A Hitchhiker’s Guide to Supplying Enzymatic Reducing Power into Synthetic Cells

Abstract

The construction from scratch of synthetic cells by assembling molecular building blocks is unquestionably an ambitious goal from a scientific and technological point of view. To realize functional life-like systems, minimal enzymatic modules are required to sustain the processes underlying the out-ofequilibrium thermodynamic status hallmarking life, including the essential supply of energy in the form of electrons. The nicotinamide cofactors NAD(H) and NADP(H) are the main electron carriers fueling reductive redox reactions of the metabolic network of living cells. One way to ensure the continuous availability of reduced nicotinamide cofactors in a synthetic cell is to build a minimal enzymatic module that can oxidize an external electron donor and reduce NAD(P) + . In the diverse world of metabolism there is a plethora of potential electron donors and enzymes known from living organisms to provide reducing power to NAD(P) + coenzymes. This perspective proposes guidelines to enable the reduction of nicotinamide cofactors enclosed in phospholipid vesicles, while avoiding high burdens of or cross-talk with other encapsulated metabolic modules. By determining key requirements, such as the feasibility of the reaction and transport of the electron donor into the cell-like compartment, we select a shortlist of potentially suitable electron donors. We review the most convenient proteins for the use of these reducing agents, highlighting their main biochemical and structural features. Noting that specificity toward either NAD(H) or NADP(H) imposes a limitation common to most of the analyzed enzymes, we discuss the need for specific enzymes�transhydrogenases�to overcome this potential bottleneck.

Biochemical and structural insight into the chemical resistance and cofactor specificity of the formate dehydrogenase from Starkeya novella

“…Particularly, Fdh SNO has recently been successfully used as a pivotal component of a minimal enzymatic pathway triggered by formate ensuring the redox regeneration in the field of bottom-up synthetic biology [14,63]. Furthermore, the manufacture of valuable enantiomers for different industries [25] could represent an application for this biocatalyst, both in in vivo and in vitro systems.…”

A Role for Bottom-Up Synthetic Cells in the Internet of Bio-Nano Things?

“…SCs then become competent for enzyme reactions, gene expression (as specified above), metabolic pathways for lipid synthesis [ 136 ], simple forms of control based on the activation of gene expression, the enzymatic production of signal molecules, sensing signal molecules, the formation of pores and gap junctions [ 137 , 138 ] in the membrane and periodic reaction–diffusion waves [ 139 ], genetic material replication, and the production of toxins [ 140 , 141 , 142 ]. Additional relevant functions, like bio-energy generation, have recently been achieved [ 105 , 143 , 144 , 145 ], while others are under preliminary analyses (e.g., reducing power for metabolism [ 146 ]; chemical neural networks for computation and control [ 114 ]). These functions have generally been implemented individually, one at a time.…”

Chemical Systems for Wetware Artificial Life: Selected Perspectives in Synthetic Cell Research

“…SCs with smaller internal compartments are used as models of eukaryotic cells, or just to achieve specific and more complex behaviors [ 131 ]. Interested readers should refer to recent reviews, e.g., [ 132 , 133 , 134 , 135 , 136 , 137 , 138 , 139 , 140 , 141 , 142 , 143 , 144 , 145 , 146 , 147 , 148 , 149 ].…”