Pichia pastoris MutS strains are prone to misincorporation of O-methyl-l-homoserine at methionine residues when methanol is used as the sole carbon source
| Autor: | Saskia De Keyser, Peter Schotte, Isabelle Dewerte, Manu De Groeve, Veronique De Brabandere, Patrick Stanssens |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2016 |
| Předmět: |
0301 basic medicine
Spectrometry Mass Electrospray Ionization Genotype Homoserine Bioengineering Applied Microbiology and Biotechnology Pichia Microbiology Pichia pastoris Fungal Proteins 03 medical and health sciences chemistry.chemical_compound Methionine Chromatography High Pressure Liquid Fungal protein 030102 biochemistry & molecular biology biology Research Methanol Wild type Modifications Single-Domain Antibodies biology.organism_classification Yeast Carbon Aldehyde Oxidase 030104 developmental biology chemistry Biochemistry Nanobody Fermentation O-methyl-l-homoserine Biotechnology Plasmids |
| Zdroj: | Microbial Cell Factories |
| ISSN: | 1475-2859 |
| Popis: | Background Over the last few decades the methylotrophic yeast Pichia pastoris has become a popular host for a wide range of products such as vaccines and therapeutic proteins. Several P. pastoris engineered strains and mutants have been developed to improve the performance of the expression system. Yield and quality of a recombinant product are important parameters to monitor during the host selection and development process but little information is published regarding quality differences of a product produced by different P. pastoris strains. Results We compared titer and quality of several Nanobodies® produced in wild type and MutS strains. Titer in fed-batch fermentation was comparable between all strains for each Nanobody but a significant difference in quality was observed. Nanobodies expressed in MutS strains contained a product variant with a Δ−16 Da mass difference that was not observed in wild type strains. This variant showed substitution of methionine residues due to misincorporation of O-methyl-l-homoserine, also called methoxine. Methoxine is likely synthesized by the enzymatic action of O-acetyl homoserine sulfhydrylase and we confirmed that Nanobodies produced in the corresponding knock-out strain contained no methoxine variants. We could show the incorporation of methoxine during biosynthesis by its addition to the culture medium. Conclusion We showed that misincorporation of methoxine occurs particularly in P. pastoris MutS strains. This reduction in product quality could outweigh the advantages of using Mut strains, such as lower oxygen and methanol demand, heat formation and in some cases improved expression. Methoxine incorporation in recombinant proteins is likely to occur when an excess of methanol is present during fermentation but can be avoided when the methanol feed rate protocol is carefully designed. Electronic supplementary material The online version of this article (doi:10.1186/s12934-016-0499-2) contains supplementary material, which is available to authorized users. |
| Databáze: | OpenAIRE |
| Externí odkaz: |
|
Full text from SpringerLink