High throughput 13 C-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
Fina Romero, Albert (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Millard, Pierre (Université de Toulouse)
Albiol i Sala, Joan (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Ferrer, Pau (Universitat Autònoma de Barcelona. Departament d'Enginyeria Química, Biològica i Ambiental)
Heux, Stephanie (Université de Toulouse)

Data:	2023
Resum:	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 an understanding on the metabolic constraints underlying these observations, the fluxome (metabolic flux phenotype) of ten 3-HP-producing P. pastoris strains has been characterized using a high throughput 13 C-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 of P. pastoris. We 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. This study shows that existing fluoxomics workflows for high-throughput analyses of metabolic phenotypes can be adapted to investigate P. pastoris, providing valuable information on the impact of genetic manipulations on the metabolic phenotype of this yeast. Specifically, our results highlight the metabolic robustness of P. 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 of P. pastoris to an acidic pH have also been obtained, showing the capability of the fluoxomics workflow to assess the metabolic impact of environmental changes. The online version contains supplementary material available at 10. 1186/s12934-023-02123-0.
Ajuts:	Agencia Estatal de Investigación PID2019-104666GB-I00 Agència de Gestió d'Ajuts Universitaris i de Recerca 2017/SGR-1462 Ministerio de Ciencia e Innovación FPU17/05434
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original.
Llengua:	Anglès
Document:	Article ; recerca ; Versió publicada
Matèria:	Fluxomics ; 13 C-Metabolic flux analysis ; Pichia pastoris ; Komagataella phaffii ; High throughput ; 3-hydroxypropionic acid ; Acetyl-CoA
Publicat a:	Microbial cell factories, Vol. 22 (June 2023) , art. 117, ISSN 1475-2859
Obra relacionada:	Fina Romero, Albert; Millard, Pierre; Albiol, Joan; Ferrer, Pau; Heux, Stephanie, 2023, "Supplementary materials of research article "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"", CORA.Repositori de Dades de Recerca https://doi.org/10.34810/data673

DOI: 10.1186/s12934-023-02123-0
PMID: 37380999

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