一株丁二酸产生菌的选育及代谢分析

Screening, breeding and metabolic analysis of a succinic-acid-producing strain

摘要:

【目的】了解细胞代谢过程,并最终选育出高产突变株，对丁二酸的工业生物转化有重要意义。【方法】在菌株生化及分子鉴定基础上，讨论了菌株的代谢途径，便于实施有针对性的诱变选育，利用矩阵计算了流量分布以及用扰动法分析了代谢节点。【结果】菌株S.JST经鉴定为产丁二酸放线杆菌，酶活检测表明磷酸烯醇式丙酮酸羧激酶、苹果酸脱氢酶在丁二酸代谢过程中具有较高酶活，出发株的流量分布显示副产物乙醇的流量仅次于丁二酸的流量，选育获得的突变株乙醇脱氢酶酶活显著降低，丁二酸与乙醇的流量分别有34%升高与93%的降低，序列分析发现突变株的乙醇脱氢酶酶基因中存在一个突变位点，且生物信息学表明该位点编码的氨基酸序列与该酶的NADH连接活性有联系。【结论】对产丁二酸放线杆菌采用定向选育的方法能够有效改善细胞代谢，并最终提高丁二酸产量。

Abstract:

[objective] In order to obtain high yield mutant strains for the industrial bioconversion of succinic acid, we analyzed the metabolic networks of the strain Actinobacillus succinogenes S.JST in the course of screening and breeding. [methods] We previously identified the wild-type strain by API biochemical reactions and 16S r RNA sequence analysis. Following the discussion of the metabolic pathway, we calculated the flux by matrix and disturbed the node by intermediate. [results] A succinic-acid-producing strain S.JST isolated from bovine rumen was identified as Actinobacillus succinogenes. Enzyme determination showed that the activities of phosphoenolpyruvate carboxykinase and malate dehydrogenase were very high. Metabolic flux from parent strain indicated that the flux of by-product ethanol was 1.51 mmol·g-1·h-1 in the second place of those end products. After being mutated, the alcohol dehydrogenase activity of the mutant-strain S.JSTA decreased markedly, furthermore the flux of succinic acid increased by 34% and the flux of ethanol decreased by 93%. By analyzing the Adh gene, we found a mutated site. Bioinformatics showed that the corresponding amino acid sequence acted as the key active site binding with NADH. [conclusion] In succinic acid synthesis, directed breeding method was effective for improving the whole cell metabolism of Actinobacillus succinogenes, and succinic acid yield was increased.