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Fe(II)/2-ketoglutarate-dependent dioxygenase (Fe(II)/2-KG DO)-mediated hydroxylation is a critical type of C–H bond functionalization for synthesizing hydroxy amino acids used as pharmaceutical raw materials and precursors. However, DO activity requires 2-ketoglutarate (2-KG), lack of which reduces the efficiency of Fe(II)/2-KG DO-mediated hydroxylation. Here, we conducted multi-enzymatic syntheses of hydroxy amino acids. Using (2<i><span style="font-variant: small-caps;">s</span></i>,3<i><span style="font-variant: small-caps;">r</span></i>,4<i><span style="font-variant: small-caps;">s</span></i>)-4-hydroxyisoleucine (4-HIL) as a model product, we coupled regio- and stereo-selective hydroxylation of <span style="font-variant: small-caps;">l</span>-Ile by the dioxygenase IDO with 2-KG generation from readily available <span style="font-variant: small-caps;">l</span>-Glu by <span style="font-variant: small-caps;">l</span>-glutamate oxidase (LGOX) and catalase (CAT). In the one-pot system, H₂O₂ significantly inhibited IDO activity and elevated Fe²⁺ concentrations of severely repressed LGOX. A sequential cascade reaction was preferable to a single-step process as CAT in the former system hydrolyzed H₂O₂. We obtained 465 mM 4-HIL at 93% yield in the two-step system. Moreover, this process facilitated C–H hydroxylation of several hydrophobic aliphatic amino acids to produce hydroxy amino acids, and C–H sulfoxidation of sulfur-containing <span style="font-variant: small-caps;">l</span>-amino acids to yield <span style="font-variant: small-caps;">l</span>-amino acid sulfoxides. Thus, we constructed an efficient cascade reaction to produce 4-HIL by providing prerequisite 2-KG from cheap and plentiful <span style="font-variant: small-caps;">l</span>-Glu and developed a strategy for creating enzymatic systems catalyzing 2-KG-dependent reactions in sustainable bioprocesses that synthesize other functional compounds.