ À¯µµÃ¼.


ÃÖ±Ù, ½ÃÄö½Ì ±â¼úÀÇ ¹ß´Þ·Î õ¿¬ È­ÇÕ¹° »ýÇÕ¼º À¯ÀüÀÚÀÇ DNA ¼­¿­Á¤º¸°¡ dzºÎÇØÁü¿¡ µû¶ó À̸¦ ÀÌ¿ëÇÏ¿© »õ·Î¿î Á¾·ùÀÇ À¯¿ë È­ÇÕ¹°À» ¸¸µé ¼ö ÀÖ´Â ±âȸ°¡ Á¡Â÷ ´Ã¾î³ª°í ÀÖ´Ù18. ÀÌ¿¡ ÇÕ¼º »ý¹°ÇÐÀû Á¢±Ù¹æ¹ýÀ» ÅëÇØ Á» ´õ ºü¸£°í Á¤È®ÇÏ°Ô Á¶ÇÕ»ýÇÕ¼ºÀ» ¼öÇàÇÒ ¼ö ÀÖ°Ô µÇ¾ú´Âµ¥, ƯÈ÷ ´Ù¾çÇÑ »ýÇÕ¼º °úÁ¤À» ÀçÁ¶ÇÕ Çϱâ À§ÇÑ ´Ù¾çÇÑ DNA assembly ¹æ¹ýÀÌ º¸°íµÇ°í ÀÖ´Ù.


±× Áß homology ±â¹ÝÀÇ ¹æ¹ýÀº õ¿¬¹° »ýÇÕ¼º À¯ÀüÀÚ Áý´ÜÀÇ ÀçÁ¶ÇÕ ¹× ÀÌÁ¾¼÷ÁÖ ¹ßÇöÀ» À§ÇØ ±¤¹üÀ§ÇÏ°Ô »ç¿ëµÇ°í ÀÖ´Ù. ÀüÅëÀûÀÎ in vivo Red/ET recombineeringÀº Escherichia coli ³»¿¡¼­ coliphage ¥ë Red system À¯·¡ÀÇ Red¥á¥â¥ã ¶Ç´Â Rac prophage À¯·¡ÀÇ truncated RecE-T¿¡ ÀÇÇØ linear vector Á¶°¢°ú circular DNA »çÀÌ¿¡ recombination (linear-plus-circular homologous recombination, LCHR)ÀÌ ÀϾ´Â ¹æ¹ýÀ¸·Î19 Áö³­ 10¿©³â °£ ÀÌ ¹æ¹ýÀ» ÅëÇØ ´Ù¾çÇÑ Ãµ¿¬ È­ÇÕ¹°ÀÌ ÀÌÁ¾¼÷ÁÖ ³»¿¡¼­ »ý»êµÇ¾úÀ¸³ª, °Å´ëÇÑ À¯ÀüÀÚ Áý´Ü¿¡ Àû¿ëÇϱ⿡´Â ¾î·Á¿òÀÌ ÀÖ¾ú´Ù. Á»´õ ÃÖ±Ù¿¡´Â full-length RecE-T¿¡ ÀÇÇØ linear vector Á¶°¢°ú linear DNA »çÀÌ¿¡ recombination (linear-plus-linear homologous recombination, LLHR)ÀÌ ÀϾ´Â ¹æ¹ýÀÌ »ç¿ëµÇ¾ú´Âµ¥20, LLHRÀº LCHR¿¡ ºñÇØ 20¹è Á¤µµ Çâ»óµÈ È¿À²À» º¸¿©ÁÖ°í ÀÖ´Ù (±×¸² 3).

±×¸² 3. õ¿¬¹° »ýÇÕ¼º À¯ÀüÀÚ Áý´ÜÀÇ assembly ¹æ¹ý.


À§ÀÇ phage-mediated homologous recombination ¹æ¹ý ¿ÜÀÇ, ¶Ç ´Ù¸¥ recombination ¹æ¹ýÀÎ transformation-associated recombination (TAR)Àº Saccharomyces cerevisiaeÀÇ ³»¿¡¼­ environmental DNA »ùÇÃÀ̳ª genomic DNA·ÎºÎÅÍ ¿øÇÏ´Â À¯ÀüÀÚ ºÎºÐÀ» ¹Ù·Î °¡Á®¿Ã ¼ö ÀÖ´Â ÀåÁ¡À» ÀÖ´Ù21 (±×¸² 3). ÇÑ ¿¹·Î, À̸¦ ÀÀ¿ëÇÏ¿© cosmid ±â¹ÝÀÇ S. cerevisiae-E. coli shuttle-actinobacterial chromosome integrative capture vector (pCAP01)°¡ °³¹ßµÇ¾ú´Ù. ÀÌ vector¸¦ ÀÌ¿ëÇÏ¿© S. cerevisiae ³»¿¡¼­ ¿øÇÏ´Â À¯ÀüÀÚ¸¦ capture ÇÏ°í, E. coli ³»¿¡¼­ ÃæºÐÇÑ ¾çÀÇ DNA¸¦ È®º¸ÇÒ ¼ö ÀÖÀ¸¸ç, ÀÌÁ¾¼÷ÁÖ ³»¿¡¼­ ¹Ù·Î ¹ßÇöÀÌ °¡´ÉÇÏ´Ù. ÀÌ ¹æ¹ýÀ¸·Î ÇØ¾ç ¹æ¼±±Õ Saccharomonospora sp. CNQ-490 À¯·¡ 67-kb Å©±âÀÇ ºñÈ°¼º NRPS À¯ÀüÀÚ Áý´ÜÀ» È®º¸ÇÏ¿´À¸¸ç, ÀÌÁ¾¼÷ÁÖ Streptomyces coelicolor¿¡¼­ ¹ßÇöÇÏ¿© daptomycin À¯µµÃ¼ÀÎ taromycin A (È­ÇÕ¹° 19)¸¦ »ý»êÇÒ ¼ö ÀÖ¾ú´Ù22 (±×¸² 4). TAR ±â¹ÝÀÇ ¡°DNA assembler¡± ¹æ¹ýÀº »ýÇÕ¼º¿¡ °ü·ÃµÈ ¿©·¯ DNA Á¶°¢µéÀ» S. cerevisiae ³»¿¡¼­ Çѹø¿¡ Á¶¸³ÇÒ ¼ö ÀÖÀ¸¸ç, mutationµÈ °¢°¢ DNA Á¶°¢µéÀÇ Á¶ÇÕÀ» ÅëÇØ ´Ù¾çÇÑ À¯µµÃ¼¸¦ »ý»êÇÒ ¼ö ÀÖ´Â ¹æ¹ýÀÌ´Ù (±×¸² 3)23.

±×¸² 4. ÇÕ¼º »ý¹°ÇÐÀ» ÀÀ¿ëÇÑ DNA assembly ¹æ¹ýÀ» ÅëÇØ »ý»êµÈ cryptic õ¿¬È­ÇÕ¹° ¶Ç´Â À¯µµÃ¼.


´ëÇ¥ÀûÀÎ in vitro homology ±â¹ÝÀÇ ¹æ¹ýÀÎ ¡°Gibson assembly¡±´Â24, ÀÌÁß °¡´Ú DNA Á¶°¢µéÀ» T5 exonuclease¸¦ ÀÌ¿ëÇØ 5¡¯ ¸»´ÜÀ» ºÐÇØÇÏ°í, ÀûÀýÇÑ ¿Âµµ¿¡¼­ anneling µÈ ºÎºÐÀ» Phusion polymerase¿Í Taq ligase¸¦ ÀÌ¿ëÇØ ¿¬°áÇØ ÁÖ´Â ¹æ¹ýÀÌ´Ù. ¶Ç ´Ù¸¥ in vitro ¹æ¹ýÀÎ sequence- and ligation-independent cloning (SLIC)Àº25 T4 DNA polymerase°¡ »ç¿ëµÇ¾î DNA Á¶°¢ÀÇ 3¡¯ ¸»´ÜÀ» ºÐÇØÇÏ°í ¶Ç ÀÌ DNA Á¶°¢À» ¿¬°áÇÑ´Ù. ÀÌ ¹æ¹ýÀ» ÅëÇØ mutationµÈ premonensin »ýÇÕ¼º DNA Á¶°¢À» shuttle vector¿¡ Ŭ·Î´×ÇÏ°í homologous recombinationÀ» ÅëÇØ chromosomal PKS¿¡ »ðÀÔÇÏ¿© ´Ù¾çÇÑ premonensin À¯µµÃ¼¸¦ ¸¸µé¾úÀ¸¸ç, ÀϺΠÀ¯µµÃ¼ (È­ÇÕ¹° 20-22)´Â Ç×±ÕÈ°¼ºÀÌ Áõ°¡µÇ´Â °ÍÀ» ¾Ë ¼ö ÀÖ¾ú´Ù26 (±×¸² 4).


ÇÕ¼º »ý¹°ÇÐÀ» ÀÀ¿ëÇÑ homology ±â¹ÝÀÇ DNA assembly ¹æ¹ýµéÀ» ÀÌ¿ëÇϸé Á»´õ ½±°í ºü¸¥ ¹æ¹ýÀ¸·Î »ýÇÕ¼º À¯ÀüÀÚµéÀ» Á¶ÇÕÇÒ ¼ö ÀÖ´Ù. ºñ±³ÀûÀ¸·Î in vivo ¹æ¹ýÀº in vitro ¹æ¹ýº¸´Ù Å« »çÀÌÁîÀÇ À¯ÀüÀÚµéÀ» Á¶ÇÕÇϱ⿡ Á»´õ ¿ëÀÌÇÏÁö¸¸, in vitro ¹æ¹ýÀº º¸´Ù ªÀº ½Ã°£¿¡ construct¸¦ ¸¸µé ¼ö ÀÖ´Ù´Â ÀåÁ¡ÀÌ ÀÖ´Ù.


´Ù¾çÇÏ°í À¯¿ëÇÑ DNA assembly ±â¹ýÀÌ ¹ßÀüÇÔ¿¡ µû¶ó, À̸¦ ÀÌÁ¾¼÷ÁÖ¿¡¼­ È¿À²ÀûÀ¸·Î ¹ßÇö½ÃÅ°±â À§ÇØ ÀûÀýÇÑ ÀÌÁ¾¼÷ÁÖ¸¦ ã°í Àû¿ëÇÏ´Â ÀÏ ¶ÇÇÑ Áß¿äÇØÁö°í ÀÖ´Ù. ÀϹÝÀûÀ¸·Î ³Î¸® ¾²ÀÌ´Â ÀÌÁ¾¼÷ÁÖ´Â Streptomyces Á¾°ú E. coliÀÌ´Ù. ƯÈ÷, Streptomyces´Â »ýÇÕ¼º Àü±¸Ã¼°¡ dzºÎÇÏ°í genome sequencingÀ» ÅëÇÑ ´ë»çüµéÀÌ Àß ¾Ë·ÁÁ® ÀÖ´Â µîÀÇ ÀÌÁ¾¼÷Áַμ­ÀÇ ÀåÁ¡À» µÎ·ç °®Ãß°í ÀÖ´Ù. ÀÌÁ¾¼÷ÁÖÀÇ ÀÌÂ÷´ë»ç»ê¹° »ýÇÕ¼º À¯ÀüÀÚ¸¦ Á¦°ÅÇÔÀ¸·Î½á, ¹ßÇö½ÃÄÑÁØ ¿Ü·¡ »ýÇÕ¼º À¯ÀüÀڷκÎÅÍ »ý»êµÇ´Â È­ÇÕ¹°·Î Àü±¸Ã¼³ª ¿¡³ÊÁöÀÇ °ø±ÞÀÌ ¸¹¾ÆÁö°í, »ý¼º È­ÇÕ¹°ÀÇ °ËÃâ ¹× ºÐ¼®µµ ¿ëÀÌÇØÁø´Ù. À̸¦ À§ÇØ S. coelicolor27¿Í S. avermitilis28ÀÇ °æ¿ì, È¿À²ÀûÀÎ ÀÌÁ¾¼÷ÁÖ·Î »ç¿ëÀ» À§ÇØ ÀÌÂ÷´ë»ç »ýÇÕ¼º À¯ÀüÀÚ Áý´ÜÀÌ Á¦°ÅµÈ ´Ù¾çÇÑ strainÀÌ ¸¸µé¾îÁ³À¸¸ç, À̸¦ ÀÌ¿ëÇØ ´Ù¸¥ ¹æ¼±±ÕÀ̳ª ½Ä¹° À¯·¡ÀÇ Ãµ¿¬È­ÇÕ¹°À» ¼º°øÀûÀ¸·Î »ý»êÇÒ ¼ö ÀÖ¾ú´Ù27-30.


¶Ç ´Ù¸¥ Àü·«À¸·Î´Â, ÀüÅëÀûÀÎ mutation ¹æ¹ýÀ¸·Î ¸¸µé¾îÁø °ú»ý»ê »ê¾÷±ÕÁÖ¸¦ »ç¿ëÇÏ´Â °ÍÀÌ´Ù. ´ëÇ¥ÀûÀÎ ¿¹·Î, Saccharopolyspora erythraea »ê¾÷ ±ÕÁÖ¿¡¼­ ÆÄ»ýµÈ ¡®clean host¡¯¸¦ ÀÌ¿ëÇÏ¿© erythromycin À¯µµÃ¼ÀÇ »ý»ê·®À» 10¹è °¡·® Áõ°¡½ÃÄ×´Ù31.


ÀÌÁ¾¼÷Áַνá E. coli´Â »ýÀå ¼Óµµ°¡ ºü¸£°í À¯¿ëÇÑ genetic toolÀÌ ´Ù¾çÇÏ´Ù´Â ÀåÁ¡ÀÌ ÀÖÁö¸¸, õ¿¬¹° »ýÇÕ¼º¿¡ ÇÊ¿äÇÑ È¿¼ÒÀÇ ºÎÀ糪 »ýÇÕ¼º Àü±¸Ã¼ÀÇ °ø±ÞÀÌ ¾î·Æ´Ù´Â ´ÜÁ¡ÀÌ ÀÖ´Ù. ±×·³¿¡µµ ºÒ±¸ÇÏ°í, PKS ÀÛµ¿ÀÌ ¿ëÀÌÇϵµ·Ï E. coli¸¦ °³·®ÇÏ°í »ýÇÕ¼º Àü±¸Ã¼¸¦ °ø±ÞÇÔÀ¸½á ÃÖÃÊ·Î polyketide 6-deoxyerythronolide B (erythromycinÀÇ aglycone)ÀÇ »ý»êÀ» °¡´ÉÇϵµ·Ï ÇÏ¿´´Ù32. ±× ÀÌÈÄ·Î erythromycin ¹× ±× À¯µµÃ¼»Ó¸¸ ¾Æ´Ï¶ó33 nonribosomal peptideµµ E. coli¿¡¼­ ¼º°øÀûÀ¸·Î »ý»êÇÏ¿´´Ù34-37.


ÀÌ »Ó¸¸ ¾Æ´Ï¶ó Pseudomonas putida, Myxococcus xanthus, S. cerevisiaeµµ ÀÌÁ¾¼÷ÁÖ·Î ±¤¹üÀ§ÇÏ°Ô »ç¿ëµÇ°í ÀÖ´Ù.


±âÁ¸ÀÇ Á¶ÇÕ»ýÇÕ¼º ¹æ¹ýÀÌ °¡Áö´Â ¹®Á¦ ¹× Á¦ÇÑÁ¡À» ÇØ°áÇϱâ À§ÇØ ÇÕ¼º »ý¹°ÇÐÀ» Àû¿ëÇÔÀ¸·Î½á ½Å±Ô À¯µµÃ¼ÀÇ »ý»êÀ» È°¼ºÈ­ ½Ãų ¼ö ÀÖÀ» °ÍÀ¸·Î Àü¸ÁµÈ´Ù. ¿¹¸¦ µé¾î, (i) ÀÌÁ¾¼÷ÁÖÀÇ codon usage¿¡ ÃÖÀûÈ­µÈ ÇÕ¼º DNA¸¦ ÀÌ¿ëÇÏ¿© ÀÌÁ¾¼÷ÁÖ¿¡¼­ÀÇ ¹ßÇö¹®Á¦¸¦ ÇØ°áÇÏ°í, (ii) ÇÕ¼º scaffold¸¦ ÀÌ¿ëÇÏ¿© »ýÇÕ¼º ´Ü¹éÁúµéÀÇ °ø°£»óÀÇ ¹èÄ¡¸¦ Á¶ÀýÇϸç, (iii) promoter¿Í ribosome binding site (RBS)ÀÇ ¹Ì¼¼Á¶ÀýÀ» ÅëÇØ transcription°ú translation levelÀ» Á¶ÀýÇÏ¿©, ÀÌÁ¾¼÷ÁÖ¿¡¼­ Á¶ÇÕµÈ »ýÇÕ¼º À¯ÀüÀÚ Áý´ÜÀ» È¿À²ÀûÀ¸·Î ¹ßÇö ½Ãų ¼ö ÀÖÀ» °ÍÀÌ´Ù (±×¸² 5).

±×¸² 5. Á¶ÇÕµÈ »ýÇÕ¼º À¯ÀüÀÚ Áý´ÜÀÇ È¿À²Àû ¹ßÇöÀ» À§ÇÑ ÇÕ¼º »ý¹°ÇÐ µµ±¸.


Epothilone NRPS-PKS hybridÀÇ °æ¿ì, ÀÌÁ¾¼÷ÁÖ·Î »ç¿ëÇÒ E. coliÀÇ codon¿¡ ¸ÂÃç ÃÖÀûÈ­µÈ»ýÇÕ¼º À¯ÀüÀÚ Áý´ÜÀ» ÇÕ¼ºÇÏ¿´°í Å©±â°¡ Å« ´Ü¹éÁú EpoD¸¦ µÎ °³ÀÇ ´Ü¹éÁú·Î ³ª´©¾î E. coli¿¡¼­ ¾ÈÁ¤ÀûÀ¸·Î ¹ßÇöµÇ´Â °ÍÀ» È®ÀÎÇÏ¿´À¸¸ç, ÃÖÁ¾ÀûÀ¸·Î 1 ug/L ÀÌÇÏ ¼öÁØÀÇ epothilone C¿Í D°¡ »ý»êµÇ´Â °ÍÀ» È®ÀÎÇÏ¿´´Ù38. À¯»çÇÏ°Ô, ÀÌÁ¾¼÷ÁÖ M. xanthus¿¡¼­ ÃÖÀûÈ­µÈ ÇÕ¼º À¯ÀüÀÚ¸¦ ¹ßÇöÇÏ¿© ¾à 100 ug/LÀÇ epothilone A¿Í B°¡ »ý»êµÇ´Â °ÍÀ» È®ÀÎÇÏ¿´´Ù39.


ÇÕ¼º scaffold¸¦ ÀÌ¿ëÇÏ¸é »ýÇÕ¼º ´Ü¹éÁúµéÀÌ °ø°£»óÀ¸·Î ±ÙÁ¢ÇÑ ºÎºÐ¿¡ À§Ä¡ÇÏ°Ô µÇ°í, »ýÇÕ¼º Áß°£Ã¼µéÀÇ ¼Õ½ÇÀÌ ÃÖ¼ÒÈ­µÇ¾î ÃÖÁ¾ »ê¹°ÀÇ yield°¡ Áõ°¡ÇÒ ¼ö ÀÖ´Ù. ¸î °¡Áö ¿¹·Î, E. coli ÀÌÁ¾¼÷ÁÖ¿¡¼­ protein scaffold¸¦ ÀÌ¿ëÇØ mevalonateÀÇ »ý»ê·®ÀÌ 77¹è °¡·® Áõ°¡ÇÏ´Â °ÍÀ» È®ÀÎÇÏ¿´À¸¸ç40, DNA scaffold¸¦ ÀÌ¿ëÇÏ¿© E. coli¿¡¼­ resveratrol, 1,2-propanediol, mevalonateÀÇ »ý»ê·®ÀÌ 5¹è Áõ°¡ÇÏ¿´´Ù41. ÇÏÁö¸¸ ÀÌ·± ÇÕ¼º scaffoldÀÇ »ç¿ëÀº PKS³ª NRPS¿¡¼­´Â Àû¿ëÇϱ⠾î·Æ´Ù´Â Á¦ÇÑÁ¡ÀÌ ÀÖ´Ù.


ÃÖ±Ù¿¡´Â ³Î¸® »ç¿ëµÇ´Â constitutive promoter ermEp 1ÀÇ engineeringÀ» ÅëÇØ ¼±º°µÈ °­·ÂÇÑ promoter¸¦ »ç¿ëÇØ ÀÌÁ¾¼÷ÁÖ S. lividans¿¡¼­ flaviolin »ý»ê·®ÀÌ 3¹è °¡·®ÀÇ Áõ°¡ÇÏ¿´´Ù42.

±×¸² 6. »ý»ê ±ÕÁÖÀÇ ÃÖÀûÈ­¸¦ À§ÇÑ ÇÕ¼º»ý¹°ÇÐ µµ±¸.


À§ÀÇ ¹æ¹ý»Ó¸¸ ¾Æ´Ï¶ó, genome ÀÚü¸¦ engineering ÇÒ ¼ö ÀÖ´Â (i) clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein (CRISPR/Cas) system, (ii) multiplex automated genome engineering (MAGE), ¶Ç´Â (iii), (iv) RNA SilencingÀ» ÅëÇØ, »ý»ê ÀÌÁ¾¼÷ÁÖ¸¦ ÃÖÀûÈ­ÇÏ¿© õ¿¬¹°ÀÇ »ý»êÀ» À¯¿ëÇÏ°Ô ÇÒ ¼ö ÀÖÀ» °ÍÀÌ´Ù (±×¸² 6).


¼¼Æ÷³»·Î À¯ÀÔµÈ ¿Ü·¡ À¯ÀüÀÚ¸¦ ºÐÇØÇÏ·Á°í ÇÏ´Â ¹ÚÅ׸®¾ÆÀÇ ¸é¿ª½Ã½ºÅÛÀ» ÀÌ¿ëÇÑ CRISPR/Cas systemÀ» ÀÌ¿ëÇÏ¿© ´Ù¾çÇÑ ¼÷ÁÖµéÀÇ genomeÀ» À籸¼º ÇÏ¿´À¸¸ç43-46, single-stranded oligonucleotide¸¦ genome¿¡ µµÀÔ½ÃÅ°¸é¼­ À¯ÀüÀÚÀÇ º¯À̸¦ À¯µµÇÏ´Â MAGE ¹æ¹ýÀ» ÀÌ¿ëÇØ, E. coli¿¡¼­ 3Àϸ¸¿¡ lycopeneÀÇ »ý»ê·®ÀÌ 5¹è Áõ°¡ÇÏ´Â strainÀ» ¾òÀ» ¼ö ÀÖ¾ú´Ù47. ¶ÇÇÑ, genomeÀÇ engineering ¾øÀÌ antisense RNA³ª small regulatory RNA¸¦ ÀÌ¿ëÇÏ¿© translationÀ» Â÷´Ü ÇÒ ¼ö ÀÖ´Â RNA silencing ¹æ¹ýÀ» ÅëÇØ ¸ñÇ¥ À¯ÀüÀÚÀÇ ¹ßÇöÀ» ÀúÇؽÃÄÑ ÀÌÂ÷´ë»ç»ê¹°ÀÇ »ý»êÀ» ¾ïÁ¦½ÃÅ°°Å³ª Áõ°¡½Ãų ¼ö ÀÖ¾ú´Ù48,49.


À§¿¡¼­ ¾ð±ÞÇÑ ÇÕ¼º »ý¹°ÇÐÀû ¹æ¹ýµéÀ» »ýÇÕ¼º À¯ÀüÀÚ Áý´ÜÀÇ Á¶ÇÕ ¹× ÀÌÁ¾¼÷ÁÖ¿¡¼­ ¹ßÇö¿¡ Àû¿ëÇÔÀ¸·Î½á È¿°úÀûÀ¸·Î À¯¿ë È­ÇÕ¹°À» »ý»êÇϴµ¥ µµ¿òÀÌ µÉ °ÍÀ¸·Î ¿¹»óÇÑ´Ù.


3. ¿¬±¸ÀÇ ¼º°ú ¹× ÀÇÀÇ


°ü·Ã ºÐ¾ß ¿¬±¸ ÀÌÇØ¿Í ¹®Á¦Á¡ Áø´Ü ¹× ÇÕ¼º »ý¹°ÇÐÀû ¹æ¹ýÀÇ ÀÀ¿ë °¡´É¼ºÀ» Á¦½ÃÇÑ À̹ø ³í¹®Àº À¯¿ëÇÑ »ý¸®È°¼º ¹°Áú ¹ß±¼°ú ½Å°³³ä ½Å¾à °³¹ß µî¿¡ ´Ù°¢ÀûÀ¸·Î È°¿ëµÉ °ÍÀ¸·Î ±â´ëÇÏ°í ÀÖ´Ù.


Âü°í¹®Çå

1. Hertweck, C. The biosynthetic logic of polyketide diversity. Angew. Chem. Int. Edn Engl. 48, 4688–4716 (2009).
2. Schwarzer, D., Finking, R. & Marahiel, M.A. Nonribosomal peptides: from genes to products. Nat. Prod. Rep. 20, 275–287 (2003).
3. Nguyen, K.T. et al. Combinatorial biosynthesis of novel antibiotics related to daptomycin. Proc. Natl. Acad. Sci. USA 103, 17462–17467 (2006).
4. Baltz, R.H. Combinatorial biosynthesis of cyclic lipopeptide antibiotics: a model for synthetic biology to accelerate the evolution of secondary metabolite biosynthetic pathways. ACS Synth. Biol. 3, 748–758 (2014).
5. Sugimoto, Y., Ding, L., Ishida, K. & Hertweck, C. Rational design of modular polyketide synthases: morphing the aureothin pathway into a luteoreticulin assembly line. Angew. Chem. Int. Edn Engl. 53, 1560–1564 (2014).
6. Wang, P., Kim, W., Pickens, L.B., Gao, X. & Tang, Y. Heterologous expression and manipulation of three tetracycline biosynthetic pathways. Angew. Chem. Int. Edn Engl. 51, 11136–11140 (2012).
7. Eustáquio, A.S. et al. Biosynthesis of the salinosporamide A polyketide synthase substrate chloroethylmalonyl-coenzyme A from S-adenosyl-L-methionine. Proc. Natl. Acad. Sci. USA 106, 12295–12300 (2009).
8. Mo, S. et al. Biosynthesis of the allylmalonyl-CoA extender unit for the FK506 polyketide synthase proceeds through a dedicated polyketide synthase and facilitates the mutasynthesis of analogues. J. Am. Chem. Soc. 133, 976–985 (2011).
9. Yan, Y. et al. Multiplexing of combinatorial chemistry in antimycin biosynthesis: expansion of molecular diversity and utility. Angew. Chem. Int. Edn Engl. 52, 12308–12312 (2013).
10. Sundermann, U. et al. Enzyme-directed mutasynthesis: a combined experimental and theoretical approach to substrate recognition of a polyketide synthase. ACS Chem. Biol. 8, 443–450 (2013).
11. Thirlway, J. et al. Introduction of a non-natural amino acid into a nonribosomal peptide antibiotic by modification of adenylation domain specificity. Angew. Chem. Int. Edn Engl. 51, 7181–7184 (2012).
12. Evans, B.S., Chen, Y., Metcalf, W.W., Zhao, H. & Kelleher, N.L. Directed evolution of the nonribosomal peptide synthetase AdmK generates new andrimid derivatives in vivo. Chem. Biol. 18, 601–607 (2011).
13. Walker, M.C. et al. Expanding the fluorine chemistry of living systems using engineered polyketide synthase pathways. Science 341, 1089–1094 (2013).
14. Kapur, S. et al. Reprogramming a module of the 6-deoxyerythronolide B synthase for iterative chain elongation. Proc. Natl. Acad. Sci. USA 109, 4110–4115 (2012).
15. Gokhale, R.S., Tsuji, S.Y., Cane, D.E. & Khosla, C. Dissecting and exploiting intermodular communication in polyketide synthases. Science 284, 482–485 (1999).
16. Broadhurst, R.W. et al. The structure of docking domains in modular polyketide synthases. Chem. Biol. 10, 723–731 (2003).
17. Hahn, M. & Stachelhaus, T. Selective interaction between nonribosomal peptide synthetases is facilitated by short communication-mediating domains. Proc. Natl. Acad. Sci. USA 101, 15585–15590 (2004).
18. Ongley, S.E., Bian, X., Neilan, B.A. & Müller, R. Recent advances in the heterologous expression of microbial natural product biosynthetic pathways. Nat. Prod. Rep. 30, 1121–1138 (2013).
19. Zhang, Y., Muyrers, J.P., Testa, G. & Stewart, A.F. DNA cloning by homologous recombination in Escherichia coli. Nat. Biotechnol. 18, 1314–1317 (2000).
20. Fu, J. et al. Full-length RecE enhances linear-linear homologous recombination and facilitates direct cloning for bioprospecting. Nat. Biotechnol. 30, 440–446 (2012).
21. Kouprina, N. & Larionov, V. TAR cloning: insights into gene function, long-range haplotypes and genome structure and evolution. Nat. Rev. Genet. 7, 805–812 (2006).
22. Yamanaka, K. et al. Direct cloning and refactoring of a silent lipopeptide biosynthetic gene cluster yields the antibiotic taromycin A. Proc. Natl. Acad. Sci. USA 111, 1957–1962 (2014).
23. Shao, Z., Luo, Y. & Zhao, H. Rapid characterization and engineering of natural product biosynthetic pathways via DNA assembler. Mol. Biosyst. 7, 1056–1059 (2011).
24. Gibson, D.G. et al. Enzymatic assembly of DNA molecules up to several hundred kilobases. Nat. Methods 6, 343–345 (2009).
25. Li, M.Z. & Elledge, S.J. SLIC: a method for sequence- and ligation-independent cloning. Methods Mol. Biol. 852, 51–59 (2012).
26. Kushnir, S. et al. Minimally invasive mutagenesis gives rise to a biosynthetic polyketide library. Angew. Chem. Int. Edn Engl. 51, 10664–10669 (2012).
27. Gomez-Escribano, J.P. & Bibb, M.J. Engineering Streptomyces coelicolor for heterologous expression of secondary metabolite gene clusters. Microb. Biotechnol. 4, 207–215 (2011).
28. Komatsu, M., Uchiyama, T., Omura, S., Cane, D.E. & Ikeda, H. Genome-minimized Streptomyces host for the heterologous expression of secondary metabolism. Proc. Natl. Acad. Sci. USA 107, 2646–2651 (2010).
29. Zhou, M. et al. Sequential deletion of all the polyketide synthase and nonribosomal peptide synthetase biosynthetic gene clusters and a 900-kb subtelomeric sequence of the linear chromosome of Streptomyces coelicolor. FEMS Microbiol. Lett. 333, 169–179 (2012).
30. Komatsu, M. et al. Engineered Streptomyces avermitilis host for heterologous expression of biosynthetic gene cluster for secondary metabolites. ACS Synth. Biol. 2, 384–396 (2013).
31. Rodriguez, E. et al. Rapid engineering of polyketide overproduction by gene transfer to industrially optimized strains. J. Ind. Microbiol. Biotechnol. 30, 480–488 (2003).
32. Pfeifer, B.A., Admiraal, S.J., Gramajo, H., Cane, D.E. & Khosla, C. Biosynthesis of complex polyketides in a metabolically engineered strain of E. coli. Science 291, 1790–1792 (2001).
33. Zhang, H., Wang, Y., Wu, J., Skalina, K. & Pfeifer, B.A. Complete  biosynthesis of erythromycin A and designed analogs using E. coli as a heterologous host. Chem. Biol. 17, 1232–1240 (2010).
34. Watanabe, K. et al. Total biosynthesis of antitumor nonribosomal peptides in Escherichia coli. Nat. Chem. Biol. 2, 423–428 (2006).
35. Bian, X. et al. Direct cloning, genetic engineering, and heterologous expression of the syringolin biosynthetic gene cluster in E. coli through Red/ET recombineering. ChemBioChem 13, 1946–1952 (2012).
36. Ross, A.C., Gulland, L.E., Dorrestein, P.C. & Moore, B.S. Targeted capture and heterologous expression of the Pseudoalteromonas alterochromide gene cluster in Escherichia coli represents a promising natural product exploratory platform. ACS Synth. Biol. 4, 414–420 (2015).
37. Liu, J., Zhu, X., Seipke, R.F. & Zhang, W. Biosynthesis of antimycins with a reconstituted 3-formamidosalicylate pharmacophore in Escherichia coli. ACS Synth. Biol. 4, 559–565 (2015).
38. Mutka, S.C., Carney, J.R., Liu, Y. & Kennedy, J. Heterologous production of epothilone C and D in Escherichia coli. Biochemistry 45, 1321–1330 (2006).
39. O©¬wald, C. et al. Modular construction of a functional artificial epothilone polyketide pathway. ACS Synth. Biol. 3, 759–772 (2014).
40. Dueber, J.E. et al. Synthetic protein scaffolds provide modular control over metabolic flux. Nat. Biotechnol. 27, 753–759 (2009).
41. Conrado, R.J. et al. DNA-guided assembly of biosynthetic pathways promotes improved catalytic efficiency. Nucleic Acids Res. 40, 1879–1889 (2012).
42. Siegl, T., Tokovenko, B., Myronovskyi, M. & Luzhetskyy, A. Design, construction and characterisation of a synthetic promoter library for fine-tuned gene expression in actinomycetes. Metab. Eng. 19, 98–106 (2013).
43. Cong, L. et al. Multiplex genome engineering using CRISPR/Cas systems. Science 339, 819–823 (2013).
44. Bao, Z. et al. Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae. ACS Synth. Biol. 4, 585–594 (2014).
45. Jiang, W., Bikard, D., Cox, D., Zhang, F. & Marraffini, L.A. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat. Biotechnol. 31, 233–239 (2013).
46. Cobb, R.E., Wang, Y. & Zhao, H. High-efficiency multiplex genome editing of Streptomyces species using an engineered CRISPR/Cas system. ACS Synth. Biol. 4, 723–728 (2014).
47. Wang, H.H. et al. Programming cells by multiplex genome engineering and accelerated evolution. Nature 460, 894–898 (2009).
48. Uguru, G.C. et al. Synthetic RNA silencing of actinorhodin biosynthesis in Streptomyces coelicolor A3(2). PLoS ONE 8, e67509 (2013).
49. Na, D. et al. Metabolic engineering of Escherichia coli using synthetic small regulatory RNAs. Nat. Biotechnol. 31, 170–174 (2013).














Total:45 page:(3/2)
29 Á¤º¸ ISBC »ý¹° ģȭÀûÀÎ Fe2O3 ¿äÅ©½© ÀÔÀÚÀÇ ´ë±Ô¸ð ¿¡¾î.. 18.09.04 2712
28 Á¤º¸ ISBC Ç×»ýÁ¦ »ý»ê ÃÖÀûÈ­¸¦ À§ÇÑ ¹æ¼±±Õ À¯ÀüÀû µµ±¸ .. 18.09.04 2230
27 Á¤º¸ ISBC Àúºñ¿ë °íÈ¿À² ¸ÂÃãÇü ¹Ì»ý¹° °Ë»ö Ç÷§Æû °³¹ß 18.09.04 5078
26 Á¤º¸ ISBC ÄÚ¸®³×¹ÚÅ׸®¿ò ±Û·çŸ¹ÌÄñ¿¡¼­ Çì¹Ì¼¿·ê·Î¿À½º .. 18.09.04 3276
25 Á¤º¸ ISBC Ç×»ýÁ¦ »ýÇÕ¼º ¹æ¼±±Õ À¯ÀüÀÚ ¹ßÇö Á¶Àý ±Ô¸í 18.09.04 2457
24 Á¤º¸ ISBC NK cell¿¡¼­ NF-¥êBÀÇ ´Ü°èÀû ÀλêÈ­¿Í Á¶ÀýÀ» Åë.. 18.09.04 3051
23 Á¤º¸ ISBC Cross linker¸¦ ÅëÇØ ¾ÈÁ¤È­µÇ´Â À¶ÇÕ ¾ËÆÄÇ︯½º.. 18.09.04 3557
22 Á¤º¸ ISBC Àü»çü ºÐ¼®°ú DNA-free CRISPR ½Ã½ºÅÛÀ» ÀÌ¿ëÇÑ .. 18.09.04 3801
21 Á¤º¸ ISBC Antisense RNA ±â¹Ý Á¤Á·¼öÀÎ½Ä Â÷´ÜÈ¿¼Ò °í¼Ó´Ù.. 18.08.30 3130
20 Á¤º¸ ISBC À¯ÀüÀÚ ¹ßÇö ÆÐÅÏÀ» ÀÌ¿ëÇÑ ¾à¹°Ç¥ÀûÀûÇÕ¼ºÀÇ °³.. 18.08.30 3030
19 Á¤º¸ ISBC ´ç °¨Áö °í°¨µµ FRET ´Ü¹éÁú ¼¾¼­ ¹× ¼ÒÇü °¨Áö .. 18.08.30 4059
18 Á¤º¸ ISBC ´ëÀå±Õ ÃÑÀ¯Àüü³»ÀÇ ArgR Àü»çÀÎÀÚ¿Í DNA °áÇÕü.. 18.08.30 2690
17 Á¤º¸ ISBC Á¾¾ç Ç÷°üÀÇ ½Å»ý°ú ÁøÇàÀ» È¿À²ÀûÀ¸·Î ¾ïÁ¦ÇÏ´Â .. 18.08.30 3278
16 Á¤º¸ ISBC Æ÷À¯¼¼Æ÷¿¡¼­ CRISPR Àü»ç ¾ïÁ¦ ÀåÄ¡¿Í À¯ÀüÀÚ È¸.. 18.08.30 2398
15 Á¤º¸ ISBC ¾ËÄÚ¿Ã ºÐÇØÈ¿¼Ò III (Alcohol Dehydrogenase III.. 18.08.30 2922
14 Á¤º¸ ISBC FACS ±â¹ÝÀÇ ÇÕ¼º Àΰ£ Ç×ü ÃÊ°í¼Ó ¹ß±¼ ½Ã½ºÅÛ .. 18.08.30 3711
[1] [2] [3]