Genetic redundancy in the catabolism of methylated amines in the yeast Scheffersomyces stipitis

Abstract

The catabolism of choline as a source of nitrogen in budding yeasts is thought to proceed via the intermediates trimethylamine, dimethylamine and methylamine before the release of ammonia. The present study investigated the utilisation of choline and its downstream intermediates as nitrogen sources in the yeast Scheffersomyces stipitis using a reverse genetics approach. Six genes (AMO1, AMO2, SFA1, FGH1, PICST_49761, PICST_63000) that have previously been predicted to be directly or indirectly involved in the catabolism of methylated amines were individually deleted. The growth of each deletion mutant was assayed on minimal media with methylamine, dimethylamine, trimethylamine or choline as the sole nitrogen source. The two amine oxidase-encoding genes AMO1 and AMO2 appeared to be functionally redundant for growth on methylated amines as both deletion mutants displayed growth on all nitrogen sources tested. However, deletion of AMO1 resulted in a pronounced growth lag on all four methylated amines while deletion of AMO2 only caused a growth lag when methylamine was the sole nitrogen source. The glutathione-dependent formaldehyde dehydrogenase-encoding gene SFA1 was found to be absolutely essential for growth on all methylated amines tested while deletion of the S-formylglutathione hydrolase gene FGH1 caused a pronounced growth lag on dimethylamine, trimethylamine and choline. The putative cytochrome P450 monooxygenase-encoding genes PICST_49761 and PICST_63000 were considered likely candidates for demethylation of di- and trimethylamine but produced no discernable phenotype on any of the tested nitrogen sources when deleted. This study revealed notable instances of genetic redundancies in the choline catabolic pathway, which are discussed.

Keywords: Amine; Metabolism; Reverse genetics; Yeast.

Fig. 1

The proposed pathway for choline assimilation in budding yeasts

Fig. 2

The requirement for amine oxidase genes AMO1 and AMO2 for the utilisation of methylated amines as sole nitrogen sources. Sc. stipitis strains TLSS001 (wildtype control), TLSS005 (Δamo1) and TLSS006 (Δamo2) were cultured in 3 ml RSNLD medium supplemented with 10 mM of the indicated nitrogen source (initial OD₆₀₀ 0.005). Samples were incubated in a shaker set at 30 °C, 200 r.p.m., and OD₆₀₀ was measured after 6, 12 and 18 days. Growth assays were performed in triplicate with error bars indicating one standard deviation

Fig. 3

The requirement for the glutathione-dependent formaldehyde dehydrogenase gene SFA1 and the S-formylglutathione hydrolase gene FGH1 for the utilisation of methylated amines as sole nitrogen sources. a A simplified overview of the glutathione-dependent formaldehyde detoxification pathway. Only the thiol group of glutathione (-SH) is shown. b Sc. stipitis strains TLSS001 (wildtype control), TLSS007 (Δsfa1) and TLSS008 (Δfgh1) were cultured in 3 ml RSNLD medium supplemented with 10 mM of the indicated nitrogen source (initial OD₆₀₀ 0.005). Samples were incubated in a shaker set at 30 °C, 200 r.p.m., and OD₆₀₀ was measured after 6, 12 and 18 days. Growth assays were performed in triplicate with error bars indicating one standard deviation

Fig. 4

The requirement for the putative cytochrome P450 (CYP) monooxygenase genes PICST_49761 and PICST_63000 for the utilisation of methylated amines as sole nitrogen sources. Sc. stipitis strains TLSS001 (wildtype control), TLSS009 (Δpicst_49761) and TLSS010 (Δpicst_63000) were cultured in 3 ml RSNLD medium supplemented with 10 mM of the indicated nitrogen source (initial OD₆₀₀ 0.005). Samples were incubated in a shaker set at 30 °C, 200 r.p.m., and OD₆₀₀ was measured after 6, 12 and 18 days. Growth assays were performed in triplicate with error bars indicating one standard deviation

Fig. 5

A model of intracellular compartmentalisation of catabolism of methylated amines in Sc. stipitis based on the data presented in this study as well as previous reports (Zwart et al. , ; Green and Large 1984). In the proposed model, extracellular methylamine is demethylated by both Amo1 and Amo2 while methylamine produced through the catabolism of choline, trimethylamine or dimethylamine is predominantly demethylated by Amo1. The intracellular localisation of the Cmo1 choline monooxygenase has not yet been established and its placement in the peroxisome in the current diagram should be considered entirely speculative (see text for details)