天冬氨酸提高乳酸乳球菌Lactococcus lactis NZ9000酸胁迫抗性的作用机制

Improvement of acid-tolerance of Lactococcus lactis NZ9000 by aspartate

【背景】乳酸菌作为重要的发酵微生物在应用过程中面临广泛存在的酸胁迫。【目的】确认天冬氨酸可有效提高乳酸乳球菌的酸胁迫抗性,通过解析天冬氨酸的作用机制,为进一步提高乳酸菌酸胁迫抗性提供可借鉴的思路。【方法】通过荧光定量PCR比较胁迫条件下天冬氨酸对L.lactisNZ9000产能和氨基酸代谢途径中关键基因转录水平的影响,并通过过量表达天冬酰胺酶增加胞内天冬氨酸的含量。【结果】天冬氨酸主要是在转氨酶的作用下生成草酰乙酸和谷氨酸。草酰乙酸参与三羧酸循环,为细胞提供更多的能量;谷氨酸经谷氨酸脱羧酶途径提高细胞的酸胁迫抗性。经pH4.0胁迫处理后,天冬氨酸使糖酵解和三羧酸循环产能途径中关键基因转录上调,胞内ATP含量为对照组的42倍;胞内谷氨酸含量为对照的1.99倍。通过过量表达天冬酰胺酶获得的重组菌株,在pH3.6条件下胁迫0.5h后,存活率约为对照组的11.11倍。【结论】在L. lactis NZ9000中探究了天冬氨酸提高酸胁迫抗性的作用机理,进一步完善了氨基酸代谢提高乳酸菌酸胁迫抗性的理论基础。

[Background] As an important microorganism in fermentation,lactic acid bacteria are confronted with severe acid stress during the process of production.[Objective] We analyzed the specific mechanism how aspartic acid improved acid stress resistance of L.lactis NZ9000.[Methods] The effects of aspartate on transcription levels of key genes in energy production and amino acid metabolic pathways were analyzed by fluorescence quantitative PCR under acid stress.The content of aspartic acid was increased by overexpression of L-asparaginase.[Results] The main mechanism of aspartic acid to improve the acid stress resistance was generating oxaloacetate and glutamate in the role of transaminase.Oxaloacetate entered three carboxylic acid cycle and can provide more energy for cells; glutamate promoted acid resistance through glutamic acid decarboxylase pathway.The transcription levels of genes of glycolysis and three carboxylic acid cycle pathway were up-regulated and the content of intracellular ATP was 42 times as high as that in the control group at pH 4.0 for some time.The intracellular glutamate content was 1.99 times higher than that of the control.The survival rate of recombinant strain obtained by overexpression L-asparaginase was about 11.11 times as high as that of the control at pH 3.6 for 0.5 h.[Conclusion] Aspartic acid could improve acid stress resistance of L.lactis NZ9000.