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 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—two l-lysine 3 S -hydroxylases and four l-lysine 4 R -hydroxylases. The latter of which partially matched known hydroxylases. Subsequent characterization of these hydroxylases revealed their capacity for regio- and stereo-selective hydroxylation into either C-3 and C-4 positions of l-lysine, yielding (2 S ,3 S )-3-hydroxylysine and (2 S ,4 R )-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 Escherichia coli cells were used as a biocatalyst for l-lysine bioconversion. In batch-scale reactions, 531 mM (86.1 g/L) (2 S ,3 S )-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/L) (2 S ,4 R )-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. IMPORTANCE 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 S ,3 S )-3-hydroxylysine was hydroxylated by 4 R -hydroxylase, and (2 S ,5 R )-5-hydroxylysine was hydroxylated by both 3 S - and 4 R -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 S ,3 S )-3-hydroxylysine and (2 S ,4 R )-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.