KlebsiellaoxytocaHP1乙醇脱氢酶基因敲除对产氢的影响_英文_

KlebsiellaoxytocaHP1乙醇脱氢酶基因敲除对产氢的影响_英文_ ()第 46卷 增 刊 中山大学学报 自然科学版 Vo l146 Sup1 2007年 6月J un1 2007 ACTA SC IEN T IARUM NA TURAL IUM UN IV ER S ITA T IS SUN YA TSEN I Effec ts of a dh E Gene Knock2out on Hydrogen ? Evolut ion of Klebs ie lla oxytoca HP1 1, 2 1XU Fang 2cheng, HON G H ua 2sheng ( 1. Environm en ta l R e sea rch Cen te r, Sta te Key L abo ra to ry of M a rine Environm en ta l Sc ience s, X iam en U n ive rsity, X iam en 361005 , Ch ina; 2. D ep a rtm en t of Chem ica l and B iochem ica l Enginee ring, In stitu te )of Chem istry and Chem ica l Enginee ring, X iam en U n ive rsity, X iam en 361005 , Ch ina A b stra c t: Klebsiella oxy toca H P1 is a po ten tia l H2p roduc ing bac te rium we sc reened from a ho t sp ring. In the p re sen t study, m e tabo lic 2 enginee ring stra tegy wa s ca rried ou t to con struc t e thano l p a thway m u tan ts by homo logou s recom b ina tion, in o rde r to imp rove hyd rogen p roduc tion yie ld. M e tabo lic fluxe s of the w ild and the m u tan ts we re ana lysed and comp a red. Key word s: m e tabo lic enginee ring; hyd rogen p roduc tion; Klebsiella oxy toca H P1; adhE ( )2007 CL C n um ber: Q939197 D ocum en t code: A A r t ic le ID : 0529 26579 S120327 202 [ 1 ] K lebsiella oxy toca H P1is a po ten tia l H 2p rodu2 2 c ing bac te rium we sc reened from a ho t sp ring. Howev2 e r, the yie ld of Hfrom gluco se of th is bac te rium is le ss 2 than 2 mo l /mo l gluco se, wh ich is on ly ha lf of the m axi2 m um yie ld of da rk hyd rogen fe rm en ta tion. Fo r indu stri2 a l2sca le Hp roduc tion by K. oxy toca , enhancem en t of 2 [ 2 - 3 ] its Hp roduc tion ab ility is needed . 2 In the p re sen t study, m e tabo lic enginee ring stra te2 gy wa s ca rried ou t to con struc t e thano l p a thway m u tan ts by homo logou s recom b ina tion. 1 Exper im en ts The Con struc tion Stra tegy of the homo logou s re2 com b ina tion p la sm id p TA 2Str is show in F ig11. A n in2 te rna l fragm en t of K. oxy toca H P1 adhE gene wa s am 2 p lified by PCR from genom ic DNA w ith degene ra te o li2 gonuc leo tide p rim e rs de signed from con se rved region s of adhE gene sequence s. B y in se rting an aadA ca sse tte in to the cod ing region of op e ron gene adhE + 694 , + 695 d istric t in p TA , the homogene tic in tegra tion p la s2 F ig11 Stra tegy of vec to r con struc tion m id vec to r p TA 2Str wh ich wa s u sed to tran sfo rm K. fo r adhE gene knockou t oxy toca H P1 w ild2typ e stra in wa s con struc ted and a m u2 tan t w ith re sistance to Strep tom yc in wa s ob ta ined. M e tabo lite s we re ana lyzed by H PLC and ga s ch ro2 [ 4 ] m a tograp h . ?收稿日期 : 2007 201 210 ( ) 基金项目 : 国家自然科学基金资助项目 30470395 ( ) 作者简介 : 徐方成 1964年生 , 博士 , 副教授 ; 通讯联系人 : 洪华生 ; E2m a il: h shong@ xm u1edu1cn K. oxy toca H P1 fo r hyd rogen p roduc tion, bu t red irec2 2 Re su lts ted to lac ta te2p roduc ing p a thway. E thano l p a thway node m ay be a rigid node fo r Hp roduc tion of K. oxy toca 2 PCR ana lysis of the genom ic DNA from con struc2 H P1 on gluco se fe rm en ta tion. ted m u tan ts ind ica ted tha t the app rop ria te de le tion and in se rtion indeed occu rred. B a tch cu ltu re s we re ca rried Tab11 M e tabo lic fluxe s of the w ild and the ou t to exam m e tabo lic enginee ring op e ra tion. H2p rodu2 2 m u tan t of gluco se fe rm en ta tion c ing m e tabo lic ne two rk of K. oxytoca H P1 is showed in H M e tabo lite s A ce ta te E thano l L ac ta te B iom a ss2 F ig12. 64168 148 164 12 14 1072513178W ild 10112 3214 5218 1112 19512 m u tan t % 5615 26919 10519 21512 914 The re su lts repo rted he re rep re sen t a step towa rd u sing m e tabo lic enginee ring to deve lop ing stra in s of H2 2 p roduc ing bac te ria w ith h igh yie ld. Referen ce s: [ 1 ] LON G M N , HUAN G J L , WU X B. Iso la tion and cha r2 ac te riza tion of a h igh H2p roduc ing stra in Klebsiella oxy2 2 toca H P1 from a ho t sp ring. R e sea rch in M ic rob io logy ( ) [ J ]. 2004, 156 1 : 76 281. DA S D , V EZ IRO GLUB T N. H yd rogen p roduc tion by [ 2 ] b io logica l p roce sse s: A su rvey of lite ra tu re [ J ]. In t J H yd rogen Ene rg, 2001 , 26: 13 - 28. 12 M e tabo lic ne two rk of K. oxy toca H P1 F igGRO SS R , L EACH M , BAU EN A. P rogre ss in renen2 [ 3 ] ab le ene rgy [ J ]. Envi: ron In t, 2003 , 29: 105 - The de le tion m u tan t d isp layed sim ila r grow th com 2 122. p a red to the w ild2typ e. B iom a ss and m e tabo lite s fo rm ed XU F C, HON G H S, LON G M N. Enhanced hyd rogen [ 4 ] we re list in Tab le 1. Comp a rison to the w ild2typ e, e th2 p roduc tion of Klebsiella oxy toca H P1 by ga s bubb ling in ano l, ace ta te, lac ta te and Hof the m u tan t we re de2 2 liqu id p ha se [ J ]. Jou rna l of X iam en U n ive rsity: N a tru2 c rea se by 6919 % and inc rea sed by 5615 % , 10519 % , ( ) a l Sc ience, 2006 , 45 6: 8322835. 914 % , re sp ec tive ly. It seem ed tha t the reduc tan t saved in e thano l p a thway wa s no t cap tu red by hyd rogena se of Klebsiella oxy toca HP1乙醇脱氢酶基因敲除对产氢的影响 1 , 2 1 徐方成 , 洪华生 ( 1. 厦门大学环境科学研究中心 ?近海海洋环境科学国家重点实验室 , 福建 厦门 361005; )2. 厦门大学化学化工学院化学工程与生物工程系 , 福建 厦门 361005 摘 要 : Klebsiella oxy toca H P1是从温泉筛选得到的很有潜力的产氢菌株 。为了进一步提高产氢能力 , 应用同源重组技术 构建了乙醇途径缺少菌株 , 并分析和比较了野生菌和工程菌的代谢流量分布 。 关键词 : 代谢工程 ; 发酵产氢 ; Klebsiella oxy toca H P1; adhE基因 中图分类 号 : Q939197