The Pichia pastoris enzyme production platform: From combinatorial library screening to bench-top fermentation on residual cyanobacterial biomass

The demand for industrial enzymes is continually rising, fueled by the growing need to shift towards more sustainable industrial processes. However, making efficient enzyme production strains and identifying optimal enzyme expression conditions remains a challenge. Moreover, the production of the enzymes themselves comes with unavoidable impacts, e.g., the need to utilize secondary feedstocks. Here, we take a more holistic view of bioprocess development and report an integrative approach that allows us to rapidly identify improved enzyme expression and secretion conditions and make use of cyanobacterial waste biomass as feed for supporting Pichia pastoris fermentation. We demonstrate these capabilities by producing a phytase secreted by P. pastoris that is grown on cyanobacterium hydrolysate and buffered glycerol-complex (BMGY) medium, with genetic expression conditions identified by high-throughput screening of a randomized secretion library. When our best-performing strain is grown in a fed-batch fermentation on BMGY, we reach over 7 000 U/mL in three days.