毕赤酵母表达重组蛋白生产工艺关键技术及其机制研究
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摘要
巴斯德毕赤酵母（P. pastoris）表达系统是目前应用最广的真核表达系统之一，己有近1000种异源蛋白利用该系统获得了成功表达，市场前景广阔。但是，虽然已有三十余年的应用历史，对毕赤酵母发酵工艺的优化研究始终未取得突破性进展。发酵是一种间歇性稳态的生物学过程，涉及基因组、蛋白质组和代谢组等水平的显著改变。因此，从组学水平上对发酵过程的内在变化进行展示，找到引导稳态间迁移的核心基因、蛋白或小分子底物，可能有助于深度优化发酵工艺。然而，目前关于毕赤酵母重组表达菌株发酵工艺过程的转录组学研究鲜有开展。
本论文采用自主设计的基因芯片，试图通过筛查与毕赤酵母发酵工艺重要时间节点密切相关的关键基因，深入研究影响发酵工艺和目的蛋白表达水平的内在因素，为该经典发酵工艺的进一步优化提供数据基础和全新视角。
为实现上述研究目标，本论文主要按照如下实验方案开展了研究工作：
1.毕赤酵母发酵工艺模型的构建：
选择研究背景清晰、发酵工艺成熟的GS115毕赤酵母菌株作为表达载体，成功构建出能够稳定表达reFIP-vvo和rHSA的重组表达菌株。通过中试工艺的对比研究，发现rHSA重组菌株的发酵过程更具标志性，生物规律节点非常明确，故选作理想模型进行后续转录组学研究。
2.毕赤酵母基因芯片的制备：
使用GeneMark方法预测GS115毕赤酵母基因组中外显子，经多步筛查、验证和优化等步骤自主设计出包含5040个基因的GS115毕赤酵母基因芯片，并完成了芯片的标准化和制备工作。
3.毕赤酵母发酵工艺的基因芯片分析：
采用自主设计的基因芯片，对毕赤酵母发酵工艺中的重要环节进行了转录组学水平的研究，重点了关注碳源置换和甲醇诱导两个生物学过程。另外，鉴于氧分子在该发酵工艺中的主导作用，本研究还采用了构建氧化还原功能树的全新分析手段，特别关注了氧化还原功能相关的基因在好氧代谢流漂移当中的作用。
通过上述实验内容的开展，本论文主要获得了如下研究结果：
1.本研究设计并制备了毕赤酵母GS115菌株表达谱芯片，填补同领域国内外空白，为毕赤酵母在其他领域的转录组学研究提供了重要的研究工具。
2.研究发现在限制性碳源置换阶段，碳源饥饿这个传统的工艺环节可能并不必要，甚至可能有害。分析依据为：a.碳源饥饿造成转录调控功能受到较大抑制，菌体产生严重应激反应；b.甘油补加阶段即检测到目的蛋白表达，表明AOX1可能受到甘油的诱导。因此，使用甘油/甲醇混合补料可能更为科学，即逐渐增加甲醇的比例以实现完全替换，而且这种连续、渐变的工艺过程也更加符合发酵工程的原理。
3.筛选到两个可能主导好氧-厌氧代谢漂移的核心基因，即PAS_chr2-1_0582和PAS_chr3_0845。其中，PAS_chr2-1_0582可能是毕赤酵母中调节和控制有氧代谢的最重要转录因子之一。而PAS_chr3_0845可能通过呼吸电子传递链的子功能辅助了厌氧与好氧代谢之间的漂移。对两者的深入研究可能有助于深度优化毕赤酵母发酵工艺，提高目的蛋白的表达水平。
本论文利用了自主设计的毕赤酵母基因表达谱芯片对甲醇诱导毕赤酵母重组表达外源蛋白的发酵工艺进行了研究，在转录组学水平上充分地展示了在限制性碳源置换和甲醇诱导表达重组蛋白两个生物学过程以及在好氧代谢漂移中发挥重要作用的功能及核心基因，并对经典的发酵工艺环节与毕赤酵母细胞内生命活动变化之间的关系进行了系统地分析，为该经典发酵工艺的进一步优化提出了方向。
Pichia pastoris expression system is currently the most widely used eukaryoticexpression systems, nearly1,000kinds of heterologous proteins have been expressedusing this system, which has a broad market prospect. However, although with anapplication history of more than30years, no breaktrough has been achived in theoptimization study of Pichia fermentation process. Fermentation is an intermittentsteady biological process involving significant changes in the genome, proteome andmetabolome levels. Therefore, the display of internal changes during fermentationprocess at genomics levels to find the core genes, proteins or small moleculesubstrates leading the steady-state migration, may help to optimize the fermentationprocess. However, few transcriptome studies have been conducted on the Pichiafermentation process.
In this study, we tried to use the self-designed gene chips to screen the key genesclosely related to the important time node during the Pichia fermentation process,digging the internal factors affecting the fermentation process and the proteinexpression levels, in order to provide the optimization of the fermentation processwith classic data base and a new perspective.
To achieve the research goals, the study was carried out according to following work:
1. The construction of Pichia fermentation process mode:GS115yeast strain was selected as the expressing vector because of the clearbackground and well-established fermentation technologies. Two recombinant strains were successfully constructed and were available for stably expression ofreFIP-vvo or rHSA. In comparison study during the pilot scale fermentation test，therHSA strain exhibited a stable growth curve with an more obvious trend which wasidentical to the typical characteristics of methanol inducible expression system.Therefore, the final selection of rHSA GS115was chosen for the transcriptomestudy using gene chips.
2. Preparation of Pichia microarray:GeneMark method was used for predicting GS115Pichia genome exon, aftermulti-step screening, validation and optimization steps, we independently designed aGS115Pichia microarray contains5040genes, and completed the standardization andpreparation work.
3. Microarray analysis of Pichia fermentation process:As it was explained above, we designed a microarray independently. The microarraywas used to investigate the transcriptomics related to the key points in the classicfermentation of Pichia pastoris in this study. The study focused on two biologicalprocesses induced by carbon source replacement and methanol-induce expression.Furthermore, concerning the importance of oxygen molecules in fermentation, therole of the oxidation-reduction-related genes were specified in the aerobic metabolicflux drift by using a new tool of building the redox function trees.
This study obtained three main results as follow:
1. We designed and prepared the Pichia GS115microarray, which filled with blank inthe fields at home and abroad, thus provides an important research tool on thetranscriptome study of Pichia in various areas.
2. It was found that the transcriptional regulation changed most significantly in the phase of limiting carbon replacement. In this phase, the biological function oftranscription, translation, protein/nucleic acid synthesis were strongly inhibited.Moreover, the rHSA protein can be barely detected in the glycerol-feeding phase,indicating that AOX1may be induced by glycerol consistent with the recent findings.These results suggest that the traditional carbon starvation may be unnecessary andharmful to the cells. As suggested by a number of scholars recently, the glycerol/methanol mixed feeding process is more scientific. The gradual increasing proportionof additional methanol is used for the final completely replacement. This process canavoid the extreme stress during the fermentation process, and is consistent with theprinciples of the gradient fermentation design.
3. Two core genes, namely PAS_chr2-1_0582and PAS_chr3_0845, which possiblyled to aerobic-anaerobic metabolic drift was screened out. PAS_chr2-1_0582may beone of most important transcription factors in Pichia aerobic metabolism regulationand control. PAS_chr3_0845possibly assists the drift between anaerobic and aerobicmetabolism through sub-function of auxiliary respiratory electron transport chain.In-depth study of the two may help to optimize the Pichia fermentation process,enhancing the expression of target proteins.
In this study, the use of the microarray enabled us to investigate and demonstrate thekey genes and functions in the aerobic metabolism drift and the two biologicalprocesses, limiting carbon source replacement and methanol-induce expression,using the transcriptomic approach. The key points in the classic fermentation processof Pichia pastoris were also systematically analyzed. This study shed lights onfurther optimization of the classic fermentation process.
本论文采用自主设计的基因芯片，试图通过筛查与毕赤酵母发酵工艺重要时间节点密切相关的关键基因，深入研究影响发酵工艺和目的蛋白表达水平的内在因素，为该经典发酵工艺的进一步优化提供数据基础和全新视角。
为实现上述研究目标，本论文主要按照如下实验方案开展了研究工作：
1.毕赤酵母发酵工艺模型的构建：
选择研究背景清晰、发酵工艺成熟的GS115毕赤酵母菌株作为表达载体，成功构建出能够稳定表达reFIP-vvo和rHSA的重组表达菌株。通过中试工艺的对比研究，发现rHSA重组菌株的发酵过程更具标志性，生物规律节点非常明确，故选作理想模型进行后续转录组学研究。
2.毕赤酵母基因芯片的制备：
使用GeneMark方法预测GS115毕赤酵母基因组中外显子，经多步筛查、验证和优化等步骤自主设计出包含5040个基因的GS115毕赤酵母基因芯片，并完成了芯片的标准化和制备工作。
3.毕赤酵母发酵工艺的基因芯片分析：
采用自主设计的基因芯片，对毕赤酵母发酵工艺中的重要环节进行了转录组学水平的研究，重点了关注碳源置换和甲醇诱导两个生物学过程。另外，鉴于氧分子在该发酵工艺中的主导作用，本研究还采用了构建氧化还原功能树的全新分析手段，特别关注了氧化还原功能相关的基因在好氧代谢流漂移当中的作用。
通过上述实验内容的开展，本论文主要获得了如下研究结果：
1.本研究设计并制备了毕赤酵母GS115菌株表达谱芯片，填补同领域国内外空白，为毕赤酵母在其他领域的转录组学研究提供了重要的研究工具。
2.研究发现在限制性碳源置换阶段，碳源饥饿这个传统的工艺环节可能并不必要，甚至可能有害。分析依据为：a.碳源饥饿造成转录调控功能受到较大抑制，菌体产生严重应激反应；b.甘油补加阶段即检测到目的蛋白表达，表明AOX1可能受到甘油的诱导。因此，使用甘油/甲醇混合补料可能更为科学，即逐渐增加甲醇的比例以实现完全替换，而且这种连续、渐变的工艺过程也更加符合发酵工程的原理。
3.筛选到两个可能主导好氧-厌氧代谢漂移的核心基因，即PAS_chr2-1_0582和PAS_chr3_0845。其中，PAS_chr2-1_0582可能是毕赤酵母中调节和控制有氧代谢的最重要转录因子之一。而PAS_chr3_0845可能通过呼吸电子传递链的子功能辅助了厌氧与好氧代谢之间的漂移。对两者的深入研究可能有助于深度优化毕赤酵母发酵工艺，提高目的蛋白的表达水平。
本论文利用了自主设计的毕赤酵母基因表达谱芯片对甲醇诱导毕赤酵母重组表达外源蛋白的发酵工艺进行了研究，在转录组学水平上充分地展示了在限制性碳源置换和甲醇诱导表达重组蛋白两个生物学过程以及在好氧代谢漂移中发挥重要作用的功能及核心基因，并对经典的发酵工艺环节与毕赤酵母细胞内生命活动变化之间的关系进行了系统地分析，为该经典发酵工艺的进一步优化提出了方向。
Pichia pastoris expression system is currently the most widely used eukaryoticexpression systems, nearly1,000kinds of heterologous proteins have been expressedusing this system, which has a broad market prospect. However, although with anapplication history of more than30years, no breaktrough has been achived in theoptimization study of Pichia fermentation process. Fermentation is an intermittentsteady biological process involving significant changes in the genome, proteome andmetabolome levels. Therefore, the display of internal changes during fermentationprocess at genomics levels to find the core genes, proteins or small moleculesubstrates leading the steady-state migration, may help to optimize the fermentationprocess. However, few transcriptome studies have been conducted on the Pichiafermentation process.
In this study, we tried to use the self-designed gene chips to screen the key genesclosely related to the important time node during the Pichia fermentation process,digging the internal factors affecting the fermentation process and the proteinexpression levels, in order to provide the optimization of the fermentation processwith classic data base and a new perspective.
To achieve the research goals, the study was carried out according to following work:
1. The construction of Pichia fermentation process mode:GS115yeast strain was selected as the expressing vector because of the clearbackground and well-established fermentation technologies. Two recombinant strains were successfully constructed and were available for stably expression ofreFIP-vvo or rHSA. In comparison study during the pilot scale fermentation test，therHSA strain exhibited a stable growth curve with an more obvious trend which wasidentical to the typical characteristics of methanol inducible expression system.Therefore, the final selection of rHSA GS115was chosen for the transcriptomestudy using gene chips.
2. Preparation of Pichia microarray:GeneMark method was used for predicting GS115Pichia genome exon, aftermulti-step screening, validation and optimization steps, we independently designed aGS115Pichia microarray contains5040genes, and completed the standardization andpreparation work.
3. Microarray analysis of Pichia fermentation process:As it was explained above, we designed a microarray independently. The microarraywas used to investigate the transcriptomics related to the key points in the classicfermentation of Pichia pastoris in this study. The study focused on two biologicalprocesses induced by carbon source replacement and methanol-induce expression.Furthermore, concerning the importance of oxygen molecules in fermentation, therole of the oxidation-reduction-related genes were specified in the aerobic metabolicflux drift by using a new tool of building the redox function trees.
This study obtained three main results as follow:
1. We designed and prepared the Pichia GS115microarray, which filled with blank inthe fields at home and abroad, thus provides an important research tool on thetranscriptome study of Pichia in various areas.
2. It was found that the transcriptional regulation changed most significantly in the phase of limiting carbon replacement. In this phase, the biological function oftranscription, translation, protein/nucleic acid synthesis were strongly inhibited.Moreover, the rHSA protein can be barely detected in the glycerol-feeding phase,indicating that AOX1may be induced by glycerol consistent with the recent findings.These results suggest that the traditional carbon starvation may be unnecessary andharmful to the cells. As suggested by a number of scholars recently, the glycerol/methanol mixed feeding process is more scientific. The gradual increasing proportionof additional methanol is used for the final completely replacement. This process canavoid the extreme stress during the fermentation process, and is consistent with theprinciples of the gradient fermentation design.
3. Two core genes, namely PAS_chr2-1_0582and PAS_chr3_0845, which possiblyled to aerobic-anaerobic metabolic drift was screened out. PAS_chr2-1_0582may beone of most important transcription factors in Pichia aerobic metabolism regulationand control. PAS_chr3_0845possibly assists the drift between anaerobic and aerobicmetabolism through sub-function of auxiliary respiratory electron transport chain.In-depth study of the two may help to optimize the Pichia fermentation process,enhancing the expression of target proteins.
In this study, the use of the microarray enabled us to investigate and demonstrate thekey genes and functions in the aerobic metabolism drift and the two biologicalprocesses, limiting carbon source replacement and methanol-induce expression,using the transcriptomic approach. The key points in the classic fermentation processof Pichia pastoris were also systematically analyzed. This study shed lights onfurther optimization of the classic fermentation process.
引文
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