Co-evolution of Yeast and Microalga: Identification of mutations that improve cooperativity

Laboratory-based evolution has long been successfully implemented for the generation of desired phenotypes in microbial strain development. The approach also provides insights into evolutionary mechanisms and adaptive molecular strategies which may be too complex to unravel in natural environments. The selection pressure in most of these approaches are physical or chemical factors or stressors, and only a few projects have attempted to use dynamic biotic selection pressures as a driver of evolution. Here we investigate the formation of novel cooperative phenotypes between the yeast Saccharomyces cerevisiae and the microalga Chlorella sorokiniana. A synthetic ecology approach based on the cross-feeding of carbon and nitrogen was used to establish an obligate mutualism between these species which allowed for prolonged physical contact in a continuous co-culture system over 100 generations. Comparative genomic analysis of co-evolved yeast strains identified several potentially high impact Single Nucleotide Polymorphisms. Of these, two genes ETP1 and GAT1, were found to synergistically contribute to the cooperative phenotype between yeast and microalgae These genes are involved in carbon ( ETP1 ) and nitrogen catabolite ( GAT1 ) repression with ETP1 encoding a protein of unknown function, but implicated in ethanol tolerance and control of Hxt3p, while GAT1 encodes a regulator of nitrogen catabolite repression. CRISPR generated null mutants of the parental (ancestral) yeast strain with either ETP1 , GAT1 or both genes deleted, were shown to mimic the co-evolved phenotype with improved cooperativity observed when paired with Chlorella sorokiniana suggesting a possible role of these genes in the establishment of mutualisms between yeast and microalgae. Importance Multispecies cultures have tremendous biotechnological potential but are difficult to control and show unpredictable population dynamics. This research aims to comprehensively characterise the behaviour and attributes of co-cultured microbial species, with the aim of optimising their combined functionality in a targeted manner. Taken together, our results demonstrate the importance and efficacy of thoughtfully integrating biotic selection pressures into strain development projects. The data also provide insights into specific molecular adaptations that favour cooperative behaviour between species. The co-evolutionary dynamics between Saccharomyces cerevisiae and other microbial species hold immense promise for unlocking novel insights into evolutionary biology, biotechnological applications, and our understanding of complex microbiological systems. Finally, the molecular characterisation of ecosystem-relevant traits provides significant impetus to the annotation of microbial genomes within an evolutionary relevant, multispecies context.