弗氏柠檬酸菌1,3-丙二醇合成的代谢工程研究

Metabolic engineering of 1,3-propanediol synthesis in Citrobacter freundii CF-5

摘要:

【目的】研究弗氏柠檬酸菌(Citrobacter freundii) 1,3-丙二醇合成的代谢过程。【方法】构建甘油脱氢酶基因GSR-lacZ、1,3-丙二醇氧化还原酶基因PDO-lacZ和甘油脱水酶基因GL-lacZ等报告基因。在此基础上，构建3个相应的转座子突变文库。【结果】筛选到6株突变子，其相应关键酶表达水平提高1?11倍，1,3-丙二醇产量提高幅度为3%?50%。对转座子插入位点分析显示，5株突变子插入位点均为β-内酰胺酶(CKO_02592)编码基因，1株突变子插入位点为二氢硫辛酰胺基转移酶(CKO_02433)编码基因。进一步分析发现，β-内酰胺酶基因突变显著提高甘油脱水酶和甘油脱氢酶的表达水平，而1,3-丙二醇氧化还原酶表达水平没有变化；二氢硫辛酰胺基转移酶基因突变显著提高1,3-丙二醇氧化还原酶表达水平，其他两种关键酶基因表达水平不变。【结论】β-内酰胺酶和二氢硫辛酰胺基转移酶基因能够分别影响1,3-丙二醇合成代谢途径关键酶的表达，为构建工程菌株打下基础。

关键词:转录融合报告基因，转座子突变文库，弗氏柠檬酸菌，1,3-丙二醇

Abstract:

[Objective] The metabolism of 1,3-propanediol in Citrobacter freundii was studied. [Methods] Transcriptional fusion reporter genes of GSR-lacZ, PDO-lacZ, and GL-lacZ were constructed. Based on this, the mariner transposon libraries were constructed. [Results] Six mutants were isolated. The expression level of the corresponding key enzymes was increased from 1 to 11 folds in the mutants, and the corresponding 1,3-propanediol production was increased from 3% to 50%. Transposon insertion sites were analyzed and the results show that the β-lactamase gene was inactivated in 5 mutants and the dihydrolipoamide acyltransferase gene was inactivated in 1 mutant. Furthermore, the expression levels of glycerol dehydrogenase and glycerol dehydratase were increased significantly in the β-lactamase mutant, whereas no increase of the 1,3-propanediol oxidoreductase expression was observed. In the diydrolipoamide acyltransferase mutant, 1,3-propanediol oxidoreductase expression level was significantly improved, whereas the other two enzymes expression level remained unchanged. [Conclusion] The results provide insight into constructing engineering strains producing a high level of 1,3-propanediol.

Key words:Transcriptional fusion reporter gene, Transposon mutant library, Citrobacter freundii, 1,3-propanediol