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Naturally occurring novel promoters for recombinant protein production around pyruvate branch-point in Pichia pastoris

Çalık, Pınar
Massahi, A.
Novel strong Pichia pastoris promoter discovery is crucial in metabolic engineering for recombinant protein (r-protein) production. Transcriptome and proteome data of P. pastoris were analysed and genes having higher expressions than glyceraldehyde-3-phosphate dehydrogenase (GAP) gene under limited-oxygen-transfer conditions were identified as promoter sources. Two promoters around pyruvate-node were determined as promising candidates, and in silico analysis of putative promoter regions was conducted. Promoter of pyruvate kinase (PPYK) has one transcription start-site with a minimum promoter-score of 0.99; while, promoter of pyruvate decarboxylase (PPDC) has two start-sites each having a minimum promoter-score of 0.91. To drive gene expression, putative promoter-regions were replaced with PGAP in parent plasmid pGAPZαA::hGH, harboring human-growth-hormone (hGH) gene. P. pastoris strains carrying single-copy hGH under PPYK, PPDC, and for comparison under PGAP were tested in high-cell-density rhGH fermentations abbreviated, respectively, as BRPYK, BRPDC, and BRGAP. Promoter strength was evaluated by mRNA transcription-copy-number (mTCN) results, rhGH concentration measurements, and calculated flux distributions. PPDC and PPYK performed higher activity compared to PGAP. Maximum rhGH production was obtained in BRPDC as 122 mg dm−3 at t = 15 h, and then in BRPYK as 101 mg dm−3 at t = 12 h, and in BRGAP as 58 mg dm−3 at t = 9 h; while, mTCN of hGH was the highest with PGAP at t = 15 h, and then with PPDC with the maximum at t = 12 h, and lowest with PPYK. Flux distributions demonstrated perturbation effects of the naturally-occurring-novel-promoters in the engineered systems and validated the cross-pathway regulatory interactions.