Chemo-enzymatic Synthesis of Coenzyme A Using Copurified Enzymes from Probiotic Escherichia coli Nissle

Coenzyme A (CoA) is an indispensable cofactor carrying fundamental building blocks in cells (e.g., acetyl-CoA). However, in vivo CoA synthesis is often limited by feedback inhibition and the cost of purification; instead, in vitro enzymatic CoA synthesis represents a sustainable alternative. Herein, we present a one-pot chemo-enzymatic cascade pathway for free CoA synthesis from pantethine using recombinant enzymes purified from probiotic Escherichia coli Nissle (endotoxin-free), along with the polyphosphate kinase system for ATP regeneration and the phosphite dehydrogenase system for NADH regeneration, respectively. First, we selected the nontoxic biological reductant glutathione for chemical pantethine reduction, where the glutathione is readily reduced by the NAD(P)H-dependent glutathione reductase. The one-pot enzymatic cascade (comprising pantothenate kinase, pantetheine-phosphate adenylyltransferase, and dephospho-CoA kinase) then completely converts pantetheine into free CoA in 2 h with a titer of ∼2 mM (1.5 g/L) in a 0.1 mL reaction system. Finally, the quality of the chemoenzymatically synthesized CoA rivals that of the commercial CoA standard for acetyl-CoA synthesis. Copurifying the recombinant enzymes from probiotic E. coli Nissle and the use of inorganic phosphate buffer further reduce the environmental burdens and carbon footprint of the whole process. Therefore, the present work could serve as a paradigm for future industrial production of acyl-CoA derivatives and other valuable cofactors.