RESUMO
Major genomic deletions in independent eukaryotic lineages have led to repeated ancestral loss of biosynthesis pathways for nine of the twenty canonical amino acids. While the evolutionary forces driving these polyphyletic deletion events are not well understood, the consequence is that extant metazoans are unable to produce nine essential amino acids (EAAs). Previous studies have highlighted that EAA biosynthesis tends to be more energetically costly, raising the possibility that these pathways were lost from organisms with access to abundant EAAs. It is unclear whether present-day metazoans can reaccept these pathways to resurrect biosynthetic capabilities that were lost long ago or whether evolution has rendered EAA pathways incompatible with metazoan metabolism. Here, we report progress on a large-scale synthetic genomics effort to reestablish EAA biosynthetic functionality in mammalian cells. We designed codon-optimized biosynthesis pathways based on genes mined from Escherichia coli. These pathways were de novo synthesized in 3 kilobase chunks, assembled in yeasto and genomically integrated into a Chinese hamster ovary (CHO) cell line. One synthetic pathway produced valine at a sufficient level for cell viability and proliferation. 13C-tracing verified de novo biosynthesis of valine and further revealed build-up of pathway intermediate 2,3-dihydroxy-3-isovalerate. Increasing the dosage of downstream ilvD boosted pathway performance and allowed for long-term propagation of second-generation cells in valine-free medium at 3.2 days per doubling. This work demonstrates that mammalian metabolism is amenable to restoration of ancient core pathways, paving a path for genome-scale efforts to synthetically restore metabolic functions to the metazoan lineage.
Assuntos
Aminoácidos Essenciais , Genoma , Aminoácidos/genética , Animais , Células CHO , Cricetinae , Cricetulus , Escherichia coli/genética , Mamíferos , ValinaRESUMO
Design and large-scale synthesis of DNA has been applied to the functional study of viral and microbial genomes. New and expanded technology development is required to unlock the transformative potential of such bottom-up approaches to the study of larger mammalian genomes. Two major challenges include assembling and delivering long DNA sequences. Here, we describe a workflow for de novo DNA assembly and delivery that enables functional evaluation of mammalian genes on the length scale of 100 kilobase pairs (kb). The DNA assembly step is supported by an integrated robotic workcell. We demonstrate assembly of the 101 kb human HPRT1 gene in yeast from 3 kb building blocks, precision delivery of the resulting construct to mouse embryonic stem cells, and subsequent expression of the human protein from its full-length human gene in mouse cells. This workflow provides a framework for mammalian genome writing. We envision utility in producing designer variants of human genes linked to disease and their delivery and functional analysis in cell culture or animal models.
Assuntos
Clonagem Molecular/métodos , Engenharia Genética/métodos , Animais , DNA/genética , Técnicas de Transferência de Genes/veterinária , Técnicas Genéticas/veterinária , Genoma/genética , Genômica/métodos , Humanos , Hipoxantina Fosforribosiltransferase/genética , Camundongos , Análise de Sequência de DNA/métodos , Fluxo de TrabalhoRESUMO
We report the case of a 64-year-old man referred for optic ataxia, constructional apraxia, and spatial orientation disorders evolving for 2 months. Benson syndrome (posterior cortical atrophy) was initially suspected. Brain F-FDG PET/CT depicted an asymmetric decreased uptake pattern consistent with Creutzfeldt-Jakob disease. 14-3-3 proteins were detected in the cerebrospinal fluid. Clinical evolution was quickly unfavorable. The patient died 1 month after the PET/CT, and Creutzfeldt-Jakob disease was confirmed on postmortem examination of brain tissue.