(585at) Non-Natural Redox Cofactor-Wired Metabolic Circuits

Substance transformation and energy transfer are two tightly-connected aspects of cellular metabolism. While tremendous progresses have been achieved in regulating substance transformation, our capacity remains limited to control energy transfer. To facilitate an energy transfer process, it requires three basic components, energy supplying module (ESM), energy carrier (EC) and energy utilizing module (EUM). As ESM and EUM are usually linked to substance transformation, the process can be considered as an EC-wired metabolic circuit. Because natural EC, such as the reduced form of NAD(P), are shared by many ESM and EUM, it is intrinsically challenging to transfer energy selectively. We devised non-natural redox cofactor-wired subsystem to better control energy transfer. We used phosphite dehydrogenase as ESM, nicotinamide cytosine dinucleotide (NCD) and NAD as EC, and malic enzyme and lactate dehydrogenase as EUM. We engineered these ESM and EUM to use NCD. We showed that chemical energy in phosphite could be converted into charged EC and then selectively drive NCD– or NAD–dependent EUM. When such systems operated in engineered Escherichia coli, it reversed the flux direction of malic enzyme-catalyzed reaction and led to improved malate production by up to 38%. This synthetic system generated NCDH from phosphite to drive the metabolic pathway, which is essentially different from other known approaches linked to endogenous substance transformation reactions. Currently we are engineering other redox enzymes to use non-natural redox cofactors and constructing more synthetic pathways in different hosts.

 

Representative references

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