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Design and construction of double promoter systems and their use in pharmaceutical protein production in P. Pastoris

Demir, İrem
Intracellular phenomena such as promoter strength, mRNA secondary structure, translation efficiency and codon preference, 5′-untranslated region processing, and protein turnover, have impacts directly on the expression of heterologous genes. Design of multi-promoter expression systems with constituent strong promoters and engineered promoter variants is a novel metabolic engineering strategy for increasing the promoter strength further, and tuning the expression for recombinant protein (r-protein) production with enhanced production and productivity in the yeast P. pastoris. Double-promoter expression systems (DPESs) carrying enhanced green fluorescent protein (eGFP) and red fluorescent protein (mApple) genes were designed and constructed for the synthesis of the heterologous model proteins mApple and eGFP in order to determine and justify the expression period of each promoter that can be either simultaneously, or by consecutively stimulating the changeover from one to another in a biphasic process or via successive-iterations in methanol-free v vi media, on glucose, glycerol or ethanol. A library of expression cassettes was designed with single naturally occurring promoters (NOPs) and novel-engineered promoter variants (NEPVs) having distinct regulatory properties: i) a NEPV of alcohol dehydrogenase1 gene (ADH1), ii) a NEPV of alcohol oxidase 1 gene (AOX1), and iii) PGAP; and P. pastoris DPESs have been constructed as separate expression cassettes. Novel P. pastoris strains were constructed with the DPESs and tested in the fed-batch phase of the fermentation of the carbon sources 2% (v/v) ethanol, excess glucose, and excess glycerol. P. pastoris DPESs having double expression cassettes each having a different antibiotic resistance gene were constructed, denoted by; i) pADH2-Cat8-L2::mApple and pGAP::eGFP, ii) pADH2-Cat8-L2::mApple and pmAOX1::eGFP, iii)pADH2-Cat8-L2::mApple and pADH2-Cat8-L2::mApple, iv) pGAP::eGFP andpGAP::eGFP v) pmAOX1::eGFP and pmAOX1::eGFP. The proteins mApple and eGFP were expressed in the novel P. pastoris strains constructed with: i) pADH2-Cat8-L2::mApple and pGAP::eGFP consecutively and also simultaneously; ii) pADH2-Cat8-L2::mApple and pmAOX1::eGFP bifunctionally and simultaneously; iii)pADH2-Cat8-L2::mApple and pADH2-Cat8-L2::mApple; iv) GAP::eGFP andpGAP::eGFP; v) pmAOX1::eGFP and pmAOX1::eGFP simultaneously as identical-twin promoters. PADH2-Cat8-L2+mAOX1DPES increased the production capacity on ethanol 2.1-fold compared to that with the single NEPVs PADH2-Cat8-L2 and PmAOX1, respectively. With PADH2-Cat8-L2+mAOX1, the expression increased to 1.3-fold on ethanol compared to that with identical-twin promoters. With simultaneously-operating PADH2-Cat8-L2+mAOX1 the expression was 1.6-fold higher than the consecutively-operating PADH2-Cat8-L2+GAP vii on ethanol. Strength of the DPESs were tested in fermentations for extracellular human growth hormone (rhGH) production. Secreted rhGH yields (YP/X, mg/gDW) by novel P. pastoris strains constructed with PADH2-Cat8-L2, PmAOX1, PGAP, PADH2-Cat8-L2+mAOX1, PADH2-Cat8-L2+GAP, and PADH2-Cat8-L2+ADH2-Cat8-L2 were as 2.95, 3.00, 0.13, 4.86, 3.73 and 4.21 mg/g at t = 48 h of the fermentations on ethanol, respectively.