Cloning, heterologous expression, and functional characterization of the nicotinate dehydrogenase gene from Pseudomonas putida KT2440 |
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Authors: | Yao Yang Sheng Yuan Ting Chen Pengjuan Ma Guangdong Shang Yijun Dai |
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Institution: | (1) Nanjing Research Center for Microbial Engineering and Biotechnology, Jiangsu Key Laboratory for Biodiversity and Biotechnology, College of Life Sciences, Nanjing Normal University, Nanjing, 210046, People’s Republic of China |
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Abstract: | 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. |
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Keywords: | Nicotinate 6-Hydroxynicotinate Nicotinate dehydrogenase (NaDH) ndhSL Pseudomonas putida KT2440 |
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