删除或更新信息,请邮件至freekaoyan#163.com(#换成@)

N-端改造植物 P450 酶实现工程大肠杆菌合成甜菜黄素

本站小编 Free考研考试/2022-01-16

濞戞挴鍋撻柡澶樺灠閵堝爼鎳犻崜浣圭暠闂佽棄宕銊╁矗椤栨瑤绨板☉鏃€婢橀崺宀勬嚀閸愵亞鍩″☉鎾存尫缁楃喓鎷犻幑鎰偒闁哄倹鐟辩槐锟�
2濞戞挸娲ㄩ~鎺楁嚀閸愵亞鍩¢柣銏ゆ涧閻℃瑦绋婇敂鑲╃濡増锚缁ㄩ亶濡存担绛嬫綊濡増鍨埀顑跨閸欏繑绺藉Δ鍕偒闁哄倹鐟辩槐姘跺矗婵犲倸鍧婃鐐差嚟濠€鈩冿紣濮楀牏绀夋繛鎴犳暩濞诧拷547闁圭鍋撻梻鍕╁灪閻楋拷4濞戞挸娲g紞鎴炵▔椤忓洠鍋撻崘顏嗗煛闁兼澘鍟畷銉︾▔閹捐尙鐟圭紒澶嬪灩濞蹭即濡存担瑙e亾閸愵亞鍩¢柛蹇e墮閸欙紕鎷犻幘鍛闁衡偓閹稿簼绗夐柤鏄忕簿椤曘垽寮弶娆惧妳闁挎稑顦埀顒婃嫹40缂佸绉崇粭鎾寸▔濮橀硸鏁嬪璇″亾缁辨瑩鏌岄幋锝団偓铏规兜閺囩儑绱滈柕鍡曞簻BA闁靛棔绀佸ù妤呮⒔閸涱厽娅岄柛鏃撶磿椤㈡碍绔熼鐘亾娴h鐓€闂傚倽顔婄槐鍫曞箻椤撶媭鏁嬪璇″亖閳ь兛鑳堕妵鐐村濮橆兛绱eù锝嗙矌椤㈡碍绔熼銈囨惣闁挎稑顦埀顒婃嫹28缂侇偉顕ч幃鎾剁驳婢跺⿴鍔呴柛鏃€绋撻弫鐢垫兜閺囨氨鐟╁☉鎾村搸閳ь剨鎷�1130缂佸绉剁划锟犲礂閸涘﹥娈岄柡澶嬪姂閳ь剨鎷�
赵广荣 ,侯亚男
AuthorsHTML:赵广荣 1, 2,侯亚男 1, 2
AuthorsListE:Zhao Guangrong,Hou Yanan
AuthorsHTMLE:Zhao Guangrong1, 2,Hou Yanan1, 2
Unit:1. 天津大学化工学院,天津 300350;
2. 教育部合成生物学前沿科学中心和系统生物工程重点实验室,天津 300350

Unit_EngLish:1. School of Chemical Engineering and Technology,Tianjin University,Tianjin 300350,China;
2. Frontier Science Center for Synthetic Biology and Key Laboratory of System Bioengineering (Ministry of Education),Tianjin 300350,China

Abstract_Chinese:甜菜黄素是安全的植物源水溶性天然色素,主要应用于食品行业.甜菜黄素还具有抗氧化活性和光学特 性,在医疗保健、荧光成像等领域具有潜在应用价值.目前,甜菜黄素的主要来源是植物提取分离,但存在甜菜黄 素含量低、种类少等问题.本文改造植物 P450 氧化酶,研发大肠杆菌异源合成甜菜黄素的新方法.首先,对甜菜 的 P450 氧化酶 CYP76AD1LL和 P450 还原酶(cytochrome P450 reductase,CPR)进行 N-端截短和改造,研究不同改 造序列和表达策略对合成 L-多巴的影响.结果表明,截去 N-端跨膜序列,利用 3 种不同 N-端序列修饰 P450 氧化 酶,MA 序列修饰 CPR(MA-tCPR),工程大肠杆菌中均能以 L-酪氨酸为底物合成 L-多巴.其中亲水性 2B1 序列 (MAKKTSS)修饰 P450 氧化酶,采用双顺反子表达策略优于单顺反子,工程大肠杆菌 BDP10 合成 L-多巴的产量最 高,为 40.42 mg/L.进一步表达紫茉莉的 4,5-多巴双加氧酶基因,构建了工程大肠杆菌 BTA11,利用 L-酪氨酸为前 体,合成了甜菜醛氨酸.最后,在培养基添加 3 种氨基酸(对氨基苯甲酸、L-组氨酸、L-亮氨酸)和 3 种胺类化合物 (酪胺、吡咯烷、吲哚啉),工程大肠杆 BTA11 合成了 6 种对应的甜菜黄素.研究结果表明,采用 N-端改造策略赋 予植物 P450 氧化酶在大肠杆菌中具有催化功能,能合成天然和非天然的颜色多样、结构丰富的多种甜菜素.
Abstract_English:Betaxanthins are water-soluble and safe pigments mainly used in the food industry. With rich antioxidant and optical properties,betaxanthins have potential applications in the fields of health care and fluorescence imaging. Presently,the main source of betaxanthins is plant extraction and separation,but the obtained betaxanthin content is low and the betaxanthin varieties are few. In this study,a new method for the heterologous synthesis of betaxanthins by Escherichia coli was developed by the expression of modified plant P450 oxidase. First,to study the effects of modified sequences and expression methods on the conversion of L-tyrosine into L-dopa,beet-derived P450 enzyme CYP76AD1LL and cytochrome P450 reductase(CPR)were truncated and modified at the N-terminal. The results showed that after the truncation of the transmembrane sequences,all three different N-terminal sequence-modified P450 oxidase co-expressing with the MA sequence-modified CPR(MA-tCPR)successfully catalyzed the conversion of L-tyrosine to L-dopa in E. coli. The engineered E. coli strain BDP10 expressing N-terminal hydrophilic sequence 2B1-modified P450 oxidase used in combination with the bicistronic strategy resulted in the highest L-dopa produc\u0002tion(40.42 mg/L),which was higher than that obtained with the monocistronic strategy. Furthermore,a betalamic acid-producing strain was constructed through the co-expression of a 4,5-dopa dioxygenase extradiol gene from Mirabilis jalapa,which enabled the conversion of L-tyrosine to betalamic acid. Finally,three amino acids(paraaminobenzoic acid,L-histidine,and L-leucine)and three amines(tyramine,pyrrolidine,and indoline)were fed into the fermentation medium with the engineered E. coli strain,and six betaxanthins could be produced. The results show that N-terminal modification can endow plant P450 oxidase with catalytic function,and the resulting strain can synthesize natural and unnatural betaxanthins with various colors and rich structures.
Keyword_Chinese:细胞色素 P450 酶;甜菜黄素;大肠杆菌;合成生物学;酶的改造
Keywords_English:cytochrome P450 enzyme;betaxanthin;Escherichia coli;synthetic biology;modification of enzymes

PDF全文下载地址:http://xbzrb.tju.edu.cn/#/digest?ArticleID=6679
相关话题/工程 植物

鐎殿喒鍋撻梺顐g濠€鎵博濮楃P闁挎稒鐭粩瀛樼▔閸モ晩娼氶柤鏉垮暟閻栫儤绋夐幘鑼懝閻犲洦宕橀~瀣紣閹达附顓瑰〒姘虫硶濠€鍛存晬鐏炲墽妲ㄩ柡鍫厸缁楀宕氭0浣侯伇闁告帒妫濋幐鍫曟晬娴h櫣鍗滈悗鐢垫嚀閸ㄦ繄绮诲Δ瀣<
547闁圭鍋撻梻鍕╁灪閻楀酣鎳撻崘顏嗗煛闁兼澘鍟畷锟�1130缂佸绉电€垫氨鈧纰嶉弳鈧柡澶嬪姉濞堟垿宕¢崘褏绋囩紒澶婄Ч閸樸倖绺藉Δ鍜佹毌閹煎瓨鎸堕埀顑挎祰椤锛愰幋顖滅婵炴垹鏁稿ú濠囨嚐鏉堫偒鍤旈柕鍡曡兌缁€趁规惔娑掑亾娴g晫妲堥柛鎺撴偠閳ь兛绶氶崳楣冩懚瀹ュ啠鍋撴担鐑樺€炵€规悶鍎埀顑胯兌椤撴悂鎮堕崱鎰ㄥ亾娴f亽浠﹀ù鍏煎搸閳ь兛娴囬崒銊﹀濮樸儮鍋撴担瑙勬畬闁煎弶褰冪缓楣冩偠閸℃劏鍋撴担鐤幀闁哄倸娲㈤埀顑挎祰婢规捇寮甸妯峰亾娴h鐓€闂傚倽顔婄槐鍫曞箻椤撴壕鍋撴担鍦€婇悗娑宠礋閳ь兛绀佺亸鎵偓娑宠礋閳ь兛娴囬鍝ョ不濡や焦绨氶柕鍡曠瀹稿宕g仦鍌楀亾娴e憡鍕鹃柣鐐叉閳ь兛鐒﹂弬鍌氣柦濮瑰洠鍋撴担鍛婃噸閻庢冻璐熼埀顑挎缂嶅鎳栭懠顒冾潶缂佹冻鎷�28缂侇偉顕ч鐔虹矓閹搭垳纾�