Synthetic Biology Approaches to Engineering Nitrogen-Fixing Cyanobacteria
DOI:
https://doi.org/10.5281/zenodo.22687958Keywords:
nitrogen fixation, cyanobacteria, synthetic biology, nitrogenase, heterocyst, biofertiliserAbstract
Biological nitrogen fixation converts atmospheric dinitrogen into ammonia using an enzyme, nitrogenase, that is
irreversibly inactivated by oxygen, a constraint that has shaped both the evolution of diazotrophic cyanobacteria
and the engineering strategies now being pursued to extend nitrogen fixation to crop-associated or free-living
photosynthetic organisms that do not naturally fix nitrogen. This review synthesises progress across four
engineering strategies that have each been pursued over roughly the past decade: transfer of the complete nif
gene cluster into non-diazotrophic hosts; engineering of temporal or spatial separation between oxygenic
photosynthesis and nitrogenase activity, modelled on the heterocysts of filamentous cyanobacteria such as
Anabaena; directed evolution and rational engineering of oxygen-tolerant or oxygen-insensitive nitrogenase
variants; and synthetic control circuits that couple nitrogenase expression to environmental or metabolic cues.
Transfer of the full nif cluster has achieved measurable nitrogenase activity in several heterologous hosts but has
generally yielded fixation rates one to two orders of magnitude below native diazotrophs, and stable, long-term
expression has proven difficult to sustain outside specialist chassis organisms. Heterocyst-inspired spatial
separation strategies, engineered into unicellular cyanobacteria that do not naturally differentiate, have achieved
the largest relative gains reported in the primary literature to date, in one case restoring aerobic nitrogen fixation to
within an order of magnitude of native heterocyst-forming relatives. Oxygen-tolerant nitrogenase engineering
remains the least mature of the four strategies at the whole-cell level despite encouraging in vitro results, while
synthetic control circuits have proven useful primarily as a complement to, rather than a substitute for, the other
three approaches. We conclude by outlining what we regard as the most promising near-term combination of
these strategies for eventual agricultural application, and the regulatory and ecological questions that such an
application would need to address before field deployment could be seriously considered.
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