Abstract
In order to describe and predict the growth and expression of recombinant proteins by using a genetically modified Pichia pastoris, we developed a number of unstructured models based on growth kinetic equation, fed-batch mass balance and the assumptions of constant cell and protein yields. The growth of P. pastoris on both glycerol and methanol could be represented by Monod kinetic equation. A simple simulation methodology and developed models were shown to satisfactorily describe both growth and production of recombinant human serum albumin (rHSA) using a genetically modified P. pastoris MutS strain. The obtained parameters from curve fitting were reasonable and could be acceptable. Moreover, the same parameter sets obtained by the experiments indicated the rigidity and consistency of the developed models and fermentation approach of this study. With correlation coefficients (r2) exceeding 0.99, the models were able to simulate and predict the cell growth behavior and recombinant protein production by P. pastoris without requiring complex models. Key words: Exponential feed, growth modeling, Monod kinetic equation, Pichia pastoris, recombinant human serum albumin.
Highlights
Pichia pastoris is a methylotrophic yeast which has been successfully used for the heterologous expression of a great number of recombinant proteins
In order to describe and predict the growth and expression of recombinant proteins by using a genetically modified Pichia pastoris, we developed a number of unstructured models based on growth kinetic equation, fed-batch mass balance and the assumptions of constant cell and protein yields
Many model approaches are proposed such as model-based feeding strategy based on mass balance (d’Anjou and Daugulis, 1997; d’Anjou and Daugulis, 2001), macrokinetic modeling based on stoichiometric balance (Ren et al, 2003), model-based specific growth rate (Ren and Yuan, 2005), mix-feed modeling based on growth kinetic (Zhang et al, 2003), growth and protein production modeling based on metabolic flux and metabolic model (Jahic et al, 2002; Sohn et al, 2010; Nocon et al, 2014), growth model optimization using response surface methodology (RSM) (Zhang et al, 2004; Yu et al, 2014) and artificial neural networks (ANNs), fuzzy rule-based systems, or a combination of both (Jenzsch et al, 2006)
Summary
Pichia pastoris is a methylotrophic yeast which has been successfully used for the heterologous expression of a great number of recombinant proteins. A number of strategies have been employed to increase expression of the recombinant proteins in question, for example, optimizing the fermentation medium (Jungo et al, 2007b; Batista et al, 2013), improving the fermentation method (Bushell et al, 2003; Ohya et al, 2005), different feeding strategies (Sinha et al, 2003; Hu et al, 2007), mixed-substrate feeding (d’Anjou and Daugulis, 2000; Jungo et al, 2007a; Arnau et al, 2011; Zalai et al, 2012) and different oxygen supplementation strategies (Charoenrat et al, 2005; Zhang et al, 2005; Charoenrat et al, 2006) Modeling is another approach without more empirical experiments enhancing and optimizing the production of recombinant proteins. Simple simulation of recombinant microorganisms can be taken by kinetic modeling with the aid of computer software (Vinayagam et al, 2015)
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