Exploiting Day- and Night-Time Metabolism of Synechocystis sp. PCC 6803 for Fitness-Coupled Fumarate Production around the Clock.

Cyanobacterial cell factories are widely researched for the sustainable production of compounds directly from CO2. Their application, however, has been limited for two reasons. First, traditional approaches have been shown to lead to unstable cell factories that lose their production capability when scaled to industrial levels. Second, the alternative approaches developed so far are mostly limited to growing conditions, which are not always the case in industry, where nongrowth periods tend to occur (e.g., darkness). We tackled both by generalizing the concept of growth-coupled production to fitness coupling. The feasibility of this new approach is demonstrated for the production of fumarate by constructing the first stable dual-strategy cell factory. We exploited circadian metabolism using both systems and synthetic biology tools, resulting in the obligatorily coupling of fumarate to either biomass or energy production. Resorting to laboratory evolution experiments, we show that this engineering approach is more stable than conventional methods.

In vivo continuous evolution of metabolic pathways for chemical production.

Microorganisms have long been used as chemical plant to convert simple substrates into complex molecules. Various metabolic pathways have been optimised over the past few decades, but the progresses were limited due to our finite knowledge on metabolism. Evolution is a knowledge-free genetic randomisation approach, employed to improve the chemical production in microbial cell factories. However, evolution of large, complex pathway was a great challenge. The invention of continuous culturing systems and in vivo genetic diversification technologies have changed the way how laboratory evolution is conducted, render optimisation of large, complex pathway possible. In vivo genetic diversification, phenotypic selection, and continuous cultivation are the key elements in in vivo continuous evolution, where any human intervention in the process is prohibited. This approach is crucial in highly efficient evolution strategy of metabolic pathway evolution.