Synthetic addiction extends the productive life time of engineered Escherichia coli populations

Significance Bioproduction of chemicals offers a sustainable alternative to petrochemical synthesis routes by using genetically engineered microorganisms to convert waste and simple substrates into higher-value products. However, efficient high-yield production commonly introduces a metabolic burden that selects for subpopulations of nonproducing cells in large fermentations. To postpone such detrimental evolution, we have synthetically addicted production cells to production by carefully linking signals of product presence to expression of nonconditionally essential genes. We addict Escherichia coli cells to their engineered biosynthesis of mevalonic acid by fine-tuned control of essential genes using a product-responsive transcription factor. Over the course of a long-term fermentation equivalent to industrial 200-m3 bioreactors such addicted cells remained productive, unlike the control, in which evolution fully terminated production. Bio-production of chemicals is an important driver of the societal transition toward sustainability. However, fermentations with heavily engineered production organisms can be challenging to scale to industrial volumes. Such fermentations are subject to evolutionary pressures that select for a wide range of genetic variants that disrupt the biosynthetic capacity of the engineered organism. Synthetic product addiction that couples high-yield production of a desired metabolite to expression of nonconditionally essential genes could offer a solution to this problem by selectively favoring cells with biosynthetic capacity in the population without constraining the medium. We constructed such synthetic product addiction by controlling the expression of two nonconditionally essential genes with a mevalonic acid biosensor. The product-addicted production organism retained high-yield mevalonic acid production through 95 generations of cultivation, corresponding to the number of cell generations required for >200-m3 industrial-scale production, at which time the nonaddicted strain completely abolished production. Using deep DNA sequencing, we find that the product-addicted populations do not accumulate genetic variants that compromise biosynthetic capacity, highlighting how synthetic networks can be designed to control genetic population heterogeneity. Such synthetic redesign of evolutionary forces with endogenous processes may be a promising concept for realizing complex cellular designs required for sustainable bio-manufacturing.