钝齿棒杆菌SYPA5-5NAD激酶表达对其L-精氨酸生物合成影响的研究

论文摘要

L-精氨酸作为一种半必须氨基酸，在食品、医药、化工领域具有广泛的应用。NADPH是棒杆菌L-精氨酸合成的重要的辅因子。在谷氨酸棒杆菌中，NADPH是主要通过NADP+在脱氢酶的催化作用下受氢形成的，如磷酸戊糖途径中的葡萄糖-6-磷酸脱氢酶（G6PDH/zwf）及6-磷酸葡萄糖酸脱氢酶（6PGDH/gnd）分别催化葡萄糖-6-磷酸和6-磷酸葡萄糖酸进行脱氢反应，同时形成NADPH。然而，NAD激酶（ppnk）通过催化NAD+的磷酸化反应形成NADP+。据相关报道，NAD激酶受变构调节作用，NAD（H）和NADP（H）的平衡可能直接调控NAD激酶的活性。改变辅因子NADP+的胞内水平，可能刺激葡萄糖-6-磷酸通过磷酸戊糖途径产生NADPH及前体代谢物，因此可提高L-精氨酸的产量。钝齿棒杆菌SYPA5-5（C. crenatum）为实验室保存的一株L-精氨酸高产菌株。本论文旨在考查NAD激酶增强表达对L-精氨酸生产和菌体代谢的影响。成功构建了ppnk增强表达质粒pJC1-tac-ppnk质粒，电转入钝齿棒杆菌SYPA5-5中，挑选重组菌株C.crenatum SYPA5-5ppnk，在高溶氧（HOS）和低溶氧（LOS）的环境下对出发菌株和重组菌株进行比较评价。在HOS条件下，重组菌NAD激酶活性较出发菌株提高116%，胞内NADP+及NADPH的浓度分别提高7.3%和36.84%。在LOS的条件下，NAD激酶活性提高49%，胞内NADP+及NADPH的浓度分别提高14.67%和15%。重组菌株在HOS及LOS的条件下，L-精氨酸的产量分别为26.47g/L和11.36g/L较出发菌株（24.29g/L，7.58g/L）均有提高。综合以上结果显示：在HOS及LOS条件下，通过增强表达NAD激酶来增加NADP+的浓度，提高NADPH的含量，达到提升L-精氨酸的产量目的是行之有效的途径。

论文目录

• Abstract
• 摘要
• Index
• 1. Introduction
• 1.1 L-arginine Biosynthesis and Metabolism
• 1.2 Significance of NADPH during L-arginine Biosynthesis
• 1.3 NAD kinase and NADPH formation
• 1.4 Other Roles of NADP（H）
• 1.5 Co-factor engineering
• 1.6 Significance of Oxygen Supply during fermentation
• 1.7 Research Objective
• 2. Materials and methods
• 2.1 Medium and Culture
• 2.1.1 The media
• 2.1.2 The culture method
• 2.2 Reagents and preparation of chemicals
• 2.2.1 Common Biochemical reagents
• 2.2.2 Enzymes, kits and other consumables
• 2.3 Antibiotic preparation
• 2.4 Lsozyme preparation
• 2.5 SDS-PAGE
• 2.5.1 SDS-PAGE electrophoresis reagents preparation
• 2.5.2 SDS-PAGE electrophoresis gel preparation
• 2.6 Instruments
• 2.7 The experiment methods
• 2.7.1 The fermentation parameters analysis
• 2.7.2 Bradford method for the determination of total protein
• 2.7.3 Assay of NAD+kinase activity
• 2.7.4 Assay of NADH Kinase activity
• 2.7.5 Assay of Glucose-6-Phosphate Dehydrogenase
• 2.7.6 Determination of intracellular NAD, NADH, NADP, NADPH concentrations
• 2.8 Strains, plasmids and oligonucleotide primers
• 2.9 Construction of PCR products in T-vector
• 2.10 Preparation of competent E. coli JM109 and Transformation
• 2.11 Recombinant plasmid extraction from E. coli
• 2.12 Recombinant plasmid sequencing
• 2.13 Construction of sticky gene fragment and vector pJC1-tac
• 2.14 Corynebacterium competent cell preparation for electric shock transformation
• 3. Results and Discussion
• 3.1 Construction of recombinant plasmid pMD19-T –ppnK
• 3.2 Construction of NAD kinase expression strain
• 3.3 Corynebaterium crenatum SYPA5-5 recombinant strains screening
• 3.4 Effects of homologous NAD kinase overexpression on Cell growth and glucose consumption
• 3.5 Enzymatic activity of NAD+kinase and NADH kinase
• 3.6 Molecular weight determination
• 3.7 Enzymatic Activity of Glucose-6-Phosphate dehydrogenase
• 3.8 Intracellular concentrations of NAD （H） and NADP （H）
• 3.9 Improved L-arginine biosynthesis through NAD kinase overexpression
• 4. Conclusion and Recommendation
• 4.1 Conclusion
• 4.2 Recommendation
• Acknowledgements
• Reference
• Appendix
• Appendix1: Papers published from this work
• Appendix 2: The nucleotide sequence of the NAD kinase fromCorynebacterium crenatum SYPA5-5