The yeast Pichia pastoris enables efficient (high titer) recombinant protein production. As the molecular tools required are well established and gene specific optimizations of transcription and translation are becoming available, metabolism moves into focus as possible limiting factor of recombinant protein production in P. pastoris. To investigate the impact of recombinant protein production on metabolism systematically, we constructed strains that produced the model protein β-aminopeptidase BapA of Sphingosinicella xenopeptidilytica at different production yields. The impact of low to high BapA production on cell physiology was quantified. The data suggest that P. pastoris compensates for the additional resources required for recombinant protein synthesis by reducing by-product formation and by increasing energy generation via the TCA cycle. Notably, the activity of the TCA cycle was constant with a rate of 2.1 ± 0.1 mmol g CDW-1 h(-1) irrespective of significantly reduced growth rates in high BapA producing strains, suggesting an upper limit of TCA cycle activity. The reduced growth rate could partially be restored by providing all 20 proteinogenic amino acids in the fermentation medium. Under these conditions, the rate of BapA synthesis increased twofold. The successful supplementation of the growth medium by amino acids to unburden cellular metabolism during recombinant protein production suggests that the metabolic network is a valid target for future optimization of protein production by P. pastoris.

Glucose repression of the tricarboxylic acid (TCA) cycle in Saccharomyces cerevisiae was investigated under different environmental conditions using 13C-tracer experiments. Real-time quantification of the volatile metabolites ethanol and CO2 allowed accurate carbon balancing. In all experiments with the wild-type, a strong correlation between the rates of growth and glucose uptake was observed, indicating a constant yield of biomass. In contrast, glycerol and acetate production rates were less dependent on the rate of glucose uptake, but were affected by environmental conditions. The glycerol production rate was highest during growth in high-osmolarity medium (2.9 mmol g−1 h−1), while the highest acetate production rate of 2.1 mmol g−1 h−1 was observed in alkaline medium of pH 6.9. Under standard growth conditions (25 g glucose l−1 , pH 5.0, 30 °C) S. cerevisiae had low fluxes through the pentose phosphate pathway and the TCA cycle. A significant increase in TCA cycle activity from 0.03 mmol g−1 h−1 to about 1.7 mmol g−1 h−1 was observed when S. cerevisiae grew more slowly as a result of environmental perturbations, including unfavourable pH values and sodium chloride stress. Compared to experiments with high glucose uptake rates, the ratio of CO2 to ethanol increased more than 50 %, indicating an increase in flux through the TCA cycle. Although glycolysis and the ethanol production pathway still exhibited the highest fluxes, the net flux through the TCA cycle increased significantly with decreasing glucose uptake rates. Results from experiments with single gene deletion mutants partially impaired in glucose repression (hxk2, grr1) indicated that the rate of glucose uptake correlates with this increase in TCA cycle flux. These findings are discussed in the context of regulation of glucose repression.

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.