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Table 2 Methoxine incorporation in the MutS and O-acetyl homoserine sulfhydrylase knock-out strains

From: 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

Strain

Genotype

Source

Nanobody B

Titers (g l−1)

% Δ−16 Da variant

CBS7435MutS

aox1

[7]

2.4 ± 0.7

0.90 ± 0.17

CBS7435MutSΔOAHS

aox1 met25

This study

2.1 ± 0.2

0

  1. Nanobody B was produced in the CBS7435MutS and in the CBS7435MutS strain with an inactivation of O-acetyl homoserine sulfhydrylase gene (CBS7435MutSΔOAHS) in shake flasks using complex media. Titers of Nanobody B were analyzed via a proteinA cleanup step followed by OD280 measurement. Relative abundance of the methoxine variant was analysed via RP-HPLC followed by total mass measurement by ESI-Q-TOF-MS. The methoxine variant of Nanobody B was only produced in the MutS strain and absent when O-acetyl homoserine sulfhydrylase was inactivated. Experiments were performed in triplicate