Pathway swapping: Toward modular engineering of essential cellular processes

Abstract

Recent developments in synthetic biology enable one-step implementation of entire metabolic pathways in industrial microorganisms. A similarly radical remodelling of central metabolism could greatly accelerate fundamental and applied research, but is impeded by the mosaic organization of microbial genomes. To eliminate this limitation, we propose and explore the concept of "pathway swapping," using yeast glycolysis as the experimental model. Construction of a "single-locus glycolysis" Saccharomyces cerevisiae platform enabled quick and easy replacement of this yeast's entire complement of 26 glycolytic isoenzymes by any alternative, functional glycolytic pathway configuration. The potential of this approach was demonstrated by the construction and characterization of S. cerevisiae strains whose growth depended on two nonnative glycolytic pathways: a complete glycolysis from the related yeast Saccharomyces kudriavzevii and a mosaic glycolysis consisting of yeast and human enzymes. This work demonstrates the feasibility and potential of modular, combinatorial approaches to engineering and analysis of core cellular processes.pathway swapping | glycolysis | Saccharomyces cerevisiae | modular genomes R eplacement of petrochemistry by bio-based processes is a key element for sustainable development and requires microbes equipped with novel-to-nature capabilities. Recent developments in synthetic biology enable introduction of entire metabolic pathways and, thereby, new functionalities for product formation and substrate consumption, into microbial cells (1). However, industrial relevance of the resulting strains critically depends on optimal interaction of the newly introduced pathways with the core metabolism of the host cell. Central metabolic pathways such as glycolysis, tricarboxylic acid cycle, and pentose phosphate pathways, are essential for synthesis of precursors, for providing free energy (ATP), and for redox-cofactor balancing. Optimization of productivity, product yield, and robustness therefore requires modifications in the configuration and/or regulation of these core metabolic functions.Engineering of central metabolism is in some respects more challenging than the functional expression of heterologous product pathways. Millions of years of evolution of microorganisms have endowed their metabolic and regulatory networks with a level of complexity that cannot be efficiently reengineered by iterative, single-gene modifications. Enzymes of central metabolism are encoded by hundreds of genes that, especially in eukaryotes, are scattered across microbial genomes. Moreover, inactivation and subsequent replacement of genes involved in central metabolism is complicated by functional redundancy of isoenzymes (2, 3) as well as by the essential role of many of the corresponding biochemical reactions. Microbial platforms in which the configuration of key pathways can be remodelled in a swift, combinatorial manner would provide an invaluable asset for fundamental research and engineering of central metabolism.Wherea...

A supernumerary designer chromosome for modularin vivopathway assembly inSaccharomyces cerevisiae

“…However none of these mutations caused a decrease in the specific activity of the corresponding enzymes tested in vitro comparing IMF17 and the ancestral SwYG strain (16) (Fig 5), demonstrating that mutations in the glycolytic genes was most likely not responsible for the decreased growth rate of IMF17 and IMF18.…”

“…Using the pathway swapping concept, we used SynChs as exclusive expression platform for the essential glycolytic pathway, and evaluated the impact of SynCh-borne glycolysis expression on yeast physiology. The 50 Kb (IMF2) and 100 Kb (IMF6) "empty" SynChs were assembled in the SwYG (16) ancestor strain IMX1338, in which the set of 13 genes coding for the glycolytic pathway has been relocalized to a single genomic locus. Using CRISPR-Cas9 editing combined with in vivo assembly of 13 glycoblocs ( Fig.4), the 35 Kb glycolytic cassette was introduced in the mTurquoise2 locus of the 50 Kb and 100 Kb SynChs.…”

“…To enable easy remodelling of existing functions, Kuijpers et al (16) developed the pathway swapping concept in S. cerevisiae. The genetic reduction and relocalization of the entire Embden-Meyerhoff-Parnas pathway of glycolysis to a single chromosomal locus enabled to swap this essential pathway to any new (heterologous) design in two simple steps (16,17). Combined to an orthogonal expression platform, in the form of a supernumerary Synthetic Chromosome (SynCh), pathway swapping would offer the possibility to make the introduction and expression of large product pathways in combination with the rewiring of essential native pathways, more efficient and predictable.…”

Allele-specific genome editing using CRISPR-Cas9 causes off-target mutations in diploid yeast

“…In S. cerevisiae , double stranded DNA oligomers with 60 bp of homology are sufficient to obtain accurate gene-editing in almost 100% of transformed cells (3). By inserting sequences between the homologous regions of the repair oligonucleotide, heterozygous sequences of up to 35 Kbp could be inserted at targeted loci (7). While such gene editing approaches have been very efficient in haploid and homozygous diploid yeasts, the accurate introduction of short DNA fragments can be tedious in heterozygous yeast.…”

Human to yeast pathway transplantation: cross-species dissection of the adenine de novo pathway regulatory node

“…for secondary metabolites into yeast using the organism as factory for producing a desired compound (Borodina and Nielsen, 2014). However, with quite a few very extensive studies aimed at swapping single genes from human to yeast (Brachmann et al, 1996; Hamza et al, 2015; Kachroo et al, 2015) and also several attempts at pathway swapping (Choi et al, 2003; Kuijpers et al, 2016), full pathway transplantation between human and yeast has been elusive. We chose the adenine de novo pathway as a candidate for a full pathway transplant of a highly conserved metabolic pathway.…”