High throughput 13C-metabolic flux analysis of 3-hydroxypropionic acid producing Pichia pastoris reveals limited availability of acetyl-CoA and ATP due to tight control of the glycolytic flux

Abstract

Abstract Background Production of 3-hydroxypropionic acid (3-HP) through the malonyl-CoA pathway has yielded promising results in Pichia 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 original P. pastoris 3-HP-producing strains resulted in unexpected outcomes, e.g. significantly lower product yield and/or growth rate. To gain understanding on the metabolic constraints underlying these observations, the fluxome of ten 3-HP-producing P. pastoris strains has been characterized using a high throughput 13C-metabolic flux analysis platform. Results Results indicate that the expression of the NADH kinase leads to a reduction in the fluxes of the pentose phosphate pathway reactions. Moreover, an increase in the pentose phosphate pathway fluxes was observed when the cytosolic acetyl-CoA 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 characterize their fluxome. Similar results were obtained at pH 3.5 and pH 5. Conclusions The reported results provide valuable information on the adaptation of the fluxome of Pichia pastoris 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, the first insights into the adaptation of P. pastoris to an acidic pH are also provided, which is of high interest for the downstream processing of carboxylic acids production.