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Exploring the electron transfer pathway in the oxidation of avermectin by CYP107Z13 in Streptomyces ahygroscopicus ZB01.

Li M, Zhang Y, Zhang L, Yang X, Jiang X - PLoS ONE (2014)

Bottom Line: Streptomyces ahygroscopicus ZB01 can effectively oxidize 4″-OH of avermectin to form 4″-oxo-avermectin.A putative [3Fe-4S] ferredoxin gene fd68 and two possible NADH-dependent ferredoxin reductase genes fdr18 and fdr28 were cloned from the genomic DNA of ZB01. fd68 gene disruption mutants showed no catalytic activity in oxidation of avermectin to form 4″-oxo-avermectin.Both of the two biocatalytic systems were found to be able to mediate the oxidation of avermectin to form 4″-oxo-avermectin.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.

ABSTRACT
Streptomyces ahygroscopicus ZB01 can effectively oxidize 4″-OH of avermectin to form 4″-oxo-avermectin. CYP107Z13 is responsible for this site-specific oxidation in ZB01. In the present study, we explored the electron transfer pathway in oxidation of avermectin by CYP107Z13 in ZB01. A putative [3Fe-4S] ferredoxin gene fd68 and two possible NADH-dependent ferredoxin reductase genes fdr18 and fdr28 were cloned from the genomic DNA of ZB01. fd68 gene disruption mutants showed no catalytic activity in oxidation of avermectin to form 4″-oxo-avermectin. To clarify whether FdR18 and FdR28 participate in the electron transfer during avermectin oxidation by CYP107Z13, two whole-cell biocatalytic systems were designed in E. coli BL21 (DE3), with one co-expressing CYP107Z13, Fd68 and FdR18 and the other co-expressing CYP107Z13, Fd68 and FdR28. Both of the two biocatalytic systems were found to be able to mediate the oxidation of avermectin to form 4″-oxo-avermectin. Thus, we propose an electron transfer pathway NADH→FdR18/FdR28→Fd68→CYP107Z13 for oxidation of avermectin to form 4″-oxo-avermectin in ZB01.

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Construction and characterization of whole-cell catalytic system for oxidation of avermectin.(A) Construction of cyp107z13 gene expression vector pRET-z13, co-expression vector pDuet-fd-fdr18 and pDuet-fd-fdr28. E. coli-zfr18 was E. coli BL21 (DE3) containing pRSET-z13 and pDuet-fd-fdr18, E. coli-zfr28 was E. coli BL21 (DE3) containing pRSET-z13 and pDuet-fd-fdr28. (B) PCR analysis of cyp107z13, fd68, fdr18 and/or fdr 28 genes in E. coli-zfr18 and E. coli-zfr28. cyp107z13 with primers: z13F+z13R, fd68 with primers: RfdF+RfdR, 1,3 and 5: E. coli-zfr18; 2,4 and 6: E. coli-zfr28. fdr18 with primers: Rzre1F+Rzre1R, fdr28 with primers: Rzre1F+Rzre2R. PCR products of cyp107z13, fd68, fdr18 and fdr28 are 1920 bp, 195 bp, 1263 bp and 1344 bp respectively. (C) SDS-PAGE analysis of recombinant proteins expressed by E. coli-zfr18 and E. coli-zfr28. Mr: protein markers; 1: E. coli-fdr18; 2: E. coli-fdr28; 3: E. coli-zfr18; 4: E. coli-zfr28; 5: E. coli-z13; 6: E. coli BL21 (DE3). (D) HPLC analysis of the products of avermectin catalyzed by E. coli BL21(DE3), wild S. ahygroscopicus ZB01, E. coli-zfr18 and E. coli-zfr28. 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.
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pone-0098916-g005: Construction and characterization of whole-cell catalytic system for oxidation of avermectin.(A) Construction of cyp107z13 gene expression vector pRET-z13, co-expression vector pDuet-fd-fdr18 and pDuet-fd-fdr28. E. coli-zfr18 was E. coli BL21 (DE3) containing pRSET-z13 and pDuet-fd-fdr18, E. coli-zfr28 was E. coli BL21 (DE3) containing pRSET-z13 and pDuet-fd-fdr28. (B) PCR analysis of cyp107z13, fd68, fdr18 and/or fdr 28 genes in E. coli-zfr18 and E. coli-zfr28. cyp107z13 with primers: z13F+z13R, fd68 with primers: RfdF+RfdR, 1,3 and 5: E. coli-zfr18; 2,4 and 6: E. coli-zfr28. fdr18 with primers: Rzre1F+Rzre1R, fdr28 with primers: Rzre1F+Rzre2R. PCR products of cyp107z13, fd68, fdr18 and fdr28 are 1920 bp, 195 bp, 1263 bp and 1344 bp respectively. (C) SDS-PAGE analysis of recombinant proteins expressed by E. coli-zfr18 and E. coli-zfr28. Mr: protein markers; 1: E. coli-fdr18; 2: E. coli-fdr28; 3: E. coli-zfr18; 4: E. coli-zfr28; 5: E. coli-z13; 6: E. coli BL21 (DE3). (D) HPLC analysis of the products of avermectin catalyzed by E. coli BL21(DE3), wild S. ahygroscopicus ZB01, E. coli-zfr18 and E. coli-zfr28. 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.

Mentions: For co-expressing CYP107z13, Fd68 and FdR18/FdR28 in E. coli. pRSET-z13, pDuet-fd-fdr18 and pDuet-fd-fdr18 were constructed (Fig. 5A). pRSET-z13 and pDuet-fd-fdr18 were co-transformed into E. coli BL21 (DE3) and the resultant transformant E. coli-zfr18, pRSET-z13 and pDuet-fd-fdr18 were co-transformed into E. coli BL21 (DE3) and transformant E. coli-zfr28 were obtained. Both of E. coli-zfr28 and E. coli-zfr28 showed cyp107z13, fd68 and fdr18/28 genes by PCR amplifying (Fig. 5B). The expressed target proteins from E. coli-zfr18 and E. coli-zfr28 were analyzed by SDS-PAGE (Fig. 5C).


Exploring the electron transfer pathway in the oxidation of avermectin by CYP107Z13 in Streptomyces ahygroscopicus ZB01.

Li M, Zhang Y, Zhang L, Yang X, Jiang X - PLoS ONE (2014)

Construction and characterization of whole-cell catalytic system for oxidation of avermectin.(A) Construction of cyp107z13 gene expression vector pRET-z13, co-expression vector pDuet-fd-fdr18 and pDuet-fd-fdr28. E. coli-zfr18 was E. coli BL21 (DE3) containing pRSET-z13 and pDuet-fd-fdr18, E. coli-zfr28 was E. coli BL21 (DE3) containing pRSET-z13 and pDuet-fd-fdr28. (B) PCR analysis of cyp107z13, fd68, fdr18 and/or fdr 28 genes in E. coli-zfr18 and E. coli-zfr28. cyp107z13 with primers: z13F+z13R, fd68 with primers: RfdF+RfdR, 1,3 and 5: E. coli-zfr18; 2,4 and 6: E. coli-zfr28. fdr18 with primers: Rzre1F+Rzre1R, fdr28 with primers: Rzre1F+Rzre2R. PCR products of cyp107z13, fd68, fdr18 and fdr28 are 1920 bp, 195 bp, 1263 bp and 1344 bp respectively. (C) SDS-PAGE analysis of recombinant proteins expressed by E. coli-zfr18 and E. coli-zfr28. Mr: protein markers; 1: E. coli-fdr18; 2: E. coli-fdr28; 3: E. coli-zfr18; 4: E. coli-zfr28; 5: E. coli-z13; 6: E. coli BL21 (DE3). (D) HPLC analysis of the products of avermectin catalyzed by E. coli BL21(DE3), wild S. ahygroscopicus ZB01, E. coli-zfr18 and E. coli-zfr28. 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.
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pone-0098916-g005: Construction and characterization of whole-cell catalytic system for oxidation of avermectin.(A) Construction of cyp107z13 gene expression vector pRET-z13, co-expression vector pDuet-fd-fdr18 and pDuet-fd-fdr28. E. coli-zfr18 was E. coli BL21 (DE3) containing pRSET-z13 and pDuet-fd-fdr18, E. coli-zfr28 was E. coli BL21 (DE3) containing pRSET-z13 and pDuet-fd-fdr28. (B) PCR analysis of cyp107z13, fd68, fdr18 and/or fdr 28 genes in E. coli-zfr18 and E. coli-zfr28. cyp107z13 with primers: z13F+z13R, fd68 with primers: RfdF+RfdR, 1,3 and 5: E. coli-zfr18; 2,4 and 6: E. coli-zfr28. fdr18 with primers: Rzre1F+Rzre1R, fdr28 with primers: Rzre1F+Rzre2R. PCR products of cyp107z13, fd68, fdr18 and fdr28 are 1920 bp, 195 bp, 1263 bp and 1344 bp respectively. (C) SDS-PAGE analysis of recombinant proteins expressed by E. coli-zfr18 and E. coli-zfr28. Mr: protein markers; 1: E. coli-fdr18; 2: E. coli-fdr28; 3: E. coli-zfr18; 4: E. coli-zfr28; 5: E. coli-z13; 6: E. coli BL21 (DE3). (D) HPLC analysis of the products of avermectin catalyzed by E. coli BL21(DE3), wild S. ahygroscopicus ZB01, E. coli-zfr18 and E. coli-zfr28. 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.
Mentions: For co-expressing CYP107z13, Fd68 and FdR18/FdR28 in E. coli. pRSET-z13, pDuet-fd-fdr18 and pDuet-fd-fdr18 were constructed (Fig. 5A). pRSET-z13 and pDuet-fd-fdr18 were co-transformed into E. coli BL21 (DE3) and the resultant transformant E. coli-zfr18, pRSET-z13 and pDuet-fd-fdr18 were co-transformed into E. coli BL21 (DE3) and transformant E. coli-zfr28 were obtained. Both of E. coli-zfr28 and E. coli-zfr28 showed cyp107z13, fd68 and fdr18/28 genes by PCR amplifying (Fig. 5B). The expressed target proteins from E. coli-zfr18 and E. coli-zfr28 were analyzed by SDS-PAGE (Fig. 5C).

Bottom Line: Streptomyces ahygroscopicus ZB01 can effectively oxidize 4″-OH of avermectin to form 4″-oxo-avermectin.A putative [3Fe-4S] ferredoxin gene fd68 and two possible NADH-dependent ferredoxin reductase genes fdr18 and fdr28 were cloned from the genomic DNA of ZB01. fd68 gene disruption mutants showed no catalytic activity in oxidation of avermectin to form 4″-oxo-avermectin.Both of the two biocatalytic systems were found to be able to mediate the oxidation of avermectin to form 4″-oxo-avermectin.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.

ABSTRACT
Streptomyces ahygroscopicus ZB01 can effectively oxidize 4″-OH of avermectin to form 4″-oxo-avermectin. CYP107Z13 is responsible for this site-specific oxidation in ZB01. In the present study, we explored the electron transfer pathway in oxidation of avermectin by CYP107Z13 in ZB01. A putative [3Fe-4S] ferredoxin gene fd68 and two possible NADH-dependent ferredoxin reductase genes fdr18 and fdr28 were cloned from the genomic DNA of ZB01. fd68 gene disruption mutants showed no catalytic activity in oxidation of avermectin to form 4″-oxo-avermectin. To clarify whether FdR18 and FdR28 participate in the electron transfer during avermectin oxidation by CYP107Z13, two whole-cell biocatalytic systems were designed in E. coli BL21 (DE3), with one co-expressing CYP107Z13, Fd68 and FdR18 and the other co-expressing CYP107Z13, Fd68 and FdR28. Both of the two biocatalytic systems were found to be able to mediate the oxidation of avermectin to form 4″-oxo-avermectin. Thus, we propose an electron transfer pathway NADH→FdR18/FdR28→Fd68→CYP107Z13 for oxidation of avermectin to form 4″-oxo-avermectin in ZB01.

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