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Nucleic Acids Res. 2019 Nov 20. pii: gkz1098. doi: 10.1093/nar/gkz1098. [Epub ahead of print]

Phylogenetic debugging of a complete human biosynthetic pathway transplanted into yeast.

Author information

1
Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, NY, USA.
2
Memorial Sloan Kettering Cancer Center, New York, NY, USA.
3
Center for Genomics and Systems Biology, Department of Biology, New York University, New York, NY, USA.
4
Institute for Computational Medicine and Department of Biochemistry and Molecular Pharmacology, NYU Langone Health, New York, NY, USA.
5
Department of Biochemistry, The University of Texas Southwestern Medical Center, Dallas, TX, USA.

Abstract

Cross-species pathway transplantation enables insight into a biological process not possible through traditional approaches. We replaced the enzymes catalyzing the entire Saccharomyces cerevisiae adenine de novo biosynthesis pathway with the human pathway. While the 'humanized' yeast grew in the absence of adenine, it did so poorly. Dissection of the phenotype revealed that PPAT, the human ortholog of ADE4, showed only partial function whereas all other genes complemented fully. Suppressor analysis revealed other pathways that play a role in adenine de-novo pathway regulation. Phylogenetic analysis pointed to adaptations of enzyme regulation to endogenous metabolite level 'setpoints' in diverse organisms. Using DNA shuffling, we isolated specific amino acids combinations that stabilize the human protein in yeast. Thus, using adenine de novo biosynthesis as a proof of concept, we suggest that the engineering methods used in this study as well as the debugging strategies can be utilized to transplant metabolic pathway from any origin into yeast.

PMID:
31745563
DOI:
10.1093/nar/gkz1098

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