title: Heterologous expression of the benzoate para-hydroxylase encoding gene (CYP53B1) from Rhodotorula minuta by Yarrowia lipolytica.
abstract: There is currently an increasing number of cytochrome P450 (CYP450) monooxygenase encoding genes becoming available from various genome-sequencing projects. These enzymes require association with cytochrome P450 reductase (CPR) to achieve optimal activities. In this study, the CYP53B1 gene, which encodes a benzoate para-hydroxylase, was successfully cloned from Rhodotorula minuta and overexpressed in Yarrowia lipolytica E150. Multiple copies of the CYP53B1 cDNA were cloned under the POX2 promoter, while the Y. lipolytica CPR was cloned under the isocitrate lyase promoter. Whole cell biotransformation of benzoic acid to para-hydroxybenzoic acid (pHBA) was used to analyse the hydroxylase activity of the recombinant Y. lipolytica UOFS Y-2366. Different induction conditions were tested in shake flask cultures. The highest concentration of pHBA produced by UOFS Y-2366 was 1.6 g l(-1) after 200 h when stearic acid was repeatedly added to the media. R. minuta accumulated up to 1.8 g l(-1) of pHBA within only 24 h. Thus, the specific hydroxylase activity of Y. lipolytica UOFS Y-2366 [approximately 0.07 U (g dry wt.)(-1)] was about 30 times lower than the specific hydroxylase activity of R. minuta [2.62 U (g dry wt.)(-1)]. However, the hydroxylation activity obtained with Y. lipolytica was one of the highest hydroxylation activities thus reported for whole cell biotransformation studies carried out with yeasts expressing foreign CYP450s.
authors.affiliation: Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, P.O. Box 339, Bloemfontein, 9300, South Africa.
authors.forename: Andreas
authors.initials: A
authors.lastname: Shiningavamwe
authors.forename: George
authors.initials: G
authors.lastname: Obiero
authors.forename: Jacobus
authors.initials: J
authors.lastname: Albertyn
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Nicaud
authors.forename: Martie
authors.initials: M
authors.lastname: Smit
mesh_terms: D050565:Benzoate 4-Monooxygenase / Q000235:genetics* / Q000378:metabolism; D019817:Benzoic Acid / Q000378:metabolism; D001711:Biotransformation; D015139:Blotting, Southern; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015870:Gene Expression / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012248:Rhodotorula / Q000201:enzymology* / Q000235:genetics; D014170:Transformation, Genetic; D025062:Yarrowia / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D019817:Benzoic Acid; D050565:Benzoate 4-Monooxygenase