Synthetic activation of yeast stress response improves secretion of recombinant proteins.

Yeasts, such as Pichia pastoris (syn Komagataella spp.), are particularly suitable expression systems for emerging classes of recombinant proteins. Among them, recombinant antibody fragments, such as single-chain variable fragments (scFv) and single-domain antibodies (VHH), are credible alternatives to monoclonal antibodies. The availability of powerful genetic engineering and synthetic biology tools has facilitated improvement of this cell factory to overcome certain limitations. However, cell engineering to improve secretion often remains a trial-and-error approach and improvements are often specific to the protein produced. Where multiple genetic interventions are needed to remove bottlenecks in the process of recombinant protein secretion, this leads to a high number of combinatorial possibilities for creation of new production strains. Therefore, our aim was to exploit whole transcriptional programs (stress response pathways) in order to simplify the strain engineering of new production strains. Indeed, the artificial activation of the general stress response transcription factor Msn4, as well as synthetic versions thereof, could replace the secretion enhancing effect of several cytosolic chaperones. Greater than 4-fold improvements in recombinant protein secretion were achieved by overexpression of MSN4 or synMSN4, either alone or in combination with Hac1 or ER chaperones. With this concept we were able to successfully engineer strains reaching titers of more than 2.5 g/L scFv and 8 g/L VHH in bioreactor cultivations. This increased secretion capacity of different industrially relevant model proteins indicates that MSN4 overexpression most likely represents a general concept to improve recombinant protein production in yeast.

Pushing and pulling proteins into the yeast secretory pathway enhances recombinant protein secretion.

Yeasts and especially Pichia pastoris (syn Komagataella spp.) are popular microbial expression systems for the production of recombinant proteins. One of the key advantages of yeast host systems is their ability to secrete the recombinant protein into the culture media. However, secretion of some recombinant proteins is less efficient. These proteins include antibody fragments such as Fabs or scFvs. We have recently identified translocation of nascent Fab fragments from the cytosol into the endoplasmic reticulum (ER) as one major bottleneck. Conceptually, this bottleneck requires engineering to increase the flux of recombinant proteins at the translocation step by pushing on the cytosolic side and pulling on the ER side. This engineering strategy is well-known in the field of metabolic engineering. To apply the push-and-pull strategy to recombinant protein secretion, we chose to modulate the cytosolic and ER Hsp70 cycles, which have a key impact on the translocation process. After identifying the relevant candidate factors of the Hsp70 cycles, we combined the push-and-pull factors in a single strain and achieved synergistic effects for antibody fragment secretion. With this concept we were able to successfully engineer strains and improve protein secretion up to 5-fold for different model protein classes. Overall, titers of more than 1.3 g/L Fab and scFv were reached in bioreactor cultivations.

Detection and Elimination of Cellular Bottlenecks in Protein-Producing Yeasts.

Yeasts are efficient cell factories and are commonly used for the production of recombinant proteins for biopharmaceutical and industrial purposes. For such products high levels of correctly folded proteins are needed, which sometimes requires improvement and engineering of the expression system. The article summarizes major breakthroughs that led to the efficient use of yeasts as production platforms and reviews bottlenecks occurring during protein production. Special focus is given to the metabolic impact of protein production. Furthermore, strategies that were shown to enhance secretion of recombinant proteins in different yeast species are presented.

The impact of ERAD on recombinant protein secretion in Pichia pastoris (syn Komagataella spp.).

The yeast Pichia pastoris (syn. Komagataella spp.) is a popular cell factory for recombinant protein production. Yeasts in general provide a good starting point for cell factory engineering. They are intrinsically robust and easy to manipulate and cultivate. However, their secretory pathway is not evolutionarily adapted to high loads of secretory protein. In particular, more complex proteins, like the antibody fragment (Fab) used in this study, overwhelm the folding and secretion capacity. This triggers cellular stress responses, which may cause excessive intracellular degradation. Previous results have shown that, in fact, about 60 % of the newly synthesized Fab is intracellularly degraded. Endoplasmic reticulum-associated protein degradation (ERAD) is one possible intracellular degradation pathway for proteins aimed for secretion. We therefore targeted ERAD for cell factory engineering and investigated the impact on recombinant protein secretion in P. pastoris. Three components of the ERAD-L complex, which is involved in the degradation of luminal proteins, and a protein involved in proteasomal degradation, were successfully disrupted in Fab-secreting P. pastoris. Contrary to expectation, the effect on secretion was marginal. In the course of more detailed investigation of the impact of ERAD, we took a closer look at the intracellular variants of the recombinant protein. This enabled us to further zero in on the issue of intracellular Fab degradation and exclude an overshooting ER quality control. We propose that a major fraction of the Fab is actually degraded before entering the secretory pathway.

GoldenPiCS: a Golden Gate-derived modular cloning system for applied synthetic biology in the yeast Pichia pastoris.

BACKGROUND: State-of-the-art strain engineering techniques for the host Pichia pastoris (syn. Komagataella spp.) include overexpression of homologous and heterologous genes, and deletion of host genes. For metabolic and cell engineering purposes the simultaneous overexpression of more than one gene would often be required. Very recently, Golden Gate based libraries were adapted to optimize single expression cassettes for recombinant proteins in P. pastoris. However, an efficient toolbox allowing the overexpression of multiple genes at once was not available for P. pastoris. METHODS: With the GoldenPiCS system, we provide a flexible modular system for advanced strain engineering in P. pastoris based on Golden Gate cloning. For this purpose, we established a wide variety of standardized genetic parts (20 promoters of different strength, 10 transcription terminators, 4 genome integration loci, 4 resistance marker cassettes). RESULTS: All genetic parts were characterized based on their expression strength measured by eGFP as reporter in up to four production-relevant conditions. The promoters, which are either constitutive or regulatable, cover a broad range of expression strengths in their active conditions (2-192% of the glyceraldehyde-3-phosphate dehydrogenase promoter P GAP ), while all transcription terminators and genome integration loci led to equally high expression strength. These modular genetic parts can be readily combined in versatile order, as exemplified for the simultaneous expression of Cas9 and one or more guide-RNA expression units. Importantly, for constructing multigene constructs (vectors with more than two expression units) it is not only essential to balance the expression of the individual genes, but also to avoid repetitive homologous sequences which were otherwise shown to trigger "loop-out" of vector DNA from the P. pastoris genome. CONCLUSIONS: GoldenPiCS, a modular Golden Gate-derived P. pastoris cloning system, is very flexible and efficient and can be used for strain engineering of P. pastoris to accomplish pathway expression, protein production or other applications where the integration of various DNA products is required. It allows for the assembly of up to eight expression units on one plasmid with the ability to use different characterized promoters and terminators for each expression unit. GoldenPiCS vectors are available at Addgene.

Systems biotechnology for protein production in Pichia pastoris.

The methylotrophic yeast Pichia pastoris (syn. Komagataella spp.) is one of the most important production systems for heterologous proteins. After the first genome sequences were published in 2009, tremendous effort was made to establish systems-level analytical methods. Methylotrophic lifestyle was one of the most thoroughly investigated topics, studied at the levels of transcriptome, proteome and metabolic flux. Also the responses of P. pastoris to environmental stress conditions experienced during high cell density production processes were studied. Metabolomics and flux analysis revealed the plasticity of the cellular metabolism in its adaption to the production of foreign proteins and served as blueprints for subsequent cell engineering and/or process design. The transcriptional response elicited by overexpression of heterologous proteins seems to depend on the nature and complexity of the recombinant product. Based on these data, novel targets for strain engineering could be deduced from transcriptomics and proteomics data mining and effectively enhanced protein secretion. Transcriptional regulation data also served as a valuable resource to identify novel promoters with the desired regulatory characteristics. This review aims to provide a comprehensive overview of systems biology applications in P. pastoris ranging from increased understanding of cell physiology to improving recombinant protein production in this cell factory.