Evolution of yeast variation during continuous manufacturing processes

连续生产过程中酵母变异的演变

• 批准号: 2593725
• 金额: --
• 依托单位: University of Nottingham
• 依托单位国家: 英国
• 项目类别: Studentship
• 财政年份: 2021
• 资助国家: 英国
• 起止时间: 2021 至 无数据
• 项目状态: 未结题
• 来源: https://gtr.ukri.org/projects?ref=studentship-2593725
• 关键词: Evolution; yeast; variation; during; continuous

Yeast is a proven cell factory for the production of therapeutic proteins, vaccines and other high-value biologics. The global market for these yeast-derived products is around $40Bn p.a. Yeast is attractive because it is fast-growing, eukaryotic (e.g., suitable for mammalian-protein expression), well characterised, safe and hardy. The biologics industry is increasingly shifting towards continuous manufacturing, with no interruptions to production. This is embraced by our non-academic partner, Phenotypeca, for lowcost vaccine manufacture; with obvious economic advantages. However, it also presents a problem that has so far been side-stepped, where the producing cell population becomes heterogeneous - showing mixed phenotypes - which can lower production yields over time. Theoretically, this can occur within 40-60 generations (a few days) after a production run has started. The metabolic burden of production could explain this decline, as it may create selection pressure for faster-growing low-yield variants. However, this significant problem has not yet been addressed, so this interdisciplinary project proposal is exceptionally timely. Yeast diversity in this context also presents intriguing academic questions. Experimental Plan and Methods The project will test the hypotheses that; (i) high-yield production drives diversification and selection in yeast cultures, and (ii) natural yeast diversity could be exploitable to address this problem. The specific objectives are: How rapidly do product-yields decline during continuous yeast culture? (Months 1-9). The project will focus on Saccharomyces cerevisiae, with which we have extensive experience and resources. The student will study key products of interest (e.g. albumin, amylases, VLP vaccines), corroborating key findings with other recombinant products. We have the relevant constructs and production yeast strains. Product measurement in this objective will be at the bulk population level. Yeasts will be analysed from batch-flask and fermenter culture. Is altered product-yield due to novel genotypes and/or novel phenotypic variants (i.e. nongenotypic heterogeneity, NGH) within cultures? (Months 10-20). Production by individual cells within populations will be compared by analysis of fluoro-tagged product with time-lapse microscopy and image-streaming flow cytometry. The student will use our microfluidics capability (CellASIC) for single-cell and NGH studies, with options for fermentation scale-up. Individual cells showing low or high production will be FACS-sorted and assayed for heritability of the production phenotype, to indicate genotypic or non-genotypic bases. One risk is semi-heritable epigenetic phenotypes, which we will mitigate with appropriate deletants and assay timescale. Genome re-sequencing of selected phenotypic variants will corroborate key conclusions. Can alternative yeast genetic-backgrounds or manipulation of NGH be harnessed to stabilise production over time? (Months 21-36). The supervisors have complementary expertise in genotypic and phenotypic heterogeneity. Using their available reagents, the student will select or refine natural strain backgrounds or NGH constructs (with results from Obj 2 steering a focus on genotypic or phenotypic heterogeneity, respectively) to develop novel production strains with decreased heterogeneity. These will be profiled for their production characteristics, hence the potential for improved manufacturing.

酵母是生产治疗性蛋白质、疫苗和其他高价值生物制剂的成熟细胞工厂。这些酵母衍生产品的全球市场每年约为400亿美元。酵母是有吸引力的，因为它是快速生长的，真核的（例如，适合于大肠杆菌蛋白表达）、良好表征、安全且哈代。 生物制品行业正日益向连续生产转变，生产不会中断。我们的非学术合作伙伴Phenotypeca采用了这一方法，用于低成本疫苗生产;具有明显的经济优势。然而，它也提出了一个迄今为止一直被忽视的问题，即生产细胞群变得异质-显示混合表型-这会随着时间的推移降低产量。理论上，这可以在生产运行开始后的40-60代（几天）内发生。生产的代谢负担可以解释这种下降，因为它可能会为快速生长的低产变种创造选择压力。然而，这一重大问题尚未得到解决，因此这一跨学科项目提案非常及时。在这种背景下，酵母的多样性也提出了有趣的学术问题。 实验计划和方法该项目将测试以下假设：（i）高产生产驱动酵母培养物的多样化和选择，以及（ii）天然酵母多样性可以用来解决这个问题。 具体目标是：在连续酵母培养过程中，产品产量下降的速度有多快？（第1-9个月）。该项目将专注于酿酒酵母，我们拥有丰富的经验和资源。学生将研究感兴趣的关键产品（如白蛋白，淀粉酶，VLP疫苗），证实与其他重组产品的关键发现。我们拥有相关的构建体和生产酵母菌株。本目标中的产品测量将在批量群体水平上进行。将分析分批培养瓶和发酵罐培养物中的酵母菌。 是否由于培养物中的新基因型和/或新表型变体（即非基因型异质性，NGH）导致产品产量改变？（第10-20个月）。将通过用延时显微镜和图像流式细胞术分析荧光标记产物来比较群体内单个细胞的产量。学生将使用我们的微流体能力（CellASIC）进行单细胞和NGH研究，并可选择发酵放大。将对显示低或高产量的单个细胞进行FACS分选，并测定生产表型的遗传性，以指示基因型或非基因型基础。一个风险是半遗传表观遗传表型，我们将通过适当的缺失体和测定时间尺度来减轻。对选定的表型变异体进行基因组重新测序将证实关键结论。 可以替代酵母遗传背景或操纵NGH被利用来稳定生产随着时间的推移？（第21-36个月）。主管人员在基因型和表型异质性方面具有互补的专业知识。使用他们可用的试剂，学生将选择或改进天然菌株背景或NGH构建体（目标2的结果分别关注基因型或表型异质性），以开发异质性降低的新型生产菌株。将对这些产品的生产特性进行分析，从而了解其改进生产的潜力。