一株高效同化氨氮霉菌的筛选及其代谢组分析

Screening and metabolomic analysis of a fungal strain efficiently assimilating ammonia nitrogen

摘要:

【目的】本试验旨在通过筛选出一株可以高效同化氨氮的霉菌，揭示其在不同培养基中的代谢组差异和其发酵饲料的氨基酸含量变化，明确霉菌氨同化代谢机制。【方法】用(NH₄)₂SO₄为唯一氮源的培养基分别培养7株木霉(Trichoderma spp.)、7株黑曲霉(Aspergillus niger)和9株米曲霉(Aspergillus oryzae)，筛选氨氮利用率和谷氨酰胺合成酶(glutamine synthetase, GS)活性都较高的霉菌为供试菌株，再利用非靶向代谢组学比较供试菌株在马铃薯葡萄糖琼脂(potato dextrose agar, PDA)培养基和无机氮培养基中的代谢差异，测定发酵饲料的粗蛋白质和有机氮含量，利用氨基酸靶向代谢组学解析供试菌株发酵饲料提取液中氨基酸含量变化。【结果】结果表明，筛选到的米曲霉MQ28氨氮利用率为54.46%，GS活性为0.61 μmol/(h·g)，均显著高于其他菌株(P<0.05)。基于比较代谢组学分析，确定MQ28在氨同化过程中与多种氨基酸代谢密切相关。MQ28发酵使饲料粗蛋白质含量提高22.25% (P<0.05)，有机氮含量提高35.83% (P<0.05)，饲料提取液中的总氨基酸含量从31.86 mmol/100 g提高到57.69 mmol/100 g (P<0.05)，苏氨酸、赖氨酸和精氨酸等14种氨基酸含量均显著提高(P<0.05)，其中谷氨酸和谷氨酰胺含量分别提高3.46倍和99倍。【结论】综上所述，米曲霉MQ28具有较高的氨氮利用能力，可能是通过合成谷氨酰胺调节氨基酸代谢途径来调控氨同化过程，可以作为生产单细胞蛋白的优良菌株。

Abstract:

[Objective] To screen out a fungal strain that can efficiently assimilate ammonia nitrogen, reveal the metabolome differences of the strain in different media and the changes in the amino acid content of the feed fermented with the strain, and clarify the mechanism of its ammonia assimilation. [Methods] Seven strains of Trichoderma, 7 strains of Aspergillus niger, and 9 strains of Aspergillus oryzae were cultured in the media with (NH₄)₂SO₄ as the only nitrogen source. The strains with high ammonia nitrogen use efficiency and glutamine synthetase (GS) activity were selected for comparison of the metabolic differences in potato dextrose agar (PDA) plates and inorganic nitrogen plates by non-targeted metabonomics. Furthermore, the crude protein and organic nitrogen content in the feed fermented with different strains was determined, and the changes in amino acid content in the fermented feed extract were measured by amino acid-targeted metabolomics. [Results] The utilization rate of ammonia nitrogen and the glutamine synthetase activity of A. oryzae MQ28 were 54.46% and 0.61 μmol/(h·g), respectively, which were higher than those of other strains (P<0.05). The comparative metabonomics analysis suggested that MQ28 was associated with the metabolism of multiple amino acids during ammonia assimilation. MQ28 fermentation increased the crude protein and organic nitrogen in the feed by 22.25% and 35.83% (P<0.05), respectively. Furthermore, MQ28 fermentation increased the total amino acid content in feed extract from 31.86 mmol/100 g to 57.69 mmol/100 g (P<0.05). Specifically, it increased the content of 14 amino acids such as threonine, lysine, and arginine, glutamic acid (by 3.46 folds), and glutamine (by 99 folds) (P<0.05). [Conclusion] To sum up, A. oryzae MQ28 has high ammonia nitrogen utilization capacity. It may regulate the ammonia assimilation process through the synthesis of glutamine to regulate amino acid metabolism and can be used as an elite strain for the production of single-cell protein.