Cutinase Production in Komagataella phaffii (Pichia pastoris): Performance Differences Between Host Strains

The Pichia system has been exploited for decades as a host for recombinant protein production, but there is still an information gap regarding problems that may arise with its use. The application of strains based on the methanol-induced alcohol oxidase 1 (AOX1) promoter may represent a safety issue, and its performance varies among strains. In this study, the ability of a Komagataella phaffii MutS KM71H strain to produce recombinant cutinases was evaluated and compared to that of the more widely used Mut+ X-33 strain. The effects of the nature of the cutinase (ANCUT1 and ANCUT3, from Aspergillus nidulans), methanol level, and inoculum concentrations were evaluated in shake flasks containing a complex medium. Higher activities and volumetric cutinase productivity were observed at lower induction cell densities (0.5%) for the MutS KM71H aox1::pPICZα-A-ANCUT1 strain, while a higher one (2%) yielded better results in KM71H aox1::pPICZα-A-ANCUT3. The best inoculum and inducer conditions for both strains yielded similar results. The behavior of the different cutinases in the MutS or Mut+ genetic background was opposed: strain KM71H aox1::pPICZα-A-ANCUT3 produced 19% more activity than strain X-33 aox1::pPICZα-A-ANCUT3, while the ANCUT1 containing strain produced significantly higher activity in the X-33 Mut+ strain. These results indicate that MutS strains are viable host options without the complications of rapidly growing methanol strains. The effect of the gene structure being expressed is a phenomenon that needs further exploration.