Abstract 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 Braz₃, which produced 4. 5-fold more brazzein than the moderate-resistant Braz₇ 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.
Ravn et al. (Fri,) studied this question.