Supplementary files of research article "Metabolic engineering of Komagataella phaffii for enhanced 3-hydroxypropionic acid production from methanol". This research paper describes a combinatorial metabolic engineering strategy aiming at increasing metabolic precursors supply and 3-HP export, applied to the original 3-HP producing K. phaffii strain harboring the synthetic β-alanine pathway and a heterologous NADP-dependent formate dehydrogenase.

Several strains overexpressing the genes encoding for enzymes catalyzing reactions immediately upstream of the β-alanine pathway were constructed. To further channel flux through the heterologous NADP-dependent formate dehydrogenase, we performed the deletion of the endogenous NAD-dependent analogous enzyme. Finally, we sought to promote 3-HP export by overexpressing two monocarboxylate permeases. The constructed strains were tested on 24 deep-well plate cultivations containing buffered minimal medium (BMM) with methanol.Three independent transformants from each strain were tested. Final biomass concentration as well as methanol and 3-HP quantification were determined at the end of the cultures.

The best performing strains were further characterized in aerobic fed-batch reactors following a pre-programmed exponential feeding strategy for controlled growth rate at pH values 5 and 3.5. Offline and online state variables during the fed-batch phase of the bioreactor-scale experiments were monitored and recorded. Experiments at pH 5 were carried in duplicates, while a single cultivation replicate for each strain was performed at pH 3.5.

The files attached herein contain: the description of the molecular cloning materials and methods, the raw and processed data obtained from the bioreactor-scale experiments, the raw data of the online monitored standard process parameters, and the raw data obtained from the 24 deep-well plate cultivations.

Additional files of research article "Metabolic engineering of Komagataella phaffii for enhanced 3-hydroxypropionic acid (3-HP) production from methanol". This research paper describes a combinatorial metabolic engineering strategy aiming at increasing the supply of metabolic precursors and 3-HP export, applied to the original 3-HP producing K. phaffii strain harboring the synthetic β-alanine pathway and a heterologous NADP-dependent formate dehydrogenase.

Several strains overexpressing the genes encoding enzymes catalyzing reactions immediately upstream of the β-alanine pathway were constructed. To further channel flux through the heterologous NADP-dependent formate dehydrogenase, we deleted the endogenous NAD-dependent analogous enzyme. Finally, we sought to promote 3-HP export by overexpressing two monocarboxylate permeases. The constructed strains were tested on 24 deep-well plate cultivations containing buffered minimal medium (BMM) with methanol. Three independent transformants from each strain were tested. Final biomass concentration as well as methanol and 3-HP quantification were determined at the end of the cultures.

The best-performing strains were further characterized in aerobic fed-batch reactors following a pre-programmed exponential feeding strategy for controlled growth rate at pH 5 and 3.5. Offline and online state variables during the fed-batch phase of the bioreactor-scale experiments were monitored and recorded. Experiments at pH 5 were conducted in duplicate, while a single cultivation replicate for each strain was performed at pH 3.5.

The files attached herein contain: a description of the molecular cloning materials and methods, raw data obtained from the 24 deep-well plate cultivations, raw data of the online monitored standard process parameters, and raw and processed data obtained from the bioreactor-scale experiments.

1. Description of methods used for collection-generation of data - For the small-scale screenings, three independent transformants from each strain were grown in 24-deep well plates containing BMM with methanol at a starting OD600 of 0.1. Cultures were grown for 48 h at 25 ºC, and a relative humidity (rh) in the incubation chamber of 80 %. After 24 h, 1 % v/v pure methanol pulse was added to the cultures. All clones were inoculated in triplicate. At the end of the culture, the final biomass concentration of each deep-well was determined in duplicate with a 96-well microtiter plate using a Multiskan FC Microplate Photometer (Thermo Fisher Scientific, Waltham, MA, USA) to ensure all the cultures were grown up to a similar endpoint OD600. 3-HP and methanol were quantified using an HPLC Dionex Ultimate 3000 (Dionex Thermo Fischer Scientific) equipped with an ionic exchange column ICSep ICE-COREGEL 87H3 (Transgenomic, Omaha, NE, USA) using 6 mM sulphuric acid as mobile phase at a flow rate of 0.6 ml/min. 3-HP and methanol were quantified from the RI spectrum. - For the bioreactor cultivations, samples were taken during the 39-h fed-batch phase to measure OD600, biomass dry cell weight and supernatant metabolites. The OD600 measurements were performed in triplicate using a Lange DR 3900 spectrophotometer (Hach, Loveland, CO, USA). For the biomass DCW determination, 10 ml of distilled water with 9 g/l NaCl were used to wet the pre-weighted glass microfiber filters (APFF04700, Merck Millipore) before filtering 2 ml of culture for each triplicate. After that, the filters were washed using the same volume of the NaCl solution and dried for 24 h at 105 °C. Filters containing the dry biomass were weighted to calculate the DCW. Biomass DCW was determined in three samples throughout the fed-batch cultivations at pH 5. For the rest of the samples, the biomass DCW values were calculated from the measured OD600 values, based on the calibration curve made for the given strain. In fed-batch cultivations at pH 3.5, a single DCW biomass determination was performed at the end of the cultures. To quantify the metabolites, 1 ml was taken from samples collected throughout the fed-batch cultivations at pH 5 and centrifuged 5 min at 13,400 rpm using a MiniSpin (Eppendorf, Germany) in duplicate. The supernatant was then filtered with a 0.2 μm pore size single-use syringe filter (SLLGX13NK, Merck Millipore, CA, USA). The filtered supernatant was stored at − 20 °C until HPLC analysis for 3-HP and residual methanol quantification from the RI spectrum. For cultivations at pH 3.5, samples for metabolites quantification were prepared in triplicate.