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β-Hydroxy-α-amino acids are useful compounds for pharmaceutical development. Enzymatic synthesis of β-hydroxy-α-amino acids has attracted considerable interest as a selective, sustainable, and environmentally benign process. In this study, we identified a novel amino acid hydroxylase, AEP14369, from Sulfobacillus thermotolerans Y0017, which is included in a previously constructed CAS-like superfamily protein library, to widen the variety of amino acid hydroxylases. The detailed structures determined by nuclear magnetic resonance and X-ray crystallography analysis of the enzymatically produced compounds revealed that AEP14369 catalyzed threo -β-selective hydroxylation of l-His and l-Gln in a 2-oxoglutarate-dependent manner. Furthermore, the production of l- threo -β-hydroxy-His and l- threo -β-hydroxy-Gln was achieved using Escherichia coli expressing the gene encoding AEP14369 as a whole-cell biocatalyst. Under optimized reaction conditions, 137 mM (23.4 g liter -1 ) l- threo -β-hydroxy-His and 150 mM l- threo -β-hydroxy-Gln (24.3 g liter -1 ) were obtained, indicating that the enzyme is applicable for preparative-scale production. AEP14369, an l-His/l-Gln threo -β-hydroxylase, increases the availability of 2-oxoglutarate-dependent hydroxylase and opens the way for the practical production of β-hydroxy-α-amino acids in the future. The amino acids produced in this study would also contribute to the structural diversification of pharmaceuticals that affect important bioactivities. IMPORTANCE Owing to an increasing concern for sustainability, enzymatic approaches for producing industrially useful compounds have attracted considerable attention as a powerful complement to chemical synthesis for environment-friendly synthesis. In this study, we developed a bioproduction method for β-hydroxy-α-amino acid synthesis using a newly discovered enzyme. AEP14369 from the moderate thermophilic bacterium Sulfobacillus thermotolerans Y0017 catalyzed the hydroxylation of l-His and l-Gln in a regioselective and stereoselective fashion. Furthermore, we biotechnologically synthesized both l- threo -β-hydroxy-His and l- threo -β-hydroxy-Gln with a titer of over 20 g liter -1 through whole-cell bioconversion using recombinant Escherichia coli cells. As β-hydroxy-α-amino acids are important compounds for pharmaceutical development, this achievement would facilitate future sustainable and economical industrial applications.

Toward a sustainable synthesis of value-added chemicals, the method of CO 2 utilization attracts great interest in chemical process engineering. Biotechnological CO 2 fixation is a promising technology; however, efficient methods that can fix carbon dioxide are still limited. Instead, some parts of microbial decarboxylases allow the introduction of carboxy group into phenolic compounds using bicarbonate ion as a C1 building block. Here, we identified a unique decarboxylase from Arthrobacter sp. K8 that acts on resorcinol derivatives. A high-throughput colorimetric decarboxylase assay facilitated gene cloning of orsellinic acid decarboxylase from genomic DNA library of strain K8. Sequence analysis revealed that the orsellinic acid decarboxylase belonged to amidohydrolase 2 family, but shared low amino acid sequence identity with those of related decarboxylases. Enzymatic characterization unveiled that the decarboxylase introduces a carboxy group in a highly regio-selective manner. We applied the decarboxylase to enzymatic carboxylation of resorcinol derivatives. Using Escherichia coli expressing the decarboxylase gene as a whole cell biocatalyst, orsellinic acid, 2,4-dihydroxybenzoic acid, and 4-methoxysalicylic acid were produced in the presence of saturated bicarbonate. These findings could provide new insights into the production of useful phenolic acids from resorcinol derivatives., (Copyright © 2020 Elsevier B.V. All rights reserved.)