Modular, synthetic chromosomes as new tools for large scale engineering of metabolism.

The construction of powerful cell factories requires intensive genetic engineering for the addition of new functionalities and the remodeling of native pathways and processes. The present study demonstrates the feasibility of extensive genome reprogramming using modular, specialized de novo-assembled neochromosomes in yeast. The in vivo assembly of linear and circular neochromosomes, carrying 20 native and 21 heterologous genes, enabled the first de novo production in a microbial cell factory of anthocyanins, plant compounds with a broad range of pharmacological properties. Turned into exclusive expression platforms for heterologous and essential metabolic routes, the neochromosomes mimic native chromosomes regarding mitotic and genetic stability, copy number, harmlessness for the host and editability by CRISPR/Cas9. This study paves the way for future microbial cell factories with modular genomes in which core metabolic networks, localized on satellite, specialized neochromosomes can be swapped for alternative configurations and serve as landing pads for the addition of functionalities.

Top-Down, Knowledge-Based Genetic Reduction of Yeast Central Carbon Metabolism.

Saccharomyces cerevisiae, whose evolutionary past includes a whole-genome duplication event, is characterized 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, the pentose phosphate pathway, the 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 of 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 from 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 remodeling of yeast CCM. IMPORTANCE Fundamental questions regarding the minimal requirements for life have prompted scientists to embark on top-down efforts to reduce microbial genomes to the minimum set of genes and proteins necessary to sustain cell survival and division. While these efforts are generally focused on small, prokaryotic genomes, Saccharomyces cerevisiae, a popular industrial and model organism, has a typical eukaryotic genome characterized by a high genetic redundancy. The cellular function of redundant genes is generally poorly understood and is often investigated at the scale of a few genes. In this study, we explore genetic redundancy at large scale, encompassing the ~100 genes involved in central carbon metabolism, a part of metabolism essential for life and highly conserved among eukaryotes. This study reveals the remarkable resilience of this model eukaryote, as it was hardly affected, under a broad range of conditions, by a 32% reduction of its central carbon metabolism.