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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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Characterization of fd68 gene disruption mutants of S.ahygroscopicus ZB01.(A) Map of the fd68 knock-out plasmids pKC1139:: fd68. The 172bp fd68 fragment named Δfd68 was subcloned into the EcoR I and Hind III sitesa of lacZα MCS in plasmid pKC1139. (B) Phenotype of wild S. ahygroscopicus ZB01 and fd68 disruption mutants ZBΔfd68-3 and ZBΔfd68-6 (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 fd68 with primers AF1/AR1 for apramycin resistance gene and fF1/fR1 for fd68; Mr, DNA Marker. The line above the lane numbers indicates DNA from wild-type strain S. ahygroscopicus ZB01, mutant ZBΔfd68-3 and ZBΔfd68-6. (D) Mycelium dry weights of fd68 disruption mutants ZBΔfd68-3, and ZBΔfd68-6 and wild-type S. ahygroscopicu ZB01 at different incubation times in YEME. 108 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 S. ahygroscopicus ZB01, ZB△fd68-3 and ZB△fd68-6. 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.
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pone-0098916-g003: Characterization of fd68 gene disruption mutants of S.ahygroscopicus ZB01.(A) Map of the fd68 knock-out plasmids pKC1139:: fd68. The 172bp fd68 fragment named Δfd68 was subcloned into the EcoR I and Hind III sitesa of lacZα MCS in plasmid pKC1139. (B) Phenotype of wild S. ahygroscopicus ZB01 and fd68 disruption mutants ZBΔfd68-3 and ZBΔfd68-6 (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 fd68 with primers AF1/AR1 for apramycin resistance gene and fF1/fR1 for fd68; Mr, DNA Marker. The line above the lane numbers indicates DNA from wild-type strain S. ahygroscopicus ZB01, mutant ZBΔfd68-3 and ZBΔfd68-6. (D) Mycelium dry weights of fd68 disruption mutants ZBΔfd68-3, and ZBΔfd68-6 and wild-type S. ahygroscopicu ZB01 at different incubation times in YEME. 108 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 S. ahygroscopicus ZB01, ZB△fd68-3 and ZB△fd68-6. 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.

Mentions: To ellucidate the function of fd68 gene, a fd68 gene disruption vector pKC1139:: fd68 was constructed (Fig. 3A) and transformed into ZB01. Two stable G418 resistant transformants ZB△fd68-3 and ZB△fd68-6 were selected. The plasmid. pKC1139:: fd68 could not be extracted from these two mutants (data not shown), so fd68 and apramycin resistance genes were analyzed by PCR using the genomic DNA of the mutants as templates. There was an intact fd68 gene (about 200 bp) and no apramycin resistance gene in wild S. ahygroscopicus ZB01, while no intact fd68 gene was amplified at the presence of apramycin gene fragments (about 500 bp) in ZB△fd68-3 and ZB△fd68-6 (Fig. 3B), suggesting that pKC1139::fd68 had been integrated into the chromosome of ZB01 and disruption had occurred in ZB△fd68-3 and ZB△fd68-6.


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)

Characterization of fd68 gene disruption mutants of S.ahygroscopicus ZB01.(A) Map of the fd68 knock-out plasmids pKC1139:: fd68. The 172bp fd68 fragment named Δfd68 was subcloned into the EcoR I and Hind III sitesa of lacZα MCS in plasmid pKC1139. (B) Phenotype of wild S. ahygroscopicus ZB01 and fd68 disruption mutants ZBΔfd68-3 and ZBΔfd68-6 (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 fd68 with primers AF1/AR1 for apramycin resistance gene and fF1/fR1 for fd68; Mr, DNA Marker. The line above the lane numbers indicates DNA from wild-type strain S. ahygroscopicus ZB01, mutant ZBΔfd68-3 and ZBΔfd68-6. (D) Mycelium dry weights of fd68 disruption mutants ZBΔfd68-3, and ZBΔfd68-6 and wild-type S. ahygroscopicu ZB01 at different incubation times in YEME. 108 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 S. ahygroscopicus ZB01, ZB△fd68-3 and ZB△fd68-6. 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.
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Related In: Results  -  Collection

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pone-0098916-g003: Characterization of fd68 gene disruption mutants of S.ahygroscopicus ZB01.(A) Map of the fd68 knock-out plasmids pKC1139:: fd68. The 172bp fd68 fragment named Δfd68 was subcloned into the EcoR I and Hind III sitesa of lacZα MCS in plasmid pKC1139. (B) Phenotype of wild S. ahygroscopicus ZB01 and fd68 disruption mutants ZBΔfd68-3 and ZBΔfd68-6 (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 fd68 with primers AF1/AR1 for apramycin resistance gene and fF1/fR1 for fd68; Mr, DNA Marker. The line above the lane numbers indicates DNA from wild-type strain S. ahygroscopicus ZB01, mutant ZBΔfd68-3 and ZBΔfd68-6. (D) Mycelium dry weights of fd68 disruption mutants ZBΔfd68-3, and ZBΔfd68-6 and wild-type S. ahygroscopicu ZB01 at different incubation times in YEME. 108 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 S. ahygroscopicus ZB01, ZB△fd68-3 and ZB△fd68-6. 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.
Mentions: To ellucidate the function of fd68 gene, a fd68 gene disruption vector pKC1139:: fd68 was constructed (Fig. 3A) and transformed into ZB01. Two stable G418 resistant transformants ZB△fd68-3 and ZB△fd68-6 were selected. The plasmid. pKC1139:: fd68 could not be extracted from these two mutants (data not shown), so fd68 and apramycin resistance genes were analyzed by PCR using the genomic DNA of the mutants as templates. There was an intact fd68 gene (about 200 bp) and no apramycin resistance gene in wild S. ahygroscopicus ZB01, while no intact fd68 gene was amplified at the presence of apramycin gene fragments (about 500 bp) in ZB△fd68-3 and ZB△fd68-6 (Fig. 3B), suggesting that pKC1139::fd68 had been integrated into the chromosome of ZB01 and disruption had occurred in ZB△fd68-3 and ZB△fd68-6.

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.

Show MeSH
Related in: MedlinePlus