Pichia pastoris Mut(S) strains are prone to misincorporation of O-methyl-L-homoserine at methionine residues when methanol is used as the sole carbon source.
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.


CONCLUSIONS
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.
Ablynx NV, Technologiepark 21, 9052, Zwijnaarde, Belgium. peter.schotte@ablynx.com.
Peter
P
Schotte
Ablynx NV, Technologiepark 21, 9052, Zwijnaarde, Belgium.
Isabelle
I
Dewerte
Ablynx NV, Technologiepark 21, 9052, Zwijnaarde, Belgium.
Manu
M
De Groeve
Ablynx NV, Technologiepark 21, 9052, Zwijnaarde, Belgium.
Saskia
S
De Keyser
Ablynx NV, Technologiepark 21, 9052, Zwijnaarde, Belgium.
Veronique
V
De Brabandere
Ablynx NV, Technologiepark 21, 9052, Zwijnaarde, Belgium.
Patrick
P
Stanssens
D042931:Aldehyde Oxidase / Q000235:genetics; D002244:Carbon / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D005656:Fungal Proteins / Q000235:genetics; D005838:Genotype; D006714:Homoserine / Q000031:analogs & derivatives* / Q000096:biosynthesis / Q000737:chemistry; D000432:Methanol / Q000378:metabolism*; D008715:Methionine / Q000737:chemistry / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D010957:Plasmids / Q000235:genetics / Q000378:metabolism; D061905:Single-Domain Antibodies / Q000032:analysis / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
D005656:Fungal Proteins; D061905:Single-Domain Antibodies; C037855:methoxine; D006714:Homoserine; D002244:Carbon; D008715:Methionine; D042931:Aldehyde Oxidase; D000432:Methanol
Methionine; Modifications; Nanobody; O-methyl-L-homoserine; Pichia pastoris