Directed evolution and characterisation of light harvesting complexes with altered spectral properties in purple non-sulfur bacteria

Abstract

Purple non-sulfur bacteria (PNSB) are metabolically versatile microorganisms that inhabit diverse environments taking advantage of a remarkably efficient photosynthetic machinery. In this work, we describe the creation of a workflow for experimental studies aiming to compare the light absorption properties of structural variants of the light-harvesting complex 2 (LH2) of PNSB. Through the creation of a library of LH2 variants using site-directed mutagenesis, we engineered proteins with different spectral properties to be expressed in a LH2-defficient mutant of the model PNSB Rhodobacter sphaeroides. We validated this approach by reproducing a previously described mutant with a blue-shift in the B850 absorption peak, in addition to identifying a novel mutant exhibiting a red-shift of this peak. We characterised the fluorescence lifetime of the purified LH2 spectral variants in vitro. Our results suggest that directed evolution approaches can be used to systematically explore the landscape of light absorption in LH2s variants.