Cloning, heterologous expression, and functional characterization of the nicotinate dehydrogenase gene from Pseudomonas putida KT2440.
6-hydroxynicotinate can be used for the production of drugs, pesticides and intermediate chemicals. Some Pseudomonas species were reported to be able to convert nicotinic acid to 6-hydroxynicotinate by nicotinate dehydrogenase. So far, previous reports on NaDH in Pseudomonas genus were confused and contradictory each other. Recently, Ashraf et al. reported an NaDH gene cloned from Eubacterium barkeri and suggested some deducted NaDH genes from other nine bacteria. But they did not demonstrate the activity of recombinant NaDH and did not mention NaDH gene in Pseudomonas. In this study we cloned the gene of NaDH, ndhSL, from Pseudomonas putida KT2440. NdhSL in P. putida KT2440 is composed of two subunits. The small subunit contains [2Fe2S] iron sulfur domain, while the large subunit contains domains of molybdenum cofactor and cytochrome c. Expression of recombinant ndhSL in P. entomophila L48, which lacks the ability to produce 6-hydroxynicotinate, enabled the resting cell and cell extract of engineering P. entomophila L48 to hydroxylate nicotinate. Gene knockout and recovery studies further confirmed the ndhSL function.
Yang
Yao
Y
Nanjing Research Center for Microbial Engineering and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, People's Republic of China.
Yuan
Sheng
S
Chen
Ting
T
Ma
Pengjuan
P
Shang
Guangdong
G
Dai
Yijun
Y
D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D055786:Gene Knockout Techniques; D005798:Genes, Bacterial; D005822:Genetic Vectors; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000235:genetics*; D016133:Polymerase Chain Reaction; D016958:Pseudomonas putida / Q000201:enzymology / Q000235:genetics*
D017931:DNA Primers; C021862:nicotinate dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors