Top-down, knowledge-based genetic reduction of yeast central carbon metabolism

Abstract

Saccharomyces cerevisiae, whose evolutionary past includes a whole-genome duplication event, is characterised by a mosaic genome configuration with substantial apparent genetic redundancy. This apparent redundancy raises questions about the evolutionary driving force for genomic fixation of minor paralogs and complicates modular and combinatorial metabolic engineering strategies. While isoenzymes might be important in specific environments, they could be dispensable in controlled laboratory or industrial contexts. The present study explores the extent to which the genetic complexity of the central carbon metabolism (CCM) in S. cerevisiae, here defined as the combination of glycolysis, pentose phosphate pathway, tricarboxylic acid cycle and a limited number of related pathways and reactions, can be reduced by elimination of (iso)enzymes without major negative impacts on strain physiology. Cas9-mediated, groupwise deletion of 35 from the 111 genes yielded a minimal CCM strain, which despite the elimination of 32 % of CCM-related proteins, showed only a minimal change in phenotype on glucose-containing synthetic medium in controlled bioreactor cultures relative to a congenic reference strain. Analysis under a wide range of other growth and stress conditions revealed remarkably few phenotypic changes of the reduction of genetic complexity. Still, a well-documented context-dependent role of GPD1 in osmotolerance was confirmed. The minimal CCM strain provides a model system for further research into genetic redundancy of yeast genes and a platform for strategies aimed at large-scale, combinatorial remodelling of yeast CCM.

Proteome dynamics during transition from exponential to stationary phase under aerobic and anaerobic conditions in yeast

“…The central carbon metabolism (CCM) consists of key pathways required for the conversion of carbon sources into the 12 building blocks for the synthesis of cellular components and encompasses ca. 150 transport proteins and enzymes [25]. The flow of carbon and electrons via the CCM therefore responds to the carbon source nature and abundance.…”

“…Many genes, more particularly those involved in metabolism, have orthologues with similar functions [23], but often with a poorly understood physiological role. In view of minimal genomes, several studies have explored the requirement for these redundant genes and implemented top-down approaches to reduce genetic redundancy [24][25][26]. Such minimised genomes have the potential to facilitate the complete redesign and construction of entirely synthetic yeast genomes.…”