| 霉酚酸微生物合成和P450单加氧酶与还原伴侣蛋白的相互作用机制研究 |
| 齐凤霞
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| 2016-05
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| 关键词 | 微生物合成
蛋白纯化
霉酚酸
P450
还原伴侣
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| 学科分类 | 生物学
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| 报告类别 | 专题报告
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| 中文摘要 | The report is focused on the microbial biosynthesis of mycophenolic acid (MPA) and National Excellent Youth Project "The interaction mechanism of the microbial biosynthesized P450 oxygenase and reducing chaperones”. This report provides a platform for further research of the subjects.
There are mainly three aspects:
1. Expression of genes involved in MPA biosynthesis from Penicillium brevicompactum using Saccharomyces cerevisiae as a host
A) A homologous recombination based DNA assembler technology was used for construction of large DNA fragments in Saccharomyces cerevisiae. This method is proved feasible, efficient and reproducible for the rational design of metabolic engineering pathways.
B) The mpaA, mpaDE and mpaC gene in for MPA synthesis in Penicillium brevicompactum strain NRRL864 were molecularly clonned. Through fermentation experiments, the mpaC mutant strain showed no production of the 5-MOA through HPLC/LC-MS detection. To detect protein expression of the three proteins, SDS-PAGE and Western blot were carried out.
C) mpaA was codon optimized and the protein was purified and verified.
2. The projiect "The interaction mechanism of the microbial biosynthesized P450 oxygenase and reducing chaperones”.
A) Molecular cloning and protein purification of the 6 reduced chaperone Fdx from the cyanobacterium strain Synechococcus elongatus PCC7942.
B) Cloning and expression of the 18 P450 genes and 6 ferredoxins (Fdx), and 4 ferredoxin reductases (FDRs) from Streptomyces coelicolor A3(2). Fdx6 was codon optimized. The P450s were verified by CO-assay. The four FDR were analyzed using a spectral analysis.
C) Progesterone, testosterone, tanshinone IIA, diosgenin, mycinamicin IV (M-IV), and YC17 were used as substrates for the reaction by P450 and reducing partners (based on HPLC screening). It was discovered for the first time that the reductive chaperone protein of Streptomyces sp. are able to support PikC to catalyze YC17. And it is proved that the different combinations of the reducing partners have various catalytic efficiencies and can achieve different products. This helps to the understanding of the regularity of the interaction and recognition mechanism of the "Fdx - FdR - P450 system.
3. The expression vector in Aspergillus oryzae, pTAex3, was sequenced and the map was achieved. |
| 英文摘要 | 本报告围绕微生物生物合成霉酚酸（MPA）和 “微生物生物合成P450单加氧酶与还原伴侣蛋白的相互作用机制”研究课题展开工作，本报告为为下一步的课题进展提供了平台。
内容包括以下三个方面：
1. 在酿酒酵母宿主中表达来自短密青霉的霉酚酸合成相关基因
a) 利用酿酒酵母同源重组系统组装大片段DNA的技术路线，证明了该技术在理性设计组装外源DNA和代谢工程改造酿酒酵母的可行性、高效性和可重复性；
b) 分子克隆了来自短密青霉的霉酚酸（MPA）合成相关基因mpaA，mpaDE和mpaC的。针对mpaC突变株开展了发酵实验，通过HPLC未能检测到代谢产物5-MOA；三个蛋白均利用SDS-PAGE和Western Blot检测了蛋白表达情况。
c) 对MPA合成相关基因mpaA进行了密码子优化并蛋白纯化鉴定。
2. 国家优青项目“微生物生物合成P450单加氧酶与还原伴侣蛋白的相互作用机制”研究课题
a) 蓝细菌来源的6个还原伴侣Fdx的分子克隆和蛋白纯化。
b）克隆表达来自天然色链霉菌的18个P450酶基因和6个铁氧化还原蛋白Fdx、4个铁氧化还原蛋白还原酶FDR，对Fdx6密码子优化，并进行了蛋白纯化鉴定. P450和FDR进行了光谱分析。
c）分别采用黄体酮、睾酮、薯蓣皂苷元、丹参酮IIA、麦新米星IV(M-IV)和YC17等多种底物对这些P450和还原伴侣进行了酶催化反应筛选鉴（HPLC检测）。首次发现了天蓝色链霉菌的还原伴侣蛋白用于催化支撑PikC催化YC17合成产物。并且证明在支撑PikC的效力层面，不同组合的还原伴侣可以达到不同的催化效率，合成不同的目的产物。为进一步获得对“P450酶—Fdx—FdR”三蛋白系统相互作用和识别机制的规律性认识做出积累。
3. 其他：对序列未知的米曲霉表达载体pTAex3进行了全测序并获得了图谱等。 |
| 文献类型 | 研究报告
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| 条目标识符 | http://ir.qibebt.ac.cn/handle/337004/9804
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| 专题 | 酶工程研究组
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推荐引用方式
GB/T 7714 |
齐凤霞. 霉酚酸微生物合成和P450单加氧酶与还原伴侣蛋白的相互作用机制研究. 2016.
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霉酚酸微生物合成和P450单加氧酶与还原伴侣蛋白的相互作用机制研究.pdf
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