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

Tomas Linder

doi:10.1007/s10482-017-0963-y

Tomas Linder

Open Access

https://doi.org/10.1007/s10482-017-0963-y

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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.

Highlights

Amines are organic nitrogen compounds common in the environment and most microorganisms have evolved pathways to assimilate amines as a source of metabolic nitrogen
Very little reverse genetics have been done in Sc. stipitis to date due to a scarcity of selection markers and the low targeting frequency of integration constructs due to the domination of the non-homologous end-joining (NHEJ) pathway over homologous recombination
The development of a Sc. stipitis strain auxotrophic for histidine that lacks a functional NHEJ pathway through deletion of the YKU80 gene has enabled reverse genetic investigations in this yeast (Maassen et al 2008)

Summary

Introduction

Amines are organic nitrogen compounds common in the environment and most microorganisms have evolved pathways to assimilate amines as a source of metabolic nitrogen. The development of a Sc. stipitis strain auxotrophic for histidine that lacks a functional NHEJ pathway through deletion of the YKU80 gene has enabled reverse genetic investigations in this yeast (Maassen et al 2008).

Results

Conclusion