Chapter 3 Development of cellular engineering strategy for improved production
3.1. Background
The dawn of metabolic engineering has ushered the utilization of microorganisms as the platform for producing biopharmaceutical based products. Over last few decades tremendous efforts have been made in developing a recombinant products to meet the criteria for increasing demands in market(Meehl and Stadheim, 2014). In recent years methylotrophic yeast Pichia pastrois emerged as an established host and used as platform for producing numerous heterologous proteins. More than 500 proteins have been successfully produced using Pichia pastoris (Cereghino and Cregg, 2000). Plethora of studies showed that Pichia can express certain proteins in grams per liter level, but some of the proteins are even expressed at very low level, since many genetic as well as physiological factors are detrimental in protein production (Hohenblum et al., 2004).
Over expression of heterologous proteins sometimes hinders the expression mechanism of the host system and results in metabolic burden (Mattanovich et al., 2004). In cells the production rate of protein can be reduced by various physiological limitations such as poor codon usage bias (Hu et al., 2006; Li et al., 2008), cloning under weak promoters (Hohenblum et al., 2004), low gene copy number (Clare et al. 1991), translocation determined by the secretion signal peptide (Koganesawa et al., 2001), inefficiency of protein folding and assembly in the endoplasmic reticulum (ER) (Xu et al., 2005).
In eukaryotic organisms such as Pichia pastoris, the secreted proteins enters secretory pathway in endoplasmic reticulum (ER) and Cytoplasm, ER provides an oxidized condition for posttranslational modifications such as glycosylation, phosphorylation and disulphide bond formation, only protein which surpasses these check points enters exocytic pathway(Samuel et al., 2013). Accumulation of unfolded proteins in ER will triggers the unfolded protein responses (UPR), which in turns aids in proper folding and efficient secretion of heterologous proteins (Guerfal et al., 2010). Several studies showed
that overexpression of heterologous proteins exerts metabolic burden/overloading of host machinery, which leads to suboptimal folding. Later the unfolded protein in ER were subjected to Endoplasmic reticulum associated degradation (ERAD), ultimately contributing in low expression levels of protein (Zhang et al., 2006).
To overcome the problems regarding protein folding and translocation, which tends to be a major bottleneck in protein expression, several studies have been carried out and the research suggests that the molecular chaperones tend to modulate the folded state of the protein. Hsp 70 and Hsp 40 chaperons play a vital role in folding and translocation of proteins in ER and cytoplasm, both chaperons acts in a synergistic pattern. Hsp 70 harbor protein folding through ATP-dependent cycle and the Hsp 40 regulates this cycle by modulating the ATPase activity of Hsp 70. Chaperons such as Ssa1p (Hsp 70) and YDJ1p (Hsp 40), assists in the transportation of intermediate proteins to the ER (Caplan et al., 1992). In yeast Ssa1p aids in protein folding biogenesis through its ability of facilitating foreign protein confirmation, it plays an active role in binding and conformational adjustment of newly synthesized proteins there by helping in the translocation of these proteins to ER. The functionality of Ssa1P is dependent on the presence of its cooperating partner protein YDJ1p(Becker et al., 1996; Chirico et al., 1988; Kang et al., 1990). YDJ1p is a DnaJ homologue, localized in cytosol. It directly interacts with Ssa1p to regulate its chaperone activity by stimulating the ATPase activity of Hsp 70, leading to dissociation of HSP 70-polypeptide complexes(Caplan et al., 1992). Binding immunoglobulin protein (BiP) in the endoplasmic reticulum is a class of Hsp 70 chaperons, BiP is ATP dependent and is assisted by its nucleotide exchanging factor Lhs1p, these chaperons binds to the nascent polypeptide chain, thereby preventing interactions between unfolding regions and neighboring proteins(Yu et al., 2014). BiP is also involved in the ER-Associated Degradation (ERAD) and Unfolded Protein
Response (UPR) pathways generated in protein folding process during stress conditions(Puxbaum et al., 2015). Other major chaperons in ER membrane is Sec complex, which acts as a membrane receptors, helps in directing the translocation of polypeptide chain based on the association of appropriate signal sequence and transferring these sequences to the Sec61 translocation channel (Zhang et al., 2006). Bip is a j partner for Sec63P of Sec complex, both of these proteins work synergistically by binding to the poly peptide chain assisting in proper translocation and prevents its backwards movement through the channel (Sadler et al., 1989; Scidmore et al., 1993).
Protein disulfide isomerase (PDI) chaperons are secreted during UPR conditions, these chaperons are responsible for the formation and isomerization of the disulfide bond and it also involved in catalytically accelerating the proper folding of heterologous proteins (Powers and Robinson, 2007; Sadler et al., 1989; Zhang et al., 2006).
To overcome the barriers of recombinant protein production various strategies like high copy number of the heterologous gene, adopting an appropriate signal peptide, usage of high efficient strong promoters, optimization of cell cultivation were used(Delroisse et al., 2005; Mansur et al., 2005; MURASUGI and TOHMA-AIBA, 2001; Sreekrishna et al., 1997; Villatte et al., 2001). One of the promising techniques for the high yield of heterologous proteins is codon optimization. In many studies it was shown that the expression host have significant impact on the expression level of recombinant protein when there is a difference in codon usage between the native gene sequences(Chang et al., 2006). This codon optimized gene has resulted in higher fold increase in the production of heterologous protein in Pichia. Only very few reports on the production of hIFN-γ in Pichia pastoris are available.
In the present study, the bottleneck regarding the folding, translocation was addressed by over expression of HSP 70 and 40 family chaperons and also by adapting codon
optimized gene the translation related problems can be reduced, further the effect of process parameters viz., temperature, pH, Methanol concentration, Inoculum size and agitation rate on rhIFN-γ production yield were investigated.