Engineering Analysis Of Pichia Pastoris Fermentation

Abstract

In recent years, several industrial yeasts, owing to their robust growth and certain other characteristics, have been developed as recombinant host systems for commercial production of heterologous proteins. One such yeast Pichia pastoris has proven to be an excellent host for production of secreted and intracellular proteins (Cereghino and Cregg. 2000). The increasing popularity of this particular expression system can be attributed to several factors, most importantly: (1) the simplicity of techniques needed for the molecular genetic manipulation of Pichia pastoris and their similarity to those of Saccharomyces cerevisiae, one of the most well-characterized experimental systems in modern biology;(2) the ability of Pichia pastoris to produce foreign proteins at high levels, either intracellularly or extracellularly; and (3) the capability of performing many eukaryotic post-translational modifications, such as glycosylation, disulfide bond formation and proteolytic processing. The expression level for a given recombinant protein produced by Pichia pastoris seems to be determined largely by its inherent properties such as amino acid sequence, the tertiary structure and the site for expression (Sreekrishna et al.,1997). The attempts on increasing the protein expression levels by far are focused on genetic manipulations to enhance the gene expression and protein stability. Although this is crucial, there is ample scope to improve the productivity of Pichia pastoris fermentations by undertaking a systematic program of optimizing the entire fermentation process. This work aims at undertaking such a program by focusing on strategy to identify and to characterize trends in the behavior of the system. It can be expected that by addressing the process as a whole, rather than narrowly focusing on the protein expression alone, the methodology proposed here can simplify process scale-up and can be applied to several products made by the same host. Pichia pastoris is methylotrophic yeast. In the Pichia pastoris fermentation, the limiting carbon source is glycerol, method or mixture of both. It can grow on methanol as a sole carbon and energy source. It possesses a highly inducible methanol utilization pathway. The first step in the metabolism of methanol is the oxidation of methanol to formaldehyde using molecular oxygen by alcohol oxidase (AOX). AOX, the first enzyme of the pathway, accounts for up to 35% of the total protein in cells grown on limited amounts of methanol. The enzymes undetectable in cells grown on glucose, ethanol or glycerol. There are two genes in Pichia pastoris that code for AOX: AOXI. The AOXI gene product accounts for the majority of alcohol oxidase activity in the cell. This highly inducible and stringently regulated AOXI promoter has been used to construct expression vectors for the production of heterologous proteins in Pichia pastoris. Although some foreign proteins have expressed well in shake-flask cultures, expression levels are typically low compared to fomenter cultures. There are several key aspects of Pichia pastoris fermentations: 1. Fed-batch operation – Controlled addition of glycerol, methanol or mixture thereof. In general, strains are grown initially in a defined medium containing glycerol as its carbon source (growth phase). During this phase, biomass accumulates but heterogonous gene expression is fully repressed. Upon depletion of glycerol, a transition phase is initiated in which additional glycerol is fed to the culture at a growth-limiting rate. Finally, method a mixture of glycerol and methanol is fed to the culture to induce expression (induction phase). The duration of individual substrate feeds, the amount and mode of feeding are critical to optimal fermentation performance. 2.Online measurement and control-One of the most important key parameters in Pichia pastor is expression system is the methanol concentration. Monitoring and controlling this variable are important because high levels of this inductor substrate can be toxic to the cells and low levels of methanol may not be enough to initiate the AOX transcription (Cereghino and Cregg, 2000) This research work aims at investigation the above mentioned aspects by conduction an in depth engineering analysis of the Pichia Pastoris fermentations.