Identifying and overcoming protein secretion bottlenecks in yeast and filamentous fungal cell factories

识别并克服酵母和丝状真菌细胞工厂中的蛋白质分泌瓶颈

• 批准号: BB/F004389/1
• 负责人: David Archer
• 金额: $ 46.2万
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Research Grant
• 财政年份: 2008
• 资助国家: 英国
• 起止时间: 2008 至 无数据
• 项目状态: 已结题
• 来源: https://gtr.ukri.org/projects?ref=BB%2FF004389%2F1
• 关键词: Identifying; overcoming; protein; secretion; bottlenecks

Yeasts and moulds are both types of fungi. While yeasts such as Saccharomyces cerevisiae are widely regarded as useful because of its roles in making beer and wine, and in making bread, most moulds are regarded as a nuisance because they cause visible spoilage of foods, even though some species have uses in making valuable chemicals (e.g. penicillin). In fact, both yeasts and moulds are very important and are used commercially to make proteins that are not naturally produced by that fungus. In those cases, the gene that is necessary to make the protein is introduced into the fungus. Examples are insulin (for diabetics), chymosin (for cheese production) and a variety of enzymes used in food processing. Also, basic information about how enzymes work requires a plentiful supply of protein which usually means that those proteins are produced by a 'cell factory' such as a fungus. A common difficulty in making proteins from cell factories is that the yields are lower than desired. That is because the cell senses that it is being asked to make, and secrete to the outside of the cell, a protein which it wouldn't ordinarily make. The cell becomes stressed and it initiates a series of events within the cell aimed at protecting itself which, in so-doing, reduces the yield of protein secreted to the outside of the cell. Some proteins give rise to a bigger stress than others but we do not know the rules that govern the stress. This project aims to identify the rules and, thereby, understand how to predict which proteins will cause most stress and then to take steps with the 'cell factory' organisms to counteract the stress. In that way, we hope to be able to improve the cell factories to make more of the desired proteins. In particular, we aim to improve the capacity of fungal cell factories to produce antibody molecules that could be useful clinically.

酵母和霉菌都是真菌的类型。尽管酿酒酵母等酵母在制作啤酒和葡萄酒中的作用以及制作面包的作用而被广泛认为是有用的，但大多数霉菌被视为滋扰，因为它们会引起食物的明显变质，即使某些物种在制造有价值的化学物质方面具有用途（例如，青霉素）。实际上，酵母和霉菌都非常重要，并且在商业上用于制造并非由这种真菌自然产生的蛋白质。在这种情况下，将蛋白质引入真正的基因被引入真菌。例子是胰岛素（用于糖尿病患者），芝士蛋白（用于奶酪生产）和用于食品加工的多种酶。同样，有关酶的工作方式的基本信息需要大量蛋白质供应，这通常意味着这些蛋白质是由“细胞工厂”（例如真菌）产生的。从细胞工厂制造蛋白质的常见困难是，产量低于所需的产量。那是因为细胞被要求制作，并分泌到细胞的外部，这种蛋白质通常不会产生。细胞变得压力，并启动了旨在保护自身的细胞内的一系列事件，这些事件在So-oding中降低了分泌到细胞外部的蛋白质的产率。一些蛋白质会产生比其他蛋白质更大的压力，但我们不知道控制压力的规则。该项目旨在确定规则，从而了解如何预测哪种蛋白质会导致最大的压力，然后采取“细胞工厂”生物的措施来抵消压力。这样，我们希望能够改善细胞工厂，以制造更多所需的蛋白质。特别是，我们旨在提高真菌细胞工厂生产可能在临床上有用的抗体分子的能力。

项目成果

Activation of the unfolded protein response in Pichia pastoris requires splicing of a HAC1 mRNA intron and retention of the C-terminal tail of Hac1p.

毕赤酵母中未折叠蛋白反应的激活需要 HAC1 mRNA 内含子的剪接和 Hac1p C 末端尾部的保留。

• DOI: 10.1016/j.febslet.2011.02.036
• 发表时间: 2011
• 期刊: FEBS letters
• 影响因子: 3.5
• 作者: Whyteside G

A protocol for the subcellular fractionation of Saccharomyces cerevisiae using nitrogen cavitation and density gradient centrifugation.

使用氮空化和密度梯度离心对酿酒酵母进行亚细胞分级的方案。

• DOI: 10.1002/yea.3002
• 发表时间: 2014
• 期刊: Yeast (Chichester, England)
• 作者: Wang Y

Native-state stability determines the extent of degradation relative to secretion of protein variants from Pichia pastoris.

天然状态的稳定性决定了与毕赤酵母分泌的蛋白质变体相关的降解程度。

• DOI: 10.17863/cam.56475
• 发表时间: 2011
• 作者: Whyteside G

Identification and characterisation of eroA and ervA, encoding two putative thiol oxidases from Aspergillus niger.

eroA 和 ervA 的鉴定和表征，编码两种假定的黑曲霉硫醇氧化酶。

• DOI: 10.1016/j.gene.2010.04.011
• 发表时间: 2010
• 期刊: Gene
• 影响因子: 3.5
• 作者: Harvey AR

Investigating the physiological response of Pichia (Komagataella) pastoris GS115 to the heterologous expression of misfolded proteins using chemostat cultures.

• DOI: 10.1007/s00253-013-5186-1
• 发表时间: 2013-11
• 期刊: APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
• 影响因子: 5
• 作者: Hesketh, Andrew R.;Castrillo, Juan I.;Sawyer, Trevor;Archer, David B.;Oliver, Stephen G.
• 通讯作者: Oliver, Stephen G.