Supplementary files of research article "A rewired NADPH-dependent redox shuttle for testing peroxisomal compartmentalization of synthetic metabolic pathways in Komagataella phaffii". This study describes the establishment of the synthetic malonyl-CoA pathway in the peroxisome of Komagataella phaffii for 3-HP production using renewable carbon sources. Several strains were constructed and tested on 24-deep-well plate cultivations containing Buffered Minimal medium supplemented with the appropriate carbon source: glucose (BMD), glycerol (BMG), methanol (BMM), and oleic acid (BMO). Three independent transformants for each strain were cultivated.

A series of strains representing the cytosolic counterparts of three peroxisomal strains were also cultivated in BMD, BMG, BMM, and BMO. An individual clone for each cytosolic strain was cultivated in triplicate in 24-deep-well plates. Full consumption of the substrates and 3-HP concentrations were checked at the end of the cultures.

The attached files contain a description of the molecular cloning materials and methods, and the raw and processed data obtained from the 24-deep-well plate cultivations.

METHODOLOGICAL INFORMATION

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

2. For peroxisomal variants, three independent transformants for each strain were cultured in 24-deep-well plates containing 2 mL of medium (BMD, BMG, BMM, and BMO) at a starting OD600 of 0.1. Cultures were incubated at 25ºC and 220 rpm in an incubator shaker. The cultures on BMD, BMG, and BMM were incubated for 48 h, while cultures on BMO were harvested after 72 h. In the case of BMM, a pulse of 1% v/v of pure methanol was added to each well after 24 h of cultivation.

At the end of the culture, 2 mL samples were centrifuged at 12,000 g for 10 min using a MiniSpin (Eppendorf, Germany). The supernatants were filtered through a 0.20-µm filter (Millex SLLGX13, Millipore, CA, USA).

For 3-HP quantification, a previously described HPLC-MS method was used (Fina et al., 2021). A Prominence HPLC (Shimadzu, Japan) with a single-quadrupole Shimadzu-2010A mass spectrometry (MS) detector with an electrospray ionization source was used. An ICSep 87H USP L17 column (Transgenomic, NE, USA) was used to separate the metabolites in the supernatant. The flow rate of the mobile phase (16 mM formic acid) was set to 0.15 mL/min. The injection volume was set to 2 µL. The MS analyser was set to 89 m/z for negatively charged molecules. The detector settings were as follows: curved desolvation line (CDL) temperature at 200°C, heat block temperature at 200°C, voltage of the detector at 1.5 kV, nebulizing gas (nitrogen) flow at 1.5 L/min, and drying gas (nitrogen) flow at 10 L/min. 3-HP standards were re-injected before each set of samples, and a new calibration curve was determined each time. All samples were analyzed in duplicate. The Savitzky-Golay (SG) filter was used in peak integration to smooth chromatographic signals and improve peak detection accuracy. The chromatographic peaks corresponding to 3-HP were manually integrated.

• Each cytosolic strain was cultivated in triplicate in 24-deep-well plates containing 2 mL of medium (BMD, BMG, BMM, and BMO) at a starting OD600 of 0.1. Culture conditions and sample processing were performed as for the peroxisomal variants.

In all cases, full consumption of the substrates was checked using a previously described HPLC protocol (Fina et al., 2021). An HPLC Ultimate3000 (Dionex – Thermo Fisher Scientific, Waltham, MA, USA) equipped with an UV detector at 210 nm and a Refractive Index (RI) detector (Dionex – Thermo Fisher Scientific) was used together with an ionic exchange column ICSep ICE-COREGEL 87H3 (Transgenomic, Omaha, NE, USA) to separate the compounds in the supernatant. The flow rate of the mobile phase (6 mM sulphuric acid) was set to 0.6 mL/min and the injection volume was set to 20 µL. Initial concentrations of glucose, glycerol, and methanol in BMD, BMG, and BMM, respectively, were also verified by HPLC analysis.

3-HP quantification in the samples derived from cytosolic variants was performed by HPLC analysis, specifically from the RI spectrum. The chromatographic peaks corresponding to 3-HP were manually integrated.

References Fina, A., Brêda, G.C., Pérez-Trujillo, M., Freire, D.M.G., Almeida, R.V., Albiol, J., Ferrer, P. Benchmarking recombinant Pichia pastoris for 3-hydroxypropionic acid production from glycerol. Microb. Biotechnol. 2021;14:1671–1682. doi: 10.1111/1751-7915.13833

1. Methods for processing the data:

2. For LC-MS: Unless otherwise specified, each standard curve was generated using only the first three 3-HP standards, corresponding to 10, 25, and 50 mg/L of 3-HP, as 3-HP concentrations in the samples fell within the range covered by these points. The peak areas from the sample injections were interpolated onto the corresponding standard curve to determine the concentration of 3-HP in each sample. Concentrations from biological triplicates and technical duplicates were averaged to calculate the mean 3-HP concentration for each strain, together with the standard deviation (SD) to assess variability. All averaged 3-HP concentrations were reported in relevant units (i.e., mg/L and mM). Concentrations were normalized to substrate consumption, obtaining the average 3-HP yields with SD for each strain and substrate (mC-mol/C-mol). Statistical analyses between the average 3-HP titers of the different strains across multiple substrates were conducted using a two-tailed unpaired Student's t-test to determine significant differences in production levels.

3. For HPLC: Concentrations from biological triplicates were averaged to calculate the mean 3-HP concentration per strain in g/L and mM. Concentrations were normalized to substrate consumption, obtaining the average 3-HP yields and SD per strain for each substrate (C-mol/C-mol). The standard error (SE) for the means was also calculated. Statistical analyses between the average 3-HP titers of the different strains across multiple substrates were conducted using a two-tailed unpaired Student's t-test to determine significant differences in production levels.

4. Instrument- or software- specific information needed to interpret the data: The software used to integrate the chromatographic peak corresponding to 3-HP in LC-MS was LabSolutions v3.

The software used for peak integration in HPLC was Chromeleon.