Macromolecular and elemental composition analysis and extracellular metabolite balances of Pichia pastoris growing at different oxygen levels.

BACKGROUND: Analysis of the cell operation at the metabolic level requires collecting data of different types and to determine their confidence level. In addition, the acquired information has to be combined in order to obtain a consistent operational view. In the case of Pichia pastoris, information of its biomass composition at macromolecular and elemental level is scarce particularly when different environmental conditions, such as oxygen availability or, genetic backgrounds (e.g. recombinant protein production vs. non production conditions) are compared. RESULTS: P. pastoris cells growing in carbon-limited chemostat cultures under different oxygenation conditions (% O2 in the bioreactor inlet gas: 21%, 11% and 8%, corresponding to normoxic, oxygen-limiting and hypoxic conditions, respectively), as well as under recombinant protein (antibody fragment, Fab) producing and non-producing conditions, were analyzed from different points of view. On the one hand, the macromolecular and elemental composition of the biomass was measured using different techniques at the different experimental conditions and proper reconciliation techniques were applied for gross error detection of the measured substrates and products conversion rates. On the other hand, fermentation data was analyzed applying elemental mass balances. This allowed detecting a previously missed by-product secreted under hypoxic conditions, identified as arabinitol (aka. arabitol). After identification of this C5 sugar alcohol as a fermentation by-product, the mass balances of the fermentation experiments were validated. CONCLUSIONS: After application of a range of analytical and statistical techniques, a consistent view of growth parameters and compositional data of P. pastoris cells growing under different oxygenation conditions was obtained. The obtained data provides a first view of the effects of oxygen limitation on the physiology of this microorganism, while recombinant Fab production seems to have little or no impact at this level of analysis. Furthermore, the results will be highly useful in other complementary quantitative studies of P. pastoris physiology, such as metabolic flux analysis.

Metabolic flux profiling of Pichia pastoris grown on glycerol/methanol mixtures in chemostat cultures at low and high dilution rates.

The metabolic pathways associated with the tricarboxylic acid cycle intermediates of Pichia pastoris were studied using biosynthetically directed fractional 13C labelling. Cells were grown aerobically in a chemostat culture fed at two dilution rates (1.39x10(-5) s-1 and 4.44x10(-5) s-1) with varying mixtures of glycerol and methanol as sole carbon sources. The results show that, with co-assimilation of methanol, the common amino acids are synthesized as in P. pastoris cells grown on glycerol only. During growth at the lower dilution rate, when both substrates are entirely consumed, the incorporation of methanol into the biomass increases as the methanol fraction in the feed is increased. Moreover, the co-assimilation of methanol impacts on how key intermediates of the pentose phosphate pathway (PPP) are synthesized. In contrast, such an impact on the PPP is not observed at the higher dilution rate, where methanol is only partially consumed. This finding possibly indicates that the distribution of methanol carbon into assimilatory and dissimilatory (direct oxidation to CO2) pathways are different at the two dilution rates. Remarkably, distinct flux ratios were registered at each of the two growth rates, while the dependency of the flux ratios on the varying fraction of methanol in the medium was much less pronounced. This study brings new insights into the complex regulation of P. pastoris methanol metabolism in the presence of a second carbon source, revealing important implications for biotechnological applications.

Measurement of the sign and the magnitude of heteronuclear coupling constants from spin-state-edited J-cross-polarization NMR experiments.

New spin-state-selective (S3) NMR pulse sequences exclusively applying cross-polarization schemes to achieve optimum homonuclear and heteronuclear 1H-X coherence transfer are reported for the simple and accurate measurement of the magnitude and sign of heteronuclear coupling constants for samples at natural abundance. The proposed spin-edited HCP-TOCSY experiments are based on clean heteronuclear S3 excitation, generated by simultaneous co-addition of two independent in-phase and anti-phase components created during the mixing heteronuclear J-cross-polarization (HCP) step, which is finally transferred to other protons by a conventional homonuclear TOCSY mechanism. Selective 1D and non-selective 2D approaches for the easy determination of long-range proton-carbon and proton-nitrogen coupling constants on any protonated and non-protonated heteronuclei are presented and discussed for several organic molecules.

Improved multiplicity-edited ADEQUATE experiments.

A very simple strategy is proposed to extract carbon multiplicity information along with the classic knowledge of carbon-carbon connectivities in ADEQUATE experiments without affecting the sensitivity ratios of the original pulse schemes. These new multiplicity-edited ADEQUATE experiments prove to be highly helpful for complete 1H and 13C resonance assignment and also for automated and easy spin system characterization of samples at natural abundance, using a single NMR experiment.