Cloning of an epoxide hydrolase-encoding gene from Rhodotorula mucilaginosa and functional expression in Yarrowia lipolytica.
Epoxide hydrolases (EHs), especially those of fungal origin, have the ability to catalyse the enantioselective hydrolysis of epoxides to their corresponding diols. Recombinant DNA technology has been used extensively to overproduce these catalysts for the efficient hydrolytic kinetic resolution of epoxides, which serve as high-value intermediates in the fine chemicals and pharmaceutical industries. Degenerate primers, based on data from available EH-encoding gene sequences, in conjunction with inverse PCR, were used to amplify the genomic EH-encoding gene from Rhodotorula mucilaginosa. The 2347 bp genomic sequence revealed a 1979 bp ORF containing nine introns. The cDNA sequence revealed an 1185 bp EH-encoding gene that translates into a 394 amino acid protein exhibiting low sequence homology towards the known EH proteins. The EH gene from R. mucilaginosa was functionally expressed in Yarrowia lipolytica using a constitutive integrative expression cassette. Whole-cell biotransformation of (2,3-epoxypropyl)benzene, using the recombinant EH, revealed activity and selectivity far superior to any other activity and selectivity reported in literature using wild-type organisms. The GenBank Accession No. for the R. mucilaginosa EH gene is AY627310.
Labuschagne
Michel
M
Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, PO Box 339, Bloemfontein 9300, South Africa.
Albertyn
Jacobus
J
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004851:Epoxide Hydrolases / Q000096:biosynthesis / Q000235:genetics* / Q000378:metabolism; D004852:Epoxy Compounds / Q000378:metabolism*; D008969:Molecular Sequence Data; D016254:Mutagenesis, Insertional; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism; D012248:Rhodotorula / Q000201:enzymology* / Q000235:genetics*; D016415:Sequence Alignment; D025062:Yarrowia / Q000201:enzymology / Q000235:genetics*
D004271:DNA, Fungal; D004852:Epoxy Compounds; D011994:Recombinant Proteins; D004851:Epoxide Hydrolases; C497708:(2,3-epoxypropyl)benzene