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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 Mut S  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 Mut S  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  Mut S  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.

microbial cell factories

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