Development of a plasmid addicted system that is independent of co-inducers, antibiotics and specific carbon source additions for bioproduct (1-butanol) synthesis in Escherichia coli

Synthetic biology approaches for the synthesis of value-based products provide interesting and potentially fruitful possibilities for generating a wide variety of useful compounds and biofuels. However, industrial production is hampered by the costs associated with the need to supplement large microbial cultures with expensive but necessary co-inducer compounds and antibiotics that are required for up-regulating synthetic gene expression and maintaining plasmid-borne synthetic genes, respectively. To address these issues, a metabolism-based plasmid addiction system, which relies on lipopolysaccharide biosynthesis and maintenance of cellular redox balance for 1-butanol production; and utilizes an active constitutive promoter, was developed in Escherichia coli. Expression of the plasmid is absolutely required for cell viability and 1-butanol production. This system abrogates the need for expensive antibiotics and co-inducer molecules so that plasmid-borne synthetic genes may be expressed at high levels in a cost-effective manner. To illustrate these principles, high level and sustained production of 1-butanol by E. coli was demonstrated under different growth conditions and in semi-continuous batch cultures, in the absence of antibiotics and co-inducer molecules.