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 by taking advantage of a remarkably efficient photosynthetic machinery. In this work, we describe the creation of a workflow for experimental studies that aim to compare the energy transfer mechanisms occurring within 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 exhibitng a blue-shift, in addition to identifying a novel mutant exhibiting a red-shift of the B850 absorption peak. We characterised the fluorescence lifetime of the purified LH2 spectral variants in vitro , and performed a bacterial growth assay to assess the fitness of the LH2 variants in vivo under oversaturating light conditions. Our results suggest that the LH2s variants expressed by PNSB in nature reflect the intricate tunning of their quantum properties not towards the fastest energy transfer but towards the optimum light-harvesting efficiency which is defined by diverse environmental factors. Statement of significance In this work we report a platform for the systematic investigation of the mutational landscape of light harvesting complexes forming part of the photosynthetic machinery of purple non-sulfur bacteria. By conducting directed evolution of selected residues of the light-harvesting antenna (LH2) of Rhodobacter sphaeroides we identified a novel mutant with distinct and red-shifted spectral properties. We characterised the energy transfer dynamics of this and a previous characterised mutant and demonstrated that the new variant confers a phenotypic growth advantage when cultured with high-intensity light. Our findings offer new insights into the mechanisms of light capture and energy transfer also bridging the in vitro observations with quantifiable fitness advantages under the conditions tested.