【作者】 史硕博；

【导师】 赵学明；

【作者基本信息】 天津大学 ， 生物化工， 2009， 博士

【摘要】 本文系统分析了三株不同核黄素产量B. subtilis工程菌株的代谢特征,揭示了菌株产核黄素的内在遗传机理,同时构建了一系列产核黄素B. subtilis基因工程菌,得到的主要结果如下:发现purF基因采用不同的整合方式对菌体代谢的影响有明显差异,原因在于采用双交换整合机理构建的系列工程菌中仅purF基因表达水平提高;而采用单交换整合机理构建的系列工程菌中不仅purF基因而且其下游purM、purN、purH、purD基因的表达水平皆有不同程度的提高,这些基因的编码产物可催化更多的嘌呤前体物谷氨酰胺、甘氨酸、10-甲醛四氢叶酸(10-Formyl-THF)进入嘌呤途径,故其对菌体代谢影响较大。从基因表达水平上揭示产核黄素工程菌的主要特点,确定了产核黄素菌株应具有的有利表型:表达增强显著的核黄素操纵子,将加强核黄素合成途径;citZ基因及副产物形成有关的基因的下调,可避免溢流代谢、减少副产物生成;采取谷氨酸脱氢酶催化的途径利用氮源(NH4+)能节约菌的能量消耗;葡萄酸支路可提供充足的核黄素合成起始物5-磷酸核酮糖;采用能量耦合效率高的aa3氧化酶来产生能量,可有效改善能量供给。以工程菌RH33和RH44为亲株,增强表达PRPP合成酶及核糖-5-磷酸异构酶可提高胞内PRPP浓度,促进PurR调控基因的表达,进而促使胞内更多的嘌呤核苷酸、谷氨酰胺、一碳单位、甘氨酸、二氧化碳和天冬氨酸等核黄素前体物合成途径加强并参与到核黄素合成途径,核黄素合成能力分别提高20%和3.4%。核黄素合成过程中,需要1分子的DARPP(长途径提供)和2分子的DHPB(短途径提供)生成1分子的核黄素。增加PRPP浓度虽然一方面加强了前体物的供给,另一方面解除了对嘌呤及甘氨酸合成途径等PurR调控基因的抑制,但这两方面作用仅仅在于提高GTP合成途径(长途径)的通量,没有缓解存在的前体物供给不平衡,RH44对此尤为敏感。为平衡前体物供给以提高核黄素生产,在RH44中增强表达3,4-二羟基磷酸丁酮(DHPB)合成酶以增加DHPB供给,缓解存在的前体物供给不平衡,从而构建RH44-RB。RH44-RB与其亲株RH44相比,菌体生长变慢,糖耗减少,核黄素产量在摇瓶培养下达到6.0 g/l,产率达到0.061(g riboflavin/g glucose),与出发菌RH44相比分别提高了17%和19%。更多还原

【Abstract】 Based on system biology and metabolic engineering, here we reported the characterization of the riboflavin biosynthesis ability in three riboflavin overproducers. The metabolism and regulation contributed to the riboflavin overproducing was studied. Consequently, a series of genetic modified B. subtilis strains were constructed and the major findings are:In this study, the purF gene was introduced into the amyE gene locus by a double crossover mechanism and the purF locus by a single like mechanism. However, as demonstrated in this work, a significant different effect on the purine pathway and riboflavin production was found according to the used integration mechanism. It was reasoned that the double crossover mechanism would singly up-regulate the expression of purF, in contrast, the single crossover mechanism would simultaneously up-regulate purF and its downstream genes purM, purN, purH, purD, which efficiently increased the supply of the purine precursors into purine pathway.A comparative transcriptome profiling between riboflavin producing strains and the wild type strain was performed, and it found the genotype that contributed to the riboflavin overproducing trait: The strongly up-regulated transcription of rib operon would enforce riboflavin biosynthesis pathway; Down-regulation of gene citZ and the byproducts formation genes would facilitate reducing overflow metabolism; Adoption of the glutamate dehydrogenase system could save one molecule of ATP for the assimilation of one molecule of ammonium; The up-regulation of gdh and gntK modulated carbon flow through the gluconate bypass to provide more precursor ribulose-5-P; Redirection electron flow to high coupling efficiency of terminal oxidase could enhance energy generation.In RH33 and RH44, we selected and co-overexpressed prs and ywlF genes simultaneously, which are involved in the biosynthetic pathway of PRPP from ribulose-5-phosphate. This co-amplification led to an elevated PRPP pool and thus the increased transcript abundances of PurR-regulated genes participated in riboflavin precursor biosynthesis, including purine nucleotides, glycine, glutamine etc. The riboflavin biosynthetic ability was enhanced by 20% and 3.4%, respectively.Since two DHBP molecules and one DARPP molecule were needed for the formation of one riboflavin molecule, DHBP was consumed at a higher rate than DARPP, which resulted in an imbalance supply of riboflavin synthesis precursors. However, enhancing purine biosynthesis pathway or PurR-regulated genes only facilitated the formation DARPP, which is provided by the long and normally tight-regulated metabolic pathway. Therefore, lacking of DHBP, the imbalance of precursors supply would limit the riboflavin overproducing in RH44. To solve this imbalance problem, ribB gene from E. coli that only coding the DHBP synthase was selected and overexpressed. As a result, a 17% increase in the riboflavin production and a 19% increase in the yield were obtained, which suggested that the expression of ribB gene from E. coli would produce more DHBP to recover the balance of the precursors supply.更多还原