Metabolic and genome engineering efforts to increase membrane protein yield
In recent years, whole genome methods to increase recombinant protein production have emerged. This has been mostly reported for E. coli . However, S. cerevisiae still remains one of the more popular eukaryotic expression platforms. Both model microbe systems are well-characterized and contain reasonably-sized genomes that enable genetic manipulation to be feasible. As our understanding of the interplay between multiple intracellular processes continues to evolve, this has shed light on additional indirect factors that influence the outcome of protein expression. This requires a top-down systems level analysis of simultaneous cell processes that take place under specific environmental conditions. Metabolic engineering aims to address these challenges. To look at how this approach can be applied to MP expression requires a recognition and understanding of the processes not only directly involved in but also those affected downstream by their synthesis, folding, PTM, and translocation. Transcriptomics and metabolomics analyses are two ways that the effects of growth conditions can be assessed by looking at changes in transcript levels, mRNA, of genes involved in certain metabolic pathways (R. Carlson et al., 2002; Chae et al., 2017; Dromms & Styczynski, 2012; Guan et al., 2018; Park et al., 2005; Trethewey, 2004; Yuan et al., 2018). Similarly, metabolomics is used to assess signature metabolites and their relative quantities, which are by-products from these pathways and interconnected processes. Information can be gleaned from these studies about the pathways that are most affected by expression conditions. Metabolic engineering aims to divert resources that support identified systems away from non-essential pathways and redirecting them toward recombinant expression. The rationale is that this results in prolonged expression leading toward increased production levels. Employing a rational approach to achieve this requires a systems level understanding of the host metabolism including those involved in transcription, translation and flux of metabolites, which also includes proteins. An analysis of the host expression platform helps in deciding which pathways are critically effected during protein expression. As a result of these analyses one strategy is to engineer KO yeast strains that are lacking pathways least critical for survival of the organism. Also, targeting genes responsible for the translation bottleneck created when strong promoters generate higher rates of mRNA synthesis helps alleviate this stress and associated degradation of mRNA. This was successfully demonstrated in E. coli when 36 genes responsible for non-essential functions were selectively knocked out (Sharma et al., 2020; L. Zhou et al., 2022). The results showed that protein expression could be increased by 1.5-fold when carried out at lower temperatures, 25 °C.
Alternatively, knockin (KI) strains also show promise as a means of increasing production. In this way, strains are engineered to carry out important processes that lead to proper protein function. This is a common strategy employed in the latest gene editing technologies such as CRISPR/Cas9. This allows for targeted gene insertion to coax the host into producing gene products that are either non-native to the host organism or to restore gene function that has been lost (Giuliano et al., 2019; Ye et al., 2021). Recently, CRISPR/Cas9 technology was used successfully to introduce the T7 RNA polymerase gene, an established robust expression system, into a strain of E. coli naturally lacking this system (Ye et al., 2021). While some genome-wide strategies have been used successfully in E. coli , an extensive exploration of these methods in S. cerevisiae has not yet been reported. The addition of folding chaperones such as Hsp150 and PDI1 has seen some success, mostly for the recombinant expression of soluble proteins (Kim et al., 2014). Yeast strains deficient in lysosomal compartment proteases along with other organellar proteases is another strategy that has been implemented to circumvent protein degradation, truncation and increase yields by at least 10-fold (Tomimoto et al., 2013). Processes such as glycosylation also have significant effects on protein yield and quality control. Mutagenesis efforts have been directed toward humanizing yeast strains to produce glycosylation patterns on recombinantly expressed proteins similar to those produced in humans (Hamilton & Gerngross, 2007) (Figure 2). As proof of concept, humans, for example, lack the ability to produce sialic acid, N -glycolylneuraminic acid, while other mammals can produce it. This leads to aberrant sialylation. Knocking out its production in mammalian and other cell lines can potentially alleviate this problem. This is an important consideration in the design and production of therapeutic proteins where posttranslational differences in the resulting protein can have severe immunological effects. For MPs along with other non-therapeutic proteins, glycosylation affects folding, stability, half-life and function (Helenius & Aebi, 2001; Mitra et al., 2006). All of which are important considerations for high-yield, high-quality recombinant expression.