Discovery of Lysine Hydroxylases in the Clavaminic Acid Synthase-Like Superfamily for Efficient Hydroxylysine Bioproduction

• Published in: Applied and environmental microbiology 2017-09, Vol.83 (17)
• Main Authors: Hara, Ryotaro, Yamagata, Kai, Miyake, Ryoma, Kawabata, Hiroshi, Uehara, Hisatoshi, Kino, Kuniki
• Format: Article
• Language: English
• Subjects: Acids; Amino acids; Bacteria - chemistry; Bacteria - classification; Bacteria - enzymology; Bacteria - genetics; Biocatalysts; Bioconversion; E coli; Enzymology and Protein Engineering; Escherichia coli - genetics; Escherichia coli - metabolism; Hydrogen bonds; Hydroxylase; Hydroxylation; Hydroxylysine - chemistry; Hydroxylysine - metabolism; Lysine; Lysine - metabolism; Microorganisms; Mixed Function Oxygenases - chemistry; Mixed Function Oxygenases - genetics; Mixed Function Oxygenases - metabolism; Multigene Family; Oxidation; Phylogenetics; Phylogeny; Procollagen-lysine 5-dioxygenase; Reagents; Substrate Specificity
• ISSN: 0099-2240; 1098-5336
• DOI: 10.1128/aem.00693-17

Hydroxylation via C-H bond activation in the absence of any harmful oxidizing reagents is technically difficult in modern chemistry. In this work, we attempted to generate pharmaceutically important hydroxylysine from readily available l-lysine with l-lysine hydroxylases from diverse microorganisms. Clavaminic acid synthase-like superfamily gene mining and phylogenetic analysis led to the discovery of six biocatalysts, namely two l-lysine 3 -hydroxylases and four l-lysine 4 -hydroxylases, the latter of which partially matched known hydroxylases. Subsequent characterization of these hydroxylases revealed their capacity for regio- and stereoselective hydroxylation into either C-3 or C-4 positions of l-lysine, yielding (2 ,3 )-3-hydroxylysine and (2 ,4 )-4-hydroxylysine, respectively. To determine if these factors had industrial application, we performed a preparative production of both hydroxylysines under optimized conditions. For this, recombinant l-lysine hydroxylase-expressing cells were used as a biocatalyst for l-lysine bioconversion. In batch-scale reactions, 531 mM (86.1 g/liter) (2 ,3 )-3-hydroxylysine was produced from 600 mM l-lysine with an 89% molar conversion after a 52-h reaction, and 265 mM (43.0 g/liter) (2 ,4 )-4-hydroxylysine was produced from 300 mM l-lysine with a molar conversion of 88% after 24 h. This report demonstrates the highly efficient production of hydroxylysines using lysine hydroxylases, which may contribute to future industrial bioprocess technologies. The present study identified six l-lysine hydroxylases belonging to the 2-oxoglutarate-dependent dioxygenase superfamily, although some of them overlapped with known hydroxylases. While the substrate specificity of l-lysine hydroxylases was relatively narrow, we found that (2 ,3 )-3-hydroxylysine was hydroxylated by 4 -hydroxylase and (2 ,5 )-5-hydroxylysine was hydroxylated by both 3 - and 4 -hydroxylases. Moreover, the l-arginine hydroxylase VioC also hydroxylated l-lysine, albeit to a lesser extent. Further, we also demonstrated the bioconversion of l-lysine into (2 ,3 )-3-hydroxylysine and (2 ,4 )-4-hydroxylysine on a gram scale under optimized conditions. These findings provide new insights into biocatalytic l-lysine hydroxylation and thus have a great potential for use in manufacturing bioprocesses.