Current challenges in recombinant membrane protein production
Determining the optimal expression cassette and culture conditions for maximum MP yield is largely an empirical process. Finding a singular, universal approach is far from realistic when considering the nuances of expression in any microbial platform including yeast. Yeast is the most commonly used eukaryotic system for recombinant protein expression. Prior knowledge of structural features of the target protein is primarily what drives the decision to use yeast over its common prokaryotic counterpart, E. coli . Bacterial and yeast strains, are selected for their well-characterized and robust transcription-translation machinery, ease of culturing and genetic alteration, and their ability to be scaled up to industrial levels. High culture densities can be attained while having direct implications for high-yield protein production. Protein expression targets that have posttranslational modifications (PTMs) critical to their structure and function may benefit from heterologous expression in eukaryotic yeast species. Yeasts are phenotypically better equipped to handle proteins that require PTMs as they are mostly unable to be carried out in prokaryotes. Eukaryotic yeast species such as Saccharomyces cerevisiae (S. cerevisiae ) are equipped with a developed secretory pathway that carries out posttranslational processing such as the addition of sugar residues at asparagine within the consensus motif, Asn-X-Ser/Thr (N -linked glycosylation) and serine and threonine residues (O -linked glycosylation). Glycosylation occurs both during (co-translation) and after (post-translation) synthesis of the polypeptide chain. It can have important ramifications for proper structure and function. Glycosylation is often required for proper protein folding and transport of MPs through the secretory pathway. In some circumstances, glycosylation reportedly increases protein secretion leading to enhanced expression, and it facilitates proper folding by destabilizing the unfolded polypeptide state (Han & Yu, 2015; Shental-Bechor & Levy, 2008). Further, glycosylation reportedly enhances the stability, function and resistance to proteolytic degradation in cellulases (Greene et al., 2015). When MPs are not properly glycosylated and folding is negatively affected, proteins may become prematurely degraded through the ER-associated degradative pathway (Han & Yu, 2015). In S. cerevisiae , improper glycosylation can lead to irreversible misfolding, aggregation and degradation, impacting protein yields. This commonly occurs in S. cerevisiae where proteins often suffer from hyperglycosylation through the addition of excess mannose residues (Conde et al., 2004; Kastberg et al., 2022; Nakamura et al., 1993). Differences in glycosylation between mammalian and yeast cells give rise to changes in glycosylation patterning through differences in the identity of sugar residues attached. This can also impact protein yield through misfolding and premature degradation. Hyperglycosylation can be avoided altogether through site-directed mutagenesis of glycosylated residues, however, amino acid substitutions also introduce some risk of improper folding and degradation (Han & Yu, 2015). Other yeast species have been explored as an alternative to S. cerevisiae since they do not suffer from the same hyperglycosylation effects. Species such as methylotrophic, Pichia pastoris (P. pastoris, syn. Komagataella phaffii ), have been used successfully for high-yield expression. Yarrowia lipolytica (Y. lipolytica ) is an emerging species with potential for industrial applications due to its ability to produce commodity compounds and hydrocarbon-based compounds for biofuels, among others. Though, it has not been extensively studied for its MP production potential. The secretory pathway is considered the rate-limiting step in protein synthesis in yeast. During expression, if proteins become misfolded, they accumulate in the ER and impede the synthesis of new proteins triggering a stress-related response called the unfolded protein response (UPR). When this happens it can induce premature degradation and cell death. These are just some of the factors that must be considered when deciding on the best expression platform when maximum yields are required. E. coli , S. cerevisiae , and P. pastoris (syn. K. phaffii ) are among the best characterized organism genomes reported enabling different recombinant expression conditions to be tested and optimized.
Disulfide bonding is another type of PTM important for the stabilization of tertiary structural contacts in some IMPs. Bacterial expression systems lack an oxidative intracellular environment where disulfide bonds form. S. cerevisiae provides a better platform to support this type of PTM. Where certain PTMs are critical for structure formation and stabilization, S. cereivisiae is principally the better choice. Mammalian proteins are often post-translationally modified compared to prokaryotic proteins, reinforcing the importance of selecting the proper host (Macek et al., 2019).
Another challenge to successful MP expression is establishing optimal cell growth and culture conditions. High-yield recombinant protein production is achieved by applying rigorous growth conditions to coax the host into producing as much protein as possible. Under such conditions, deleterious effects can lead to truncated, misfolded, or degraded protein. For IMPs, overexpression also leads to an overabundance of the target protein in the plasma membrane after trafficking from the secretory pathway. This results in molecular crowding and alters both protein conformation and the morphology of the membrane itself (Chen et al., 2016; Guigas & Weiss, 2016; Löwe et al., 2020; H.-X. Zhou, 2009). The latter can have serious destabilizing effects and potentially lead to premature cell death (Figure 1).
The goals of synthetic biology are directly aimed at addressing challenges in expression and other compounds of industrial importance. It is for this reason that the next section is devoted to discussing techniques that have gained traction in the field. It is meant to suggest a practical set of parameters that can be used to guide the design of gene expression constructs to increase MP yields in yeast.