Production of (R)-mandelic acid from styrene, L-phenylalanine, glycerol, or glucose via cascade biotransformations

Abstract

Abstract(R)-mandelic acid is an industrially important chemical, especially used for producing antibiotics. Its chemical synthesis often uses highly toxic cyanide to produce its racemic form, followed by kinetic resolution with 50% maximum yield. Here we report a green and sustainable biocatalytic method for producing (R)-mandelic acid from easily available styrene, biobased L-phenylalanine, and renewable feedstocks such as glycerol and glucose, respectively. An epoxidation-hydrolysis-double oxidation artificial enzyme cascade was developed to produce (R)-mandelic acid at 1.52 g/L from styrene with > 99% ee. Incorporation of deamination and decarboxylation into the above cascade enables direct conversion of L-phenylalanine to (R)-mandelic acid at 913 mg/L and > 99% ee. Expressing the five-enzyme cascade in an L-phenylalanine-overproducing E. coli NST74 strain led to the direct synthesis of (R)-mandelic acid from glycerol or glucose, affording 228 or 152 mg/L product via fermentation. Moreover, coupling of E. coli cells expressing L-phenylalanine biosynthesis pathway with E. coli cells expressing the artificial enzyme cascade enabled the production of 760 or 455 mg/L (R)-mandelic acid from glycerol or glucose. These simple, safe, and green methods show great potential in producing (R)-mandelic acid from renewable feedstocks.

Engineering of Cascade Reactions and Alditol Oxidase for High‐Yielding Synthesis of (R)‐Phenylethanolamine from Styrene, l‐Phenylalanine, Glycerol or Glucose

“…These results for the first time clearly demonstrated the feasibility of the use of AldO and CvTA for cascade conversion of ( R )‐PED to ( R )‐PEA. Although H 2 O 2 was produced from the AldO‐catalyzed oxidation reaction, no obvious inhibition was observed, probably due to the endogenous catalase that decomposes H 2 O 2 in E. coli cells [22a,31] . In comparison, cascade biotransformation of ( R )‐PED via 4‐enzyme system (AcCO6‐Ch1AmDH‐catalase‐CbFDH) gave 14.4 mM ( R )‐PEA in 48 h with 72 % conversion [21] …”

“…AldO is a flavin‐dependent oxidase, which uses cheap and clean molecular oxygen as electron acceptor to selectively oxidize a broad range of alcohols, producing H 2 O 2 as a co‐product [27b] . Recently, AldO was found to enantioselectively oxidize ( R )‐PED to ( R )‐mandelic acid (MA) via ( R )‐MALD [22a,27a] . In order to quickly convert ( R )‐MALD into ( R )‐PEA without the formation of ( R )‐MA, an enzyme for efficient amination of ( R )‐MALD is required.…”

Optimization of the medium composition and product extraction for R-mandelic acid using recombinant Escherichia coli expressing Alcaligenes sp. nitrilase

“…1,2 Biocatalysts are inexpensive, environmentally responsible and highly efficient alternatives to chemical catalysts, and have attracted increasing attention in industrial settings. 3 Due to the importance of R -MA as a chiral building block, numerous biosynthetic routes for R -MA production have been reported, including examples based on lipase, 4,5 esterase, 6 a biocatalyst for the asymmetric reduction of phenyl glyoxylic acid (PGA) to R -mandelic acid, 7 the production of R -MA from styrene, L-phenylalanine, glycerol, or glucose via cascade biotransformations, 8 the synthesis of R -MA and R -MA amide by utilizing recombinant E. coli strains expressing an R -specific oxynitrilase and an arylacetonitrilase, 9 and biotransformation of mandelonitrile with nitrilase. 2,10…”

“…1,2 Biocatalysts are inexpensive, environmentally responsible and highly efficient alternatives to chemical catalysts, and have attracted increasing attention in industrial settings. 3 Due to the importance of R-MA as a chiral building block, numerous biosynthetic routes for R-MA production have been reported, including examples based on lipase, 4,5 esterase, 6 a biocatalyst for the asymmetric reduction of phenyl glyoxylic acid (PGA) to R-mandelic acid, 7 the production of R-MA from styrene, L-phenylalanine, glycerol, or glucose via cascade biotransformations, 8 the synthesis of R-MA and R-MA amide by utilizing recombinant E. coli strains expressing an R-specific oxynitrilase and an arylacetonitrilase, 9 and biotransformation of mandelonitrile with nitrilase. 2,10 Among the enzyme sources, nitrilases have been employed frequently as a powerful tool for the enantioselective hydrolysis of racemic mandelonitrile, and the nitrilase-mediated pathway is superior because of excellent enantioselectivity, low-cost starting materials, and the possibility of performing a dynamic kinetic resolution of the racemic substrate, which offers a theoretical yield of 100%.…”

Cascade Biotransformations for Enantioconvergent Conversion of Racemic Styrene Oxides to (R)-Mandelic Acids

“…Enzymatic cascades involving multiple enzymes in one pot allow for direct syntheses of valuable enantiopure chemicals from cheap starting materials. − In addition to the use of nonchiral substrates, the use of easily available racemic substrates for enantioconvergent cascade conversion to enantiopure chemicals , has been receiving increasing attention. We are interested in developing a new enantioconvergent cascade to convert racemic epoxides into the corresponding enantiopure hydroxy acids.…”