Evolution of yeast variation during continuous manufacturing processes

酵母是一种成熟的细胞工厂，可用于生产治疗性蛋白质、疫苗和其他高价值生物制剂。这些酵母衍生产品的全球市场年销售额约为400亿美元。酵母是很有吸引力的，因为它生长迅速，真核生物(例如，适合哺乳动物蛋白质表达)，良好的特性，安全和耐寒。生物制品行业正越来越多地转向连续生产，生产不会中断。这受到了我们的非学术合作伙伴Phenotyeca的欢迎，用于低成本疫苗制造；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回避的问题，即产生细胞群体变得异质性--表现出混合的表型--这可能会随着时间的推移而降低产量。从理论上讲，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，因为它可能会给生长更快的低产变种带来选择压力。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。酵母多样性在这一背景下也提出了有趣的学术问题。实验计划和方法该项目将检验以下假设：(I)高产生产推动酵母培养的多样化和选择性，以及(Ii)可利用天然酵母多样性来解决这一问题。具体目标是：在连续的酵母培养过程中，产品产量下降的速度有多快？(1-9个月)该项目将专注于酿酒酵母，我们在这方面有丰富的经验和资源。学生将学习感兴趣的关键产品(例如白蛋白、淀粉酶、VLP疫苗)，并用其他重组产品证实关键发现。我们有相关的结构和生产酵母菌株。这一目标中的产品计量将在总体人口水平上进行。酵母菌将从分批摇瓶和发酵罐培养中分析。产品产量是否因培养物中新的基因类型和/或新的表型变异(即非基因异质性，NGH)而改变？(10-20个月)。种群内单个细胞的产量将通过用延时显微镜和图像流式细胞术分析氟标记产物来进行比较。学生将使用我们的微流体能力(CELLAIC)进行单细胞和NGH研究，并选择发酵放大。显示低或高产量的单个细胞将被FACS分类，并分析生产表型的遗传力，以表明遗传或非遗传基础。一种风险是半可遗传的表观遗传表型，我们将通过适当的缺失和检测时间表来缓解这种风险。对选定的表型变异进行基因组重新测序将证实关键结论。能否利用替代酵母菌的遗传背景或NGH的操纵来稳定产量？(21-36个月)。监管者在基因和表型异质性方面有互补的专业知识。利用他们可用的试剂，学生将选择或提炼自然菌株背景或NGH结构(OBJ 2分别将重点放在基因或表型异质性上)，以开发异质性较低的新生产菌株。这些将根据它们的生产特点进行分析，因此有可能改进制造。

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.