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Current manufacturing methods based on mammalian cell culture do not allow for the precise control of glycosylation and produce a mixture of different glycoforms, some of which are beneficial and some of which are problematic or of little benefit.

By mutating the Pichia pastoris OCH1 gene and introducing heterologous enzyme activities, Pichia has been engineered to produce human-like glycoproteins. This allows for the identification of the most biologically active glycoforms and creates the opportunity to control and optimize the profile of a final product whether it is used as a therapeutic, a research reagent, or some other purpose.

All the operating parameters were kept constant as reported in the reference along the cultivations except the pH, which was controlled at different values of 5, 6 and 7. Main details regarding medium composition, operational parameters and process strategies are described in previous works [ 45 ]. Furthermore, off-line methanol determinations by HPLC were used to validate the control system. Further details about the methanol control system are described elsewhere [ 54 , 55 ].

Biomass concentration was measured by DCW using a protocol described elsewhere [ 56 ]. The amounts of both carbon sources glycerol, methanol and potential fermentation by products were analysed by HPLC. The chromatography procedure and the column specifications were described elsewhere [ 57 ]. Whole cell screenings using diclofenac as substrate were carried out in well microtiter plates.

Fluorescent metabolite formation was quantified measuring the fluorescence of the product 7-hydroxy trifluoromethyl -2 H -chromenone HFC. For bioreactor samples, the enzymatic assays were performed in 50 mL falcon tubes. In this case, the washed cells were diluted to OD of 5 as a standard OD for all reactions. The product-related results of the fermentations are presented in terms of activity units AU.

The mobile phase was composed of Trifluoroacetic acid 0. Gradient-elution was performed at 0. Retention times were For calculations, peak areas of the UV chromatograms recorded at nm were used.

The mobile phase was composed of water acidified with 0. Gradient-elution was performed at 1. To allow a flow rate of 1. Preparative scale conversions were performed as described previously [ 6 , 58 ]. Conversion was performed in a baffled 2 L shake flask over a period of 86 h. To facilitate the interpretation, the C-spectra were proton decoupled to gain better identification of the peaks.

For the signal multiplicities the following abbreviations were most commonly used: s singlet , bs broad singlet , d doublet , t triplet , q quadruplet , m multiplet.

Quaternary carbons are labelled as Cq. This publication reflects the author's view and the Agency is not responsible for any use that may be made of the information it contains. JGM performed all the cultures in bench-scale bioreactors.

CR performed the preparative scale demonstration. All authors read and approved the final manuscript. The authors of TU Graz and bisy declare that they have commercial interests in the use of bidirectional promoters for protein coexpression.

Bisy GmbH owns and commercializes tools for recombinant bidirectional coexpression of genes. Publisher's Note. Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. National Center for Biotechnology Information , U. Journal List Microb Cell Fact v. Microb Cell Fact. Published online Apr Author information Article notes Copyright and License information Disclaimer.

Anton Glieder, Email: ta. Corresponding author. Received Sep 9; Accepted Apr 7. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

This article has been cited by other articles in PMC. Whole cell hydroxylation of diclofenac by P. Additional file 2. Figure S2A. Figure S2B. Product 1, 13 C NMR Figure S2C. Figure S2D. Product 2, 13 C NMR Abstract Background Currently, the numerous and versatile applications in pharmaceutical and chemical industry make the recombinant production of cytochrome P enzymes CYPs of great biotechnological interest.

Results A scalable bioprocess for fine-tuned co-expression of hCYP2C9 and its essential complementary human cytochrome P reductase hCPR in the yeast Pichia pastoris Komagataella phaffii is presented. Conclusions For the first time a scalable bioprocess for the production of hCYP2C9 whole cell catalysts was successfully designed and implemented in bioreactor cultures, and as well, further tested in a preparative-scale biotransformation of interest.

Supplementary Information The online version contains supplementary material available at Background In order to avoid adverse drug reactions ADRs not only the drug as such, but also the impact of its metabolites on the organism are considered of great importance [ 1 ]. Results and discussion Strain generation Expression systems based on engineered bidirectional promoters offer a simple and quick solution to enable multi-gene co-expression in which the expression of each gene can be optimally tuned towards to achieve the desired objective [ 37 ].

Open in a separate window. Production of P. Preparative scale demonstration Biomass produced by controlled cultivation in bioreactors was applied to evaluate its activity for preparative biotransformation of ibuprofen to its respective CYP2C9 metabolites. Table 2 Main parameters obtained for the preparative scale conversion of ibuprofen. Conclusions Recombinant production of hCYPs has been intensively investigated and it is expected to continue attracting high attention by the pharmaceutical and chemical industry.

Whole cell cultivation High-throughput small scale cultures Microscale 96 deep well cultivation was performed based on Weis et al. Chemostat cultures Chemostat cultures were performed based on the methodology previously described by Garcia-Ortega et al.

Fed-batch cultures Main details regarding medium composition, operational parameters and process strategies are described in previous works [ 45 ]. Analytical methods Biomass determination Biomass concentration was measured by DCW using a protocol described elsewhere [ 56 ]. Carbon source and by-products quantification The amounts of both carbon sources glycerol, methanol and potential fermentation by products were analysed by HPLC.

Whole cell bioconversions High throughput screening HTS Whole cell screenings using diclofenac as substrate were carried out in well microtiter plates. Bioconversion reactor For bioreactor samples, the enzymatic assays were performed in 50 mL falcon tubes. Preparative scale conversion of ibuprofen Preparative scale conversions were performed as described previously [ 6 , 58 ]. Supplementary Information Additional file 1: Figure S1.

Disclaimer This publication reflects the author's view and the Agency is not responsible for any use that may be made of the information it contains. Production of a recombinant carrot antifreeze protein by Pichia pastoris GS and its cryoprotective effects on frozen dough properties and bread quality. Frontiers in Laboratory Medicine , 2 1 , 23— Cultivation strategies to enhance productivity of Pichia pastoris : A review.

Biotechnology Advances , 33 6 , — Journal of Biotechnology , , 54— Heterologous expression of the plant cysteine protease bromelain and its inhibitor in Pichia pastoris. Biotechnology Progress , 33 1 , 54— Vaccine , 25 22 , — Heterologous protein production using the Pichia pastoris expression system. Yeast , 22 4 , — Vaccine , 34 28 , — Expression, purification and characterization of a recombinant antimicrobial peptide Hispidalin in Pichia pastoris.

Protein Expression and Purification , , 19— Scedosporium boydii CatA1 and SODC recombinant proteins, new tools for serodiagnosis of Scedosporium infection of patients with cystic fibrosis. Diagnostic Microbiology and Infectious Disease , 89 4 , — Live attenuated vaccines: Historical successes and current challenges. Virology , , — Infection, Genetics and Evolution , 39 , — Description of Komagataella mondaviorum sp. Antonie Van Leeuwenhoek , , 1— Chinese Journal of Microbiology and Immunology , 36 4 , — Yeast , 18 9 , — Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris.

BMC Biotechnology , 11 1 , Biotechnology and Applied Biochemistry , 52 Pt 3 , — Frontiers in Microbiology , 8 , Patents in therapeutic recombinant protein production using mammalian cells. Recent Patents on Biotechnology , 8 2 , — Biotechnological production of the cell penetrating antifungal PAF peptide in Pichia pastoris. Frontiers in Microbiology , Process Biochemistry , 51 6 , — Expression of nitrile hydratase gene of mutant 4D strain of Rhodococcus rhodochrous PA 34 in Pichia pastoris.

Biocatalysis and Biotransformation , 35 1 , 19— Microbial Cell Factories , 16 1 , Bioresource Technology , , — Photosynthetic biomanufacturing in green algae; production of recombinant proteins for industrial, nutritional, and medical uses. Photosynthesis Research , 3 , — Is Pichia pastoris a realistic platform for industrial production of recombinant human interferon gamma? Biologicals , 45 , 52— Recombinant protein expression in Escherichia coli : Advances and challenges.

Frontiers in Microbiology , 5 , Pichia pastoris expression system: A potential candidate to express protein in industrial and biopharmaceutical domains. Biomedical Letters , 4 1 , 1— Effect of methanol induction and incubation time on expression of human erythropoietin in methylotropic yeast Pichia pastoris.

Makara Journal of Technology , 16 1 , 29— Generation of a functionally distinct Rhizopus oryzae lipase through protein folding memory. PLOS One , 10 5 , 1— Raising expectations for subunit vaccine.

The Journal of Infectious Diseases , 9 , — Applied Microbiology and Biotechnology , 99 24 , — Biotechnology and Bioengineering , 4 , — Yeast quality assessment, management and culture maintenance, Brewing Microbiology pp.

Edinburgh, Scotland, UK: Elsevier. Synthetic and Systems Biotechnology , 2 2 , — Journal of Immunological Methods , , 50— The Pichia pastoris formaldehyde dehydrogenase gene FLD1 as a marker for selection of multicopy expression strains of P.

Gene , , 39— Development of simple random mutagenesis protocol for the protein expression system in Pichia pastoris. Biotechnology for Biofuels , 9 1 , Iranian Journal of Basic Medical Sciences , 21 2 , — T cell memory to vaccination. Vaccines , 6 4 , 84 Frontiers in Microbiology , 6 , Microbiological Research , , 12— Applied Microbiology and Biotechnology , 97 7 , — Functional expression of recombinant goat chymosin in Pichia pastoris bioreactor cultures: A commercially viable alternate.

PLOS One , 12 1 , 1— Microbial Cell Factories , 11 1 , Heterologous expression of the antimyotoxic protein DM64 in Pichia pastoris. Applied Microbiology and Biotechnology , 3 , — Recent advances in the production of recombinant subunit vaccines in Pichia pastoris. Bioengineered , 7 3 , — Efficient production of recombinant glycoprotein D of herpes simplex virus type 2 in Pichia pastoris and its protective efficacy against viral challenge in mice.

Archives of Virology , 3 , — Fused IgY Fc and polysaccharide adjuvant enhanced the immune effect of the recombinant VP2 and VP5 subunits—A prospect for improvement of infectious bursal disease virus subunit vaccine. Protein Expression and Purification , , 67— Bioresource Technology , 4 , — Heterologous expression of three Camellia sinensis small heat shock protein genes confers temperature stress tolerance in yeast and Arabidopsis thaliana.

Plant Cell Reports , 36 7 , — Toxins , 9 9 , Constitutive and secretory expression of the AiiA in Pichia pastoris inhibits Amorphophallus konjac soft rot disease.

American Journal of Molecular Biology , 6 2 , 79— High efficiency transformation by electroporation of Pichia pastoris pretreated with lithium acetate and dithiothreitol. Biotechniques , 36 1 , — Applied Biochemistry and Biotechnology , 5 , — Experimental and Therapeutic Medicine , 12 4 , — Enhanced production of recombinant secretory proteins in Pichia pastoris by optimizing Kex2 P1'site. PLOS One , 8 9 :e Expression of recombinant transglutaminase gene in Pichia pastoris and its uses in restructured meat products.

Food Chemistry , , — Select what you need: A comparative evaluation of the advantages and limitations of frequently used expression systems for foreign genes. Journal of Biotechnology , 3 , — Core—shell protein clusters comprising haemoglobin and recombinant feline serum albumin as an artificial O2 carrier for cats.

Journal of Materials Chemistry B , 6 16 , — Protein Expression and Purification , , 69— Systems biotechnology for protein production in Pichia pastoris. Biotechnology and Bioengineering , 70 1 , 1—8. Bioresource Technology , , 81— Vaccine , 33 20 , — Protective immunity induced by the vaccination of recombinant Proteus mirabilis OmpA expressed in Pichia pastoris. Protein Expression and Purification , , 33— Expression of plectasin in Pichia pastoris and its characterization as a new antimicrobial peptide against Staphyloccocus and Streptococcus.

Protein Expression and Purification , 78 2 , — Functional recombinant human Legumain protein expression in Pichia pastoris to enable screening for Legumain small molecule inhibitors. Protein Expression and Purification , , 12— Characterization of bioactive recombinant antimicrobial peptide parasin I fused with human lysozyme expressed in the yeast Pichia pastoris system.

Enzyme and Microbial Technology , 77 , 61— Microbial Cell Factories , 13 , High level expression and purification of recombinant human serum albumin in Pichia pastoris. Contact RCT: Over 5, Proteins Made in Pichia pastoris. More Than 70 commercial products made in Pichia pastoris on the Market. Pichia Expression Platform.

Frequently Asked Questions The Pichia Expression Platform is the most economical eukaryotic expression system for both secretion and intracellular expression.