DOI: 10.34810/data1747
title: Replication data for: "A glance into growth-decoupled promoters for recombinant protein production in Komagataella phaffii"

description: Dataset of the transcriptional analyses performed with OpenArray technology to study the effects of different cultivating conditions to the expression of key genes involved in Komagataella phaffii metabolism in a recombinant protein producing strain. In particular, 27 genes were studied from glycolysis/gluconeogenesis, pentose phosphate pathway, glyoxylate cycle, oxidative stress response, lipids biosynthetic metabolism, the recombinant gene of interest and four more relevant genes related to heterologous protein production.
The dataset contains information of the genes studied, the sequences used for amplification and the results of both the no template control (NTC) and the samples. The samples results are expressed in terms of relative expression and log2fold.
description: Microsoft Excel, null
description: METHODOLOGICAL INFORMATION

1. Description of methods used for collection-generation of data:
The samples anlyazed by OpenArray were obtained from Komagataella phaffii bioreactor cultivations on glucose or glycerol limiting conditions, or methanol pulses, with four strains. All strains produced the lipases B from Candida antarctica (CalB) but differed in the promoter used, being Pdh, Pmv1, Pmv2 or Pdf. The fed-batch strategies were defined as growth-coupled and growth-decoupled considering the protein production. The growth-decoupled strategy consisted of a glucose or glycerol fed-bach cultivation at a specific growth rate of 0.025 1/h. While the growth-decoupled one was splited in twho phases, in the first one biomass grew at a specific growth rate of 0.15 1/h until reaching about 70 g/l of dry cell weight, where then CalB production was induced by reducing the feding to a very low constant rate. The conditions achieved in this last phase were called pseudo-starving. Finally, methanol pulses were performed to a glucose-grown cultivation with about 70 g/l of dry cell weight. Samples were taken just before starting the fed-bath phase in all cultivations, after 17 h and 52 h in the growth-coupled strategies, and in the growth-decoupled one at 8.5 h (finalization of growth phase), 16 h and 45 h. The samples were stored at -80 ºC as pellets after mixing 750 ul of broth with 500 ul of ethanol:phenol solution (20:1 ratio), centrifuging at max speed for 2 min and descarting the supernatant.

The SV Total RNA Isolation System kit (Promega, Madison, WI, United States) was used for total RNA extraction following the manufacturers instructions. One additional step was performed at the beginning of the extraction process to lysate the cells, which was done by adding 300 ul of glassbeads to the eppendorf tubes and shaking them in two cycles of 1 min at 30 Hz in the TissueLyser II. RNA concentration and pureness was measured by NanoDrop Spectrophotometer and diluted to 200 ng/ul, which was retrotranscribed to cDNA by iScript cDNA Synthesis kit (Bio-rad, Hercules, CA, United States).

OpenArray plate was manufactured by Thermo Fisher Scientific according to the provided primers and probes sequences, which were designed with IDT PrimerQuest Tool. These plates were loaded with the cDNA and TaqMan OpenArray Real-Time Master Mix using the QuantStudio OpenArray AccuFill System, which were sealed for 90 s with the OpenArray Cas Lid. Then, the standard protocol for running QuantStudio 12K Flex was followed.

2. Methods for processing the data:
Data was visualized with Thermo Fisher Connect platform, which enabled the determination of the Cq of the samples and genes. The expression levels of the genes were calculated with the fold effect method, as reported by:
- Ruiz-Villalba A, Ruijter JM, van den Hoff MJB. Use and misuse of Cq in qPCR data analysis and reporting. Life. 2021;11:496. 10.3390/life11060496
The reference gene was actin (Act1), while the results at the end of the fast-growing cultivation with glucose were used to normalize the expression levels. Finally, the expression levels were expressed as the mean log2 fold change of the four strains.

3. Instrument- or software- specific information needed to interpret the data:
MS Excel

4. Insutrements, calibration and standards information:
QuantStudio 12K Flex, QuantStudio OpenArray AccuFill Sytem, OpenArray Case Lid are form Thermo Fisher Scientific (Waltham, MA, United States).

5. Environmental or experimental conditions:
All reactions were performed according to the manufacturer.