高产核黄素枯草芽孢杆菌代谢工程研究-化学工程与技术专业论文.docxVIP

Deregulation of purine pathway was attempted to improve purine nucleotides supply, based on a riboflavin producer B. subtilis strain with modification of its rib operon. To eliminate transcription repression, the pur operon repressor PurR and the 5’-UTR of pur operon containing a guanine-sensing Riboswitch were disrupted. Quantitative RT-PCR analysis revealed that the relative transcription levels of purine genes were up-regulated about 380 times. Furthermore, site-directed mutagenesis was successfully introduced into PRPP amidotransferase (encoded by purF) to remove feedback inhibition by homologous alignment and analysis. Overexpression of the novel mutant PurF (D293V, K316Q and S400W) significantly increased PRPP amidotransferase activity and triggered a strong refractory effect on purine nucleotides mediated inhibition. Intracellular metabolite target analysis indicated that the purine nucleotides supply in engineered strains was facilitated by a stepwise gene-targeted deregulation. With these genetic manipulations, we managed to enhance the metabolic flow through purine pathway and consequently increased riboflavin production 3-fold (826.52 mg/L) in the purF-VQW mutant strain. KEY WORDS：Bacillus subtilis; riboflavin; markerless mutation delivery system; inverse metabolic engineering; reconstruction; purine biosynthetic pathway; 目 录 HYPERLINK \l _bookmark0 第一章 文献综述1 HYPERLINK \l _bookmark1 1.1 核黄素的理化性质、功能、用途及工业化生产 1 HYPERLINK \l _bookmark2 1.1.1 核黄素的理化性质、功能和用途1 HYPERLINK \l _bookmark3 1.1.2 核黄素的工业化生产2 HYPERLINK \l _bookmark4 1.2 B. subtilis 核黄素生物合成研究进展 3 HYPERLINK \l _bookmark5 1.2.1 B. subtilis 核黄素生产菌株 3 HYPERLINK \l _bookmark6 1.2.2 B. subtilis 核黄素生物合成代谢工程策略 4 HYPERLINK \l _bookmark7 1.3 核黄素生物合成途径代谢调节机制10 HYPERLINK \l _bookmark8 1.3.1 rib 操纵子与核黄素合成途径 10 HYPERLINK \l _bookmark9 1.3.2 Riboswitch 调控机制 13 HYPERLINK \l _bookmark10 1.3.3 RFN 元件与 rib 操纵子转录调控 14 HYPERLINK \l _bookmark11 1.3.4 ribC 和 ribR 基因 16 HYPERLINK \l _bookmark12 1.4 嘌呤生物合成途径代谢