  Protein engineering is an established method for tailoring enzymatic
reactivity. A commonly used method is directed evolution, where the mutagenesis
and natural selection process is mimicked and accelerated in the laboratory.
Here, we describe a reliable method for generating saturation mutagenesis
libraries by golden gate cloning in a broad host range plasmid containing the
pBBR1 replicon. The applicability is demonstrated by generating a mutant
library of the iron nitrogenase gene cluster (anfHDGK) of Rhodobacter
capsulatus, which is subsequently screened for the improved formation of
molecular hydrogen.
