Elevated temperatures do not trigger a conserved metabolic network response among thermotolerant yeasts.

BACKGROUND: Thermotolerance is a highly desirable trait of microbial cell factories and has been the focus of extensive research. Yeast usually tolerate only a narrow temperature range and just two species, Kluyveromyces marxianus and Ogataea polymorpha have been described to grow at reasonable rates above 40 °C. However, the complex mechanisms of thermotolerance in yeast impede its full comprehension and the rare physiological data at elevated temperatures has so far not been matched with corresponding metabolic analyses. RESULTS: To elaborate on the metabolic network response to increased fermentation temperatures of up to 49 °C, comprehensive physiological datasets of several Kluyveromyces and Ogataea strains were generated and used for 13C-metabolic flux analyses. While the maximum growth temperature was very similar in all investigated strains, the metabolic network response to elevated temperatures was not conserved among the different species. In fact, metabolic flux distributions were remarkably irresponsive to increasing temperatures in O. polymorpha, while the K. marxianus strains exhibited extensive flux rerouting at elevated temperatures. CONCLUSIONS: While a clear mechanism of thermotolerance is not deducible from the fluxome level alone, the generated data can be valued as a knowledge repository for using temperature to modulate the metabolic activity towards engineering goals.

A comprehensive evaluation of constraining amino acid biosynthesis in compartmented models for metabolic flux analysis.

Recent advances in the availability and applicability of genetic tools for non-conventional yeasts have raised high hopes regarding the industrial applications of such yeasts; however, quantitative physiological data on these yeasts, including intracellular flux distributions, are scarce and have rarely aided in the development of novel yeast applications. The compartmentation of eukaryotic cells adds to model complexity. Model constraints are ideally based on biochemical evidence, which is rarely available for non-conventional yeast and eukaryotic cells. A small-scale model for 13C-based metabolic flux analysis of central yeast carbon metabolism was developed that is universally valid and does not depend on localization information regarding amino acid anabolism. The variable compartmental origin of traced metabolites is a feature that allows application of the model to yeasts with uncertain genomic and transcriptional backgrounds. The presented test case includes the baker's yeast Saccharomyces cerevisiae and the methylotrophic yeast Hansenula polymorpha. Highly similar flux solutions were computed using either a model with undefined pathway localization or a model with constraints based on curated (S. cerevisiae) or computationally predicted (H. polymorpha) localization information, while false solutions were found with incorrect localization constraints. These results indicate a potentially adverse effect of universally assuming Saccharomyces-like constraints on amino acid biosynthesis for non-conventional yeasts and verify the validity of neglecting compartmentation constraints using a small-scale metabolic model. The model was specifically designed to investigate the intracellular metabolism of wild-type yeasts under various growth conditions but is also expected to be useful for computing fluxes of other eukaryotic cells.

The trade-off of availability and growth inhibition through copper for the production of copper-dependent enzymes by Pichia pastoris.

BACKGROUND: Copper is an essential chemical element for life as it is a part of prosthetic groups of enzymes including super oxide dismutase and cytochrome c oxidase; however, it is also toxic at high concentrations. Here, we present the trade-off of copper availability and growth inhibition of a common host used for copper-dependent protein production, Pichia pastoris. RESULTS: At copper concentrations ranging from 0.1 mM (6.35 mg/L) to 2 mM (127 mg/L), growth rates of 0.25 h(-1) to 0.16 h(-1) were observed with copper uptake of as high as 20 mgcopper/gCDW. The intracellular copper content was estimated by subtracting the copper adsorbed on the cell wall from the total copper concentration in the biomass. Higher copper concentrations led to stronger cell growth retardation and, at 10 mM (635 mg/L) and above, to growth inhibition. To test the determined copper concentration range for optimal recombinant protein production, a laccase gene from Aspergillus clavatus [EMBL: EAW07265.1] was cloned under the control of the constitutive glyceraldehyde-3-phosphate (GAP) dehydrogenase promoter for expression in P. pastoris. Notably, in the presence of copper, laccase expression improved the specific growth rate of P. pastoris. Although copper concentrations of 0.1 mM and 0.2 mM augmented laccase expression 4 times up to 3 U/mL compared to the control (0.75 U/mL), while higher copper concentrations resulted in reduced laccase production. An intracellular copper content between 1 and 2 mgcopper/gCDW was sufficient for increased laccase activity. The physiology of the yeast could be excluded as a reason for the stop of laccase production at moderate copper concentrations as no flux redistribution could be observed by (13)C-metabolic flux analysis. CONCLUSION: Copper and its pivotal role to sustain cellular functions is noteworthy. However, knowledge on its cellular accumulation, availability and distribution for recombinant protein production is limited. This study attempts to address one such challenge, which revealed the fact that intracellular copper accumulation influenced laccase production and should be considered for high protein expression of copper-dependent enzymes when using P. pastoris. The results are discussed in the context of P. pastoris as a general host for copper -dependent enzyme production.