{"id":223,"date":"2024-01-08T10:31:28","date_gmt":"2024-01-08T02:31:28","guid":{"rendered":"https:\/\/shandong.nugene.com.cn\/?p=223"},"modified":"2024-01-09T16:36:43","modified_gmt":"2024-01-09T08:36:43","slug":"%e4%bb%a3%e8%b0%a2%e5%b7%a5%e7%a8%8b%e6%94%b9%e9%80%a0%e9%85%bf%e9%85%92%e9%85%b5%e6%af%8d%e5%8f%91%e9%85%b5%e7%94%9f%e4%ba%a7%e5%92%96%e5%95%a1%e9%85%b8","status":"publish","type":"post","link":"https:\/\/shandong.nugene.com.cn\/index.php\/%e4%bb%a3%e8%b0%a2%e5%b7%a5%e7%a8%8b%e6%94%b9%e9%80%a0%e9%85%bf%e9%85%92%e9%85%b5%e6%af%8d%e5%8f%91%e9%85%b5%e7%94%9f%e4%ba%a7%e5%92%96%e5%95%a1%e9%85%b8\/","title":{"rendered":"\u4ee3\u8c22\u5de5\u7a0b\u6539\u9020\u917f\u9152\u9175\u6bcd\u53d1\u9175\u751f\u4ea7\u5496\u5561\u9178"},"content":{"rendered":"\n<p class=\"wp-block-paragraph\">\u8881\u8c46\u8c46\uff0c\u5468\u79c0\u742a\uff0c\u5e9e\u96ea\u6674\uff0c\u675c\u5bb6\u8273\uff0c\u5468\u840d\u840d<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">*\uff08\u626c\u5dde\u5927\u5b66\u00a0\u751f\u7269\u79d1\u5b66\u4e0e\u6280\u672f\u5b66\u9662\uff0c\u6c5f\u82cf\u00a0\u626c\u5dde\uff0c225009\uff09<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">*\u901a\u4fe1\u4f5c\u8005 \u5468\u840d\u840d\uff0c\u526f\u6559\u6388\uff0cE-mail\uff1appzhou@yzu.edu.cn<\/p>\n\n\n\n<p class=\"wp-block-paragraph\"><strong>\u6458\u8981\u00a0<\/strong> \u5496\u5561\u9178\u662f\u4e00\u79cd\u5929\u7136\u915a\u9178\u7c7b\u5316\u5408\u7269\uff0c\u5177\u6709\u6297\u6c27\u5316\u3001\u6297\u80bf\u7624\u3001\u6297\u708e\u7b49\u591a\u79cd\u751f\u7269\u5b66\u6d3b\u6027\uff0c\u5496\u5561\u9178\u4e5f\u662f\u8ff7\u8fed\u9999\u9178\u3001\u7eff\u539f\u9178\u548c\u5496\u5561\u9178\u82ef\u4e59\u916f\u7b49\u9ad8\u9644\u52a0\u503c\u5316\u5408\u7269\u5408\u6210\u7684\u91cd\u8981\u524d\u4f53\uff0c\u5728\u533b\u836f\u3001\u98df\u54c1\u548c\u5316\u5986\u54c1\u7b49\u884c\u4e1a\u5177\u6709\u8f83\u5927\u7684\u5e94\u7528\u4ef7\u503c\u3002\u4e3a\u5b9e\u73b0\u5496\u5561 \u9178 \u7eff \u8272 \u53ef \u6301 \u7eed \u751f \u4ea7 \uff0c \u5728 \u524d \u671f \u6784 \u5efa \u7684 \u4ea7 \u5496 \u5561 \u9178 \u917f \u9152 \u9175 \u6bcd \u83cc \u682a \u4e2d \uff0c \u6572 \u9664 \u82ef \u4e19 \u6c28 \u9178 \u9014 \u5f84 \u4e2d \u7684 \u9884 \u82ef \u9178 \u8131 \u6c34 \u9176 \u57fa \u56e0\uff08PHA2\uff09\uff0c\u5c06\u5496\u5561\u9178\u4ea7\u91cf\u63d0\u9ad8 42%\u3002\u800c\u6572\u9664\u8272\u6c28\u9178\u9014\u5f84\u4e2d\u90bb\u6c28\u57fa\u82ef\u7532\u9178\u5408\u6210\u9176\u57fa\u56e0\uff08TRP2\uff09\uff0c\u5bf9\u5496\u5561\u9178\u79ef\u7d2f\u5f71\u54cd\u4e0d\u5927\u3002\u4e4b\u540e\uff0c\u56de\u8865\u4e86 URA3\u3001HIS3\u3001MET15 \u8425\u517b\u6807\u8bb0\u57fa\u56e0\uff0c\u7136\u540e\u5c06\u5de5\u7a0b\u83cc\u682a\u5728 5 \u00a0L \u7684\u53d1\u9175\u7f50\u4e2d\u8fdb\u884c\u4e86\u8865\u6599\u5206\u6279\u53d1\u9175\uff0c\u4f7f\u5f97\u5496\u5561\u9178\u4ea7\u91cf\u8fbe\u5230\u4e86 9.3 g\/L\uff0c\u662f\u76ee\u524d\u6587\u732e\u4e2d\u62a5\u9053\u5fae\u751f\u7269\u53d1\u9175\u751f\u4ea7\u5496\u5561\u9178\u7684\u6700\u9ad8\u6c34\u5e73\uff0c\u8be5\u7814\u7a76\u4e3a\u5496\u5561\u9178\u7684\u7eff\u8272\u751f\u4ea7\u4ee5\u53ca\u5176\u884d\u751f\u7269\u7684\u9ad8\u6548\u5fae\u751f\u7269\u5408\u6210\u5960\u5b9a\u4e86\u57fa\u7840\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\"><strong>\u5173\u952e\u8bcd<\/strong>\u00a0\u917f\u9152\u9175\u6bcd\uff1b\u5496\u5561\u9178\uff1b\u9884\u82ef\u9178\u8131\u6c34\u9176\uff1b\u90bb\u6c28\u57fa\u82ef\u7532\u9178\u5408\u6210\u9176\uff1b\u751f\u7269\u5408\u6210<\/p>\n\n\n\n<p class=\"wp-block-paragraph\"><strong>Abstract <\/strong>\u00a0\u00a0Caffeic \u00a0acid \u00a0is \u00a0a \u00a0natural \u00a0phenolic \u00a0acid \u00a0with \u00a0various \u00a0pharmaceutical \u00a0properties, \u00a0including \u00a0antioxidant, \u00a0anti -tumor and \u00a0anti-inflammatory \u00a0effects. \u00a0It \u00a0is \u00a0also \u00a0an \u00a0important \u00a0precursor \u00a0for \u00a0biosynthesis \u00a0of \u00a0other \u00a0valuable \u00a0compounds \u00a0such \u00a0as rosmarinic acid, chlorogenic acid and phenethyl caffeate. Therefore, caffeic acid has significant value in pharmaceutical, fo od, cosmetic \u00a0and \u00a0other \u00a0industries. \u00a0To \u00a0achieve \u00a0green \u00a0and \u00a0sustainable \u00a0production \u00a0of \u00a0caffeic \u00a0acid, \u00a0the \u00a0prephenate \u00a0dehydratase \u00a0gene (PHA2), \u00a0which \u00a0is \u00a0involved \u00a0in \u00a0the \u00a0biosynthesis \u00a0of \u00a0phenylalanine, \u00a0was \u00a0knocked \u00a0out \u00a0in \u00a0a \u00a0previously \u00a0constructed \u00a0caffeic \u00a0acid produced Saccharomyces cerevisiae, resulting in a 42% improvement in caffeic acid production. Additionally, the anthranilate synthase gene (TRP2), which is responsible for tryptophan biosynthesis, was deleted. However, this had little effect on coffee acid \u00a0production. \u00a0The \u00a0engineered \u00a0strain \u00a0was \u00a0further \u00a0complemented \u00a0with \u00a0auxotrophic \u00a0marker \u00a0URA3, \u00a0HIS3, \u00a0MET15. \u00a0In \u00a0a subsequent \u00a0fed-batch \u00a0fermentation \u00a0process \u00a0conducted \u00a0in \u00a0a \u00a05 \u00a0L \u00a0bioreactor, \u00a0the \u00a0engineered \u00a0strain \u00a0achieved \u00a0a \u00a0caffeic \u00a0acid production \u00a0of \u00a09.3 \u00a0g\/L, \u00a0this \u00a0is \u00a0currently \u00a0the \u00a0highest \u00a0reported \u00a0titer \u00a0of \u00a0caffeic \u00a0acid \u00a0produced \u00a0by \u00a0engineered \u00a0microbial \u00a0cells. \u00a0T his study provides a foundation for the green production of caffeic acid and its derivatives.<\/p>\n\n\n\n<p class=\"wp-block-paragraph\"><strong>Key words<\/strong> \u00a0\u00a0Saccharomyces cerevisiae; caffeic acid; prephenate dehydratase; anthranilate synthase; biosynthesis<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u5496\u5561\u9178\uff08caffeic &nbsp;acid\uff09\uff0c\u53c8\u79f0 3, &nbsp;4-\u4e8c\u7f9f\u57fa\u8089\u6842\u9178\uff0c\u662f\u5b58\u5728\u4e8e\u591a\u79cd\u690d\u7269\u4e2d\u7684\u4e00\u79cd\u5929\u7136\u915a\u9178\u7c7b\u5316\u5408\u7269\uff0c\u5177\u6709\u6297\u6c27\u5316\u3001\u6297\u708e\u3001\u6297\u80bf\u7624\u7b49\u591a\u79cd\u751f\u7269\u5b66\u6d3b\u6027[1-3]\u3002\u6b64\u5916\uff0c\u5496\u5561\u9178\u662f\u7eff\u539f\u9178[4]\u3001\u8ff7\u8fed\u9999\u9178[5]\u3001\u4e39\u915a\u9178 B[6]\u3001\u5496\u5561\u9178\u82ef\u4e59\u916f[7]\u7b49\u9ad8\u9644\u52a0\u503c\u5316\u5408\u7269\u7684\u524d\u4f53\uff0c\u5728\u98df\u54c1\u3001\u836f\u54c1\u548c\u5316\u5986\u54c1\u7b49\u9886\u57df\u5177\u6709\u5e7f\u6cdb\u7684\u5e94\u7528[8]\u3002\u7531\u4e8e\u5496\u5561\u9178\u5b58\u5728\u4e8e\u5496\u5561\u4ee5\u53ca\u8335\u9648\u3001\u83dc\u84df\u3001\u91d1\u94f6\u82b1\u7b49\u591a\u79cd\u690d\u7269\u4e2d\uff0c\u56e0\u6b64\uff0c\u53ef\u4ee5\u4ece\u8fd9\u4e9b\u690d\u7269\u4e2d\u76f4\u63a5\u63d0\u53d6\u6765\u5236\u5907\u3002\u4f46\u662f\u690d\u7269\u751f\u957f\u5468\u671f\u957f\u3001\u4ea7\u7269\u79ef\u7d2f\u91cf\u4f4e\uff0c\u4e14\u5728\u690d\u7269\u63d0\u53d6\u8fc7\u7a0b\u4e2d\u9700\u8981\u7528\u5230\u591a\u79cd\u6eb6\u5a92\uff0c\u9650\u5236\u4e86\u5176\u5927\u89c4\u6a21\u7684\u751f\u4ea7\u3002\u5496\u5561\u9178\u4e5f\u53ef\u4ee5\u7531\u82ef\u915a\u6216\u53d6\u4ee3\u82ef\u915a\u7ecf\u8fc7\u5316\u5b66\u50ac\u5316\u5242\u53cd\u5e94\u751f\u6210\uff0c\u4f46\u5316\u5b66\u5408\u6210\u8fc7\u7a0b\u4e2d\u53cd\u5e94\u6761\u4ef6\u8981\u6c42\u9ad8\u3001\u5236\u5907\u5de5\u827a\u590d\u6742\u3001\u6210\u672c\u9ad8\uff0c\u5e76\u4e0d\u662f\u4e00\u6761\u7eff\u8272\u7ecf\u6d4e\u7684\u751f\u4ea7\u8def\u7ebf\u3002\u968f\u7740\u5408\u6210\u751f\u7269\u6280\u672f\u7684\u53d1\u5c55\uff0c\u4ee3\u8c22\u6539\u9020\u5fae\u751f\u7269\u5408\u6210\u5496\u5561\u9178\u6210\u4e3a\u4e00\u79cd\u6709\u6548\u7684\u66ff\u4ee3\u65b9\u6cd5\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u5728\u690d\u7269\u4e2d\uff0c\u82ef\u4e19\u6c28\u9178\u89e3\u6c28\u9176\uff08phenylalamine \u00a0ammonia \u00a0lyase\uff0cPAL\uff09\u50ac\u5316 L-\u82ef\u4e19\u6c28\u9178\u8131\u6c28\u751f\u6210\u53cd\u5f0f\u8089\u6842\u9178\uff0c\u53cd\u5f0f\u8089\u6842\u9178\u5728\u8089\u6842\u9178-4-\u7f9f\u5316\u9176\uff084H\uff09\u50ac\u5316\u4e0b\u751f\u6210\u5bf9\u9999\u8c46\u9178\uff0c\u6700\u540e\u5bf9\u9999\u8c46\u9178\u5728\u5bf9\u9999\u8c46\u9178-3-\u7f9f\u5316\u9176\uff08C3H\uff09\u50ac\u5316\u4e0b\u751f\u6210\u5496\u5561\u9178\u3002\u690d\u7269\u6765\u6e90\u7684 C3H \u548c C4H \u5c5e\u4e8e P450 \u9176\uff0c\u5728\u5fae\u751f\u7269\u4e2d\u96be\u4ee5\u9ad8\u6548\u8868\u8fbe[9]\u3002BERNER \u7b49\u9996\u6b21\u62a5\u544a\u4e86\u897f\u73ed\u7259\u7cd6\u4e1d\u83cc\uff08Saccharothrix \u00a0espanaensis\uff09\u6765\u6e90\u7684 4-\u9999\u8c46\u9178-3-\u7f9f\u57fa\u5316\u9176\uff08\u7531 Sam5 \u7f16\u7801\uff09\u548c\u916a\u6c28\u9178\u89e3\u6c28\u9176\uff08\u7531Sam8 \u7f16\u7801\uff09\uff0c\u5728\u5f17\u6c0f\u94fe\u9709\u83cc\u4e2d\u8868\u8fbe\u53ef\u4ee5\u5c06 L-\u916a\u6c28\u9178\u8f6c\u5316\u4e3a\u5496\u5561\u9178[10]\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">CHOI \u7b49\u5c06\u8fd9\u4e24\u4e2a\u6765\u6e90\u4e8e\u897f\u73ed\u7259\u7cd6\u4e1d\u83cc\u7684 Sam8 \u548c Sam5 \u57fa\u56e0\u5728\u5927\u80a0\u6746\u83cc\u8868\u8fbe\uff0c\u6210\u529f\u5c06\u5bf9\u9999\u8c46\u9178\u8f6c\u5316\u4e3a\u5496\u5561\u9178[11]\u3002LIN \u7b49\u53d1\u73b0\u6765\u81ea\u5927\u80a0\u6746\u83cc\u81ea\u8eab\u7684 4-\u7f9f\u57fa\u82ef\u4e59\u9178 3-\u52a0\u6c27\u9176\uff08Hpa B\uff09\u548c 4-\u7f9f\u57fa\u82ef\u4e59\u9178 3-\u8fd8\u539f\u9176\uff08Hpa C\uff09\u53ef\u4ee5\u66ff\u6362\u5fae\u751f\u7269\u4e2d\u96be\u8868\u8fbe\u7684 C3H\u3002\u901a\u8fc7\u5c06\u5927\u80a0\u6746\u83cc\u6765\u6e90\u7684 Hpa BC \u4e0e\u835a\u819c\u7ea2\u7ec6\u83cc\uff08Rhodobacter \u00a0capsulatus\uff09\u6765\u6e90\u7684\u916a\u6c28\u9178\u89e3\u6c28\u9176\uff08tyrosine \u00a0ammonia-lyase\uff0cTAL\uff09\u5728\u5927\u80a0\u6746\u83cc\u4e2d\u5171\u8868\u8fbe\uff0c\u6210\u529f\u6784\u5efa\u51fa\u4e86\u4e00\u6761\u5496\u5561\u9178\u5f02\u6e90\u5408\u6210\u9014\u5f84\uff0c\u5e76\u901a\u8fc7\u89e3\u9664\u916a\u6c28\u9178\u7684\u53cd\u9988\u6291\u5236\u6b65\u9aa4\uff0c\u83b7\u5f97\u4e86 50.2 \u00a0mg\/L\u7684\u5496\u5561\u9178[12]\u3002\u8fdb\u4e00\u6b65\u6572\u9664\u5927\u80a0\u6746\u83cc\u4e2d\u82ef\u4e19\u6c28\u9178\u5408\u6210\u9014\u5f84\u4ee5\u53ca\u7cfb\u7edf\u4f18\u5316\u916a\u6c28\u9178\u5408\u6210\u9014\u5f84\u7b49\uff0c\u4f7f\u5f97\u5496\u5561\u9178\u7684\u4ea7\u91cf\u8fbe\u5230\u4e86 766.7 \u00a0mg\/L[13]\u30022022 \u5e74\uff0c\u7814\u7a76\u4eba\u5458\u5229\u7528\u5927\u80a0\u6746\u83cc\u4e3a\u51fa\u53d1\u83cc\u682a\uff0c\u8868\u8fbe\u4e86\u7ea6\u6c0f\u9ec4\u6746\u83cc\uff08Flavobacterium \u00a0johnsoniaeu\uff09\u6765\u6e90\u7684 Fj TAL \u548c\u5927\u80a0\u6746\u83cc\u5185\u6e90\u7684 Hpa BC\uff0c\u5e76\u901a\u8fc7\u4f18\u5316\u57fa\u56e0\u62f7\u8d1d\u6570\u3001\u6572\u9664\u916a\u6c28\u9178\u5408\u6210\u7684\u7ade\u4e89\u9014\u5f84\u3001\u5f3a\u5316\u8f85\u56e0\u5b50 FAD \u7684\u5408\u6210\u4ee5\u53ca\u8fc7\u8868\u8fbe\u8f6c\u8fd0\u86cb\u767d ycj P\uff0c\u4f7f\u5f97\u5496\u5561\u9178\u6447\u74f6\u4ea7\u91cf\u8fbe\u5230\u4e86 775.7 \u00a0mg\/L\uff0c\u901a\u8fc7\u5728 5 \u00a0L \u53d1\u9175\u7f50\u8fdb\u884c\u53d1\u9175\u6761\u4ef6\u4f18\u5316\u540e\uff0c\u53d1\u9175 66 \u00a0h \u5496\u5561\u9178\u4ea7\u91cf\u8fbe\u5230\u4e86 7.9 g\/L[14]\uff0c\u8fd9\u662f\u76ee\u524d\u62a5\u9053\u7684\u5fae\u751f\u7269\u5408\u6210\u5496\u5561\u9178\u7684\u6700\u9ad8\u4ea7\u91cf\u3002\u5728\u4e0a\u8ff0\u6784\u5efa\u7684\u5927\u80a0\u6746\u83cc\u5de5\u7a0b\u83cc\u4e2d\uff0c\u4ee3\u8c22\u9014\u5f84\u4e2d\u7684\u57fa\u56e0\u5e38\u5e38\u901a\u8fc7\u591a\u4e2a\u6e38\u79bb\u8d28\u7c92\u8fdb\u884c\u8868\u8fbe\uff0c\u83cc\u682a\u7a33\u5b9a\u6027\u5dee\uff0c\u5728\u57f9\u517b\u8fc7\u7a0b\u4e2d\u9700\u8981\u6dfb\u52a0\u591a\u79cd\u6297\u751f\u7d20\uff0c\u4e14\u57f9\u517b\u8fc7\u7a0b\u4e2d\u9700\u8981\u6dfb\u52a0\u5f02\u4e19\u57fa-\u03b2-D-\u786b\u4ee3\u534a\u4e73\u7cd6\u82f7\uff08isopropyl-beta-D-thiogalactopyranoside\uff0cIPTG\uff09\u8fdb\u884c\u8bf1\u5bfc\uff0c\u5f71\u54cd\u4e86\u540e\u671f\u7684\u63d0\u53d6\u5de5\u827a\uff0c\u4e0d\u5177\u5907\u98df\u54c1\u5b89\u5168\u6027\u548c\u751f\u4ea7\u7ecf\u6d4e\u6027\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u917f\u9152\u9175\u6bcd\u4f5c\u4e3a\u57fa\u56e0\u64cd\u4f5c\u7b80\u5355\u7684\u771f\u6838\u5fae\u751f\u7269\uff0c\u4e0e\u5927\u80a0\u6746\u83cc\u76f8\u6bd4\uff0c\u4e0d\u6613\u67d3\u566c\u83cc\u4f53\uff0c\u4e14\u5728\u751f\u7269\u5b89\u5168\u6027\u65b9\u9762\u548c\u690d\u7269\u57fa\u56e0\u7684\u529f\u80fd\u8868\u8fbe\u65b9\u9762\u6709\u5176\u72ec\u7279\u7684\u4f18\u52bf\uff0c\u662f\u690d\u7269\u5929\u7136\u4ea7\u7269\u5f02\u6e90\u5408\u6210\u7684\u6781\u4f73\u5bbf\u4e3b\u3002\u5728\u917f\u9152\u9175\u6bcd\u4e2d\uff0cLIU \u7b49\u7b5b\u9009\u4e86\u4e0d\u540c\u7269\u79cd\u6765\u6e90\u7684 Hpa B \u548c Hpa C\uff0c\u5e76\u901a\u8fc7\u7ec4\u5408\u8868\u8fbe\u53d1\u73b0\u94dc\u7eff\u5047\u5355\u80de\u83cc\u7684 Hpa B \u548c\u80a0\u6c99\u95e8\u6c0f\u83cc\u7684 Hpa C \u7684\u7ec4\u5408\u6548\u679c\u6700\u4f73\uff0c\u53ef\u4ee5\u6709\u6548\u5730\u5c06\u5bf9\u9999\u8c46\u9178\u8f6c\u5316\u5230\u5496\u5561\u9178\u3002\u5e76\u7ed3\u5408\u5706\u7ea2\u51ac\u5b62\u9175\u6bcd\uff08Rhodotorula \u00a0toruloides\uff09\u6765\u6e90\u7684 Rt TAL \u8868\u8fbe\uff0c\u5728\u917f\u9152\u9175\u6bcd\u4e2d\u5b9e\u73b0\u4e86\u5496\u5561\u9178\u7684\u4ece\u5934\u5408\u6210\uff0c\u4ea7\u91cf\u8fbe\u5230289.4 \u00a0mg\/L[15]\u30022022 \u5e74\uff0cCHEN \u7b49\u901a\u8fc7\u6539\u9020\u4e2d\u5fc3\u4ee3\u8c22\u63d0\u9ad8 NADPH \u4f9b\u5e94\uff0c\u901a\u8fc7\u6784\u5efa\u80de\u6d46 FAD(H2)\u5408\u6210\u9014\u5f84\u4ee5\u53ca\u5c06\u7ebf\u7c92\u4f53 FAD(H2)\u5bfc\u5230\u80de\u6d46\u4ee5\u63d0\u9ad8 FAD(H2)\u4f9b\u5e94\uff0c\u663e\u8457\u63d0\u9ad8\u4e86\u5496\u5561\u9178\u751f\u7269\u5408\u6210\u6548\u7387\uff0c\u6447\u74f6\u4ea7\u91cf\u8fbe\u5230\u4e86 1.6 g\/L\uff0c\u5728 5 L\u53d1\u9175\u7f50\u4e2d\u8865\u6599\u5206\u6279\u53d1\u9175\u4ea7\u91cf\u8fbe\u5230\u4e86 5.5 g\/L[16]\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">2021 \u5e74\uff0c\u672c\u8bfe\u9898\u7ec4\u4ee5\u917f\u9152\u9175\u6bcd\u5185\u6e90 L-\u916a\u6c28\u9178\u4e3a\u524d\u4f53\uff0c\u5c06\u7c98\u7ea2\u9175\u6bcd\uff08Rhodotorula \u00a0glutinis\uff09\u6765\u6e90\u7684Rg TAL \u57fa\u56e0\u3001\u94dc\u7eff\u5047\u5355\u80de\u83cc\u6765\u6e90\u7684 Hpa B \u57fa\u56e0\u548c\u6c99\u95e8\u6c0f\u83cc\u6765\u6e90\u7684 Hpa C \u57fa\u56e0\u7ec4\u5408\uff0c\u5728\u917f\u9152\u9175\u6bcd\u4e2d\u6784\u5efa\u4e86\u4e00\u6761\u5496\u5561\u9178\u751f\u7269\u5408\u6210\u9014\u5f84[17]\uff0c\u5e76\u901a\u8fc7\u6572\u9664\u82ef\u4e19\u6c28\u9178\u53cd\u9988\u6291\u5236\u7684 Aro3\u3001\u8fc7\u8868\u8fbe\u916a\u6c28\u9178\u53cd\u9988\u6291\u5236\u4e0d\u654f\u611f\u7a81\u53d8\u4f53 Aro4K229L \u548c Aro7G141S\u3001\u6572\u9664\u4e19\u916e\u9178\u8131\u7fa7\u9176 Aro10\u548c\u8868\u8fbe\u8fd0\u52a8\u53d1\u9175\u5355\u80de\u83cc\u6765\u6e90\u7684\u73af\u5df1\u4e8c\u70ef\u8131\u6c22\u9176 Tyr C\uff08\u56fe 1\uff09\uff0c\u6784\u5efa\u7684\u917f\u9152\u9175\u6bcd\u83cc\u682a YCA113-8B\uff0c\u5176\u5496\u5561\u9178\u4ea7\u91cf\u63d0\u9ad8\u4e86\u7ea6 2.6 \u500d\uff0c\u6447\u74f6\u4ea7\u91cf\u8fbe\u5230\u4e86 568.5 \u00a0mg\/L\u3002\u4e3a\u4e86\u8fdb\u4e00\u6b65\u63d0\u9ad8\u5496\u5561\u9178\u5408\u6210\u4ea7\u91cf\uff0c\u63d0\u9ad8\u524d\u4f53\u4f9b\u5e94\u81f3\u5173\u91cd\u8981\u3002\u5728\u4e0a\u8ff0\u5408\u6210\u8fc7\u7a0b\u4e2d\uff0c\u524d\u4f53\u5206\u652f\u9178\u4f1a\u5728\u90bb\u6c28\u57fa\u82ef\u7532\u9178\u5408\u9176\uff08TRP2\uff09\u50ac\u5316\u4e0b\u751f\u6210\u90bb\u6c28\u57fa\u82ef\u7532\u9178\uff0c\u5373\u8fdb\u5165\u8272\u6c28\u9178\u652f\u8def\uff0c\u800c\u9884\u82ef\u9178\u5219\u5728\u9884\u82ef\u9178\u8131\u6c34\u9176\uff08PHA2\uff09\u7684\u50ac\u5316\u4e0b\u5408\u6210\u82ef\u4e19\u916e\u9178\uff0c\u800c\u82ef\u4e19\u916e\u9178\u4f1a\u8fdb\u4e00\u6b65\u7ecf\u9176\u50ac\u5316\u8f6c\u5316\u751f\u6210\u82ef\u4e19\u6c28\u9178\uff0c\u5373\u8fdb\u5165\u82ef\u4e19\u6c28\u9178\u9014\u5f84\uff08\u56fe 1\uff09\u3002\u56e0\u6b64\uff0c\u4e3a\u4f7f\u66f4\u591a\u7684\u78b3\u4ee3\u8c22\u6d41\u6d41\u5411\u5496\u5561\u9178\u5408\u6210\u9014\u5f84\uff0c\u672c\u7814\u7a76\u5728\u524d\u671f\u6784\u5efa\u7684\u5496\u5561\u9178\u5408\u6210\u83cc\u682a\u57fa\u7840\u4e0a\uff0c\u5229\u7528 CRISPR\/Cas9 \u7f16\u8f91\u7cfb\u7edf\u6572\u9664 PHA2 \u548c TRP2 \u57fa\u56e0\uff0c\u8003\u5bdf\u4e86\u5176\u5bf9\u917f\u9152\u9175\u6bcd\u5496\u5561\u9178\u5408\u6210\u7684\u5f71\u54cd\u3002\u5e76\u5bf9\u5de5\u7a0b\u83cc\u682a\u4e2d\u5bfc\u81f4\u6c28\u57fa\u9178\u8425\u517b\u7f3a\u9677\u7684 URA3\u3001HIS3\u3001MET15 \u6807\u8bb0\u57fa\u56e0\u8fdb\u884c\u4e86\u56de\u8865\u3002\u6700\u540e\u5c06\u5de5\u7a0b\u83cc\u682a\u5728 5 \u00a0L \u53d1\u9175\u7f50\u4e2d\u8fdb\u884c\u4e86\u8865\u6599\u5206\u6279\u53d1\u9175\uff0c\u4ee5\u671f\u63d0\u9ad8\u5496\u5561\u9178\u7684\u4ea7\u91cf\u3002\u672c\u7814\u7a76\u4e3a\u53d1\u9175\u751f\u4ea7\u5496\u5561\u9178\u5960\u5b9a\u4e86\u57fa\u7840\uff0c\u4e5f\u4e3a\u5176\u4ed6\u5929\u7136\u915a\u9178\u7c7b\u5316\u5408\u7269\u7684\u751f\u7269\u5408\u6210\u63d0\u4f9b\u4e86\u65b9\u6cd5\u53c2\u8003\u3002<\/p>\n\n\n\n<figure class=\"wp-block-image size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"554\" height=\"349\" src=\"https:\/\/shandong.nugene.com.cn\/wp-content\/uploads\/2024\/01\/image-5.png\" alt=\"\" class=\"wp-image-224\" srcset=\"https:\/\/shandong.nugene.com.cn\/wp-content\/uploads\/2024\/01\/image-5.png 554w, https:\/\/shandong.nugene.com.cn\/wp-content\/uploads\/2024\/01\/image-5-300x189.png 300w\" sizes=\"auto, (max-width: 554px) 100vw, 554px\" \/><\/figure>\n\n\n\n<p class=\"wp-block-paragraph\">\u56fe 1 &nbsp;\u917f\u9152\u9175\u6bcd\u4e2d\u5496\u5561\u9178\u751f\u7269\u5408\u6210\u9014\u5f84<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">Fig. 1 The biosynthetic pathway of caffeic acid in Saccharomyces cerevisiae<\/p>\n\n\n\n<p class=\"wp-block-paragraph\"><strong>1 \u00a0\u6750\u6599\u4e0e\u65b9\u6cd5<\/strong><\/p>\n\n\n\n<p class=\"wp-block-paragraph\"><strong>1.1 \u00a0\u5b9e\u9a8c\u6750\u6599<\/strong><\/p>\n\n\n\n<p class=\"wp-block-paragraph\"><strong>1.1.1 \u00a0\u83cc\u682a\u4e0e\u8d28\u7c92<\/strong><\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u7814\u7a76\u4f7f\u7528\u7684\u5f15\u7269\u3001\u8d28\u7c92\u548c\u83cc\u682a\u5206\u522b\u89c1\u8868 1~\u8868 3\u3002\u5927\u80a0\u6746\u83cc\uff08Escherichia \u00a0coli\uff09DH5\u03b1\u7528\u4e8e\u91cd\u7ec4\u8d28\u7c92\u7684\u514b\u9686\u548c\u6269\u589e\u5b9e\u9a8c\u3002\u524d\u671f\u6784\u5efa\u7684\u917f\u9152\u9175\u6bcd\u5496\u5561\u9178\u5408\u6210\u83cc\u682a YCA113-8B \u4e3a\u672c\u5b9e\u9a8c\u7684\u51fa\u53d1\u83cc\u682a\u3002\u83cc\u682a\u3001\u8d28\u7c92\u5168\u90e8\u4fdd\u85cf\u5728\u672c\u5b9e\u9a8c\u5ba4\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u88681 \u00a0\u672c\u7814\u7a76\u6240\u4f7f\u7528\u7684\u5f15\u7269<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">Table 1 Primers used in this study<\/p>\n\n\n\n<figure class=\"wp-block-image size-full is-resized\"><img loading=\"lazy\" decoding=\"async\" width=\"554\" height=\"224\" src=\"https:\/\/shandong.nugene.com.cn\/wp-content\/uploads\/2024\/01\/image-6.png\" alt=\"\" class=\"wp-image-225\" style=\"width:554px;height:auto\" srcset=\"https:\/\/shandong.nugene.com.cn\/wp-content\/uploads\/2024\/01\/image-6.png 554w, https:\/\/shandong.nugene.com.cn\/wp-content\/uploads\/2024\/01\/image-6-300x121.png 300w\" sizes=\"auto, (max-width: 554px) 100vw, 554px\" \/><\/figure>\n\n\n\n<p class=\"wp-block-paragraph\">\u88682 &nbsp;\u672c\u7814\u7a76\u6240\u7528\u7684\u8d28\u7c92<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">Table 2 Plasmids used in this study<\/p>\n\n\n\n<figure class=\"wp-block-image size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"554\" height=\"288\" src=\"https:\/\/shandong.nugene.com.cn\/wp-content\/uploads\/2024\/01\/image-7.png\" alt=\"\" class=\"wp-image-226\" srcset=\"https:\/\/shandong.nugene.com.cn\/wp-content\/uploads\/2024\/01\/image-7.png 554w, https:\/\/shandong.nugene.com.cn\/wp-content\/uploads\/2024\/01\/image-7-300x156.png 300w\" sizes=\"auto, (max-width: 554px) 100vw, 554px\" \/><\/figure>\n\n\n\n<p class=\"wp-block-paragraph\"><strong>1.1.2 \u6bcd\u6db2\u4e0e\u57f9\u517b\u57fa<\/strong><\/p>\n\n\n\n<p class=\"wp-block-paragraph\">&nbsp;100\u00d7\u6c28\u57fa\u9178\u6bcd\u6db2\uff1aL-\u7532\u786b\u6c28\u9178\u3001L-\u7ec4\u6c28\u9178\u3001L-\u8272\u6c28\u9178\u3001L-\u82ef\u4e19\u6c28\u9178\u3001\u5c3f\u5627\u5576\u8d28\u91cf\u6d53\u5ea6\u5206\u522b\u6309\u71672L\u30012\u30012\u30015\u30012 mg\/m L \u914d\u5236\uff0c\u6dfb\u52a0\u4e8e\u57f9\u517b\u57fa\u65f6\u6309\u7167 1%\u7684\u4f53\u79ef\u6bd4\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u9057\u4f20\u9709\u7d20\uff08G418\uff09\u6bcd\u6db2\uff1a\u6309\u7167 20 mg\/m L \u914d\u5236\uff0c\u6dfb\u52a0\u4e8e\u57f9\u517b\u57fa\u65f6\u6309\u7167 1%\u7684\u4f53\u79ef\u6bd4\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u6c28\u82c4\u9752\u9709\u7d20\u6bcd\u6db2\uff1a\u6309\u7167 100 mg\/m L \u914d\u5236\uff0c\u6dfb\u52a0\u4e8e\u57f9\u517b\u57fa\u65f6\u6309\u7167 0.1%\u7684\u4f53\u79ef\u6bd4\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u5361\u90a3\u9709\u7d20\u6bcd\u6db2\uff1a\u6309\u7167 50 mg\/m L \u914d\u5236\uff0c\u6dfb\u52a0\u4e8e\u57f9\u517b\u57fa\u65f6\u6309\u7167 0.1%\u7684\u4f53\u79ef\u6bd4\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">Single-straided &nbsp;carrier &nbsp;DNA\uff08ss DNA\uff09\uff1a20 &nbsp;mg \u6eb6\u4e8e 10 &nbsp;m L &nbsp;TE &nbsp;Buffer \u4e2d\uff0c0.22 &nbsp;\u00b5m \u5fae\u5b54\u6ee4\u819c\u8fc7\u6ee4\u9664\u83cc\uff0c\u5206\u88c5\u540e-20 \u2103\u4fdd\u5b58\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">1 &nbsp;mol\/L \u918b\u9178\u9502\u6eb6\u6db2\uff1a10.2 &nbsp;g \u4e59\u9178\u9502\u4e8c\u6c34\u5b9a\u5bb9\u4e8e 100 &nbsp;m L \u84b8\u998f\u6c34\u4e2d\uff0c121 &nbsp;\u2103\u6e7f\u70ed\u706d\u83cc 15 &nbsp;min\uff0c\u51b7\u5374\u540e\u4e8e 4 \u2103\u4fdd\u5b58\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">PEG &nbsp;MV3 &nbsp;350\uff0850% &nbsp;M\/V\uff09\uff1a50 g &nbsp;PEG &nbsp;MV3 &nbsp;350 \u5b9a\u5bb9\u4e8e 100 &nbsp;m L \u84b8\u998f\u6c34\u4e2d\uff0c121 &nbsp;\u2103\u6e7f\u70ed\u706d\u83cc 15 min\uff0c\u51b7\u5374\u540e\u4e8e 4 \u2103\u4fdd\u5b58\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">Luria-Bertani\uff08LB\uff09\u6db2\u4f53\u57f9\u517b\u57fa\uff08g\/L\uff09\uff1aLB \u8089\u6c64\u57f9\u517b\u57fa 25 g\uff0c115 \u2103\u706d\u83cc 21 min\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">Luria-Bertani\uff08LB\uff09\u56fa\u4f53\u57f9\u517b\u57fa\uff08g\/L\uff09\uff1aLB \u8089\u6c64\u743c\u8102 40 g\uff0c115 \u2103\u706d\u83cc 21 min\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">Yeast \u00a0Extract \u00a0Peptone \u00a0Dextrose\uff08YPD\uff09\u6db2\u4f53\u57f9\u517b\u57fa\uff08g\/L\uff09\uff1a\u9175\u6bcd\u86cb\u767d\u80e8 20\uff0c\u65e0\u6c34\u8461\u8404\u7cd6 20\uff0c\u9175\u6bcd\u6d78\u7c89 10\uff0c115 \u2103\u706d\u83cc 21 min\uff0c\u5728\u914d\u5236\u56fa\u4f53\u57f9\u517b\u57fa\u65f6\uff0c\u6dfb\u52a0 1.5%~2%\u7684\u743c\u8102\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">10\u00d7Yeast nitrogen base\uff0810\u00d7YNB\uff09\u6bcd\u6db2\uff1aYNB 17 g, (NH4)2SO4&nbsp;50 g\uff0c\u6eb6\u4e8e 1 L \u84b8\u998f\u6c34\u4e2d\uff0c\u8fc7\u6ee4\u9664\u83cc\uff0c\u6dfb\u52a0\u4e8e\u57f9\u517b\u57fa\u65f6\u6309\u7167 10%\u7684\u4f53\u79ef\u6bd4\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">Synthetic &nbsp;Defined\uff08SD\uff09\u56fa\u4f53\u57f9\u517b\u57fa\uff1a\u8461\u8404\u7cd6 2 &nbsp;g\uff0c\u743c\u8102\u7c89 1.5 &nbsp;g\uff0c\u84b8\u998f\u6c34 90 &nbsp;m L\uff0c115 &nbsp;\u2103\u706d\u83cc 21 min, &nbsp;\u4f7f\u7528\u65f6\u5e94\u52a0\u5165 1 m L 100\u00d7\u6c28\u57fa\u9178\u6bcd\u6db2\u548c 10 m L 10\u00d7YNB \u6bcd\u6db2\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">5-\u6c1f\u4e73\u6e05\u9178\uff085-fluoroorotic &nbsp;acid\uff0cFo A\uff09\u56fa\u4f53\u57f9\u517b\u57fa\uff1a\u79f0\u53d6 0.1 &nbsp;g &nbsp;FOA \u56fa\u4f53\u6eb6\u4e8e 1 &nbsp;m L \u4e8c\u7532\u57fa\u4e9a\u781c\u4e2d\uff0c100 m L SD \u56fa\u4f53\u57f9\u517b\u57fa\u878d\u5316\u964d\u6e29\u81f3 50 \u2103\u5de6\u53f3\uff0c\u52a0\u5165 1 m L 100\u00d7\u6c28\u57fa\u9178\u6bcd\u6db2\u30010.1 g\/m L Fo A \u6eb6\u6db2\u6df7\u5300\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u7ef4\u751f\u7d20\u6eb6\u6db2\uff08g\/L\uff09\uff1a\u751f\u7269\u7d20 0.05\uff0c\u6cdb\u9178\u9499 1\uff0c\u70df\u9178 1\uff0c\u808c\u9187 25\uff0c\u76d0\u9178\u786b\u80fa\u7d20 1\uff0c\u5421\u54c6\u9187 1\uff0c\u5bf9\u6c28\u57fa\u82ef\u7532\u9178 0.2\uff0c0.22 \u00b5m \u5fae\u5b54\u6ee4\u819c\u8fc7\u6ee4\u9664\u83cc\uff0c\u5206\u88c5\u540e 4 \u2103\u4fdd\u5b58\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u5fae\u91cf\u5143\u7d20\u6eb6\u6db2\uff08g\/L\uff09\uff1aEDTA \u00a015\uff0cZn SO4\u00b77H2O \u00a010.2\uff0cMn Cl2\u00b74H2O \u00a00.5\uff0cCu SO4 0.5\uff0cCo Cl2\u00b76H2O 0.86\uff0cNa2Mo O4\u00b72H2O 0.56\uff0cCa Cl2\u00b72H2O 3.84\uff0cFe SO4\u00b77H2O 5.12\uff0c115 \u2103\u706d\u83cc 21 min\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u5206 \u6279 \u53d1 \u9175 \u8865 \u6599 \u6d53 \u7f29 \u6db2 \u2160\uff08g\/L\uff09 \uff1a \u8461 \u8404 \u7cd6 500\uff0c KH2PO4 &nbsp;9 \uff0c Mg SO4 &nbsp;2.5 \uff0cK2SO4 &nbsp;3.5 \uff0c Na2SO 0.28,115 \u2103\u706d\u83cc 21 min\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u5206\u6279\u53d1\u9175\u8865\u6599\u6d53\u7f29\u6db2\u2161\uff08g\/L\uff09\uff1a\u9175\u6bcd\u6d78\u7c89 250, 115 \u2103\u706d\u83cc 21 min\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">2.5 mol\/L Na OH \u6eb6\u6db2\uff1a30 g Na OH \u6eb6\u4e8e 300 m L \u7684\u706d\u83cc\u6c34\u4e2d\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\"><strong>1.1.3 \u00a0\u9176\u4e0e\u8bd5\u5242\u76d2<\/strong><\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u672c\u7814\u7a76\u4f7f\u7528\u7684\u5de5\u5177\u9176\u4e0e\u8bd5\u5242\u76d2\u89c1\u8868 4\u3002<\/p>\n\n\n\n<figure class=\"wp-block-image size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"554\" height=\"266\" src=\"https:\/\/shandong.nugene.com.cn\/wp-content\/uploads\/2024\/01\/image-8.png\" alt=\"\" class=\"wp-image-227\" srcset=\"https:\/\/shandong.nugene.com.cn\/wp-content\/uploads\/2024\/01\/image-8.png 554w, https:\/\/shandong.nugene.com.cn\/wp-content\/uploads\/2024\/01\/image-8-300x144.png 300w\" sizes=\"auto, (max-width: 554px) 100vw, 554px\" \/><\/figure>\n\n\n\n<p class=\"wp-block-paragraph\"><strong>1.2 \u00a0\u5b9e\u9a8c\u65b9\u6cd5<\/strong><\/p>\n\n\n\n<p class=\"wp-block-paragraph\"><strong>1.2.1 \u00a0\u91cd\u7ec4\u8d28\u7c92\u7684\u6784\u5efa<\/strong><\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u4ee5\u8d28\u7c92 M3-T[18]\u4e3a\u6a21\u677f\uff0c\u5206\u522b\u4ee5 g PHA2-F\uff08Esp3 I\uff09\u548c t RNA-R\uff08Esp3 I\uff09\u3001g TRP2-F\uff08Esp3 I\uff09\u548ct RNA-R\uff08Esp3 \u00a0I\uff09\u4e3a\u5f15\u7269\uff0c\u5206\u522b\u6269\u589e\u5f97\u5230 PHA2 \u00a0g RNA-scaffold-t RNA \u5e8f\u5217 \u3001 TRP2 \u00a0g RNA-scaffold-t RNA \u5e8f\u5217\u3002\u4f7f\u7528\u9650\u5236\u6027\u6838\u9178\u5185\u5207\u9176 Esp3 \u00a0I \u5c06\u6269\u589e\u6240\u5f97\u7684\u57fa\u56e0\u7247\u6bb5\u4e0e TYPE3-M \u8d28\u7c92[18]\u5206\u522b\u8fdb\u884c\u9176\u5207\uff0c\u4f7f\u7528 T4 \u00a0DNA \u8fde\u63a5\u9176\u5c06\u9176\u5207\u540e\u7684\u57fa\u56e0\u7247\u6bb5\u4e0e\u8d28\u7c92\u5206\u522b\u8fdb\u884c\u8fde\u63a5\uff0c\u5bfc\u5165 E.coli \u00a0DH5\u03b1 \u611f\u53d7\u6001\u7ec6\u80de\u4e2d\uff0c\u7b5b\u9009\u9633\u6027\u8f6c\u5316\u5b50\uff0c\u5206\u522b\u5f97\u5230 TYPE3-M- g PHA2 \u548cTYPE3-M-g TRP2 \u8f7d\u4f53\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\"><strong>1.2.2 \u00a0\u917f\u9152\u9175\u6bcd\u91cd\u7ec4\u83cc\u682a\u7684\u6784\u5efa<\/strong><\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u4ee5\u917f\u9152\u9175\u6bcd BY4741 \u7684\u57fa\u56e0\u7ec4\u4e3a\u6a21\u677f\uff0c\u4ee5 HAPHA2-F\/HAPHA2-R \u4e3a\u5f15\u7269\uff0c\u7528\u9ad8\u4fdd\u771f\u7684 Prime STARTM \u00a0HS \u00a0DNA \u805a\u5408\u9176\u8fdb\u884c PCR \u6269\u589e\uff0c\u83b7\u5f97\u540c\u6e90\u81c2 HAPHA2\uff1b\u540c\u7406\uff0c\u7528 HATRP2-F \u548c HATRP2-R\u5f15\u7269\u8fdb\u884c PCR \u6269\u589e\uff0c\u83b7\u5f97\u540c\u6e90\u81c2 HATRP2\u3002\u5c06\u6784\u5efa\u7684\u8d28\u7c92\u8f7d\u4f53 TYPE3-M- \u00a0g PHA2 \u4e0e\u540c\u6e90\u81c2 HAPHA2\u901a\u8fc7\u918b\u9178\u9502\u8f6c\u5316\u6cd5\u5bfc\u5165 YCA113-8B \u9175\u6bcd\u83cc\u682a\u4e2d\uff0c\u5747\u5300\u6d82\u5e03\u5728 SD+ \u00a0Ura-\u5e73\u677f\u4e0a\u3002\u57f9\u517b 3~4 \u00a0d \u540e\uff0c\u6311\u53d6\u9175\u6bcd\u8f6c\u5316\u83cc\u682a\u4e8e 10 \u00a0\u03bcL \u65e0\u83cc\u6c34\u4e2d\uff0c\u4ece\u4e2d\u53d6 2 \u00a0\u03bcL \u81f3\u53e6\u4e00\u542b\u6709 8 \u00a0\u03bcL \u65e0\u83cc\u6c34\u7684 PCR \u7ba1\u4e2d\uff0c100 \u00a0\u2103\u52a0\u70ed 5 min \u540e\u76f4\u63a5\u7528\u4f5c PCR \u6a21\u677f\u3002\u4ee5 PHA2-F \u548c HAPHA2-R \u5f15\u7269\uff0c\u5229\u7528 KOD \u00a0One TM PCR Master Mix \u805a\u5408\u9176\u8fdb\u884c PCR \u6269\u589e\uff0c\u5c06\u6269\u589e\u5f97\u5230\u7684 PCR \u4ea7\u7269\u8fdb\u884c\u9001\u6d4b\u5e8f\u9a8c\u8bc1\u3002\u5c06\u6d4b\u5e8f\u6b63\u786e\u7684\u83cc\u682a\uff0c\u540d\u4e3a YCA113-16B \u83cc\u682a\u3002\u4e3a\u4e86\u8fdb\u884c\u4e0b\u4e00\u8f6e\u57fa\u56e0\u7f16\u8f91\uff0c\u9700\u8981\u5c06\u83cc\u682a YCA113-16B \u4e2d\u542b Cas9 \u86cb\u767d\u548c g RNA \u7684\u8d28\u7c92\u53bb\u9664\u3002\u5229\u7528\u5728\u6ca1\u6709\u7b5b\u9009\u538b\u529b\u7684\u57f9\u517b\u57fa\u4e2d\uff0c\u901a\u8fc7\u4f20\u4ee3\uff0c\u8d28\u7c92\u53ef\u81ea\u7136\u4e22\u5931\u8fd9\u4e00\u539f\u7406\uff0c\u5c06\u83cc\u682a YCA113-16B \u63a5\u79cd\u4e8e 5 m L \u00a0YPD \u8bd5\u7ba1\u4e2d\uff0c30 \u00a0\u2103\u4e0b\u57f9\u517b\u8fc7\u591c\u540e\uff0c\u518d\u8f6c\u63a5\u4e00\u6b21\u5230 YPD \u8bd5\u7ba1\u57f9\u517b\u8fc7\u591c\u3002\u53d6 1 \u00a0m L \u83cc\u6db2\u65e0\u83cc\u6c34\u6c34\u6d17\u4e24\u6b21\u540e\u7a00\u91ca 10 000 \u500d\uff0c\u53d6 100 \u03bcL \u6d82\u4e8e FOA \u5e73\u677f\u4e0a\u3002\u82e5 FOA \u677f\u4e0a\u7684\u5355\u83cc\u843d\u5728 SD \u4e0d\u52a0 URA \u8bd5\u7ba1\u4e2d\u4e0d\u80fd\u751f\u957f\uff0c\u800c\u5728\u52a0\u4e86 URA \u7684 SD \u8bd5\u7ba1\u4e2d\u80fd\u751f\u957f\uff0c\u5219\u8bf4\u660e TYPE3-M- \u00a0g PHA2 \u8d28\u7c92\u5df2\u7ecf\u53bb\u9664\uff0c\u83cc\u682a\u547d\u540d\u4e3aYCA113-16B \u00a0(-)\u3002\u540c\u7406\uff0c\u5c06 TYPE3-M-g TRP2\u4e0e\u540c\u6e90\u81c2 HATRP2\uff0c\u901a\u8fc7\u918b\u9178\u9502\u8f6c\u5316\u6cd5\u5bfc\u5165 YCA113-16B (-)\u9175\u6bcd\u83cc\u682a\u4e2d\uff0c\u83b7\u5f97 YCA113-20B \u83cc\u682a\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u5c06 p UMRI-\u0394Pdc5 \u7528 Sfi \u00a0I \u8fdb\u884c\u9176\u5207\u7ebf\u6027\u5316\u540e\uff0c\u901a\u8fc7\u918b\u9178\u9502\u8f6c\u5316\u6cd5\u5bfc\u5165 YCA113-16B \u00a0(-)\u9175\u6bcd\u83cc\u682a\u4e2d\uff0c\u6784\u5efa\u5f97\u5230\u83cc\u682a YCA113-23B\u3002\u4e3a\u4e86\u56de\u8865 YCA113-23B \u4e2d HIS3 \u548c MET15 \u8425\u517b\u7f3a\u9677\u578b\u57fa\u56e0\uff0c\u4ee5\u917f\u9152\u9175\u6bcd BY4742-MO4-His[19]\u57fa\u56e0\u7ec4\u4e3a\u6a21\u677f\uff0c\u7528 His3-F1\/ \u00a0His3-R1 \u4e3a\u5f15\u7269\u7528\u9ad8\u4fdd\u771f\u7684 Prime \u00a0STARTM HS DNA \u805a\u5408\u9176\u8fdb\u884c PCR \u6269\u589e\uff0c\u83b7\u5f97 HIS3 \u8425\u517b\u7f3a\u9677\u578b\u57fa\u56e0\u3002\u901a\u8fc7\u918b\u9178\u9502\u8f6c\u5316\u6cd5\uff0c\u5c06 HIS3 \u7247\u6bb5\u5bfc\u5165\u5230YCA113-23B \u83cc\u682a\u4e2d\uff0c\u5e76\u5c06\u8f6c\u5316\u7ec6\u80de\u6d82\u5e03\u5728\u4e0d\u6dfb\u52a0\u7ec4\u6c28\u9178\u800c\u6dfb\u52a0\u4e86\u7532\u786b\u6c28\u9178\u548c\u82ef\u4e19\u6c28\u9178\u7684 SD \u56fa\u4f53\u5e73\u677f\u4e0a\uff0c\u4ece\u800c\u6784\u5efa\u51fa YCA113-23BH \u83cc\u682a\u3002\u4ee5\u917f\u9152\u9175\u6bcd BY4742 \u57fa\u56e0\u7ec4\u4e3a\u6a21\u677f\uff0c\u7528 Met15-F1\/ \u00a0Met15-R1\u4e3a\u5f15\u7269\uff0c\u7528\u9ad8\u4fdd\u771f\u7684 Prime \u00a0STARTM HS \u00a0DNA \u805a\u5408\u9176\u8fdb\u884c PCR \u6269\u589e\uff0c\u83b7\u5f97 MET15 \u8425\u517b\u7f3a\u9677\u578b\u7b5b\u9009\u6807\u8bb0\u7684\u57fa\u56e0\u7247\u6bb5\uff0c\u5e76\u5c06\u7247\u6bb5\u5bfc\u5165\u5230 YCA113-23BH \u9175\u6bcd\u83cc\u682a\u4e2d\uff0c\u5c06\u8f6c\u5316\u7ec6\u80de\u6d82\u5e03\u5728\u4e0d\u6dfb\u52a0\u7532\u786b\u6c28\u9178\u4f46\u6dfb\u52a0\u4e86\u82ef\u4e19\u6c28\u9178\u7684 SD \u5e73\u677f\u4e0a\u3002\u4ece\u800c\u6784\u5efa\u51fa YCA113-23BHM \u83cc\u682a\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\"><strong>1.2.3 \u00a0\u91cd\u7ec4\u83cc\u682a\u53d1\u9175\u5b9e\u9a8c<\/strong><\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u5728\u6447\u74f6\u53d1\u9175\u5b9e\u9a8c\u4e2d\uff0c\u5c06\u83cc\u682a\u5728 YPD \u56fa\u4f53\u57f9\u517b\u57fa\u4e2d\u5212\u7ebf\uff0c\u4e8e 30 \u2103\u6052\u6e29\u57f9\u517b\u7bb1\u4e2d\u57f9\u517b 72 h\u3002\u6311\u53d6\u5e73\u677f\u4e0a\u7684\u83cc\u843d\u63a5\u79cd\u5230\u542b 5 &nbsp;m L &nbsp;YPD \u57f9\u517b\u57fa\u7684\u8bd5\u7ba1\u4e2d\uff0c30 &nbsp;\u2103\uff0c220 &nbsp;r\/min \u57f9\u517b 15 &nbsp;h\uff0c\u53d6\u4e00\u5b9a\u7684\u79cd\u5b50\u6db2\u63a5\u81f3\u542b 50 m L YPD \u57f9\u517b\u57fa\u7684 250 m L \u6447\u74f6\u4e2d\uff0c\u4f7f\u521d\u59cb OD600\u4e3a 0.05\uff0c30 \u2103\uff0c220 r\/min \u57f9\u517b 72 h\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u5728\u8865\u6599\u5206\u6279\u53d1\u9175\u8bd5\u9a8c\u4e2d\uff0c\u5c06\u83cc\u682a YCA113-23BHM \u5728 YPD \u56fa\u4f53\u57f9\u517b\u57fa\u4e2d\u5212\u7ebf\uff0c\u4e8e 30 \u00a0\u2103\u6052\u6e29\u57f9\u517b\u7bb1\u4e2d\u57f9\u517b 72 \u00a0h\u3002\u6311\u53d6\u5e73\u677f\u4e0a\u7684\u83cc\u843d\u63a5\u79cd\u5230\u542b 5 \u00a0m L \u00a0YPD \u57f9\u517b\u57fa\u7684\u8bd5\u7ba1\u4e2d\uff0c30 \u00a0\u2103\uff0c220 \u00a0r\/min \u57f9\u517b 15 h\uff0c\u8f6c\u63a5 1 \u00a0m L \u83cc\u6db2\u81f3\u542b\u6709 125 \u00a0m L \u00a0YPD \u00a0\u57f9\u517b\u57fa\u7684 500 \u00a0m L \u6447\u74f6\u4e2d\uff0c30 \u00a0\u2103\uff0c220 \u00a0r\/min \u57f9\u517b 20 \u00a0h\u3002\u5c06\u4e24\u74f6 250 \u00a0m L \u79cd\u5b50\u6db2\u5168\u90e8\u63a5\u79cd\u5230\u542b\u67092.25 \u00a0L \u00a0YPD \u57f9\u517b\u57fa\u7684 5 \u00a0L \u53d1\u9175\u7f50\u4e2d\uff0c\u53d1\u9175\u8fc7\u7a0b\u521d\u59cb\u901a\u6c14\u91cf\u8bbe\u5b9a\u4e3a2.5 \u00a0L\/min\uff0c\u6405\u62cc\u901f\u7387\u8bbe\u5b9a\u4e3a 300 \u00a0r\/min\uff0c\u6b64\u65f6\u76f8\u5bf9\u6eb6\u6c27\u503c\u8bbe\u5b9a\u4e3a 100\u3002\u53d1\u9175\u8fc7\u7a0b\u4e2d\u901a\u8fc7\u8c03\u8282\u6405\u62cc\u8f6c\u901f\u548c\u7a7a\u6c14\u6d41\u91cf\uff0c\u5c06\u6eb6\u6c27\u7ef4\u6301\u5728 20 \u5de6\u53f3\u3002\u57f9\u517b\u8fc7\u7a0b\u4e2d\u901a\u8fc7\u81ea\u52a8\u8865\u52a0 2.5 \u00a0mol\/L \u7684\u6c22\u6c27\u5316\u94a0\u6eb6\u6db2\uff0c\u5c06 PH \u503c\u7a33\u5b9a\u5728 5.5 \u5de6\u53f3\uff0c\u6e29\u5ea6\u6052\u5b9a\u5728 30 \u2103\u3002\u53d1\u9175\u521d\u671f\uff0c\u6bcf\u9694 12 h\uff0c\u8865 250 g\/L \u9175\u6bcd\u7c89\u6eb6\u6db2 50 m L\uff0c\u76f4\u5230\u83cc\u682a\u751f\u957f\u8fdb\u5165\u5e73\u7a33\u671f\u3002\u8461\u8404\u7cd6\u6d41\u52a0\u901f\u7387\u5728 0~20 m L\/h \u4e4b\u95f4\uff0c\u4f7f\u53d1\u9175\u6db2\u4e2d\u4e59\u9187\u542b\u91cf\u63a7\u5236\u5728 5 g\/L \u4ee5\u4e0b\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\"><strong>1.2.4 \u00a0\u53d1\u9175\u6837\u54c1\u5904\u7406<\/strong><\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u53d1\u9175\u7ed3\u675f\u540e\uff0c\u53d6 600 \u00a0\u03bcL \u53d1\u9175\u6db2\uff0c\u4e0e 600 \u00a0\u03bcL \u7532\u9187\u8d85\u58f0 15 \u00a0min\uff0c\u5145\u5206\u6df7\u5300\u3002\u7136\u540e\uff0c12 \u00a0000 \u00a0r\/min \u79bb\u5fc3 1 min\uff0c\u53d6\u4e0a\u6e05\u6db2 100 \u03bcL\uff0c\u4e0e 900 \u03bcL 50%\u7532\u9187-\u6c34\u6df7\u5300\uff0c\u7528 0.22 \u03bcm\u6709\u673a\u7cfb\u9488\u5f0f\u8fc7\u6ee4\u5668\u8fc7\u6ee4\uff0c\u7528\u4e8e\u540e\u7eed\u7684\u6db2\u76f8\u5206\u6790\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\"><strong>1.2.5 \u00a0\u68c0\u6d4b\u65b9\u6cd5<\/strong><\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u5b9e\u9a8c\u4e2d\u4f7f\u7528 Agilent \u00a01200 \u9ad8\u6548\u6db2\u76f8\u8272\u8c31\u4eea\uff0cPntulips \u00a0QS-C18 \u00a0plus\uff084.6 \u00a0mm\u00d7250 \u00a0mm\uff0c5 \u00a0\u00b5m\uff0cPuningtech\uff09\u6db2\u76f8\u8272\u8c31\u67f1\uff0c\u6240\u7528\u6d41\u52a8\u76f8\u4e3a 0.1%\u7532\u9178\uff08A \u6cf5\uff09\u548c\u4e59\u8148\uff08B \u6cf5\uff09\uff0c\u6d41\u901f 1 \u00a0m L\/min\uff0c\u68af\u5ea6\u6d17\u8131\u7a0b\u5e8f\u8bbe\u5b9a\u5982\u4e0b\uff1a0~30 \u00a0min\uff0cA \u6eb6\u5242 90%~50%\uff0cB \u6eb6\u5242 10%~50%\u3002\u67f1\u6e29 35 \u00a0\u2103\u3002\u6bcf\u6b21\u8fdb\u6837 20 \u00a0\u03bcL\uff0c\u6bcf\u4e2a\u6837\u54c1\u8fd0\u884c 15 min\u3002\u5496\u5561\u9178\u7684\u68c0\u6d4b\u6ce2\u957f\u4e3a 320 nm\uff0c\u51fa\u5cf0\u65f6\u95f4\u7ea6\u4e3a 11 min\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\"><strong>2 \u00a0\u7ed3\u679c\u4e0e\u5206\u6790<\/strong><\/p>\n\n\n\n<p class=\"wp-block-paragraph\"><strong>2.1 \u00a0\u6572\u9664 PHA2 \u5bf9\u5496\u5561\u9178\u5408\u6210\u7684\u5f71\u54cd<\/strong><\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u5728\u524d\u671f\u7684\u7814\u7a76\u4e2d\uff0c\u4ee5 L-\u916a\u6c28\u9178\u4e3a\u524d\u4f53\uff0c\u901a\u8fc7\u5f15\u5165\u5496\u5561\u9178\u5408\u6210\u6240\u9700\u7684\u57fa\u56e0\u4ee5\u53ca\u6572\u9664\u82ef\u4e19\u6c28\u9178\u53cd\u9988\u6291\u5236\u7684 Aro3\u3001\u8fc7\u8868\u8fbe\u916a\u6c28\u9178\u53cd\u9988\u6291\u5236\u4e0d\u654f\u611f\u7a81\u53d8\u4f53 Aro4K229L \u548c Aro7G141S\u3001\u6572\u9664\u4e19\u916e\u9178\u8131\u7fa7\u9176 Aro10 \u548c\u8868\u8fbe\u8fd0\u52a8\u53d1\u9175\u5355\u80de\u83cc\u6765\u6e90\u7684\u73af\u5df1\u4e8c\u70ef\u8131\u6c22\u9176 Tyr C\uff0c\u6784\u5efa\u4e86\u4e00\u682a\u4ea7\u5496\u5561\u9178\u7684\u917f\u9152\u9175\u6bcd\u83cc\u682a YCA113-8B[17]\u3002\u5728\u8be5\u83cc\u682a\u4e2d\uff0cL-\u82ef\u4e19\u6c28\u9178\u548c L-\u916a\u6c28\u9178\u90fd\u662f\u7531\u9884\u82ef\u9178\u8f6c\u5316\u5f62\u6210\uff0cL-\u82ef\u4e19\u6c28\u9178\u9014\u5f84\u662f L-\u916a\u6c28\u9178\u884d\u751f\u7269\u7684\u4e3b\u8981\u7ade\u4e89\u9014\u5f84\u3002\u963b\u65ad\u8fd9\u6761\u7ade\u4e89\u9014\u5f84\u53ef\u4ee5\u4f7f\u5f97\u78b3\u4ee3\u8c22\u6d41\u8f6c\u5411 L-\u916a\u6c28\u9178\u884d\u751f\u7269\u7684\u751f\u7269\u5408\u6210\u3002\u5728\u82ef\u4e19\u6c28\u9178\u5408\u6210\u9014\u5f84\u4e2d\uff0c\u9884\u82ef\u9178\u8131\u6c34\u9176\uff08PHA2\uff09\u53ef\u4ee5\u50ac\u5316\u9884\u82ef\u9178\u751f\u6210\u82ef\u4e19\u916e\u9178\uff0c\u82ef\u4e19\u916e\u9178\u5728\u82b3\u9999\u65cf\u6c28\u57fa\u9178\u8f6c\u79fb\u9176\uff08Aro8\/Aro9\uff09\u4f5c\u7528\u4e0b\u5408\u6210\u82ef\u4e19\u6c28\u9178\u3002\u4e3a\u4e86\u8fdb\u4e00\u6b65\u63d0\u9ad8\u5496\u5561\u9178\u4ea7\u91cf\uff0c\u672c\u7814\u7a76\u5229\u7528 CRISPR\/Cas9\u57fa\u56e0\u7f16\u8f91\u7cfb\u7edf\u6572\u9664\u4e86 YCA113-8B \u83cc\u682a\u57fa\u56e0\u7ec4\u4e2d\u7684 PHA2 \u57fa\u56e0\uff0c\u6784\u5efa\u4e86 YCA113-16B \u83cc\u682a\u3002\u7ecf\u6447\u74f6\u57f9\u517b 72 \u00a0h \u540e\uff0c\u7ed3\u679c\u5982\u56fe 2 \u6240\u793a\uff0c\u76f8\u6bd4\u4e8e YCA113-8B \u83cc\u682a\uff0c\u5496\u5561\u9178\u7684\u4ea7\u91cf\u63d0\u9ad8\u4e86 42%\uff0c\u8fbe\u5230\u4e86 807.3 mg\/L\u3002\u6b64\u5916\uff0c\u6572\u9664 PHA2 \u540e\uff0c\u7ec6\u80de\u7684\u751f\u7269\u91cf\u6ca1\u6709\u660e\u663e\u53d8\u5316\u3002\u7ed3\u679c\u8868\u660e\u6572\u9664 PHA2 \u57fa\u56e0\u80fd\u6709\u6548\u5730\u964d\u4f4e\u524d\u4f53\u5411\u82ef\u4e19\u6c28\u9178\u652f\u8def\u9014\u5f84\u7684\u8f6c\u5316\uff0c\u4f7f\u78b3\u4ee3\u8c22\u66f4\u591a\u5730\u6d41\u5411\u5496\u5561\u9178\u5408\u6210\u9014\u5f84\u3002<\/p>\n\n\n\n<figure class=\"wp-block-image size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"519\" height=\"369\" src=\"https:\/\/shandong.nugene.com.cn\/wp-content\/uploads\/2024\/01\/image-9.png\" alt=\"\" class=\"wp-image-228\" srcset=\"https:\/\/shandong.nugene.com.cn\/wp-content\/uploads\/2024\/01\/image-9.png 519w, https:\/\/shandong.nugene.com.cn\/wp-content\/uploads\/2024\/01\/image-9-300x213.png 300w\" sizes=\"auto, (max-width: 519px) 100vw, 519px\" \/><\/figure>\n\n\n\n<p class=\"wp-block-paragraph\"><strong>2.2 \u00a0\u6572\u9664 TRP2 \u5bf9\u5496\u5561\u9178\u5408\u6210\u7684\u5f71\u54cd<\/strong><\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u5728\u917f\u9152\u9175\u6bcd\u4e2d\uff0c\u7531\u83bd\u8349\u9178\u9014\u5f84\u751f\u6210\u7684\u5206\u652f\u9178\uff0c\u4e00\u65b9\u9762\u8f6c\u5316\u751f\u6210\u9884\u82ef\u9178\u7528\u4e8e L-\u82ef\u4e19\u6c28\u9178\u548c L-\u916a\u6c28\u9178\u7684\u5408\u6210\uff0c\u53e6\u4e00\u65b9\u9762\uff0c\u4f1a\u5728\u90bb\u6c28\u57fa\u82ef\u7532\u9178\u5408\u9176\uff08TRP2\uff09\u50ac\u5316\u4e0b\uff0c\u751f\u6210\u90bb\u6c28\u57fa\u82ef\u7532\u9178\uff0c\u5c06\u78b3\u4ee3\u8c22\u6d41\u8f6c\u5411 L-\u8272\u6c28\u9178\u5408\u6210\u9014\u5f84\u3002\u4e3a\u4e86\u8fdb\u4e00\u6b65\u63d0\u9ad8\u5496\u5561\u9178\u7684\u4ea7\u91cf\uff0c\u672c\u7814\u7a76\u5229\u7528 CRISPR\/Cas9\u57fa\u56e0\u7f16\u8f91\u7cfb\u7edf\u8fdb\u4e00\u6b65\u5c06 YCA113-16B \u83cc\u682a\u57fa\u56e0\u7ec4\u4e2d\u7684 TRP2 \u57fa\u56e0\u8fdb\u884c\u6572\u9664\uff0c\u83b7\u5f97 YCA113-20B \u83cc\u682a\u3002\u7ecf\u6447\u74f6\u57f9\u517b\u540e\uff0c\u6572\u9664 TRP2 \u57fa\u56e0\u540e\uff0cYCA113-20B \u83cc\u682a\u5496\u5561\u9178\u4ea7\u91cf\u7565\u5fae\u4e0b\u964d\uff08\u56fe 2\uff09\u3002\u4e4b\u524d\u62a5\u9053\u7684\u9175\u6bcd\u5408\u6210\u916a\u9187\u7684\u7814\u7a76\u4e2d\uff0cLIU \u7b49\u53d1\u73b0\u6572\u9664 TRP2 \u57fa\u56e0\u540e\uff0c\u916a\u9187\u7684\u4ea7\u91cf\u53cd\u800c\u4e0b\u964d\u4e86 19.7%\uff0c\u5176\u63a8\u6d4b\u662f\u7531\u4e8e\u6572\u9664 TRP2 \u57fa\u56e0\u540e\uff0c\u5f71\u54cd\u4e86\u9175\u6bcd\u751f\u957f\u6240\u5bfc\u81f4\u7684[20]\u3002\u800c\u5728\u672c\u7814\u7a76\u4e2d\uff0c\u6572\u9664 TRP2 \u540e\uff0c\u65e0\u8bba\u5bf9\u9175\u6bcd\u751f\u7269\u91cf\u548c\u5496\u5561\u9178\u4ea7\u91cf\u7684\u5f71\u54cd\u4e0d\u662f\u7279\u522b\u660e\u663e\u3002\u63a8\u6d4b\u539f\u56e0\u5982\u4e0b\uff1a\u8272\u6c28\u9178\u5206\u652f\u9014\u5f84\u76f8\u5bf9\u8f83\u590d\u6742\uff0c\u6709 TRP1-TRP5 \u4e94\u4e2a\u9176\u901a\u8fc7\u516d\u6b65\u53cd\u5e94\u5c06\u5206\u652f\u9178\u8f6c\u5316\u5230\u8272\u6c28\u9178\u3002\u5176\u4e2d\u5728\u7b2c\u4e00\u6b65\u53cd\u5e94\u4e2d\uff0c\u9664\u4e86 TRP2 \u80fd\u50ac\u5316\u5206\u652f\u9178\u8f6c\u5316\u6210\u90bb\u6c28\u57fa\u82ef\u7532\u9178\u5916\uff0cTRP3\u4f5c\u4e3a\u4e00\u4e2a\u53cc\u529f\u80fd\u7684\u9176\uff0c\u542b\u6709\u8c37\u6c28\u9170\u80fa\u9170\u80fa\u8f6c\u79fb\u9176\u548c\u5432\u54da -3-\u7518\u6cb9\u78f7\u9178\u5408\u9176\u4e24\u4e2a\u72ec\u7acb\u7684\u50ac\u5316\u7ed3\u6784\u57df\uff0c\u53ef\u4e0e TRP2 \u534f\u540c\uff0c\u50ac\u5316\u5206\u652f\u9178\u5411\u90bb\u6c28\u57fa\u82ef\u7532\u9178\u7684\u8f6c\u5316[21]\uff0c\u56e0\u6b64\u4ec5\u6572\u9664 TRP2 \u65e0\u6cd5\u5b8c\u5168\u963b\u65ad\u8272\u6c28\u9178\u652f\u8def\u3002\u540c\u6837\u5730\uff0c\u5728 GUO \u7b49\u7684\u7814\u7a76\u4e2d\uff0c\u53ea\u6572\u9664 TRP3\uff0c\u9175\u6bcd\u83cc\u682a\u751f\u7269\u91cf\u548c\u916a\u9187\u7684\u4ea7\u91cf\u4e5f\u65e0\u660e\u663e\u53d8\u5316[22]\u3002\u56e0\u6b64\uff0c\u5728\u540e\u7eed\u7684\u7814\u7a76\u4e2d\uff0c\u53ef\u4ee5\u540c\u65f6\u6572\u9664 TRP2 \u548c TRP3\uff0c\u8003\u5bdf\u5176\u5bf9\u5496\u5561\u9178\u5408\u6210\u7684\u5f71\u54cd\u3002\u6b64\u5916\uff0c\u4e0a\u8ff0\u7684\u7814\u7a76\u4e5f\u6697\u793a TRP2 \u6216\u8005 TRP3 \u53ef\u4ee5\u4f5c\u4e3a\u9175\u6bcd\u5916\u6e90\u57fa\u56e0\u6574\u5408\u7684\u4f4d\u70b9\u3002<\/p>\n\n\n\n<p class=\"wp-block-paragraph\"><strong>2.3 \u00a0\u91cd\u7ec4\u83cc\u682a\u8865\u6599\u5206\u6279\u53d1\u9175<\/strong><\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u4e3a\u4e86\u8fdb\u4e00\u6b65\u63d0\u9ad8\u5496\u5561\u9178\u7684\u4ea7\u91cf\uff0c\u8ba1\u5212\u9009\u7528\u6447\u74f6\u4e2d\u5496\u5561\u9178\u4ea7\u91cf\u76f8\u5bf9\u8f83\u9ad8\u7684\u83cc\u682a\u8fdb\u884c\u8865\u6599\u5206\u6279\u53d1\u9175\u5b9e\u9a8c\u3002\u7136\u800c\uff0cYCA113-16B \u662f HIS3 \u548c MET15 \u8425\u517b\u7f3a\u9677\u578b\u83cc\u682a\uff0c\u4e14 YCA113-16B \u83cc\u682a\u4e2d\uff0c\u643a\u5e26\u6709 URA3\u8425\u517b\u7f3a\u9677\u57fa\u56e0\u7684 TYPE3-M- \u00a0g PHA2 \u8d28\u7c92\u5bb9\u6613\u5728\u4f20\u4ee3\u4e2d\u4e22\u5931\u3002\u4e3a\u4e86\u5728\u8865\u6599\u5206\u6279\u53d1\u9175\u4e2d\uff0c\u907f\u514d\u7ec4\u6c28\u9178\u3001\u7532\u786b\u6c28\u9178\u548c\u5c3f\u5627\u5576\u7f3a\u4e4f\u5f71\u54cd\u7ec6\u80de\u751f\u957f\uff0c\u9996\u5148\uff0c\u5728\u53bb\u9664\u4e86 TYPE3-M-g 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\u83cc\u682a\u3002\u4e3a\u4e86\u8003\u5bdf\u8fd9\u4e9b\u8425\u517b\u7f3a\u9677\u578b\u57fa\u56e0\u56de\u8865\u540e\uff0c\u5bf9\u83cc\u682a\u5496\u5561\u9178\u548c\u751f\u7269\u91cf\u7684\u5f71\u54cd\uff0c\u5148\u8fdb\u884c\u4e86\u6447\u74f6\u53d1\u9175\u5b9e\u9a8c\u3002\u7ed3\u679c\u663e\u793a\uff0c\u5728\u6447\u74f6\u53d1\u9175\u4e2d\uff0c\u8fdb\u884c\u4e0a\u8ff0\u8425\u517b\u7f3a\u9677\u57fa\u56e0\u56de\u8865\uff0c\u5bf9\u751f\u7269\u91cf\u548c\u4ea7\u91cf\u6ca1\u6709\u9020\u6210\u660e\u663e\u7684\u5f71\u54cd\uff08\u56fe 3\uff09\u3002\u56e0\u6b64\uff0c\u6700\u7ec8\u9009\u7528\u4e86 YCA113-23BHM \u83cc\u682a\u5728 5 \u00a0L \u7684\u53d1\u9175\u7f50\u4e2d\u8fdb\u884c\u4e86\u8865\u6599\u5206\u6279\u53d1\u9175\uff0c\u6d4b\u8bd5\u4e86\u5176\u751f\u4ea7\u6027\u80fd\u3002\u7ed3\u679c\u5982\u56fe 4 \u6240\u793a\uff0c\u5728\u53d1\u9175 8 \u00a0h \u5de6\u53f3\uff0c\u8461\u8404\u7cd6\u8017\u5c3d\uff0c\u6b64\u65f6\u53d1\u9175\u6db2\u4e2d\u4e59\u9187\u542b\u91cf\u8fbe\u5230\u4e8613.9 g\/L\uff0c\u4ece 10 h \u5f00\u59cb\u4ee5 1 g\/(L\u00b7h)\u7684\u901f\u7387\u8865\u6599\uff0c12.5 h \u65f6\uff0c\u4e59\u9187\u6d53\u5ea6\u4e0b\u964d\u5230\u4e86 8.5 g\/L\uff0c\u6b64\u65f6\uff0c\u5c06\u8461\u8404\u7cd6\u7684\u8865\u6599\u901f\u5ea6\u63d0\u9ad8\u5230\u4e86 2 \u00a0g\/(L\u00b7h)\uff0c\u83cc\u682a\u8fdb\u5165\u4e86\u5bf9\u6570\u751f\u957f\u671f\uff0c\u53d1\u9175 24.5 \u00a0h\u540e\uff0c\u4e59\u9187\u7684\u542b\u91cf\u53ea\u67090.2 g\/L\u3002\u56e0\u6b64\u5c06\u8461\u8404\u7cd6\u7684\u8865\u6599\u901f\u5ea6\u63d0\u9ad8\u5230\u4e86 4 g\/(L\u00b7h)\u3002\u4f46\u4e4b\u540e\uff0c\u83cc\u682a\u7684\u751f\u957f\u53d8\u6162\u3002\u56e0\u6b64\u53d1\u9175 36 h \u4e4b\u540e\u5c06\u8865\u7cd6\u901f\u5ea6\u8c03\u4e3a 2 \u00a0g\/(L\u00b7h)\u3002\u53d1\u9175 12.5 \u00a0h \u540e\uff0c\u5496\u5561\u9178\u5feb\u901f\u79ef\u7d2f\uff0c\u76f4\u5230\u53d1\u9175 84 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class=\"wp-image-229\" srcset=\"https:\/\/shandong.nugene.com.cn\/wp-content\/uploads\/2024\/01\/image-10.png 554w, https:\/\/shandong.nugene.com.cn\/wp-content\/uploads\/2024\/01\/image-10-300x163.png 300w\" sizes=\"auto, (max-width: 554px) 100vw, 554px\" \/><\/figure>\n\n\n\n<figure class=\"wp-block-image size-full\"><img loading=\"lazy\" decoding=\"async\" width=\"554\" height=\"344\" src=\"https:\/\/shandong.nugene.com.cn\/wp-content\/uploads\/2024\/01\/image-11.png\" alt=\"\" class=\"wp-image-230\" srcset=\"https:\/\/shandong.nugene.com.cn\/wp-content\/uploads\/2024\/01\/image-11.png 554w, https:\/\/shandong.nugene.com.cn\/wp-content\/uploads\/2024\/01\/image-11-300x186.png 300w\" sizes=\"auto, (max-width: 554px) 100vw, 554px\" \/><\/figure>\n\n\n\n<p class=\"wp-block-paragraph\"><strong>3 \u7ed3\u8bba\u4e0e\u8ba8\u8bba<\/strong><\/p>\n\n\n\n<p class=\"wp-block-paragraph\">\u5496\u5561\u9178\u4f5c\u4e3a\u4e00\u79cd\u91cd\u8981\u7684\u6d3b\u6027\u915a\u9178\u7c7b\u5316\u5408\u7269\uff0c\u5177\u6709\u6297\u6c27\u5316\u3001\u6297\u764c\u3001\u6297\u83cc\u7b49\u751f\u7269\u5b66\u6d3b\u6027\uff0c\u5728\u98df\u54c1\u3001\u533b\u836f\u3001\u5316\u5986\u54c1\u7b49\u9886\u57df\u5177\u6709\u5e7f\u6cdb\u7684\u5e94\u7528\u3002\u8fd1\u5e74\u6765\uff0c\u968f\u7740\u5408\u6210\u751f\u7269\u6280\u672f\u7684\u53d1\u5c55\uff0c\u4ee5\u5927\u80a0\u6746\u83cc\u548c\u917f\u9152\u9175\u6bcd\u4e3a\u5e95\u76d8\u7ec6\u80de\uff0c\u901a\u8fc7\u5f02\u6e90\u4ee3\u8c22\u9014\u5f84\u7684\u8868\u8fbe\u4e0e\u4f18\u5316\uff0c\u5df2\u6210\u529f\u5b9e\u73b0\u4e86\u5496\u5561\u9178\u7684\u751f\u7269\u5408\u6210\uff0c\u4f46\u4ea7\u91cf\u4ecd\u666e\u904d\u8f83\u4f4e\uff0c\u8ddd\u5de5\u4e1a\u5316\u5e94\u7528\u4ecd\u6709\u8f83\u5927\u7684\u8ddd\u79bb\u3002\u5728\u524d\u671f\u7684\u7814\u7a76\u4e2d\uff0c\u672c\u8bfe\u9898\u7ec4\u4ee5\u917f\u9152\u9175\u6bcd\u4e2dL-\u916a\u6c28\u9178\u4e3a\u524d\u4f53\uff0c\u6784\u5efa\u4e86\u4e00\u682a\u4ea7\u5496\u5561\u9178\u83cc\u682a 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