Engineering sweetness: zeocin-based strain screening to identify high-titer Komagataella phaffii clones for brazzein...

Brazzein is a heat-stable sweet protein with strong potential as a sugar alternative. Here, we leveraged precision fermentation to produce high titers of recombinant brazzein in the yeast Komagataella phaffii. As the brazzein expression cassette included a zeocin resistance marker, we investigated whether high zeocin resistance could serve as a predictive indicator of high brazzein production. A microscale screen across 100-1000 µg mL^-1 zeocin rapidly identified clones with varying resistance, including the high-resistant Braz_3, which produced 4.5-fold more brazzein than the moderate-resistant Braz_7 in fed-batch bioreactors (61.5 vs. 13.7 mg L^-1). Long-read genome sequencing revealed that five copies of the expression cassette were integrated into the genome of the high-producing strain compared to a single copy in the moderate-producing strain. Consequently, our results suggest a positive correlation between zeocin resistance and brazzein titers, demonstrating that antibiotic resistance phenotyping can guide strain selection for scalable production of functional food proteins.

The online version contains supplementary material available at 10.1007/s10068-026-02150-8.