Evolution of yeast variation during continuous manufacturing processes

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。酵母具有吸引力，因为它是快速生长的真核生物（例如，适合哺乳动物蛋白表达），具有良好的特性，安全和耐受性。生物制剂行业正日益转向连续生产，生产不会中断。这被我们的非学术合作伙伴Phenotypeca所接受，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回避的问题，即生产细胞群体变得异质-显示混合表型-随着时间的推移可能降低产量。理论上，这可能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，因为它可能对生长更快的低产量变种造成选择压力。然而，这一重大问题尚未得到解决，因此，这个跨学科的项目提案是异常及时的。酵母多样性在这种情况下也提出了有趣的学术问题。实验计划与方法本项目将测试以下假设：(1)高产推动了酵母培养物的多样化和选择，(2)天然酵母的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？(1 - 9个月)。该项目将以酿酒酵母为重点，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），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。本目标中的产品测量将在总体人口水平上进行。将从批量瓶和发酵培养中分析酵母。产品产量的改变是由于新的基因型和/或新的表型变异（即非基因型异质性，NGH）在培养中？(月10 - 20)。将通过延时显微镜和流式细胞术分析氟标记产品来比较群体内单个细胞的产量。该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。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性，以表明基因型或非基因型基础。一种风险是半遗传的表观遗传表型，我们将通过适当的删除和分析时间尺度来减轻这种风险。所选表型变异的基因组重测序将证实关键结论。随着时间的推移，是否可以利用替代酵母遗传背景或操纵NGH来稳定生产？(月21-36)。督导人员在基因型和表型异质性方面具有互补的专业知识。利用他们现有的试剂，学生将选择或改进自然菌株背景或NGH结构（根据Obj 2的结果，分别关注基因型或表型异质性），以开发具有降低异质性的新型生产菌株。这些将被描述为他们的生产特点，因此有可能改善制造。

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.