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- (P-PYK) and pyruvate decarboxylase- (P-PDC) 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 pGAPZ alpha A::hGH harboring human growth hormone (hGH) gene, instead of P-GAP. Single-copy hGH gene carrying strains under P-PYK P-PDC, and for comparison under P-GAP 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. P-PDC and P-PYK exhibited higher activity compared to P-GAP. 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 P-PDC and P-GAP 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. (C) 2018 Elsevier B.V. All rights reserved.