Development of a fed-batch process for the production of recombinant proteins in Pichia pastoris under GAP promoter

The most basic operation step for biotechnological products originating from microorganisms is the fermentation process where production is carried out. Fermentation can be defined as a controlled development of cell in a closed area (bioreactor). The bioreactor allows the cells to proliferate aseptically in a defined development environment (media) where important parameters such as dissolved oxygen concentration (DO), pH, redox potential and feed rate can be adjusted. In such a process where many variables play a role, all of the variables can be the target of optimization separately. In bioprocesses where protein production is carried out, expensive raw materials are usually used, energy consumption is high and requires high capital investment. Therefore, even the slightest improvement in the production process will positively affect the total cost of production. Various technical constraints are encountered in high cell density fermentations especially in Pichia pastoris expression system. The most important of these limitations is oxygen transfer. During the systematic analysis of the oxygen limitation response of recombinant P. pastoris strain, it has been found that hypoxic conditions increase the specific product formation rate (qp) by 2.5 fold. In the fed-batch hypoxic feeding protocol that was developed based on this detection, the substrate feed is adjusted according to the ethanol concentration produced by cells from glucose under oxygen limitation. In this study, the effects of specific growth rate (μ) and oxygen restriction (hypoxia) on growth and production parameters of recombinant P. pastoris strain expressing xylanase enzyme under the GAP promoter were investigated. For this purpose, the glucose limited fed-batch productions were performed at 4 different specific growth rates (μmax, 0.8μmax, 0.4μmax and 0.2μmax) under normoxic conditions where %DO value was controlled over %30 with the addition of pure oxygen when necessary. At the same time, hypoxic fed-batch fermentations were carried out where the concentration of ethanol in the culture were kept constant at 4 different values (1.0%, 0.75%, 0.5%, 0.25% v/v) assuming different hypoxia levels were created. In hypoxic fermentations, culture was supplied only with air, %DO was not controlled and allowed to fall to zero. In normoxic fermentations, the specific growth rate remained constant at a certain value (constant-μ strategy) by the addition of the substrate to the medium in a predefined amount (exponential feeding strategy) to allow the cells to develop at the specified rate (exponential growth). For constant-ethanol feeding strategy under hypoxic conditions, the feed rate is automatically adjusted by a sensor which has a probe immersed in the broth and a control unit connected to the feed pump. The sensor retained the ethanol concentration in the broth around the setpoint by controlling directly the action of feed pump. After performing all fermentations, performance of the processes were evaluated comparatively on the basis of specific rates, yields and coefficients related to cell growth, enzyme production and substrate consumptions. In the scope of this study, it was found that hypoxic conditions increased the cellular yield (YPX), specific product formation rate (qp) and volumetric efficiency (Qp) of the process by 2 fold while limiting the biomass formation (lowering the specific growth rate) in the glucose-limited fed-batch production under the control of GAP promoter. In addition to reducing the biomass waste and increasing the production efficiency, hypoxic conditions, especially for large-scale industrial production, have very important advantages such as the lack of pure oxygen supply to the system during the process, reduced heat generation and reduced cooling requirement due to the decrease in the amount of cells formed. In summary, it was shown in detail that fed-batch production under hypoxic conditions has several technical and economical advantages over the standart normoxic strategies which were most commonly applied and considered to be the most efficient for the recombinant productions under GAP promoter.