Influence of different factors on the nitrogenase activity of the engineered Escherichia coli 78-7.

The engineered Escherichia coli 78-7 is a derivative of E. coli JM109 carrying a nitrogen fixation (nif) gene cluster composed of nine genes (nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV) and its own σ(70)-dependent nif promoter from a gram-positive bacterium Paenibacillus sp. WLY78. The physiological and biochemical characteristics of the engineered E. coli 78-7 were analyzed by using Biolog GEN III MicroPlate, with E. coli JM109 and JM109/pHY300PLK (E. coli JM109 carrying empty vector) as controls. Analysis of 94 phenotypic tests: 71 carbon source utilization assays and 23 chemical sensitivity tests showed that the engineered E. coli 78-7, E. coli JM109 and JM109/pHY300PLK gave similar patterns of utilization of various substrates as carbon and energy sources. Furthermore, the effect of carbon source, nitrogen source, culture temperature on the nitrogenase activity of the engineered E. coli 78-7 was investigated. Our study demonstrates that the nif capacity of E. coli 78-7 was affected significantly by the different culture condition. The significant nitrogenase activity of E. coli 78-7 were obtained when cells were cultivated in the medium containing 4 g/l glucose (carbon source) and 2 mM glutamate (nitrogen source) and at 30 °C.