An upstream platform for the production of high grade heterologous proteins in the yeast Pichia pastoris
An upstream platform for the production of high grade heterologous proteins in the yeast Pichia pastoris
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发表时间：
2016-02
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通讯作者：
S. Woodhouse
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作者：
S. Woodhouse
巴斯德毕赤酵母是一种甲基营养型酵母，并且是用于生产治疗性蛋白质和工业酶的良好建立的表达系统。其特征在于其严格调控的启动子、分泌重组蛋白的天然能力以及生长至非常高的细胞密度以对抗低产物滴度的潜力。然而，这些高细胞密度与需要甲醇作为诱导剂相结合，导致细胞活力受到负面影响的问题，导致宿主细胞蛋白质的释放，所述宿主细胞蛋白质包括降低产物质量和使纯化复杂化的蛋白酶。本论文介绍了一种简单的发酵策略，通过利用非阻遏底物山梨醇作为诱导过程中与甲醇共进料，减少发酵过程中所需的甲醇量。这使得细胞生长曲线保持与当前方案所见的相似，最终细胞密度达到120-140 g/L干细胞重量（DCW）。细胞活力和产物产量不受新策略的影响，但由于宿主细胞蛋白质杂质谱的变化，蛋白酶释放减少。发酵的可扩展性也大大增加，因为在诱导过程中产热减少了60%。最后，使用自适应聚焦声学成功开发了用于确定细胞稳健性的方法：这用于证明甲醇诱导不会对细胞稳健性产生负面影响，并且降低的生长速率是增强细胞稳健性的关键要求。这些结果均已用两种巴斯德毕赤酵母菌株证明，一种是表达分泌型胚胎碱性磷酸酶（SEAP）的内部GS 115菌株，另一种是表达抑肽酶的工业用菌株CLD 804。
Pichia pastoris is a methylotrophic yeast and well established expression system for the production of therapeutic proteins and industrial enzymes. It is characterised by its tightly regulated promoters, natural ability to secrete recombinant proteins and potential to grow to very high cell densities in order to combat low product titres. These high cell densities, in combination with the requirement of methanol as an inducing agent, lead to problems, however, with cell viability being negatively influenced, resulting in the release of host cell proteins, which include proteases that both reduce product quality and complicate purification. This thesis introduces a simple fermentation strategy that reduces the amount of methanol required during fermentation by utilising the non-repressing substrate sorbitol as a co-feed with methanol during induction. This allowed cell growth profiles to remain similar to those seen with current protocols, with final cell densities of 120-140 g/L dry cell weight (DCW) being attained. Cell viability and product yields were unaffected by the new strategy, but protease release was reduced due to a shift in host cell protein impurity profiles. The scalability of fermentations was also greatly increased due to a 60% reduction in heat generation during induction. Finally, a methodology for the determination of cellular robustness was successfully developed using adaptive focussed acoustics: this was used to demonstrate that methanol induction did not negatively impact cellular robustness, and that reduced growth rates were a key requirement of enhanced cellular robustness. These results have all been demonstrated with two strains of P. pastoris, an in-house GS115 strain expressing secreted embryonic alkaline phosphatase (SEAP) and an industrially utilised strain, CLD804 expressing aprotinin.