Using synthetic biology to distinguish and overcome regulatory and functional barriers related to nitrogen fixation.
Biological nitrogen fixation is a complex process requiring multiple genes working in concert. To date, the Klebsiella pneumoniae nif gene cluster, divided into seven operons, is one of the most studied systems. Its nitrogen fixation capacity is subject to complex cascade regulation and physiological limitations. In this report, the entire K. pneumoniae nif gene cluster was reassembled as operon-based BioBrick parts in Escherichia coli. It provided ~100% activity of native K. pneumoniae system. Based on the expression levels of these BioBrick parts, a T7 RNA polymerase-LacI expression system was used to replace the σ(54)-dependent promoters located upstream of nif operons. Expression patterns of nif operons were critical for the maximum activity of the recombinant system. By mimicking these expression levels with variable-strength T7-dependent promoters, ~42% of the nitrogenase activity of the σ(54)-dependent nif system was achieved in E. coli. When the newly constructed T7-dependent nif system was challenged with different genetic and physiological conditions, it bypassed the original complex regulatory circuits, with minor physiological limitations. Therefore, we have successfully replaced the nif regulatory elements with a simple expression system that may provide the first step for further research of introducing nif genes into eukaryotic organelles, which has considerable potentials in agro-biotechnology.
State Key Laboratory of Protein and Plant Gene Research, College of Life Sciences, Peking University, Beijing, China.
Xia
X
Wang
Jian-Guo
JG
Yang
Li
L
Chen
Ji-Long
JL
Wang
Qi
Q
Cheng
Ray
R
Dixon
Yi-Ping
YP
Wang
D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism; D012321:DNA-Directed RNA Polymerases / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics* / Q000378:metabolism; D007711:Klebsiella pneumoniae / Q000235:genetics* / Q000378:metabolism; D056968:Lac Repressors / Q000235:genetics* / Q000378:metabolism; D060847:Metabolic Engineering / Q000379:methods*; D005810:Multigene Family; D009586:Nitrogen Fixation / Q000235:genetics*; D009591:Nitrogenase / Q000235:genetics / Q000378:metabolism; D009876:Operon; D011401:Promoter Regions, Genetic; D058615:Synthetic Biology; D014764:Viral Proteins / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D056968:Lac Repressors; C539064:LacI protein, E coli; D014764:Viral Proteins; D009591:Nitrogenase; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases