Specific flavoenzyme oxidases catalyze oxidative decarboxylation in addition to their classical oxidation reactions in the same active sites. The mechanisms underlying oxidative decarboxylation by these enzymes and how they control their two activities are not clearly known. This article reviews the current state of knowledge of four enzymes from the l‐amino acid oxidase and l‐hydroxy acid oxidase families, including l‐tryptophan 2‐monooxygenase, l‐phenylalanine 2‐oxidase and l‐lysine oxidase/monooxygenase and lactate monooxygenase which catalyze substrate oxidation and oxidative decarboxylation. Apart from specific interactions to allow substrate oxidation by the flavin cofactor, specific binding of oxidized product in the active sites appears to be important for enabling subsequent decarboxylation by these enzymes. Based on recent findings of l‐lysine oxidase/monooxygenase, we propose that nucleophilic attack of H2O2 on the imino acid product is the mechanism enabling oxidative decarboxylation.

Cofactor imbalance obstructs the productivities of metabolically engineered cells. Herein, we employed a minimally perturbing system, xylose reductase and lactose (XR/lactose), to increase levels of a pool of sugar-phosphates which are connected to the biosynthesis of NAD(P)H, FAD, FMN and ATP in Escherichia coli. The XR/lactose system could increase the amounts of the precursors of these cofactors and was tested with three different metabolically engineered cell systems (fatty alcohol biosynthesis, bioluminescence light generation and alkane biosynthesis) with different cofactor demands. Productivities of these cells were increased 2-4-fold by the XR/lactose system. Untargeted metabolomic analysis revealed different metabolite patterns among these cells; demonstrating that only metabolites involved in relevant cofactor biosynthesis were altered. The results were also confirmed by transcriptomic analysis. Another sugar reducing system (glucose dehydrogenase, GDH) could also be used to increase fatty alcohol production but resulted in less yield enhancement than XR. This work demonstrates that the approach of increasing cellular sugar phosphates can be a generic tool to increase in vivo cofactor generation upon cellular demand for synthetic biology.

Microbial production of hydrogen (future ideal fuel and important gas for industries) under anoxic conditions has limited ATP availability and low efficiency. We engineered E. coli K12 to acquire a flavin-based electron bifurcation (FBEB) system, a bioenergetic route typically found in strict anaerobes, which uses NADH to generate low potential reduced ferredoxin and high potential butyryl-CoA. The oxygen-tolerant FBEB-E. coli showed higher H2 and succinate production (2-4 folds), lower cellular reduction potentials, greater accumulation of cellular reductants and various metabolites, including ATP (up to a 7-fold increase). It could better tolerate prolonged and recycled usage of the engineered cell for H2 and succinate production than the native strain. FBEB-E. coli could also use various substrates such as formate, D-glucose and food waste for H2 and succinate production. This is a promising pathway to sustainable H2 and succinate production. This work also demonstrates that E. coli with an extra electron bifurcation system is a robust synthetic biology host.