BackgroundProduction of 3-hydroxypropionic acid (3-HP) through the malonyl-CoA pathway has yielded promising results inPichia pastoris(Komagataella phaffii), demonstrating the potential of this cell factory to produce this platform chemical and other acetyl-CoA-derived products using glycerol as a carbon source. However, further metabolic engineering of the originalP. pastoris3-HP-producing strains resulted in unexpected outcomes, e.g., significantly lower product yield and/or growth rate. To gain an understanding on the metabolic constraints underlying these observations, the fluxome (metabolic flux phenotype) of ten 3-HP-producingP. pastorisstrains has been characterized using a high throughput13C-metabolic flux analysis platform. Such platform enabled the operation of an optimised workflow to obtain comprehensive maps of the carbon flux distribution in the central carbon metabolism in a parallel-automated manner, thereby accelerating the time-consuming strain characterization step in the design-build-test-learn cycle for metabolic engineering ofP. pastoris.ResultsWe generated detailed maps of the carbon fluxes in the central carbon metabolism of the 3-HP producing strain series, revealing the metabolic consequences of different metabolic engineering strategies aimed at improving NADPH regeneration, enhancing conversion of pyruvate into cytosolic acetyl-CoA, or eliminating by-product (arabitol) formation. Results indicate that the expression of the POS5 NADH kinase leads to a reduction in the fluxes of the pentose phosphate pathway reactions, whereas an increase in the pentose phosphate pathway fluxes was observed when the cytosolic acetyl-CoA synthesis pathway was overexpressed. Results also show that the tight control of the glycolytic flux hampers cell growth due to limited acetyl-CoA biosynthesis. When the cytosolic acetyl-CoA synthesis pathway was overexpressed, the cell growth increased, but the product yield decreased due to higher growth-associated ATP costs. Finally, the six most relevant strains were also cultured at pH 3.5 to assess the effect of a lower pH on their fluxome. Notably, similar metabolic fluxes were observed at pH 3.5 compared to the reference condition at pH 5.ConclusionsThis study shows that existing fluoxomics workflows for high-throughput analyses of metabolic phenotypes can be adapted to investigateP. pastoris, providing valuable information on the impact of genetic manipulations on the metabolic phenotype of this yeast. Specifically, our results highlight the metabolic robustness ofP. pastoris’s central carbon metabolism when genetic modifications are made to increase the availability of NADPH and cytosolic acetyl-CoA. Such knowledge can guide further metabolic engineering of these strains. Moreover, insights into the metabolic adaptation ofP. pastoristo an acidic pH have also been obtained, showing the capability of the fluoxomics workflow to assess the metabolic impact of environmental changes.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12934-023-02123-0.