5-thiohistidine N-acetyltransferase from Proteiniphilum saccharofermentans.

Ovothiol A is a 5-thiohistidine derivative biosynthesized by a broad range of prokaryotic and eukaryotic organisms. Its redox-active mercaptoimidazole side chain is believed to protect cells from oxidative stress. The three enzymes that produce ovothiol A from histidine, cysteine, and S-adenosyl methionine have been identified and characterized.

Development of an enzymatic cascade for semi de novo ATP production using thermophilic enzymes.

Industrial production of ATP has mostly relied on extraction from living cells. Although microbial and enzymatic ATP production have also been developed, the former suffers from complexity in product separation, while the latter requires expensive substrates, making their practical use difficult. To tackle these problems, we newly developed an enzymatic cascade for ATP production, which does not use expensive substrates, by assembling 16 thermophilic enzymes prepared through a heat-purification from the crude extract of recombinant Escherichia coli.

Cell-Free Production and Regeneration of Cofactors.

Cofactors, such as adenosine triphosphate, nicotinamide adenine dinucleotide, and coenzyme A, are involved in nearly 50% of enzymatic reactions and widely used in biocatalytic production of useful chemicals. Although commercial production of cofactors has been mostly dependent on extraction from microbial cells, this approach has a theoretical limitation to achieve a high-titer, high-yield production of cofactors owing to the tight regulation of cofactor biosynthesis in living cells. Besides the cofactor production, their regeneration is also a key challenge to enable continuous use of costly cofactors and improve the feasibility of enzymatic chemical manufacturing.

One-Step Preparation of Cell-Free ATP Regeneration Module Based on Non-Oxidative Glycolysis Using Thermophilic Enzymes.

Adenosine triphosphate (ATP) is an essential cofactor for energy-dependent enzymatic reactions that occur during in vitro biochemical conversion. Recently, an enzyme cascade based on non-oxidative glycolysis, which uses starch and orthophosphate as energy and phosphate sources, respectively, for the regeneration of ATP from adenosine diphosphate, has been developed (Wei et al., ChemCatChem 2018, 10, 5597-5601).

Production of Coenzyme A Using Thermophilic Enzymes.

Coenzyme A (CoA) is an essential cofactor present in all domains of life and is involved in numerous metabolic pathways, including fatty acid metabolism, pyruvate oxidation through the tricarboxylic acid (TCA) cycle, and the production of secondary metabolites. This characteristic makes CoA a commercially valuable compound in the pharmaceutical, cosmetic, and clinical industries. However, CoA is difficult to accumulate in living cells at a high level, since it is consumed in multiple metabolic pathways, hampering its manufacturing by typical cell cultivation and extraction approaches.