Engineering of Hydroxymandelate Oxidase and Cascade Reactions for High-Yielding Conversion of Racemic Mandelic Acids to Phenylglyoxylic Acids and (R)- and (S)-Phenylglycines

An alcohol oxidase (AOx) for the ( S )-enantioselective oxidation of 4-hydroxymandelic acid (4-HMA) to 4-hydroxyphenylglyoxylic acid (4-HPGA) with enhanced activity was developed by directed evolution of hydroxymandelate oxidase (HMO) through three rounds of iterative saturation mutagenesis. The engineered HMO mutant A80G-T159S-T162Q (HMOTM) catalyzed the oxidation of ( S )-4-HMA to 4-HPGA with a 23-fold enhancement in catalytic efficiency ( k cat / K M ). Substrate docking simulation on HMOTM suggested that A80G reoriented FMN, while T159S and T162Q formed hydrogen bonds with the carboxylic group of the substrate, thus facilitating substrate binding and catalysis. ( S )-Enantioselective HMOTM was used together with mandelic acid racemase (MR) and catalase (KatE) to achieve high-yielding oxidation of rac -4-HMA to 4-HPGA with either purified enzymes (up to 93% yield and 426 mM) or Escherichia coli (HMC) cells expressing the three enzymes (up to 93% yield and 140 mM). The HMOTM-MR-KatE cascades were applied for the oxidation of seven other substituted MAs, producing the corresponding phenylglyoxylic acids with 90–99% conversions using purified enzymes or whole cells. Efficient conversion of racemic α-hydroxy acids to ( S )- or ( R )-α-amino acids was achieved by combining HMOTM-MR-KatE with ( S )-enantioselective transaminase (EcαTA) or ( R )-enantioselective transaminase (DpgAT), together with glutamate dehydrogenase (GluDH). Coupling of E. coli (HMC) with E. coli (E-G) expressing EcαTA and GluDH for one-pot biotransformation of five rac -MAs produced the corresponding ( S )-phenylglycines (PGs) in 91–99% ee with 73–98% conversions. Using E. coli (HMC) and E. coli (D-G) expressing DpgAT and GluDH enabled the production of five ( R )-PGs with 93–99% ee and 73–98% conversions from the corresponding rac -MAs. The engineered AOx, AOx-MR-KatE cascades, and AOx-MR-Kat-GluDH-( S )- or ( R )-TA cascades provide useful tools for highly active and enantioselective oxidation of racemic hydroxyacids and high-yielding conversion of racemic hydroxyacids to ketoacids and enantiopure ( S )- or ( R )-aminoacids, respectively, which are challenging and useful chemical reactions.