Cutinase Production in <i>Komagataella phaffii</i> (<i>Pichia pastoris</i>): Performance Differences Between Host Strains

The <i>Pichia</i> 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 <i>Komagataella phaffii</i> Mut^S 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 <i>Aspergillu</i>s <i>nidulans)</i>, 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 Mut^S KM71H <i>aox1</i>::pPICZα-A-<i>ANCUT1</i> strain, while a higher one (2%) yielded better results in KM71H <i>aox1</i>::pPICZα-A-<i>ANCUT3</i>. The best inoculum and inducer conditions for both strains yielded similar results. The behavior of the different cutinases in the Mut^S or Mut⁺ genetic background was opposed: strain KM71H <i>aox1</i>::pPICZα-A-<i>ANCUT3</i> produced 19% more activity than strain X-33 <i>aox1</i>::pPICZα-A-<i>ANCUT3</i>, while the ANCUT1 containing strain produced significantly higher activity in the X-33 Mut⁺ strain. These results indicate that Mut^S 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.