Abstract
BackgroundThe methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production.ResultsThe metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA) using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol) in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h−1 using a glucose:methanol 80:20 (w/w) mix as carbon source.The MFA performed in this study reveals a significant redistristribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates.ConclusionsOverall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic yeasts when growing on mixed methanol:multicarbon sources has been implemented, thus providing a new tool for the investigation of the relationships between central metabolism and protein production. Specifically, the study points at a limited but significant impact of the conformational stress associated to secretion of recombinant proteins on the central metabolism, occurring even at modest production levels.
Highlights
The methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins
This enzyme has proven to be an attractive model for physiological studies of protein production in P. pastoris; its over expression triggers the unfolded protein response [23] and it has a negative impact on cell growth parameters, in strains expressing multiple copies of the ROL gene [12]
The selected dilution rate is below the maximum specific growth rate of the original Rolproducing strain growing on glucose (0.18 h−1) [5], and slightly above the μmax of this strain growing on methanol (0.07 h−1) [6], as unique carbon sources
Summary
The methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, when growing this yeast on mixed carbon sources, hampering future metabolic network engineering strategies for improved protein production. Several studies have reported on the impact of recombinant protein over expression on different growth parameters of yeast, such as maximum growth rate, biomass. Mixed carbon strategies (mixing methanol with a multicarbon source such as sorbitol or glycerol) have proven to boost productivity levels significantly [2,20], suggesting that metabolic burden caused by recombinant protein production can be reduced [21]
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