Department of Microbial and Cell Culture Development, Research and Development, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA, 19406, USA.
Department of Bioanalytical Sciences, Research and Development, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA, 19406, USA.
Microb Cell Fact. 2018 Dec 20;17(1):195. doi: 10.1186/s12934-018-1044-2.
Scaling up of bioprocesses represents a crucial step in the industrial production of biologicals. However, our knowledge about the impact of scale-up on the organism's physiology and function is still incomplete. Our previous studies have suggested the existence of morphological changes during the scale-up of a yeast (Saccharomyces cerevisiae) fermentation process as inferred from the volume fraction occupied by yeast cells and exometabolomics analyses. In the current study, we noticed cell morphology changes during scale-up of a yeast fermentation process from bench (10 L) to industrial scale (10,000 L). We hypothesized that hypoxia observed during scale-up partially impaired the availability of N-acetyl-glucosamine, a precursor of chitin synthesis, a key polysaccharide component of yeast mother-daughter neck formation.
Using a combination of flow cytometry with two high throughput cell imaging technologies, Vi-CELL and Flow Imaging, we found changes in the distribution of cell size and morphology as a function of process duration at the industrial scale of the production process. At the end of run, concomitantly with lowest levels of dissolved oxygen (DO), we detected an increase in cell subpopulations exhibiting low aspect ratio corresponding to morphologies exhibited by large-single-budded and multi-budded cells, reflecting incomplete cytokinesis at the M phase of the yeast mitotic cycle. Metabolomics from the intracellular milieu pointed to an impaired supply of precursors for chitin biosynthesis likely affecting the septum formation between mother and daughter and cytokinesis. Inducing hypoxia at the 10 L bench scale by varying DO levels, confirmed the existence and impact of hypoxic conditions on yeast cell size and morphology observed at the industrial scale.
We conclude that the observed increments in wet cell weight at the industrial scale correspond to morphological changes characterized by the large diameter and low aspect ratio exhibited by cell subpopulations comprising large single-budded and multi-budded cells. These changes are consistent with impairment of cytokinesis triggered by hypoxia as indicated by experiments mimicking this condition at DO 5% and 10 L scale. Mechanistically, hypoxia impairs N-acetyl-glucosamine availability, a key precursor of chitin synthesis.
生物制品的工业化生产中，扩大生物工艺规模是至关重要的一步。然而，我们对于规模放大对生物体生理和功能影响的了解仍不完整。我们之前的研究表明，在酵母（酿酒酵母）发酵过程的规模放大过程中，细胞形态发生了变化，这可以从细胞占据的体积分数和外代谢组学分析中推断出来。在本研究中，我们注意到在从实验室规模（10L）到工业规模（10000L）的酵母发酵过程的规模放大过程中细胞形态发生了变化。我们假设，在规模放大过程中观察到的缺氧部分削弱了 N-乙酰葡萄糖胺的可用性，N-乙酰葡萄糖胺是几丁质合成的前体，几丁质是酵母母-子颈形成的关键多糖成分。
我们使用流式细胞术与两种高通量细胞成像技术 Vi-CELL 和 Flow Imaging 相结合，发现随着生产过程工业规模的进行，细胞大小和形态的分布发生了变化。在运行结束时，伴随着最低的溶解氧（DO）水平，我们检测到细胞亚群的增加，这些细胞亚群表现出低纵横比，对应于大单芽和多芽细胞的形态，这反映了酵母有丝分裂周期 M 期的不完全细胞分裂。来自细胞内环境的代谢组学研究表明，几丁质生物合成前体的供应受到影响，可能影响母细胞和子细胞之间的隔膜形成和细胞分裂。通过改变 DO 水平在 10L 实验室规模上诱导缺氧，证实了在工业规模上观察到的缺氧条件对酵母细胞大小和形态的存在和影响。
我们得出结论，在工业规模上观察到的湿细胞重量增加对应于细胞形态的变化，其特征是细胞亚群的大直径和低纵横比，这些细胞亚群包含大的单芽和多芽细胞。这些变化与缺氧引起的细胞分裂受损一致，这可以通过在 DO 5%和 10L 规模下模拟这种情况的实验来证明。从机制上讲，缺氧会削弱 N-乙酰葡萄糖胺的可用性，N-乙酰葡萄糖胺是几丁质合成的关键前体。
