Modular electron transfer circuits for synthetic biology: insulation of an engineered biohydrogen pathway

Abstract

Electron transfer is central to a wide range of essential metabolic pathways, from photosynthesis to fermentation. The evolutionary diversity and conservation of proteins that transfer electrons makes these pathways a valuable platform for engineered metabolic circuits in synthetic biology. Rational engineering of electron transfer pathways containing hydrogenases has the potential to lead to industrial scale production of hydrogen as an alternative source of clean fuel and experimental assays for understanding the complex interactions of multiple electron transfer proteins in vivo. We designed and implemented a synthetic hydrogen metabolism circuit in Escherichia coli that creates an electron transfer pathway both orthogonal to and integrated within existing metabolism. The design of such modular electron transfer circuits allows for facile characterization of in vivo system parameters with applications toward further engineering for alternative energy production.

Keywords: electron transfer; ferredoxin; hydrogen; iron-sulfur cluster; modularity; synthetic biology.

Figure 1

(A) Schematic of the synthetic hydrogen production pathway. The circuit is analogous to an amorphous electronic circuit. Electrons enter the circuit at −520 mV through the function of pyruvate-ferredoxin oxidoreductase (PFOR), which thus behaves as a “battery.” Electrons are transferred to ferredoxin, an electron carrier that behaves as a “resistive wire.” Ferredoxin passes electrons to the hydrogenase, which in turn produces hydrogen at −420 mV that can be measured as as the “current” through the circuit. The circuit can be insulated from competing cellular electron metabolism through: (B) deletion of reactions that can interact with any of the circuit components leading to “short circuits”, (C) improvement of the ferredoxin-hydrogenase binding surface and (D) through direct protein fusion of the ferredoxin and hydrogenase. This fusion provides physical “circuit board” structure that increases the local concentration of electrons available to the hydrogenase.