Tyo Keith E J, Liu Zihe, Magnusson Ylva, Petranovic Dina, Nielsen Jens
Novo Nordisk Foundation Center for Biosustainability, Department of Chemical and Biological Engineering, Chalmers University of Technology, 41296, Gothenburg, Sweden.
Appl Microbiol Biotechnol. 2014 Aug;98(16):7149-59. doi: 10.1007/s00253-014-5783-7. Epub 2014 May 10.
Protein titers, a key bioprocessing metric, depend both on the synthesis of protein and the degradation of protein. Secreted recombinant protein production in Saccharomyces cerevisiae is an attractive platform as minimal media can be used for cultivation, thus reducing fermentation costs and simplifying downstream purification, compared to other systems that require complex media. As such, engineering S. cerevisiae to improve titers has been then the subject of significant attention, but the majority of previous efforts have been focused on improving protein synthesis. Here, we characterize the protein uptake and degradation pathways of S. cerevisiae to better understand its impact on protein secretion titers. We do find that S. cerevisiae can consume significant (in the range of 1 g/L/day) quantities of whole proteins. Characterizing the physiological state and combining metabolomics and transcriptomics, we identify metabolic and regulatory markers that are consistent with uptake of whole proteins by endocytosis, followed by intracellular degradation and catabolism of substituent amino acids. Uptake and degradation of recombinant protein products may be common in S. cerevisiae protein secretion systems, and the current data should help formulate strategies to mitigate product loss.
蛋白质滴度是生物加工的一个关键指标，它既取决于蛋白质的合成，也取决于蛋白质的降解。与其他需要复杂培养基的系统相比，酿酒酵母中分泌的重组蛋白生产是一个有吸引力的平台，因为可以使用基本培养基进行培养，从而降低发酵成本并简化下游纯化过程。因此，对酿酒酵母进行工程改造以提高滴度一直是备受关注的课题，但此前的大多数努力都集中在改善蛋白质合成方面。在此，我们对酿酒酵母的蛋白质摄取和降解途径进行了表征，以更好地了解其对蛋白质分泌滴度的影响。我们确实发现酿酒酵母能够消耗大量（每天1克/升左右）的完整蛋白质。通过表征生理状态并结合代谢组学和转录组学，我们确定了与通过内吞作用摄取完整蛋白质、随后进行细胞内降解和取代氨基酸分解代谢相一致的代谢和调控标志物。重组蛋白产物的摄取和降解在酿酒酵母蛋白质分泌系统中可能很常见，目前的数据应有助于制定减轻产物损失的策略。
