Supplementary files of research article "Engineering the synthetic β-alanine pathway in Komagataella phaffii for conversion of methanol into 3-hydroxypropionic acid". This research paper performs a theoretical comparison between the the two main metabolic pathways towards 3-hydroxypropionic (3-HP) acid production, and describes the establishment of the synthetic β-alanine pathway in Komagataella phaffii for conversion of methanol into 3-HP. Several strains overexpressing the β-alanine pathway were constructed and 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. Series of growth kinetics experiments were performed by cultivating a representative clone from each strain in shake-flask cultures using BMM to determine the maximum specific growth rate of the different strains.

The best performing strains were further evaluated in aerobic fed-batch reactors following a pre-programmed exponential feeding strategy for controlled growth rate. Offline and online state variables during the fed-batch phase of the bioreactor-scale experiments were monitored and recorded. Nuclear Magnetic Resonance (NMR) spectroscopy was used to perform the exometabolome profiling analyses of the supernatant samples from early, mid, and late methanol-feeding phase. All experiments were carried in duplicates.

The files attached herein contain: the diagrams and stoichiometric balances of the malonyl-CoA and β-alanine pathways, the raw data obtained from the 24 deep-well plate cultivations and cell growth kinetics experiments, the NMR spectra for different strains' exometabolome, the raw and processed data obtained from the bioreactor-scale experiments, the description of the molecular cloning materials and methods, and the raw data of the online monitored standard process parameters.

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

2. 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 was 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 was quantified from the RI spectrum.

3. For the growth kinetics experiments, a representative clone from each strain was inoculated in shake-flasks containing BMM with methanol at a starting OD600 of 0.2 and grown during 24 h. Samples were taken every 3 h to measure the OD600 in duplicate with a 96-well microtiter plate using the SPECTROstar Nanoabsorbance microplate reader (BMG Labtech, Ortenberg, Germany).

4. For the bioreactor cultivations, samples were taken every 5.5 h 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. This parameter was quantified for three samples throughout the fed-batch.For the rest of the samples, the DCW was calculated by interpolation using an equation from a linear regression between DCW and OD600 measures of the initial fed-batch cultivations. To quantify the metabolites, 2 ml of culture samples were centrifuged 5 min at 13,400 rpm using a MiniSpin (Eppendorf, Germany). 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 quantification from both the RI and UV spectra. Residual glycerol and methanol from the batch and fed-batch phases, respectively, were quantified from the RI spectrum. Part of this filtered supernatant was used for exometabolome profiling by NMR.

5. For the NMR method, a Bruker Avance 600 MHz NMR spectrometer operating at a proton (1H) frequency of 600.13 MHz equipped with a triple-resonance Bruker TXI 5 mm room-temperature probe and an autosampler (Bruker Biospin, Rheinstetten, Germany) was utilized. The probe temperature was maintained at 298.0 K in all experiments. Once centrifuged and filtered, each supernatant aliquot (400 µl) was mixed with a D2O sodium phosphate buffer (200 µl, 0.2 M, pH 7.4) containing an internal standard (3-(trimethylsilyl)-[2,2,3,3-2H4]-propionic acid sodium salt (TSP), 1 mM) and transferred to the NMR tube. All samples were analysed conducting 1D 1H NMR experiments with presaturation of the residual water signal applying the pulse sequence commonly termed 1D NOESY-presat (Nicholson et al., 1995).

References

Nicholson JK, Foxall PJD, Spraul M, Farrant RD, Lindon JC. 750 MHz 1H and 1H-13C NMR spectroscopy of human blood plasma. Anal Chem. 1995;67:793–811. doi: 10.1021/ac00101a004

1. Methods for processing the data:

2. For the NMR method, data were collected into 32K data points during an acquisition time of 2.3 s using a recycle delay of 2 s. Spectra were recorded in the time domain as interferograms (FID) across a spectral width of 7211 Hz and as the sum of 1024 transients. Each FID was multiplied by an exponential apodisation function equivalent to a 0.2 Hz line broadening, prior to Fourier transform. The frequency-domain spectra were manually phased, baseline corrected, and referenced to the TSP resonance at δH 0.00 ppm. The identification of metabolites was carried out using the BMRB spectral database (Hoch et al., 2023) and the software Chenomix NMR Suite 8.5 (Chenomix Inc., Edmonton, Canada).

References

Hoch JC, Baskaran K, Burr H, Chin J, Eghbalnia HR, Fujiwara T, et al. Biological magnetic resonance data bank. Nucleic Acids Res. 2023;51:D368–76. doi: 10.1093/nar/gkac1050