Tezin Türü: Yüksek Lisans
Tezin Yürütüldüğü Kurum: Orta Doğu Teknik Üniversitesi, Fen Bilimleri Enstitüsü, Türkiye
Tezin Onay Tarihi: 2019
Tezin Dili: İngilizce
Öğrenci: OMAR WEHBE AL MASRI WEHBE AL MASRI
Danışman: Pınar Çalık
Özet:
In recombinant protein (r-protein)
production, the host Pichia pastoris (Komagataella
phaffii) is used with the methanol-inducible alcohol oxidase 1 promoter (PAOX1), one of the strongest naturally occurring promoters, due to its tight regulation and exceptional strength. For the pharmaceutical and food sectors,
since the use of methanol as a substrate in r-protein productions may
complicate separation processes, and required
documentations, methanol-free production by green bio-processes is encouraged but
challenging. In this
context, using the non-toxic carbon and energy source ethanol under our newly
developed alcohol
dehydrogenase 2 (ADH2)
hybrid
promotor PADH2-Cat8-L2,
ethanol feeding strategies were developed for rhGH production by P. pastoris. Prior to pilot-scale
experiments, the effects of ethanol on the growth of P. pastoris were investigated in laboratory-scale
batch-bioreactors at T=30°C and N=200 min-1 (rpm). An increase in ethanol
concentration lead to an increase in the final cell concentration, however, at
CEtOH > 3 g L-1, the substrate inhibited the growth
marked by a decrease in specific cell growth rates and the longer cultivation time
required to reach the same cell densities. This inhibition behavior was also
mathematically modelled where the Haldane model was able to clearly depict the
inhibitory effect of ethanol at high concentrations with a critical substrate
concentration of 1.97 g L‑1 and a maximum specific growth rate of
0.194 h-1. The effects of two different continuous feed
stream (CFS) operational strategies on the specific growth rate, specific
ethanol uptake rate and specific rhGH production rate were investigated as
follows: CFS
designed with three pre-determined specific growth rate (µ) values of 0.020,
0.035 and 0.050 h-1, and ethanol-stat CFS designed with three
constant CEtOH values of 0.5, 1.0, and 1.5 g L-1 in the
cultivation medium. In the ethanol-stat fed-batch bioreactors, approximately 1.6-fold higher maximum cell
concentration and 2.1-fold higher maximum rhGH concentration (91 mg L-1)
were obtained compared to those obtained by the fed-batch bioreactors
designed with pre-determined µ values.
In addition, 2-fold higher maximum volumetric productivity was obtained by the
ethanol-stat fed-batch bioreactors. The ethanol-stat strategy of CEtOH,set=0.5
g L-1 was the most favorable operational strategy due to it having
the highest rhGH titer, along with high volumetric productivity and protein on
biomass yield (Yp/x). In addition, high ethanol concentrations
inhibited the specific growth rate, the specific ethanol uptake rate and the
protein production rate, where the inhibition pattern was mathematically
described according to the Haldane model. The relationship between the specific
ethanol uptake rate and the specific growth rate was described by the linear
Pirt model, whereas the relationship between the specific protein production
rate and the specific growth rate was not linear, and thus could not be
described by the Luedeking-Piret model. Lastly, cell generation and rhGH production were simulated based on the Haldane
models relating the specific growth rate and specific rhGH production rate to
the residual ethanol concentration.