Harnessing alkaline-pH regulatable promoters for efficient methanol-free expression of enzymes of industrial interest in Komagataella phaffii

Background The yeast Komagataella phaffii has become a very popular host for heterologous protein expression, very often based on the use of the AOX1 promoter, which becomes activated when cells are grown with methanol as a carbon source. However, the use of methanol in industrial settings is not devoid of problems, and therefore, the search for alternative expression methods has become a priority in the last few years. Results We recently reported that moderate alkalinization of the medium triggers a fast and wide transcriptional response in K. phaffii . Here, we present the utilization of three alkaline pH-responsive promoters (p TSA1 , p HSP12 and p PHO89 ) to drive the expression of a secreted phytase enzyme by simply shifting the pH of the medium to 8.0. These promoters offer a wide range of strengths, and the production of phytase could be modulated by adjusting the pH to specific values. The TSA1 and PHO89 promoters offered exquisite regulation, with virtually no enzyme production at acidic pH, while limitation of Pi in the medium further potentiated alkaline pH-driven phytase expression from the PHO89 promoter. An evolved strain based on this promoter was able to produce twice as much phytase as the reference p AOX1 -based strain. Functional mapping of the TSA1 and HSP12 promoters suggests that both contain at least two alkaline pH-sensitive regulatory regions. Conclusions Our work shows that the use of alkaline pH-regulatable promoters could be a useful alternative to methanol-based expression systems, offering advantages in terms of simplicity, safety and economy. ### Competing Interest Statement The authors have declared no competing interest.