Jiang Shaotong, Li Xingjiang, Pan Lijun, Wei Zhaojun, Chen Xiaohui, Zhang Jianguo, Tang Xueying, Xiong Jianchun, Yuan Yuan, Zhang Yuanyuan
School of Biotechnology and Food Engineering, Hefei University of Technology, Hefei 230009, China.
Wei Sheng Wu Xue Bao. 2008 Aug;48(8):1048-55.
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.
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.
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 x g(-1) x 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.
In succinic acid synthesis, directed breeding method was effective for improving the whole cell metabolism of Actinobacillus succinogenes, and succinic acid yield was increased.
为了获得用于琥珀酸工业生物转化的高产突变菌株，我们在筛选和育种过程中分析了产琥珀酸放线杆菌S.JST菌株的代谢网络。
我们先前通过API生化反应和16S rRNA序列分析鉴定了野生型菌株。在讨论代谢途径后，我们通过矩阵计算通量，并通过中间产物干扰节点。
从牛瘤胃中分离出的产琥珀酸菌株S.JST被鉴定为产琥珀酸放线杆菌。酶活性测定表明，磷酸烯醇式丙酮酸羧激酶和苹果酸脱氢酶的活性非常高。来自亲本菌株的代谢通量表明，副产物乙醇的通量在这些终产物中排第二位，为1.51 mmol·g-1·h-1。突变后，突变菌株S.JSTA的乙醇脱氢酶活性明显降低，此外琥珀酸通量增加了34%，乙醇通量降低了93%。通过分析Adh基因，我们发现了一个突变位点。生物信息学表明，相应的氨基酸序列作为与NADH结合的关键活性位点。
在琥珀酸合成中，定向育种方法对于改善产琥珀酸放线杆菌的全细胞代谢是有效的，并且提高了琥珀酸产量。
