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

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 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.

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.