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

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 availablel -lysine withl -lysine hydroxylases from diverse microorganisms. Clavaminic acid synthase-like superfamily gene mining and phylogenetic analysis led to the discovery of six biocatalysts, namely twol -lysine 3S -hydroxylases and fourl -lysine 4R -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 ofl -lysine, yielding (2S ,3S )-3-hydroxylysine and (2S ,4R )-4-hydroxylysine, respectively. To determine if these factors had industrial application, we performed a preparative production of both hydroxylysines under optimized conditions. For this, recombinantl -lysine hydroxylase-expressingEscherichia coli cells were used as a biocatalyst forl -lysine bioconversion. In batch-scale reactions, 531 mM (86.1 g/liter) (2S ,3S )-3-hydroxylysine was produced from 600 mMl -lysine with an 89% molar conversion after a 52-h reaction, and 265 mM (43.0 g/liter) (2S ,4R )-4-hydroxylysine was produced from 300 mMl -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.IMPORTANCE The present study identified sixl -lysine hydroxylases belonging to the 2-oxoglutarate-dependent dioxygenase superfamily, although some of them overlapped with known hydroxylases. While the substrate specificity ofl -lysine hydroxylases was relatively narrow, we found that (2S ,3S )-3-hydroxylysine was hydroxylated by 4R -hydroxylase and (2S ,5R )-5-hydroxylysine was hydroxylated by both 3S - and 4R -hydroxylases. Moreover, thel -arginine hydroxylase VioC also hydroxylatedl -lysine, albeit to a lesser extent. Further, we also demonstrated the bioconversion ofl -lysine into (2S ,3S )-3-hydroxylysine and (2S ,4R )-4-hydroxylysine on a gram scale under optimized conditions. These findings provide new insights into biocatalyticl -lysine hydroxylation and thus have a great potential for use in manufacturing bioprocesses.