7 research outputs found
Structures of the avermectin and product 4″-O-avermectin.
<p>Structures of the avermectin and product 4″-O-avermectin.</p
Sequence alignments of ferredoxins and ferredoxin reductases.
<p>(A) Alignment of the amino acid sequence of Fd68 from <i>S. ahygroscopicus</i> ZB01 with different 3Fe-4S-Fds. Fd_rim(<b>ZP_20965346</b>) is from <i>S. tubercidicus</i>;Fd233(<b>AY549200</b>) is from <i>S. tubercidicus</i> R-922; Fd232(<b>AY552101</b>) is from <i>S. tubercidicus</i> I-1529; Fd_virT(<b>WP_003995703</b>) is from <i>S. Viridochromogenes</i>; Fd_virD(<b>ZP_07308348</b>) is from <i>S. viridochromogenes</i> DSM 40736; Fd_aur(<b>ZP_10545293</b>) is from <i>S. auratus</i> AGR0001; Fd_svi(<b>ZP_06921672</b>) is from <i>S. sviceus</i> ATCC 29083; Fd_cha(<b>ZP_06921672</b>) is from <i>S. sviceus</i> ATCC 29083; Fd_sp(<b>BAG55293</b>) is from <i>Streptomyces</i> sp. A-1544; Fd_amb(<b>CAJ88533</b>) is from <i>S. ambofaciens</i> ATCC 23877; Fd_gha(<b>ZP_06574442</b>) is from <i>S. ghanaensis</i> ATCC 14672. (B) Multiple sequence alignment of conserved regions in FdRs. Consensus sequences for the FAD ADP-binding motifs, the NAD (ADP)-binding motifs, and the FAD ribytil-binding motifs (Asturis 1995) are shown above the alignment. FdR18, FdR28 are the FdRs described in this study. FprD(<b>NP_826852.1)</b> is from <i>S. avermitilis</i> MA-4680. Fre3(<b>AAT45306.1)</b>, Fre14(<b>AAT45308.1)</b>, Fre16(<b>AAT45309.1)</b>, FreEA(<b>AAT45279.1</b>) are from the genome of <i>S. tubercidicus</i> (Molnar, 2005). Fdr793(<b>AJ628764.1)</b> is of <i>S. peucetius</i> ATCC27952. SCF15(<b>CAB60462.1</b>) and SC4B10(<b>CAC04223.1</b>) are of <i>S. coelicolor</i> A3(2); FdR-sau(sau, <b>EJJ04871.1</b>) is of <i>S. auratus</i> AGR0001.</p
Construction and characterization of whole-cell catalytic system for oxidation of avermectin.
<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
Expression and characterization of FdR18 and FdR28.
<p>(A) Recombinant expression vectors pRSET-<i>fdr</i>18 and pRSET-<i>fdr</i>28. (B) SDS-PAGE analysis of recombinant proteins FdR18 and FdR28 expressed by <i>E. coli</i> BL21 (DE3). Mr: protein markers. (C) UV-visible spectra of purified FdR18 and FdR28. Spectra were recorded at ambient temperature in 50 mM Tris buffer (pH 7.5). (D) DCPIP reduction activities of purified FdR18 and FdR28, measured in the presence of 200 uM NADH (â–ª) or NADPH (â–¡).</p
Microoganisms and plasmids used in this study.
<p>Microoganisms and plasmids used in this study.</p
Characterization of <i>fd</i>68 gene disruption mutants of <i>S.ahygroscopicus</i> ZB01.
<p>(A) Map of the fd68 knock-out plasmids pKC1139:: <i>fd</i>68. The 172bp <i>fd</i>68 fragment named Δfd68 was subcloned into the <i>EcoR</i> I and <i>Hind</i> III sitesa of lacZα MCS in plasmid pKC1139. (B) Phenotype of wild <i>S. ahygroscopicus</i> ZB01 and <i>fd</i>68 disruption mutants ZBΔ<i>fd</i>68-3 and ZBΔ<i>fd</i>68-6 (7 d on YMS medium at 30°C). Note the color changes of the colonies of the strains. (C) PCR analysis of apramycin resistance gene and <i>fd</i>68 with primers AF1/AR1 for apramycin resistance gene and fF1/fR1 for <i>fd</i>68; Mr, DNA Marker. The line above the lane numbers indicates DNA from wild-type strain <i>S. ahygroscopicus</i> ZB01, mutant ZBΔ<i>fd</i>68-3 and ZBΔ<i>fd</i>68-6. (D) Mycelium dry weights of <i>fd</i>68 disruption mutants ZBΔ<i>fd</i>68-3, and ZBΔ<i>fd</i>68-6 and wild-type <i>S. ahygroscopicu</i> ZB01 at different incubation times in YEME. 10<sup>8</sup> spores of strains were inoculated in 250 ml flasks with 80 ml liquid YEME medium and cultured for 8 d, the mycelium were collected and dried at 70°C for 1 d. Error bars represent the standard deviation of three replicas in three independent experiments. (E) HPLC analysis of the products of avermectin catalyzed by avermectin standard, wild <i>S. ahygroscopicus</i> ZB01, ZB△<i>fd</i>68-3 and ZB△<i>fd</i>68-6. 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 22.5 min, for 4″-oxo-avermectin B1a is 24.7 min, and for avermectin B1b is 20.7 min.</p