毕赤酵母全基因组代谢网络模型构建及其应用

发布时间：2018-06-20 19:23

本文选题：毕赤酵母 + 基因组代谢网络模型　；参考：《华东理工大学》2017年硕士论文

【摘要】：毕赤酵母是一种应用广泛的外源蛋白表达平台,高质量的全基因组代谢网络模型对于全面理解毕赤酵母的代谢功能,优化蛋白生产工艺和构建性能优良的新菌种都具有十分重要作用。针对现有毕赤酵母全基因组代谢网络模型存在的覆盖度有限、描述精度不够高的问题,本文结合最新的毕赤酵母基因注释信息和文献数据,升级了毕赤酵母全基因组代谢网络模型,并对升级后的模型进行了全面的性能验证和初步的应用研究。根据KEGG、IMG和UniProtKB等三个数据库中有关毕赤酵母基因注释信息,重构了毕赤酵母全基因组代谢网络模型。新的iRY1243模型与最近公开发表的iMT1026模型相比,iRY1243模型包含的基因数由1026增大到1243,代谢反应数量由2035增大到2407,代谢物数量由1018增大到1094。iRY1243模型中,新添加了维生素和辅因子的代谢途径,使新模型能描述的代谢功能更加完整。敏感性分析结果表明,部分细胞组分及维持能对模型的验证及菌株改造有很大的影响,本文基于最新的文献及实验更新了细胞组分和维持能的大小。分别利用转录组学、细胞生理代谢参数、可利用碳氮源和13C代谢通量等多种数据对iRY1243模型进行了验证。转录组学数据分析结果表明,在iRY1243模型所包含的1243个基因中,有79.37%的单基因及91.93%的多基因注释的反应有表达,表明模型的基因注释信息比较精确。以最大化细胞生长为目标函数,通过FBA分析发现,iRY1243模型能较好地预测细胞比生长速率(平均预测误差为9%)、氧比消耗速率(平均预测误差为11.1%)和二氧化碳比生成速率(平均预测误差为8.2%)。在文献中有报道的能够支持毕赤酵母生长的30种碳源和21种氮源中,iRY1243模型都能进行预测。将通过FBA模拟出的中心代谢的代谢通量与通过13C标记实验所计算的代谢通量进行比较,发现两者具有较好的一致性(R2=0.88)。这些结果表明,iRY1243模型能较好地描述毕赤酵母的代谢功能。利用iRY1243分别预测了毕赤酵母生长的必须基因以及能提高β-半乳糖苷酶表达量和S-腺苷甲硫氨酸生成量的潜在基因靶点。在以葡萄糖为碳源的合成培养基上,发现有123个基因是必须基因,这些基因与能量代谢、TCA循环、氨基酸代谢等相关。模拟结果表明,过表达PPP途径中的一些基因或敲除乙酸、乙醇和甘油等副产物生成途径的基因可以提高β-半乳糖苷酶表达量。插入vgb基因,敲除spe2、aaox1和cys4基因,过表达zwf1和sam2基因,都有利于S-腺苷甲硫氨酸的产物合成。这些预测结果和实验数据有较好一致性,表明iRY1243模型有较好的应用前景。综上所述,升级后的iRY1243模型能较好地描述毕赤酵母的代谢功能,对毕赤酵母表达外源蛋白的生产工艺优化和性能优良新菌种的构建都具有重要指导意义。
[Abstract]:Pichia pastoris (Pichia pastoris) is a widely used platform for the expression of exogenous proteins. High quality genome-wide metabolic network models can fully understand the metabolic function of Pichia pastoris. It is very important to optimize the production process of protein and to construct new strains with good performance. In view of the limited coverage and low precision of the existing Pichia pastoris genome metabolic network models, this paper combined with the latest gene annotation information and literature data of Pichia pastoris. The whole genome metabolic network model of Pichia pastoris was upgraded. Based on the gene annotation information of Pichia pastoris in three databases of KEGGG IMG and UniProtKB, the whole genome metabolic network model of Pichia pastoris was reconstructed. Compared with the recently published iRY1243 model, the new iRY1243 model has increased the number of genes from 1026 to 1243, the number of metabolic reactions from 2035 to 2407, and the number of metabolites from 1018 to 1094.iRY1243. The metabolic function described by the new model is more complete. The results of sensitivity analysis showed that some cell components and maintenance ability had great influence on the validation of the model and the modification of the strain. In this paper, the size of cell components and maintenance energy was updated based on the latest literature and experiments. The iRY1243 model was validated by transcriptome, cell physiological metabolic parameters, carbon and nitrogen sources and 13C metabolic flux. Transcriptome data analysis showed that 79.37% of the 1243 genes contained in the iRY1243 model and 91.93% of the responses to polygenic annotation were expressed, indicating that the gene annotation information of the model was accurate. To maximize cell growth as the objective function, Through FBA analysis, it was found that the specific cell growth rate (average prediction error was 9), oxygen consumption rate (average prediction error was 11.1) and carbon dioxide specific formation rate (average prediction error was 8.2%). In the literature, 30 carbon sources and 21 nitrogen sources which can support the growth of Pichia pastoris can be predicted by iRY1243 model. The metabolic flux of central metabolism simulated by FBA was compared with that calculated by 13C labeling experiment, and it was found that there was a good agreement between them. These results suggest that the IRY1243 model can better describe the metabolic function of Pichia pastoris. The essential genes for Pichia pastoris growth and potential gene targets for increasing the expression of 尾 -galactosidase and the production of S- adenosine methionine were predicted by iRY1243, respectively. In the synthetic medium with glucose as carbon source, 123 genes were found to be essential genes, which were related to TCA cycle and amino acid metabolism of energy metabolism. The simulation results showed that overexpression of some genes in the PPP pathway or knockout of by-product production pathways such as acetic acid, ethanol and glycerol could increase the expression of 尾 -galactosidase. Insertion of vgb gene, knockout of spe2maox1 and cys4 genes and overexpression of zwf1 and sam2 genes are all beneficial to the production of S-adenosine methionine. These predicted results are in good agreement with the experimental data, which indicates that the iRY1243 model has a good application prospect. In conclusion, the upgraded iRY1243 model can better describe the metabolic function of Pichia pastoris, and has important guiding significance for the optimization of the production process and the construction of a new strain with excellent performance for the expression of exogenous protein by Pichia pastoris.
【学位授予单位】：华东理工大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78

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