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.