Functional identification of novel genes involved in the glutathione-independent gentisate pathway in Corynebacterium glutamicum.
Corynebacterium glutamicum used gentisate and 3-hydroxybenzoate as its sole carbon and energy source for growth. By genome-wide data mining, a gene cluster designated ncg12918-ncg12923 was proposed to encode putative proteins involved in gentisate/3-hydroxybenzoate pathway. Genes encoding gentisate 1,2-dioxygenase (ncg12920) and fumarylpyruvate hydrolase (ncg12919) were identified by cloning and expression of each gene in Escherichia coli. The gene of ncg12918 encoding a hypothetical protein (Ncg12918) was proved to be essential for gentisate-3-hydroxybenzoate assimilation. Mutant strain RES167Deltancg12918 lost the ability to grow on gentisate or 3-hydroxybenzoate, but this ability could be restored in C. glutamicum upon the complementation with pXMJ19-ncg12918. Cloning and expression of this ncg12918 gene in E. coli showed that Ncg12918 is a glutathione-independent maleylpyruvate isomerase. Upstream of ncg12920, the genes ncg12921-ncg12923 were located, which were essential for gentisate and/or 3-hydroxybenzoate catabolism. The Ncg12921 was able to up-regulate gentisate 1,2-dioxygenase, maleylpyruvate isomerase, and fumarylpyruvate hydrolase activities. The genes ncg12922 and ncg12923 were deduced to encode a gentisate transporter protein and a 3-hydroxybenzoate hydroxylase, respectively, and were essential for gentisate or 3-hydroxybenzoate assimilation. Based on the results obtained in this study, a GSH-independent gentisate pathway was proposed, and genes involved in this pathway were identified.
State Key Laboratory of Microbial Resources at the Institute of Microbiology, Chinese Academy of Sciences, Beijing 100080, People's Republic of China.
Xi-Hui
XH
Shen
Cheng-Ying
CY
Jiang
Yan
Y
Huang
Zhi-Pei
ZP
Liu
Shuang-Jiang
SJ
Liu
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D048230:Corynebacterium glutamicum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D049308:Dioxygenases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005841:Gentisates / Q000378:metabolism*; D005978:Glutathione / Q000378:metabolism*; D006867:Hydrolases / Q000235:genetics / Q000378:metabolism; D062385:Hydroxybenzoates / Q000378:metabolism; D005810:Multigene Family; D019745:cis-trans-Isomerases / Q000235:genetics / Q000378:metabolism
D001426:Bacterial Proteins; D005841:Gentisates; D062385:Hydroxybenzoates; C032948:3-hydroxybenzoic acid; D049308:Dioxygenases; C026247:gentisate 1,2-dioxygenase; D006867:Hydrolases; C026249:fumarylpyruvate hydrolase; D019745:cis-trans-Isomerases; C046138:maleylpyruvate isomerase; D005978:Glutathione