本文選題：畢赤酵母 + 基因組代謝網(wǎng)絡(luò)模型��； 參考：《華東理工大學(xué)》2017年碩士論文

【摘要】：畢赤酵母是一種應(yīng)用廣泛的外源蛋白表達(dá)平臺(tái),高質(zhì)量的全基因組代謝網(wǎng)絡(luò)模型對(duì)于全面理解畢赤酵母的代謝功能,優(yōu)化蛋白生產(chǎn)工藝和構(gòu)建性能優(yōu)良的新菌種都具有十分重要作用。針對(duì)現(xiàn)有畢赤酵母全基因組代謝網(wǎng)絡(luò)模型存在的覆蓋度有限、描述精度不夠高的問題,本文結(jié)合最新的畢赤酵母基因注釋信息和文獻(xiàn)數(shù)據(jù),升級(jí)了畢赤酵母全基因組代謝網(wǎng)絡(luò)模型,并對(duì)升級(jí)后的模型進(jìn)行了全面的性能驗(yàn)證和初步的應(yīng)用研究。根據(jù)KEGG、IMG和UniProtKB等三個(gè)數(shù)據(jù)庫(kù)中有關(guān)畢赤酵母基因注釋信息,重構(gòu)了畢赤酵母全基因組代謝網(wǎng)絡(luò)模型。新的iRY1243模型與最近公開發(fā)表的iMT1026模型相比,iRY1243模型包含的基因數(shù)由1026增大到1243,代謝反應(yīng)數(shù)量由2035增大到2407,代謝物數(shù)量由1018增大到1094。iRY1243模型中,新添加了維生素和輔因子的代謝途徑,使新模型能描述的代謝功能更加完整。敏感性分析結(jié)果表明,部分細(xì)胞組分及維持能對(duì)模型的驗(yàn)證及菌株改造有很大的影響,本文基于最新的文獻(xiàn)及實(shí)驗(yàn)更新了細(xì)胞組分和維持能的大小。分別利用轉(zhuǎn)錄組學(xué)、細(xì)胞生理代謝參數(shù)、可利用碳氮源和13C代謝通量等多種數(shù)據(jù)對(duì)iRY1243模型進(jìn)行了驗(yàn)證。轉(zhuǎn)錄組學(xué)數(shù)據(jù)分析結(jié)果表明,在iRY1243模型所包含的1243個(gè)基因中,有79.37%的單基因及91.93%的多基因注釋的反應(yīng)有表達(dá),表明模型的基因注釋信息比較精確。以最大化細(xì)胞生長(zhǎng)為目標(biāo)函數(shù),通過FBA分析發(fā)現(xiàn),iRY1243模型能較好地預(yù)測(cè)細(xì)胞比生長(zhǎng)速率(平均預(yù)測(cè)誤差為9%)、氧比消耗速率(平均預(yù)測(cè)誤差為11.1%)和二氧化碳比生成速率(平均預(yù)測(cè)誤差為8.2%)。在文獻(xiàn)中有報(bào)道的能夠支持畢赤酵母生長(zhǎng)的30種碳源和21種氮源中,iRY1243模型都能進(jìn)行預(yù)測(cè)。將通過FBA模擬出的中心代謝的代謝通量與通過13C標(biāo)記實(shí)驗(yàn)所計(jì)算的代謝通量進(jìn)行比較,發(fā)現(xiàn)兩者具有較好的一致性(R2=0.88)。這些結(jié)果表明,iRY1243模型能較好地描述畢赤酵母的代謝功能。利用iRY1243分別預(yù)測(cè)了畢赤酵母生長(zhǎng)的必須基因以及能提高β-半乳糖苷酶表達(dá)量和S-腺苷甲硫氨酸生成量的潛在基因靶點(diǎn)。在以葡萄糖為碳源的合成培養(yǎng)基上,發(fā)現(xiàn)有123個(gè)基因是必須基因,這些基因與能量代謝、TCA循環(huán)、氨基酸代謝等相關(guān)。模擬結(jié)果表明,過表達(dá)PPP途徑中的一些基因或敲除乙酸、乙醇和甘油等副產(chǎn)物生成途徑的基因可以提高β-半乳糖苷酶表達(dá)量。插入vgb基因,敲除spe2、aaox1和cys4基因,過表達(dá)zwf1和sam2基因,都有利于S-腺苷甲硫氨酸的產(chǎn)物合成。這些預(yù)測(cè)結(jié)果和實(shí)驗(yàn)數(shù)據(jù)有較好一致性,表明iRY1243模型有較好的應(yīng)用前景。綜上所述,升級(jí)后的iRY1243模型能較好地描述畢赤酵母的代謝功能,對(duì)畢赤酵母表達(dá)外源蛋白的生產(chǎn)工藝優(yōu)化和性能優(yōu)良新菌種的構(gòu)建都具有重要指導(dǎo)意義。
[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.
【學(xué)位授予單位】：華東理工大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：Q78

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