DOI: 10.34810/data2146
title: Replication data for appendices in the doctoral thesis "Metabolic engineering of Komagataella phaffii for renewable methanol bioconversion into 3-hydroxypropionic acid"

description: This dataset contains the additional files associated with appendices I and III of the doctoral thesis "Metabolic engineering of Komagataella phaffii for renewable methanol bioconversion into 3-hydroxypropionic acid".

The first appendix (I) describes the construction of a Komagataella phaffii proof-of-concept strain for 3-HP production from renewable carbon sources by implementing the synthethic β-alanine pathway. The performance of this strain was evaluated in 24-deep-well plate cultivations using Buffered Minimal medium supplemented with either glycerol (BMG) or methanol (BMM) and compared to two K. phaffii strains expressing the malonyl-CoA pathway for 3-HP synthesis. The corresponding file (Additional file S1) contains the raw and processed data obtained from these cultivations.

The third appendix (III) details the experimental procedures and findings related to the study of the redox metabolism of two 3-HP-producing K. phaffii strains. The corresponding file (Additional file S3) contains raw and processed data from intracellular redox cofactor quantification, measured using a colorimetric kit. It also includes quantification of 3-HP and methanol in the supernatant samples.
description: METHODOLOGICAL INFORMATION 1. Description of methods used for collection-generation of data: APPENDIX I Three independent transformants of the K. phaffii strain engineered with the β-alanine pathway (PpXβ01) were inoculated in 24-deep-well plates containing 2 mL of Buffered Minimal medium supplemented with either glycerol (BMG) or methanol (BMM) at a starting optical density at 600 nm (OD600) of 0.1. These cultures were compared to the parental strain X-33 and representative clones of two strains expressing the malonyl-CoA pathway (PpHP2 and PpHP6), previously constructed by Fina et al. (2021). All clones were inoculated in triplicate. Cultures were incubated for 48 h at 220 rpm with 80% relative humidity (RH) in a Multitron Standard incubator shaker (Infors HT, Bottmingen, Switzerland). BMG cultures were grown at 28°C, while BMM cultures were incubated at 25°C. After 24 h, a 1% v/v pure methanol pulse was added to the BMM cultures. At the end of the cultivation, OD600 was measured in duplicate using a Multiskan FC Microplate Photometer (Thermo Fisher Scientific, Waltham, MA, USA). Afterwards, the remaining volume in each deep well was collected into 2-mL microcentrifuge tubes and centrifuged at 13,400 rpm for 5 min using a MiniSpin centrifuge (Eppendorf, Hamburg, Germany). The supernatant was filtered through a 0.20-μm pore size single-use syringe filter (SLLGX13) from Merck Millipore (Burlington, MA, USA). Filtered supernatants were analyzed by HPLC to quantify 3-HP and verify methanol or glycerol depletion. 3-HP was quantified using an HPLC Ultimate3000 (Dionex—Thermo Fischer Scientific, Waltham, MA, USA) equipped with a Refractive Index (RI) detector. The ion-exchange column ICSep ICE-COREGEL 87H3 (Transgenomic, Omaha, NE, USA) was used to separate the compounds in the supernatant. The mobile phase consisted of 6 mM sulphuric acid with a flow rate of 0.6 mL/min. Glycerol and methanol depletion was verified using the same analytical method. APPENDIX III Representative clones of the 3-HP-producing K. phaffii strains PpCβ21 and PpCβ21-P (Àvila-Cabré et al. 2023) were grown in defined mineral medium in fed-batch cultures. Four samples were taken at certain intervals during the methanol feeding phase of fed-batch cultivations to quantify the NADP+/NADPH ratio and the concentrations of 3-HP and methanol in the supernatants. The NADP+/NADPH ratio was measured using the NADP/NADPH Assay Kit (Colorimetric) (ab65349) from Abcam (Cambridge, UK). Approximately 1 mL of broth was collected rapidly and immediately mixed with 1 mL of pure methanol in a 2.5-mL syringe pre-cooled to -20°C. Cells were harvested by centrifugation at 2,000 rpm for 5 min at -9°C using the refrigerated centrifuge MPW-352R equipped with the angular rotor MPW-11760 (Biogen Científica, Madrid, Spain). After centrifugation, the supernatant was removed, and the pellet was suspended in 1 mL of cold PBS. Serial dilutions with cold PBS were performed with a minimum of two replicates in 1.5-mL microcentrifuge tubes to obtain an OD600 between 0.5 and 2.0, as an initial recommendation for the analysis. Subsequently, cells were harvested again by centrifugation at 2,000 rpm for 5 min at 0°C. The supernatant was discarded, and a volume of 650 µL of extraction buffer from the kit was added to suspend the pellets. Samples were transferred to pre-cooled 2-mL reinforced screw-cap tubes (432-0379) (Avantor, Radnor, PA, USA) containing approximately 570 mg of 0.5-mm acid-washed glass beads (Merck, Darmstadt, Germany) and kept on ice. Cells were then lysed using a Fastprep-24 5G™ bench-top homogenizer (MP Biomedicals, Santa Ana, CA, USA) equipped with the Coolprep™ cryogenic adapter at 5.0 m/s for 10 s, with 3 rounds of lysis and 2-minute resting periods between cycles. The lysed samples were centrifuged at 14,000 rpm for 5 min at 0°C. The supernatant from cell lysates was transferred to Amicon® Ultra-0.5 MWCO 3kDa centrifugal tubes (Merck) to remove proteins, following the manufacturer’s instructions. Filtered supernatants were transferred to 1.5-mL microcentrifuge tubes and kept on ice. Samples were stored at -80°C until the quantification assay was performed. All steps were carried out at a temperature not exceeding 4°C. On the day of the assay, samples were thawed on ice, and the colorimetric detection of NADP+ and NADPH was performed according to the kit’s manual. OD450 measurements were performed in a 96-well microtiter plate using the SPECTROstar Nano absorbance microplate reader (BMG Labtech, Ortenberg, Germany). For metabolite quantification, 2 mL of culture broth were centrifuged 5 min at 13,400 rpm per replicate using a MiniSpin (Eppendorf, Germany). Subsequently, the supernatant was filtered with a 0.2-μm pore size single-use syringe filter (SLLGX13NK, Merck Millipore). Methanol and 3-HP were quantified using an Ultimate3000 HPLC system (Dionex—Thermo Fisher Scientific, Waltham, MA, USA) equipped with a Refractive Index (RI) detector and an ion-exchange column ICSep ICE-COREGEL 87H3 (Transgenomic, Omaha, NE, USA) to separate the compounds in the supernatant. The mobile phase was 6 mM sulfuric acid with a flow rate of 0.6 mL/min. References Fina, A., Brêda, G.C., Pérez-Trujillo, M., Freire, D.M.G., Almeida, R.V., Albiol, J., Ferrer, P (2021). Benchmarking recombinant Pichia pastoris for 3-hydroxypropionic acid production from glycerol. Microbial Biotechnology, 14(4), 1671–1682. doi: 10.1111/1751-7915.13833 Àvila-Cabré, S., Pérez-Trujillo, M., Albiol, J., Ferrer, P. Engineering the synthetic β-alanine pathway in Komagataella phaffii for conversion of methanol into 3-hydroxypropionic acid (2023). Microbial Cell Factories, 22, 237. https://doi.org/10.1186/s12934-023-02241-9. Erratum in Microbial Cell Factories, 23, 235. https://doi.org/10.1186/s12934-024-02507-w 2. Methods for processing the data: - For HPLC: 3-HP concentrations were normalized to substrate consumption and biomass, obtaining the 3-HP yields on substrate (Yp/s) and biomass (Yp/x), respectively, for every replicate. Replicate values were averaged to calculate the mean 3-HP concentration (g/L), Yp/s (g/g and C-mol/C-mol), and Yp/x (g 3-HP/L/OD600) per strain, together with the corresponding standard deviation (SD) of the mean and the coefficient of variation (CV, %). - For NADP+/NADPH quantification: data analysis was performed based on the methods described in the instructions of the NADP/NADPH Assay Kit (Colorimetric) (ab65349) from Abcam (Cambridge, UK). 3. Instrument- or software- specific information needed to interpret the data: The software used for peak integration in HPLC was Chromeleon.
description: MS Office, null