(Keynote) Engineering Cyanobacteria for Nitrogen Reduction

Abstract

Ammonia is currently the second most widely produced chemical in the world (second to sulfuric acid). Most ammonia (over 66%) is now produced through the Haber−Bosch process, consuming approximately 5% of the global natural gas production and contributes to over 3% of global carbon dioxide emissions. In an effort to develop an electrochemical alternative, we have engineered cyanobacterium to perform nitrogen fixation under ambient conditions without consuming natural gas, in stark contrast to the Haber-Bosch process that requires extreme temperatures and pressures to the same end. To realize the heterologous expression of nitrogenase, we transformed the cyanobacterium S. elongatus PCC 7942 with the minimal nitrogen fixation (nif) gene cluster. Nitrogenase can reduce N2 to NH3 at mild conditions (< 40 oC, atmospheric pressure). There are three known types of nitrogenase, in which the central composition of their active-site metalloclusters are molybdenum(Mo), vanadium (V), and iron (Fe), respectively. Most biological nitrogen fixation is catalyzed by the Mo-based nitrogenase enzyme. Biosynthesis of Mo-nitrogenase generally requires a large number of nif genes. However, the transformation and transcription of such a larger set of gene cluster into the cyanobacterium is challenging. In this work, a minimal nif cluster with reduced genetic complexity was chosen to accomplish the transformation of the cyanobacterium S. elongatus PCC 7942. The cyanobacterium S. elongatus PCC 7942 was transformed with nif genes and it was shown to be able to express active nitrogenase. The engineered S. elongatus PCC 7942 was immobilized on an electrode to accelerate the microbial conversion of nitrogen into ammonium using electrochemical driving forces. Methyl viologen (MV) was added as an electron mediator to shuttle electrons from the electrode to the enzyme active center, since MV can go through the cell membrane and deliver continuous electrons to nitrogenase.