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

Abstract

Pathway transplantation from one organism to another represents a means to a more complete understanding of a biochemical or regulatory process. The purine biosynthesis pathway, a core metabolic function, was transplanted from human to yeast. We replaced the entire Saccharomyces cerevisiae adenine de novo pathway with the cognate human pathway components. A yeast strain was “humanized” for the full pathway by deleting all relevant yeast genes completely and then providing the human pathway in trans using a neochromosome expressing the human protein coding regions under the transcriptional control of their cognate yeast promoters and terminators. The “humanized” yeast strain grows in the absence of adenine, indicating complementation of the yeast pathway by the full set of human proteins. While the strain with the neochromosome is indeed prototrophic, it grows slowly in the absence of adenine. Dissection of the phenotype revealed that the human ortholog of ADE4, PPAT, shows only partial complementation. We have used several strategies to understand this phenotype, that point to PPAT/ADE4 as the central regulatory node. Pathway metabolites are responsible for regulating PPAT’s protein abundance through transcription and proteolysis as well as its enzymatic activity by allosteric regulation in these yeast cells. Extensive phylogenetic analysis of PPATs from diverse organisms hints at adaptations of the enzyme-level regulation to the metabolite levels in the organism. Finally, we isolated specific mutations in PPAT as well as in other genes involved in the purine metabolic network that alleviate incomplete complementation by PPAT and provide further insight into the complex regulation of this critical metabolic pathway.

Purine auxotrophy: Possible applications beyond genetic marker

“…The purine synthesis pathway and its regulation are highly conserved in all eukaryotes from fungi to mammals (Agmon et al, ). Most probably, the last common ancestor had a pathway with the same structure that diversified into the now known three eukaryotic domains (Armenta‐Medina, Segovia, & Perez‐Rueda, ; Vázquez‐Salazar, Becerra, & Lazcano, ).…”

“…The purine synthesis pathway and its regulation are highly conserved in all eukaryotes from fungi to mammals (Agmon et al, 2017).…”

“…Because purine metabolism in S. cerevisiae and human cells is functionally similar, parts of it can be “humanized” in yeast—replaced by genes coding for analogous human proteins. Humanized yeast strains can then be used as screens for specific drug targets (Agmon et al, ). We propose that purine auxotrophic yeast strains expressing both—human mutant nucleotidase and nucleoside transporters—could form a tool for drug screening against a particular subtype of relapsing ALL.…”

Superloser: A Plasmid Shuffling Vector for Saccharomyces cerevisiae with Exceedingly Low Background

“…Humanization of entire pathways or complexes in yeast is a largely unexplored frontier with few exemplars (Wildt and Gerngross 2005; Labunskyy et al 2014; Agmon et al 2017; Truong and Boeke 2017). Better tools are therefore needed to ensure the ease with which essential yeast genes can be humanized.…”

“…One important and timely potential use of this plasmid lies in the area of complex pathway engineering in yeast, especially considering increasingly large and ongoing genome construction efforts (Annaluru et al 2014; Mitchell et al 2017; Richardson et al 2017; Shen et al 2017; Wu et al 2017; Xie et al 2017; Zhang et al 2017). Because the Superloser is a centromeric plasmid, and we have extensive experience using similar centromeric vectors for host pathways and other DNA molecules of up to 120 kb (Mitchell et al 2015, 2019; Agmon et al 2017; Laurent et al 2019), we envision that it can be used directly as a vector for pathway engineering. We imagine that this tool could be used for the humanization (or from other species) of other essential genome architectural proteins such as cohesins, histone modifying complexes or chromatin remodelers, additional histone variants, and metabolic pathways.…”