Overcoming the problem of heterologous proteins folding to improve the efficiency of yeast bioproducers

In the last few decades, yeasts have been successfully engineered to be an excellent microbial cell factory for producing recombinant proteins with desired properties. This was due to their cost-effective characteristics and the successful application of genomic modification technologies. In addition, yeasts have a conserved post-translational modification pathway among eukaryotic organisms, which ensures the correct folding of recombinant proteins. However, the folding machinery cannot always cope with the load caused by the overexpression of recombinant genes, leading to the accumulation of misfolded proteins, the formation of aggregates and low production. Therefore, the protein-folding capacity of the endoplasmic reticulum (ER) remains one of the main limitations for heterologous protein production in yeast host organisms. However, thanks to many years of effective research of the fundamental mechanisms of protein folding, these limitations have been largely overcome. The study of folding in both model organisms and bioproducers has allowed to identify the molecular factors and cellular mechanisms that determine how a nascent polypeptide chain acquires its three-dimensional functional structure. This knowledge has become the basis for developing new effective techniques for engineering highly productive yeast strains. In this review, we examined the main cellular mechanisms associated with protein folding, such as ER transition, chaperone binding, oxidative folding, glycosylation, protein quality control. We discuss the effectiveness of applying this knowledge to the development of various engineering techniques aimed at overcoming bottlenecks in the protein folding system. In particular, selection of optimal signal peptides, co-expression with chaperones and foldases, modification of protein quality control, inhibition of proteolysis, and other techniques have allowed to enhance the ability of yeast bioproducers to effectively secrete heterologous proteins.