Gene clusters are controlled by the combined action of a mixture of regulatory elements, including promoters, 5′ untranslated regions, pause sites and small RNAs. In addition, regulation can be further complicated by the presence of redundancy as well as feedforward and feedback loops. The authors sought to design a new version of the 20-gene nitrogen fixation gene cluster in K. oxytoca by stripping out and replacing all of the native regulation. They began in silico by removing sequences associated with non-coding DNA and regulatory genes. They then re-coded the remaining genes to produce a sequence that was as different as it could be from wild type to eliminate as much internal regulation as possible. Any remaining functional elements were then identified and removed, and the genes were organized into artificial operons with synthetic ribosome-binding sites and T7 promoters of the appropriate strength. The authors then created a system of sensors and circuitry on a separate plasmid, the output of which was the expression of an engineered T7 RNA polymerase, which would control the refactored gene cluster in an orthogonal manner.
Having designed all of the components in silico, the authors then synthesized the refactored DNA, created half clusters using the Gibson assembly reaction and verified their function in K. oxytoca strains with the corresponding genes deleted, finding that the two half clusters supported ∼18% and ∼26%, respectively, of the nitrogen-fixing activity of wild-type cells. The full cluster was then assembled from the two half clusters and found to support ∼7% of the wild-type activity. Importantly, although the nitrogen-fixing activity was markedly lower than that of the wild-type cluster, the refactored cluster was decoupled from the native environmental signals such that nitrogen fixation continued in the presence of levels of ammonia that would normally be inhibitory.
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