Supplementary files 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 ". This research paper describes the fluxome of 3-hydroxypropionic acid producing Pichia pastoris recombinant strains. Glycerol was used as a sole carbon source. A High Throughput platform was used to simultaneously characterize the fluxome of 10 recombinant strains at pH 5 and 6 recombinant strains at pH 3.5. All the experiments were carried in triplicates. To determine the intracellular fluxes, stationary 13C-Metabolic Flux Analysis was used using the data of the isotopologue distribution of the proteinogenic amino acids.

The files attached herein contain: the description of how to obtain the models used for this study, the raw data obtained from the mini-bioreactor cultivations, the files to process the raw data, and the results (bioprocess parameters and fluxes from 13C-Metabolic flux analysis and Flux Balance Analysis).

1. Description of methods used for collection-generation of data:

The files contain relevant information and scripts which are used for: - Compression of a Genome Scale Metabolic Model of Pichia pastoris into a core model using Matlab. - Addition of the 3-hydroxypropionic acid producing pathway to the metabolic core model. - Translation of the core model into a FTBL format core model, which is the input model for the python-based software influx_si and IsoDesign. - Modification of the core FTBL model to generate an input model for IsoDesign (a software for the optimization of the C-source for 13C-Metabolic Flux Analysis). - IsoDesign results. Optimization of the C-source for the 13C-Metabolix Flux Analysis experiment. - Raw data from the NMR analyses of the supernatant of bioreactor cultures. - Calculation of the bioprocess parameters. - Raw data of the isotopoic distribution of each amino acid. - Correction of the isotopic distribution of the amino acids accounting for the natural abundance of the amino acids and the labelling of the inoculum. - 13C-Metabolic Flux Analysis results. Flux values. - Flux Balance Analysis results using 13C-Metabolic Flux Analysis results as flux boundaries.

1. Methods for processing the data:

Matlab scripts using the linear programming tools CobraToolbox and CellNetAnalyzer were used to generate the Core model of the metabolism of Pichia pastoris from its Genome Scale Model. Excel was used to simplify the stoichiometry coefficient of the biomass reaction. To do so, some intermediary reactions were manually lumped. The core model was modified in matlab to account for the lumped reactions. The model was further refined based on literature data. the 3-HP-production reactons were also manually added. The FTBL model was manually written from the previously generated core model. The C-transitions for each reaction were checked on https://metacyc.org. IsoDesign was used to optimize the labelling of the C-source for 13C-Metabolic Flux Analysis experiments using the previously generated FTBL format file. The experiments were carried in a High Throughput mini-bioreactor platform using glycerol as substrate. Glycerol was labelled accordingly to IsoDesign results (20% 1-13C-glycerol + 80% 2-13C-glycerol). The samples were withdrawn and processed as described in the main text linked to the supplementary materials attached herein. The supernatant samples were analysed using Nuclear Magnetic Resonance (NMR). The NMR and the biomass concentration data (OD600) were used to calculate the bioprocess parameters using PhysioFit. The amino acids isotopologue distribution was first analyzed using Liquid-Chromatography coupled to Mass Spectrometry (LC-MS). The derived data was processed to account for the the volume of inoculum, anc correction of the isotopologue distribution based on the abundance of the natural isotopes using IsoCor. The metabolic fluxes were calculated using influx_s for 13C-Metabolic Flux Analysis. The bioprocess parameters and the isotopologue distribution of the amino acids were used as input data for each experiment. The flux results were used as input for Flux Balance Analysis in Matlab using the CobraToolbox and the previously generated core model for 3-HP-producing Pichia pastoris strains.