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

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.