# Primary high-throughput screening of engineered phytases by online monitoring of the oxygen transfer rate of Komagataella phaffii

## TL;DR

This paper introduces a new method to screen phytase enzymes using oxygen transfer rate monitoring in yeast, allowing faster identification of high-performing variants.

## Contribution

The study introduces a novel online screening method for phytase variants using oxygen transfer rate monitoring in Komagataella phaffii.

## Key findings

- Strains with high protein concentrations show elevated metabolic burden during constitutive expression.
- A modified medium with phytic acid as the sole phosphate source enabled screening for active phytase variants via oxygen transfer rate.
- Online and offline screening results matched in 83% of cases, validating the new method's effectiveness.

## Abstract

Recombinant phytase production has recently gained increased recognition in phosphate recycling from phytate contained in plant-based side and waste streams. Until now, new phytase variants are evaluated at the end of the expression by standard offline screening procedures, where promising candidates with high activities and protein titers are identified. However, for large mutant libraries, this implies extensive laboratory work for a first screening of hundreds of clones. In this study, for the first time, two synergistic concepts for the primary screening of phytases were investigated.

The aim was to predict high recombinant protein producer strains as well as high volumetric activity phytase variants, based on the development of the respiratory activity over time of the host cell, in this case, Komagataella phaffii (Pichia pastoris). In a first step, the metabolic burden was investigated by cultivating a clone library in YPD medium in a µTOM device. It was found that strains expressing medium or high protein concentrations show clear characteristics of an elevated level of metabolic burden during constitutive expression. However, a high protein concentration does not imply a high enzymatic activity. Therefore, in a second approach, the screening was adapted to screen for phytase variants with high volumetric activity. To do so, a modified Syn6 MES medium was developed, where phytic acid was used as the only phosphate source. Thereby, only clones secreting active phytase and generating free phosphate were able to grow, which was monitored via the oxygen transfer rate. A correlation between the offline measured volumetric phytase activity and µmax was found. The clones were then ranked according to their online and offline performance and the results matched in 83% of the cases.

Online monitoring of the oxygen transfer rates in 96-well plates allowed for the evaluation of the total protein concentration and the volumetric phytase activity already during the expression. Using these results, also the specific activity can be calculated. In the future, primary screening experiments of large enzyme mutant libraries can be conducted without offline activity assays, to identify promising candidates.

The online version contains supplementary material available at 10.1186/s12934-025-02806-w.