Selektive Hydroxylierung von alpha- und beta-Ionon durch Streptomyces Stämme und molekulargenetische Arbeiten zur Identifizierung und Isolierung der Ionon-Hydroxylase aus Streptomyces fradiae Tü 27
The aim of this work was the selective conversion of α- and/or β-ionone to 3-hydroxy ionones by microbial transformation. These 3-hydroxy ionones could supply valuable intermediates for the chemoenzymatic synthesis of astaxanthin and zeaxanthin. More than 200 Streptomyces strains were screened for their capacity to regio- and stereoselectively hydroxylate β- and/or α-ionone to the respective 3-hydroxy derivatives. With β-ionone as the substrate, 19 strains showed conversion to 4-hydroxy- and
... o 4-hydroxy- and none showed conversion to the 3-hydroxy-β-ionone as desired. Among these 19 strains six converted α-ionone to 3-hydroxy-α-ionone. S. fradiae Tü 27 showed the highest hydroxylation activity (75). GLC and NMR analyses clearly established that hydroxylation of racemic α-ionone [(6R)-(-)/(6S)-(+)] resulted in the exclusive formation of only the two enantiomers (3R,6R)- and (3S,6S)-hydroxy-α-ionone. Thus, the enzymatic hydroxylation of α-ionone by the Streptomyces strains tested proceeds with both high regio- and stereoselectivity. For the isolation of the gene encoding oforthis hydroxylation enzyme, P450 gene fragments of three strains were amplified and sequenced. Two gene fragments from S. fradiae Tü 27 were identified but the isolation of the whole gene by different methods did not succeed. The two P450 homologous genes of the strain S. fradiae Tü 27 were interrupted by integrationmutagenesis, to identify the P450 gene fragment encoding for the hydroxylase which is responsible for the selective hydroxylation of α-ionone. With these mutants the biotransformation of α-ionone was tested. Whereas the mutant GMP450-27Q was capable to convert α-ionone to 3-hydroxy-α-ionone, although with by-product, the mutant GMP450-27P α-ionone could not selectively convert α-ionone to 3-hydroxy-α-ionone. Therefore, P450-27P seems to be responsible for the selective hydroxylation of α-ionone.