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

Abstract

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.

Biosynthetic Pathways to Nonproteinogenic α-Amino Acids

“…This type of sequence analysis led to characterization of six novel lysine hydroxylases with unknown biosynthetic roles (Figure 32b). 208 Two of these enzymes form (3S)-hydroxylysine (165) (K3Hs), while the other four make (4R)-4-hydroxylysine (166) (K4Hs), and each type of enzyme was used for batch production of hydroxylysine at mM concentrations in E. coli. 208 Therefore, Fe(II)-, α-KG-dependent lysine hydroxylases represent a promising source of new biocatalysts.…”

“…This conserved feature allows for novel amino acid hydroxylases to be identified based on their amino acid sequences. This type of sequence analysis led to characterization of six novel lysine hydroxylases with unknown biosynthetic roles (Figure b) . Two of these enzymes form (3 S )-hydroxylysine ( 165 ) (K3Hs), while the other four make (4 R )-4-hydroxylysine ( 166 ) (K4Hs), and each type of enzyme was used for batch production of hydroxylysine at mM concentrations in E.…”

Industrial Application of 2-Oxoglutarate-Dependent Oxygenases

“…Apart from KDO1, six additional lysine hydroxylases (K3H1, K3H2, K4H1, K4H2, K4H3, and K4H4) are known for the regio-and stereoselective hydroxylation of the C-3 or C-4 position in L-lysine [124]. To explore their biocatalytic usefulness, Hara et al conducted several multi-gram scale hydroxylation reactions: Using K3H1 from Kineococcus radiotolerans (SRS30216) and K4H4 from Chryseobacterium gleum (ATCC 35910), the desired products (2S,3S)-3-hydroxylysine (531 mM; 86.1 g/L; 88% molar conversion in 52h) and (2S,4R)-4-hydroxylysine (265 m; 43 g/L, 88% molar conversion in 24h), respectively, could be synthesized in batch-scale experiments [124]. All of these enzymes are mentioned in a patent by API Corp. protecting the production of hydroxylysine [59].…”

Reshaping the Binding Pocket of Lysine Hydroxylase for Enhanced Activity

“…In addition, hydroxylysine is present in some biotherapeutic proteins derived from mammalian cells, including activated enzymes . As a significant intermediate for pharmaceutical agents, hydroxylysine has a variety of regioisomers and stereoisomers, such as (2 S ,4 R )-4-hydroxylysine, which is generated by the hydroxylation of l -lysine at C4 position. , As a promising precursor of functionalized (2 S ,4 R )-4-hydroxypiperidinic acid, (2 S ,4 R )-4-hydroxylysine is a highly versatile intermediate for the synthesis of many medicinally important building blocks, such as peptides, N -methyl- d -aspartic acid (NMDA) antagonists, enzyme inhibitors, , and immunosuppressants . For example, (2 S ,4 R )-4-hydroxypipecolic acid is a constituent of certain cyclopeptide antibiotics and has been used as a building block for the synthesis of a potent HIV protease inhibitor, palinavir (Figure ).…”