This Work activity on L-proline, was previously reported to produce trans-5-hydroxy-L-Pip 12 in 68-78% conversion and 61% isolated yield on a preparative scale (35 mg) by using a purified enzyme or a recombinant E. coli. Our initial attempt to reproduce this hydroxylation only resulted in 39% conversion, which was ultimately improved to 100% after significant optimization of the reaction parameters (equivalence of a-KG and Fe 2+ and buffer pH, Fig. and Table in the Supporting Information). The optimized condition was amenable to gram scale synthesis, with higher substrate concentration than previously reported conditions (8 mM vs 1 mM). Reasoning that the stereochemical pattern on the hydroxylated building blocks would have significant influence on the stereochemical outcome of subsequent functionalizations, the synthesis of cis-5-hydroxy-L-Pip 13 was sought. Toward this end, an engineered a-KG-dependent L-pipecolic acid hydroxylase from Xenorhabdus doucetiae (XdPH YR) and a proline-4-hydroxylase (P4H810) developed by