One focus of synthetic biology is the assessment of minimal gene sets required for cellular life. However, in early evolution, pathways in operation may no longer be used in modern cells. Fundamentally rewiring pathways of conserved central metabolism is the next frontier for this field, and is essential to assessing their origins. Our group is using synthetic biology to test a hypothesis about the emergence of DNA genomes. Ribonucleotide reduction is universally used in biology for de novo synthesis of deoxyribonucleotides. The chemical complexity of this reaction suggests that transition to DNA genomes may have occurred relatively late, possibly after the primary lineages of modern life began to diverge. However, ongoing interdomain horizontal gene transfer obscures the evolutionary history of ribonucleotide reductases (RNRs). However, an alternative, chemically simpler, pathway for the synthesis of deoxyribonucleotides may predate ribonucleotide reduction. In this pathway, production of deoxyribose is catalysed by deoxyriboaldolase (DERA). This pathway is ubiquitous, but naturally runs in the catabolic direction. Our goal is to establish the operation of the DERA pathway in the synthetic direction in vivo, aiming for complete functional replacement of ribonucleotide reduction by reverse DERA. If achieved, this will be the only free-living cell that produces its deoxyribonucleotides de novo without RNRs, bolstering the plausibility of an earlier transition to DNA genomes by simpler chemistry. Our group has generated knockouts of all RNRs in Escherichia coli, completely abolishing ribonucleotide reduction activity, resulting in deoxyribonucleoside (dNS) auxotrophic strains. We are screening for conditions where the DERA pathway permits cells to synthesise dNS in the absence of RNRs. This research highlights the value of synthetic biology for experimentally testing hypotheses on the origin of life.