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

BackgroundIn 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.ResultsThe 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.ConclusionsThe results obtained help explain the WCW differences observed at both scales by hypoxia-triggered weakening of the yeast cell wall during the scale up.Electronic supplementary materialThe online version of this article (doi:10.1186/s12934-016-0542-3) contains supplementary material, which is available to authorized users.

Saccharomyces cerevisiae morphological changes and cytokinesis arrest elicited by hypoxia during scale-up for production of therapeutic recombinant proteins

“…These morphological alterations can explain the increase in the volume occupied by cells as reflected by WCW measurements [1, 2]. In line with our initial hypothesis of the presence of hypoxic conditions at the end of the industrial scale bioreactor [1] and has been supported by small scale experiments reported herein.…”

“…We previously reported [1, 2] a clear increase in the volume occupied by yeast cells in 10,000 L bioreactors, as revealed by wet cell weight (WCW) measurements. It was postulated that hypoxia may have played a role in the observation of increased wet cell weight, the impaired supply of intermediates for \cell wall formation as well as the release of intracellular metabolites key for biomass synthesis.…”

“…We reported physiological changes in the yeast Saccharomyces cerevisiae induced by hypoxia when a high-cell density fed-batch production process was scaled-up by 1000-fold [1]. We hypothesized that low oxygen availability at the 10,000 L scale changed the physiological state affecting yeast growth, while increased cell volume suggested compromised cell wall integrity [2]. These changes induced modifications in cell morphology, which in yeast is determined by the cell wall, a rigid structure that provides mechanical protection, dictates cell shape, and modulates the selective uptake of macromolecules throughout the different stages of the yeast’s life cycle [3–5].…”

“…Previous data showed the cell wall formation biosynthesis pathways which are affected by hypoxia in 10,000 L bioreactors. Aon and colleagues [2] reported that at the 10,000 L scale cells were exposed to more severe hypoxia triggering impairment in oxidative phosphorylation and the synthesis of precursors such as GlcNAc. It appears as though, we reported the involvement of chitin synthesis and formation of proper primary septum during cytokinesis [12], likely eliciting the arrest of cell proliferation.…”

Investigation of Bar-seq as a method to study population dynamics of Saccharomyces cerevisiae deletion library during bioreactor cultivation

“…Multiple functional genomics methods (such as transcriptomics, proteomics, metabolomics, fitness profiling, and fluxomics) have been used to examine critical aspects of strain development such as carbon source utilization, tolerance to toxic substrates or final products, and metabolic flux optimization [8][9][10][11][12][13]. However, few studies applying omics-level techniques have investigated the differences in process scales on the physiology of a microbial production strain [14][15][16][17]. Furthermore, most reports focus on individual aspects of the fermentation process, including oxygen supply [18][19][20] as well as substrate heterogeneity [21][22][23][24].…”