L-异亮氨酸生产菌株的构建及发酵条件优化-轻工技术与工程（发酵）专业业论文.docx

ABSTRACTL-isoleucine，one ABSTRACT L-isoleucine，one of the ei曲t essential amino acids for human，plays锄imponant pan in the metabolism of body．During the process of L-isoleucine Synthesis，the en巧me acetohydroxy acid synthaLse catalyzes叶ketobutyrate锄d pyruVate to generate L-isoleucine．Acetohydroxy acid synthaSe (AHAS)shows hi曲er amn时to值-ketobutyrate than to pyruVate．Therefore，increaSing the supply of intracellular 0【-ketobutyrate may lead to preferred synthesis of L-isoleucine catalyzed by acetohydroxy acid syntllase．Wrllat、s more，if the AHAS，one of the limited enzymes for L-isoleucine驴t11esis，is stren舀hened，锄d result，the limited reactions will be accelerated with more L—isoleucine accumulation． Tb inVestigate efrects of increaLsing supplies of intracellular 0【-ketobutyrate and streng廿lened AHAS L-isoleucine fementation，L-isoleucine producer C∥甜f日历fc姗?YILw waS used t}le o“ginal strain for molecular constmction． C ∥甜触坍耙z删 YILWpXMJ l 9cf蒯 waS constmcted through the oVerexpression of c加翻，c．∥“f硎配z删YILwPtac waS constmcted by the insertion of Ptac，just in the upstreaIll of肌田in genome． C ∥甜f铡记聊?YII，wnacpxMJ 1 9c泐爿waS cons仃ucted by the oVerexpressiOn 0f plaSmid baSed C g缸f翻啊配堋YII胛taC．The real—time PCR showed the relatiVe traIlscription of cf，，纠and f，沾waS increaLsed by 920．0锄d 1 2．5 fold，respectiVely．The feaSibil时of c西州 gene oVerexpression in c∥砌m记踟YILwpxMJ 1 9cf—翻waLs detemined by sDs·Pf～GE． Fed_batch fementation experiment waS perfIo珊ed to compare the L-isoleucine production of original s仃ain to recombinallt strai凼． The result discoVered that the L-is01eucine production of YlLwpxMJl 9cf，，翻and YILwPtac increaLsed by 8．9％aIld l 4．8％with the yield increaSed by 1 7．8％锄d 1 8．6％，respectiVely，compared to original stmin YILW．The u11it ceU pmduction waS 15．1％and 20．3％ hi曲er tllan that of YILW．YILWpXMJl9c西刎锄d YII，WPtac excreted 25．0％and 29．9％lower L．alaJline thaJl that行om original strain．Compared to YILW YII WPtacpXMJ 1 9c砌爿e)【Ilibited 1 4．5％ hi曲er L-isoleucine produ“0n with 42．