【作者】 吴世根；

【导师】 王芳；

【作者基本信息】 北京化工大学 ， 化学工程与技术， 2011， 硕士

【摘要】 富马酸是一种重要的化工原料和精细化工产品,在医药、化工、树脂等领域有着非常广泛的应用前景,其生物生产方法已经受到了广泛的重视,如微生物发酵法。本文以毕赤酵母作为出发菌株,研究考察米根霉富马酸酶基因、毕赤酵母丙酮酸羧化酶基因在毕赤酵母细胞中的过表达对富马酸胞质途径的影响,以及敲除副产物乳酸代谢过程中的关键酶乳酸脱氢酶基因,使得更多碳源流向富马酸胞质途径。本实验克隆了毕赤酵母(Pichia pastoris GS115)中编码丙酮酸羧化酶的基因(PC),在毕赤酵母中进行了强化表达,以研究PC基因在毕赤酵母中的过量表达对TCA还原途径碳源分流及草酰乙酸、L-苹果酸生产的影响。为此,研究构建了表达载体pPIC3.5K-PC,并转化了毕赤酵母GS115。转化子经过表型鉴定,PCR分析和G418浓度梯度筛选获得了高拷贝的重组子(pas-01)。甲醇诱导表达后,经SDS-PAGE分析及PC活性检测,发现酶活提高了3倍,说明PC在毕赤酵母中获得了成功表达。以pPIC3.5K转化菌为对照,对该重组子进行了草酰乙酸、苹果酸的发酵研究,结果显示草酰乙酸产量(177.4 mg.L-1)提高了109.6%,L-苹果酸的产量(127.45 mg.L-1)提高33.7%,菌体生物量提高15.7%,表明PC的过量表达有助于L-苹果酸、草酰乙酸的积累,并且对菌体的生长有一定的促进作用。为克隆米根霉细胞中的富马酸酶基因,本实验先后更换了二十对引物,尝试通过逆转录聚合酶链式反应(RT-PCR)扩增富马酸酶基因片段,未果。随后根据米根霉富马酸酶基因的保守序列设计引物,扩增得到250bp长度的保守序列,与模板相比同源性只有76%；说明目前所使用的这株米根霉3.2686细胞中的富马酸酶基因序列与1959年以色列学者报导的模板序列具有较大差异,该菌中的fumR是一个新的基因。克隆了毕赤酵母中编码乳酸脱氢酶的基因LDH,同时也克隆了博来霉素抗性基因zeocin,构建了LDH敲除载体pMD18-T-LDHup-Zeocin-LDHdown,其中上游同源臂有900bp左右,下游同源臂包含500bp大小的序列,通过同源重组敲除LDH基因。更多还原

【Abstract】 Fumaric acid is an important chemical raw materials and fine chemical products, has a very wide range of applications in pharmaceuticals, chemicals, resins and other fields, and its biological production methods have received wide attention, such as microbial fermentation. In this paper, we took Pichia pastoris as the starting strain and investigated influence of the expression of fumarase gene from Rhizopus oryzae CGMCC3.2686 and pyruvate carboxylase gene from Pichia pastoris on the cytoplasmic pathway of fumarate. We also knocked out lactate dehydrogenase gene, which is a key enzyme in the metabolism of lactic acid, to make way for more carbon flow of cytoplasmic fumarate.In this study, we overexpressed PC gene in Pichia pastoris GS115, and investigated its influence on the diversion of carbon to the reductive TCA pathway and the yields of the oxaloacetate and L-malic acid. The expression vector pPIC3.5K-PC was constructed, and was introduced in Pichia pastoris GS115. A high-copy recombinant (pas-01) was screened out from 30 positive transformants by phenotypic identification, PCR analysis and G418 concentration gradient selection. After methanol induction, recombinant protein was analysed by SDS-PAGE and PC activity was detected. Results showed that PC was overexpressed in the Pichia pastoris, and the activity was increased 3-folds. Compared to the control (pPIC3.5K transformant), oxaloacetate production(177.4 mg.L-1) of the recombinant (pas-01) was increased by 109.6%, L-malic acid production (127.45 mg.L-1) increased by 33.7% and cell biomass increased by 10% through the fermentation. These results indicated that overexpression of PC is propitious to the accumulation of oxaloacetate and L-malic acid, which also play a significant role in promoting the growth of strain.In order to clone fumarase gene from Rhizopus oryzae CGMCC3.2686, in this experiment we designed twenty pairs of primers and tried to clone fumarase gene by reverse transcription polymerase chain reaction (RT-PCR), but failed. Subsequently, according to the conserved sequence of fumarase gene from Rhizopus oryzae, we designed a pair of primers and amplified 250 bp length conserved sequence, compared with the template is only 76% homology; that this strain is currently used by the cells of Rhizopus oryzae CGMCC 3.2686 The gene sequence of fumarase 1959 template sequence Israeli scholars have reported large differences in the fumR bacteria is a new gene. The LDH gene which encodes lactate dehydrogenase was cloned, and the bleomycin resistance gene zeocin was also cloned. At the same time, the knockout vector of LDH was constructed, named pMD18-T-LDHup-Zeocin-LDHdown, which has 900 bp upstream homologous arm and about 500 bp downstream homology arm in order to knockout the LDH genes from the genome of Pichia pastoris by homologous recombination.更多还原