title: Hypoxia-elicited impairment of cell wall integrity, glycosylation precursor synthesis, and growth in scaled-up high-cell density fed-batch cultures of Saccharomyces cerevisiae.
abstract: BACKGROUND
  In this study we examine the integrity of the cell wall during scale up of a yeast fermentation process from laboratory scale (10 L) to industrial scale (10,000 L). In a previous study we observed a clear difference in the volume fraction occupied by yeast cells as revealed by wet cell weight (WCW) measurements between these scales. That study also included metabolite analysis which suggested hypoxia during scale up. Here we hypothesize that hypoxia weakens the yeast cell wall during the scale up, leading to changes in cell permeability, and/or cell mechanical resistance, which in turn may lead to the observed difference in WCW. We tested the cell wall integrity by probing the cell wall sensitivity to Zymolyase. Also exometabolomics data showed changes in supply of precursors for the glycosylation pathway.
  
  
  RESULTS
  The results show a more sensitive cell wall later in the production process at industrial scale, while the sensitivity at early time points was similar at both scales. We also report exometabolomics data, in particular a link with the protein glycosylation pathway. Significantly lower levels of Man6P and progressively higher GDP-mannose indicated partially impaired incorporation of this sugar nucleotide during co- or post-translational protein glycosylation pathways at the 10,000 L compared to the 10 L scale. This impairment in glycosylation would be expected to affect cell wall integrity. Although cell viability from samples obtained at both scales were similar, cells harvested from 10 L bioreactors were able to re-initiate growth faster in fresh shake flask media than those harvested from the industrial scale.
  
  
  CONCLUSIONS
  The results obtained help explain the WCW differences observed at both scales by hypoxia-triggered weakening of the yeast cell wall during the scale up.
authors.affiliation: Department of Microbial and Cell Culture Development, Research and Development, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA, 19406, USA. juan.c.aon@gsk.com.
authors.forename: Juan C
authors.identifier: http://orcid.org/0000-0001-8030-1273
authors.initials: JC
authors.lastname: Aon
authors.affiliation: Department of Microbial and Cell Culture Development, Research and Development, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA, 19406, USA.
authors.forename: Jianxin
authors.initials: J
authors.lastname: Sun
authors.affiliation: Department of Microbial and Cell Culture Development, Research and Development, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA, 19406, USA.
authors.forename: Julie M
authors.initials: JM
authors.lastname: Leighton
authors.affiliation: Department of Microbial and Cell Culture Development, Research and Development, GlaxoSmithKline, 709 Swedeland Road, King of Prussia, PA, 19406, USA.
authors.forename: Edward R
authors.initials: ER
authors.lastname: Appelbaum
mesh_terms: D000693:Anaerobiosis; D061249:Batch Cell Culture Techniques; D019149:Bioreactors; D002473:Cell Wall / Q000502:physiology*; D005285:Fermentation; D006031:Glycosylation; D006155:Guanosine Diphosphate Mannose / Q000378:metabolism; D006867:Hydrolases / Q000378:metabolism; D007218:Industrial Microbiology; D008360:Mannosephosphates / Q000378:metabolism; D055442:Metabolome; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000254:growth & development* / Q000378:metabolism*
chemical_list: D008360:Mannosephosphates; D006155:Guanosine Diphosphate Mannose; C027693:mannose-6-phosphate; C037833:zymolyase; D006867:Hydrolases
keywords: Cell wall; Exometabolomics; Fermentation process; Hypoxia, glycosylation pathway; Metabolite profiling; Saccharomyces cerevisiae; Scale-up