Pichia pastoris'in etanol tüketim yolunda yapılan metabolik mühendisliğinin rekombinant protein üretimi ve merkezi metabolizmadaki transkripsiyon düzeylerine etkisi

The aim of this MSc thesis is to increase the ethanol uptake rate and decrease by-product formation in the ethanol utilization (EUT) pathway and acquire a smoothly operating intracellular reaction network in the target microorganism. A metabolically engineered novel host, Pichia pastoris, was developed by overexpression of the two EUT pathway enzymes, alcohol dehydrogenase 2 (ADH2) and Acetyl-CoA synthetase (ACS1) that catalyze rate-limiting reactions, one by one and together, to understand how they affect the ethanol uptake rate, cell growth, and by-product formation. The metabolically engineered novel Pichia pastoris expression systems constructed were denoted by; i) ADH2-OE, ii) ACS1-OE, iii) ADH2-OE + ACS1-OE. The influence of ADH2 overexpression on the growth of P. pastoris strains constructed with PADH2-wt::ADH2 was investigated in batch cultivations on 2% (v/v) ethanol, with two separate colonies carrying 3 and 9 ADH2 gene copies, named as ADH2-OE-C3 and ADH2-OE-C9, respectively. At the end of the fermentation, compared to P. pastoris X-33 strain, 1.1-fold and 1.35-fold lower cell concentrations were obtained with ADH2-OE-C3 and ADH2-OE-C9, respectively. At t = 36 h of the fermentation, compared to P. pastoris X-33, acetic acid concentrations were 1.46-fold and 3.97-fold higher in ADH2-OE-C3 and ADH2-OE-C9, respectively; while 1.18-fold and 2.28-fold higher acetaldehyde concentrations were obtained with ADH2-OE-C3 and ADH2-OE-C9, respectively. The effects of ADH2 and ACS1 overexpression on the growth of P. pastoris were investigated in batch cultivations on 1% (v/v) ethanol using metabolically engineered strains: i) ADH2-OE, ii) ACS1-OE, and iii) ADH2-OE and ACS1-OE. At the end of the fermentation, compared to ADH2-OE and ADH2-OE+ACS1-OE, 1.08-fold and 1.12-fold higher cell concentrations were obtained with ACS1-OE. Furthermore, specific ethanol uptake rate (qEtOH) at t = 9 h for ADH2-OE and ADH2-OE + ACS1-OE strains were 0.52 and 0.35 g g-1 h-1, respectively. Compared to P. pastoris X-33 (qEtOH = 0.19 gg-1h-1), showing that ADH2 overexpression significantly increased ethanol uptake rate. 1.49-fold and 1.78-fold decrease in excreted acetic acid concentrations compared to P. pastoris X-33 and ADH2-OE were obtained with ACS1-OE at t = 36 h of the fermentation. The effects of overexpression of ADH2 and ACS1 on transcription levels of central metabolism were also investigated. Three genes encoding crucial enzymes in ethanol utilization, ADH2, ALD4, and ACS1, were upregulated in all three metabolically engineered strains. Finally, to investigate the effect of ACS1 overexpression on r-protein production, a novel host strain was constructed in which the gene of the reporter r-protein mApple was expressed under the control of PADH2-Cat8-L2, and ACS1 was overexpressed under PAOX1/Cat8-L3. ACS1-OE-C4 increased mApple production 1.2-fold at t= 24 h of the fermentation, and 1.32-fold at t=45 h of the fermentation.