Improving tolerance to fluctuating light through adaptive laboratory evolution in the cyanobacterium Synechocystis

Fluctuating light (FL) poses a serious challenge to photosynthetic organisms like cyanobacteria, disrupting carbon assimilation and damaging photosystems. While key components of cyanobacterial FL tolerance have been identified, their genetic enhancement remains unexplored. We applied adaptive laboratory evolution to Synechocystis PCC 6803 under two complex FL regimes, including one lethal to the starter strain (LT), to generate FL-adaptive alleles. Our analysis revealed 44 fully segregated novel mutations in 24 monoclonal evolved strains, 28 of which affected proteins or structural RNAs. We focused on three mutations for further study. Mutations in Pam68, involved in photosystem II (PSII) assembly, and Sll0518 were present in all evolved strains, indicating early emergence. These mutations increased tolerance to non-lethal FL conditions when introduced into LT, with the Pam68 mutation possibly protecting PSI by increasing the proportion of less active PSII monomers. A gain-of-function mutation in RpaB, regulator of phycobilisome association B, was found in three strains tolerant to lethal FL. When introduced into LT, this mutation significantly increased tolerance to both lethal FL and high light conditions, associated with downregulation of photosystem accumulation and light harvesting. As RpaB has plant homologs, this finding could potentially be used to improve agricultural productivity under variable light conditions.