Combining Protein and Strain Engineering for the Production of Glyco-Engineered Horseradish Peroxidase C1A in Pichia pastoris.

Horseradish peroxidase (HRP), conjugated to antibodies and lectins, is widely used in medical diagnostics. Since recombinant production of the enzyme is difficult, HRP isolated from plant is used for these applications. Production in the yeast Pichia pastoris (P. pastoris), the most promising recombinant production platform to date, causes hyperglycosylation of HRP, which in turn complicates conjugation to antibodies and lectins. In this study we combined protein and strain engineering to obtain an active and stable HRP variant with reduced surface glycosylation. We combined four mutations, each being beneficial for either catalytic activity or thermal stability, and expressed this enzyme variant as well as the unmutated wildtype enzyme in both a P. pastoris benchmark strain and a strain where the native α-1,6-mannosyltransferase (OCH1) was knocked out. Considering productivity in the bioreactor as well as enzyme activity and thermal stability, the mutated HRP variant produced in the P. pastoris benchmark strain turned out to be interesting for medical diagnostics. This variant shows considerable catalytic activity and thermal stability and is less glycosylated, which might allow more controlled and efficient conjugation to antibodies and lectins.

Development of a mixed feed strategy for a recombinant Pichia pastoris strain producing with a de-repression promoter.

BACKGROUND: Recombinant protein production in the yeast Pichia pastoris is usually based on the alcohol oxidase promoters pAOX1 and pAOX2, which are regulated by methanol and strongly repressed by other C-sources, like glycerol and glucose. However, the use of methanol brings several disadvantages, which is why current trends in bioprocess development with P. pastoris are focussing on minimizing the required amount of methanol or even avoid its employment. In this respect novel promoter systems which do not rely on methanol have been investigated and promoter variants were designed to fine-tune gene expression. Amongst these novel promoter systems, mutated AOX promoters, which are regulated by available carbon source concentration (so-called de-repressed promoters), are currently raising attention. However, the main disadvantage of such a production system is that expression and growth usually cannot happen concomitantly resulting in low space-time-yields. RESULTS: Here we show the development of a mixed-feed strategy for an industrial recombinant P. pastoris de-repression strain aiming at increased productivity and maximum space-time-yield. By doing dynamic experiments we determined a ratio between the specific substrate uptake rates of glycerol and sorbitol allowing a more than 2-fold increased productivity compared to the conventional single substrate de-repression strategy. CONCLUSION: Based on our results we recommend adjusting q(s glycerol) = 0.04 g g(-1) h(-1) and q(s sorbitol) = 0.055 g g(-1) h(-1) to obtain highest productivity with a P. pastoris de-repression strain. Our methodological approach of designing mixed-feed strategies based on physiological strain characterization using dynamic experiments proved to be beneficial.

Optimizing cofactor availability for the production of recombinant heme peroxidase in Pichia pastoris.

BACKGROUND: Insufficient incorporation of heme is considered a central impeding cause in the recombinant production of active heme proteins. Currently, two approaches are commonly taken to overcome this bottleneck; metabolic engineering of the heme biosynthesis pathway in the host organism to enhance intracellular heme production, and supplementation of the growth medium with the desired cofactor or precursors thereof to allow saturation of recombinantly produced apo-forms of the target protein. In this study, we investigated the effect of both, pathway engineering and medium supplementation, to optimize the recombinant production of the heme protein horseradish peroxidase in the yeast Pichia pastoris. RESULTS: In contrast to studies with other hosts, co-overexpression of genes of the endogenous heme biosynthesis pathway did not improve the recombinant production of active heme protein. However, medium supplementation with hemin proved to be an efficient strategy to increase the yield of active enzyme, whereas supplementation with the commonly used precursor 5-aminolevulinic acid did not affect target protein yield. CONCLUSIONS: The yield of active recombinant heme peroxidase from P. pastoris can be easily enhanced by supplementation of the cultivation medium with hemin. Thereby, secreted apo-species of the target protein are effectively saturated with cofactor, maximizing the yield of target enzyme activity.

Glyco-variant library of the versatile enzyme horseradish peroxidase.

When the glycosylated plant enzyme horseradish peroxidase (HRP) is conjugated to specific antibodies, it presents a powerful tool for medical applications. The isolation and purification of this enzyme from plant is difficult and only gives low yields. However, HRP recombinantly produced in the yeast Pichia pastoris experiences hyperglycosylation, which impedes the use of this enzyme in medicine. Enzymatic and chemical deglycosylation are cost intensive and cumbersome and hitherto existing P. pastoris strain engineering approaches with the goal to avoid hyperglycosylation only resulted in physiologically impaired yeast strains not useful for protein production processes. Thus, the last resort to obtain less glycosylated recombinant HRP from P. pastoris is to engineer the enzyme itself. In the present study, we mutated all the eight N-glycosylation sites of HRP C1A. After determination of the most suitable mutation at each N-glycosylation site, we physiologically characterized the respective P. pastoris strains in the bioreactor and purified the produced HRP C1A glyco-variants. The biochemical characterization of the enzyme variants revealed great differences in catalytic activity and stability and allowed the combination of the most promising mutations to potentially give an unglycosylated, active HRP C1A variant useful for medical applications. Interestingly, site-directed mutagenesis proved to be a valuable strategy not only to reduce the overall glycan content of the recombinant enzyme but also to improve catalytic activity and stability. In the present study, we performed an integrated bioprocess covering strain generation, bioreactor cultivations, downstream processing and product characterization and present the biochemical data of the HRP glyco-library.

Microbials for the production of monoclonal antibodies and antibody fragments.

Monoclonal antibodies (mAbs) and antibody fragments represent the most important biopharmaceutical products today. Because full length antibodies are glycosylated, mammalian cells, which allow human-like N-glycosylation, are currently used for their production. However, mammalian cells have several drawbacks when it comes to bioprocessing and scale-up, resulting in long processing times and elevated costs. By contrast, antibody fragments, that are not glycosylated but still exhibit antigen binding properties, can be produced in microbial organisms, which are easy to manipulate and cultivate. In this review, we summarize recent advances in the expression systems, strain engineering, and production processes for the three main microbials used in antibody and antibody fragment production, namely Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli.