[2S06a] 23.Protein and Metabolic Engineering

3:15 PM - 3:55 PM JST(6:15 AM - 6:55 AM UTC)

[2MBT-01-KL] Orthogonal energy supply and transfer for metabolism

○Zongbao K. ZHAO¹ (1. Dalian University of Technology (China))

Keywords:

Energy metabolism,Non-natural redox cofactor,Metabolic engineering,C1 biotechnology,Redox enzymes

Reduced nicotinamide cofactors including NAD(P)H are ubiquitous energy carriers that fuel biosynthesis and the respiratory chain for ATP production. Thus, fluctuation in NAD(P)H level leads to systemic yet unpredictable biological consequences. To enable more rational control over energy metabolism, we created a non-natural cofactor, nicotinamide cytosine dinucleotide (NCD) and employed its reduced form NCDH for energy transfer in vivo. Specifically, we constructed NCD-dependent dehydrogenases that can abstract energy from phosphite, formate, methanol or formaldehyde, thus enable generation of energy carrier orthogonal to endogenous systems. Next, we engineered enzymes including malic enzyme, lactate dehydrogenase and cytochrome P450 BM3 to favor NCDH over NAD(P)H. In vitro, it was found that NCDH-mediated reductive reactions can operate regardless the presence of NAD(P)H. When implanted in Escherichia coli cells, the flux direction of malic enzyme-catalyzed reaction was reversed driving by NCDH at the expense of formate or phosphite. Interestingly, it was found that NCDH can be generated via transhydrogenation process from NAD(P)H, which greatly expand the potential to use NCDH for targeted energy supply. Taken together, NCD(H) and associated enzymes offer unique tools to regulate energy supply that should be widely applicable in synthetic biology and chemical biology.