Linder Tomas
Department of Molecular Sciences, Swedish University of Agricultural Sciences, Box 7015, 750 07, Uppsala, Sweden.
Antonie Van Leeuwenhoek. 2018 Mar;111(3):401-411. doi: 10.1007/s10482-017-0963-y. Epub 2017 Oct 30.
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.
在出芽酵母中，胆碱作为氮源的分解代谢被认为是在释放氨之前，通过三甲胺、二甲胺和甲胺等中间体进行的。本研究采用反向遗传学方法，研究了在树干毕赤酵母中胆碱及其下游中间体作为氮源的利用情况。先前预测与甲基化胺分解代谢直接或间接相关的6个基因（AMO1、AMO2、SFA1、FGH1、PICST_49761、PICST_63000）被逐一删除。在以甲胺、二甲胺、三甲胺或胆碱作为唯一氮源的基本培养基上，对每个缺失突变体的生长情况进行了测定。两个编码胺氧化酶的基因AMO1和AMO2在利用甲基化胺生长方面似乎功能冗余，因为两个缺失突变体在所有测试的氮源上均能生长。然而，删除AMO1导致在所有四种甲基化胺上均出现明显的生长延迟，而删除AMO2仅在甲胺作为唯一氮源时导致生长延迟。发现编码谷胱甘肽依赖性甲醛脱氢酶的基因SFA1对于在所有测试的甲基化胺上生长绝对必要，而删除S-甲酰谷胱甘肽水解酶基因FGH1则导致在二甲胺、三甲胺和胆碱上出现明显的生长延迟。假定的编码细胞色素P450单加氧酶的基因PICST_49761和PICST_63000被认为可能参与二甲胺和三甲胺的去甲基化，但删除后在任何测试的氮源上均未产生可识别的表型。本研究揭示了胆碱分解代谢途径中显著的基因冗余实例，并对其进行了讨论。
