Quantitative physiology of Pichia pastoris during glucose‐limited high‐cell density fed‐batch cultivation for recombinant protein production

Abstract

Pichia pastoris has become one of the major microorganisms for the production of proteins in recent years. This development was mainly driven by the readily available genetic tools and the ease of high-cell density cultivations using methanol (or methanol/glycerol mixtures) as inducer and carbon source. To overcome the observed limitations of methanol use such as high heat development, cell lysis, and explosion hazard, we here revisited the possibility to produce proteins with P. pastoris using glucose as sole carbon source. Using a recombinant P. pastoris strain in glucose limited fed-batch cultivations, very high-cell densities were reached (more than 200 g(CDW) L(-1)) resulting in a recombinant protein titer of about 6.5 g L(-1). To investigate the impact of recombinant protein production and high-cell density fermentation on the metabolism of P. pastoris, we used (13)C-tracer-based metabolic flux analysis in batch and fed-batch experiments. At a controlled growth rate of 0.12 h(-1) in fed-batch experiments an increased TCA cycle flux of 1.1 mmol g(-1) h(-1) compared to 0.7 mmol g(-1) h(-1) for the recombinant and reference strains, respectively, suggest a limited but significant flux rerouting of carbon and energy resources. This change in flux is most likely causal to protein synthesis. In summary, the results highlight the potential of glucose as carbon and energy source, enabling high biomass concentrations and protein titers. The insights into the operation of metabolism during recombinant protein production might guide strain design and fermentation development.

Cultivation strategies to enhance productivity of Pichia pastoris: A review

“…The highest final biomass concentration reported for P. pastoris grown on methanol, continuously added in fedbatch culture, was 150 g l -1 (Curvers et al, 2001a). In fedbatch with glucose, a biomass concentration of more than 200 g CDW l -1 was achieved (Heyland et al, 2010). Generally, high productivity and a high final titre (Table 4) are reached if cultivation occurs at a high biomass concentration, for long periods, at a desired µ for product formation, before system boundaries are reached (i.e.…”

Quantification of metabolic limitations during recombinant protein production in Escherichia coli

“…To quantify the metabolic burden, quantitative physiology, including the determination of intracellular fluxes by 13 C-based metabolic flux analysis is a powerful approach (Celik et al, 2010;Gheshlaghi et al, 2007;Heyland et al, 2010b;Jin et al, 1997;Ozkan et al, 2005;Sauer et al, 1997;Stephan et al, 2006;Wittmann et al, 2007). For 13 C-based metabolic flux analysis it is important that the analytes chosen fulfil the pseudo-steady-state assumption, i.e., that in the time of label introduction the investigated rates do not change (Gombert, 2001).…”

Synergistic modular promoter and gene optimization to push cellulase secretion by Pichia pastoris beyond existing benchmarks

“…The values in Table 1 indicate that, if the gene dosage is too high protein production will drop down as seen for gene variant V04, V09 and reported for other proteins in previous studies (Mellitzer et al, 2012b). Due to the strong promoter, the optimized gene sequence and the high gene dosage, the increased transcript level might overload the folding and secretion machinery of the cells (Glick, 1995;Heyland et al, 2010;Mattanovich et al, 2004). This is even more evident in the case of methanol induced TrCBH2 expression.…”

“…For class B and especially for class C genes we emphasize that the gene variants should be optimized in a non-optimal way. By doing so, fewer transcripts will be expressed but still more correctly protein might be produced by reduced translational efficiency (Gasser et al, 2007;Heyland et al, 2010Heyland et al, , 2011. These are typically also genes where weaker promoters can help to obtain more biologically active expressed protein.…”

“…Almost pure proteins were produced in the supernatant of Pichia strains compared to commercially available supernatants of other fungi host systems producing lignocellulolytic enzymes. Moreover, it was already known that presence of methanol can lead to cell lysis which further on negatively affects protein quality (Heyland et al, 2010;Sinha et al, 2005). Besides this glycerol (or glucose)-based cultivations are highly interesting for industrial application since a 10-fold decreased heat production, significantly reduced oxygen consumption and higher protein synthesis rates as compared to methanol-based processes can be obtained (Heyland et al, 2010).…”