Design and Construction of an Artificial Nonmevalonate Operon of Escherichia coli

So far it has been possible to construct bacterial genome from chemically synthesis DNAs, but no example of designer genome emerged, due to non strategy for design genome. Toward creation of a novel genome, we should start to construct a readily simplified genome in terms of engineering that is far apart from the naturally occurring bacterial genome because it is too complicated. In a bacterium with the simplified genome, it can be regarded as an assembly of metabolic pathways. To design each metabolic pathway, certain systems for balancing quantity of multiple kinds of enzymes are required. We have been proposed that polycistronic operon architecture that is well observed in bacterial genome contains hints for the genome designing. For a experimental demonstration for construction of a metabolic pathway by a designed polycistronic operon, 9 of Escherichia coli genes for nonmevalonate pathway, one of the essential metabolic pathways, were rendered. According to the possible operon rule, the genes were connected under a sole promoter from highest to lowest of the mRNA abundance in the wild type cell in order by OGAB assembly. This prototype operon plasmid was introduced into wild type E. coli cell to knock out relevant nonmevalonate genes in the genome, but two of the relevant genes weren’t able to remove from the genome. In case of ispE, the ORF contains a promoter sequence for essential gene located downstream. In case of dxr, expression level is lower than expected in the operon. By taking account of these facts, finally we could construct new operon that doesn’t require any of nonmevalonate genes in the genome.