Naturally occurring novel promoters around pyruvate branch-point for recombinant protein production in Pichia pastoris (Komagataella phaffii): Pyruvate decarboxylase- and pyruvate kinase- promoters

Abstract

Novel strong promoter discovery is crucial for the design of novel strains of the industrial yeast Pichia pastoris for recombinant protein (r-protein) production. In order to remedy the deficiency, transcriptome and proteome data of P. pastoris were analysed. Genes having higher expression levels than glyceraldehyde-3-phosphate-dehydrogenase (GAP) gene were identified as promoter sources. Pyruvate kinase- (PPYK) and pyruvate decarboxylase- (PPDC) promoters around pyruvate-node were determined as promising candidates, and in silico analysis of the putative promoter regions was performed. The putative promoters were introduced into the plasmid pGAPZαA::hGH harboring human growth hormone (hGH) gene, instead of PGAP. Single-copy hGH gene carrying strains under PPYK, PPDC, and for comparison under PGAP were tested in high-cell-density rhGH fermentations, respectively, abbreviated as BRPYK, BRPDC, and BRGAP. Comparative promoter strength assessment was made by the mRNA-transcription-copy-number (mTCN) and r-protein concentration measurements, and by constrained flux analysis. PPDC and PPYK exhibited higher activity compared to PGAP. Highest rhGH titer was obtained in BRPDC as 122 mg dm−3 at t = 15 h, in BRPYK as 101 mg dm−3 at t = 12 h, and in BRGAP as 58 mg dm−3 at t = 9 h; while, with PPDC and PGAP the mTCNs were high and close. The flux distributions demonstrated regulatory effects of the novel promoters in the engineered strains and validated cross-pathway regulatory interactions.