基于非靶向代谢组学研究砷酸盐胁迫下库德毕赤酵母代谢物的变化

Metabolite changes of Pichia kudriavzevii under arsenate stress based on untargeted metabolomics

砷酸盐[arsenate,As(Ⅴ)]作为水体中砷的主要存在形态之一,通过食物链影响食品安全。微生物砷脱除技术以其安全经济的优势成为解决食品砷污染途径之一。对砷的脱除能力是微生物脱砷技术的前提和基础。库德毕赤酵母(Pichia kudriavzevii)作为一种多抗性酵母,可耐盐、高温和重金属(镉、锌和铜)等。该文研究了As(Ⅴ)对P.kudriavzevii A16生长的影响,并运用非靶向代谢组学技术研究了砷酸盐胁迫与正常培养条件下P.kudriavzevii A16之间的差异表达代谢物及代谢通路的变化。砷酸盐胁迫与正常培养相比,正离子模式下检测到317个差异代谢物,169个显著上调表达,148个代谢物显著下调表达;负离子模式下,检测到差异代谢物329个,其中171个显著上调表达,158个代谢物显著下调表达;差异代谢物的KEGG(Kyoto Encyclopedia of Genes and Genomes)富集通路主要涉及谷胱甘肽代谢、氨酰tRNA合成等代谢通路,As(Ⅴ)促进了酵母细胞能量代谢,增强了其抗氧化性能。该文从代谢组学水平解析了砷酸盐胁迫下库德毕赤酵母代谢物的变化,为指导微生物法脱砷技术的开发提供参考。

The toxicity of arsenic in the natural environment and in human food chain is a major current public health concern.Arsenate[As(V)]is one of the main forms of arsenic present in water and is absorbed easily by marine organisms and enters food chain.As(V)affects food safety because it threatens human health.Recently,there were incidents of excessive arsenic levels in food products.Therefore,the control of arsenic is imminent.Microbial arsenic removal technology stands out among many other arsenic removal technologies for its high safety,low price and no other pollution.The excellent arsenic removal capacity of microorganisms is the basis and prerequisite for the widespread application of this technology,and is an important guarantee for the removal of arsenic from active cells.Pichia kudriavzevii,usually isolated from fermented foods,is a multi-resistant yeast that is tolerant of complex environments such as salt,high temperatures and heavy metals(cadmium,zinc and copper),while having a wide range of promising applications in the food industry.Metabolomics aims to measure the global dynamic metabolic response of living systems to biological stimuli or genetic manipulation,seeks analytical descriptions of complex biological samples.And it also aims to characterize and quantify all small molecules differences between samples,enabling a more direct and more accurate reflection of the physiological state of an organism.It was found that As(V)inhibited the growth of P.kudriavzevii A16 and that the rate of arsenic removal was closely related to the growth of yeast cells.The arsenic removal rate of P.kudriavzevii A16 was 61.84%in As(V)at a concentration of 5 mg/L medium.The metabolites of P.kudriavzevii A16 under As(V)stress were significantly altered compared to the normal culture group.In positive ion mode,317 differential metabolites were detected,of which 169 metabolites were significantly up-regulated in expression,including guanine and L-cysteine;148 metabolites were significantly down-regulated in expression,including DL-glutamine and nicotinamide adenine dinucleotide(NAD+).In the negative ion mode,329 differential metabolites were detected,of which 171 metabolites were significantly up-regulated in expression,including bile acids and flavin mononucleotides;158 metabolites were significantly down-regulated in expression,including D-(-)-glutamine and citric acid.The KEGG enrichment pathway for differential metabolites in the positive ion mode is mainly involved in glutathione metabolism and aminyl tRNA biosynthesis metabolism.However,the KEGG enrichment pathway for differential metabolites in the negative ion mode mainly is involved in the citric acid cycle,glyoxalate and dicarboxylic acid metabolism.Thus,P.kudriavzevii A16 increases the antioxidant properties of yeast cells by upregulating metabolic pathways such as glutathione metabolism as a means of resisting As(V)-induced intracellular oxidative stress.Moreover,P.kudriavzevii A16 also enhances energy supply in response to toxic effects caused by As(V)exposure by regulating intracellular glycolytic reactions,the TCA cycle,oxidative phosphorylation and amino acid metabolism.In this study,the metabolites of P.kudriavzevii A16 were screened under normal and arsenic stress conditions using advanced non-targeted metabolomics techniques.By comparing the differences in metabolism between arsenic stress and no arsenic present conditions,the relevant metabolites were screened out,which will provide a reference for the development of microbial arsenic removal technology.