Construction and characterization of whole-cell catalytic system for oxidation of avermectin.
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Abstract
<p>(A) Construction of <i>cyp107z13</i> gene expression vector pRET-z13, co-expression vector pDuet-<i>fd</i>-<i>fdr</i>18 and pDuet-<i>fd</i>-<i>fdr</i>28. <i>E. coli-zfr</i>18 was <i>E. coli</i> BL21 (DE3) containing pRSET-z13 and pDuet-<i>fd</i>-<i>fdr</i>18, <i>E. coli-zfr</i>28 was <i>E. coli</i> BL21 (DE3) containing pRSET-z13 and pDuet-<i>fd</i>-<i>fdr</i>28. (B) PCR analysis of <i>cyp107z13</i>, <i>fd</i>68, <i>fdr</i>18 and/or <i>fdr</i> 28 genes in <i>E. coli-zfr</i>18 and <i>E. coli-zfr</i>28. <i>cyp107z13</i> with primers: z13F+z13R, <i>fd</i>68 with primers: RfdF+RfdR, 1,3 and 5: <i>E. coli-zfr</i>18; 2,4 and 6: <i>E. coli-zfr</i>28. <i>fdr</i>18 with primers: Rzre1F+Rzre1R, <i>fdr</i>28 with primers: Rzre1F+Rzre2R. PCR products of <i>cyp107z13</i>, <i>fd</i>68, <i>fdr</i>18 and <i>fdr</i>28 are 1920 bp, 195 bp, 1263 bp and 1344 bp respectively. (C) SDS-PAGE analysis of recombinant proteins expressed by <i>E. coli-zfr</i>18 and <i>E. coli-zfr</i>28. Mr: protein markers; 1: <i>E. coli-fdr</i>18; 2: <i>E. coli-fdr</i>28; 3: <i>E. coli-zfr</i>18; 4: <i>E. coli-zfr</i>28; 5: <i>E. coli-z13</i>; 6: <i>E. coli</i> BL21 (DE3). (D) HPLC analysis of the products of avermectin catalyzed by <i>E. coli</i> BL21(DE3), wild <i>S. ahygroscopicus</i> ZB01, <i>E. coli-zfr</i>18 and <i>E. coli-zfr</i>28. The peaks of avermectin B1a and metabolites are indicated. The 1 represents the peak of avermectin B1a, 2 represents the peak of 4″-oxo-avermectin, and 3 represents the peak of avermectin B1b. The retention times for avermectin B1a is 21.6 min, for 4″-oxo-avermectin B1a is 24.8 min, and for avermectin B1b is 20.3 min.</p