Transcriptional reprogramming with endogenous cis- and trans-elements for efficient food protein expression from safe substrates in Komagataella phaffii.

Sustainable economy and social development require a paradigm shift in the modes of production of edible proteins. Komagataella phaffii is a preferred host for food protein expression. However, its methanol-dependent production mode and host improvement with exogenous bioelements pose challenges for the manufacturing and approval of bulk food proteins. In this study, we report an endogenous streamlined promoter-regulator integrated transcriptional (ESPRIT) system for efficient expression on non-methanol substrates in K. phaffii. A strong and simplified PAOX1 variant, PAA13-t13, was obtained through core promoter mining and streamlining of upstream regulatory sequences, with a 57% reduction in promoter length and expression increased to 3.4-fold that of PAOX1 under methanol conditions. Overexpression of the transactivator Mit1 enabled PAA13-t13 to drive high-level expression on glucose, glycerol, and ethanol. Endogenous zinc-finger transactivators were screened from genome, and their activation regions were fused with the DNA-binding domain of Mit1, resulting in 25 synthetic transactivators. Combinatory function of the engineered endogenous promoter and transactivator established the ESPRIT system, which achieved 6.8-fold and 10.1-fold expression levels on glucose that of methanol-inducible PAOX1 on methanol and constitutive PGAP on glucose, respectively. It also allowed efficient production of three food proteins under methanol-free conditions. In the bioreactor, the ESPRIT system produced 5.14 g/L β-lactoglobulin and 1.95 g/L brazzein on glucose, achieving 3.0-fold and 1.9-fold as compared to the commercial methanol-dependent PAOX1 system, respectively. These results established the ESPRIT system as an alternative expression platform for efficient food protein production from non-methanol substrates in K. phaffii.