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Designed biosynthesis of 25-methyl and 25-ethyl ivermectin with enhanced insecticidal activity by domain swap of avermectin polyketide synthase.

Zhang J, Yan YJ, An J, Huang SX, Wang XJ, Xiang WS - Microb. Cell Fact. (2015)

Bottom Line: Moreover, the insecticidal activity of the mixture of 25-methyl and 25-ethyl ivermectin was 2.5-fold and 5.7-fold higher than that of milbemycin A3/A4 against C. elegans and the second-instar larva of Mythimna separate, respectively.Two new avermectin derivatives 25-methyl and 25-ethyl ivermectin were generated by the domain swap of avermectin PKS.The enhanced insecticidal activity of 25-methyl and 25-ethyl ivermectin implied the potential use as insecticide in agriculture.

View Article: PubMed Central - PubMed

Affiliation: School of Life Science, Northeast Agricultural University, No. 59 Mucai Street, Xiangfang District, Harbin, 150030, China. zhangji@neau.edu.cn.

ABSTRACT

Background: Avermectin and milbemycin are important 16-membered macrolides that have been widely used as pesticides in agriculture. However, the wide use of these pesticides inevitably causes serious drug resistance, it is therefore imperative to develop new avermectin and milbemycin analogs. The biosynthetic gene clusters of avermectin and milbemycin have been identified and the biosynthetic pathways have been elucidated. Combinatorial biosynthesis by domain swap provides an efficient strategy to generate chemical diversity according to the module polyketide synthase (PKS) assembly line.

Results: The substitution of aveDH2-KR2 located in avermectin biosynthetic gene cluster in the industrial avermectin-producing strain Streptomyces avermitilis NA-108 with the DNA regions milDH2-ER2-KR2 located in milbemycin biosynthetic gene cluster in Streptomyces bingchenggensis led to S. avermitilis AVE-T27, which produced ivermectin B1a with high yield of 3450 ± 65 μg/ml. The subsequent replacement of aveLAT-ACP encoding the loading module of avermectin PKS with milLAT-ACP encoding the loading module of milbemycin PKS led to strain S. avermitilis AVE-H39, which produced two new avermectin derivatives 25-ethyl and 25-methyl ivermectin (1 and 2) with yields of 951 ± 46 and 2093 ± 61 μg/ml, respectively. Compared to commercial insecticide ivermectin, the mixture of 25-methyl and 25-ethyl ivermectin (2:1 = 3:7) exhibited 4.6-fold increase in insecticidal activity against Caenorhabditis elegans. Moreover, the insecticidal activity of the mixture of 25-methyl and 25-ethyl ivermectin was 2.5-fold and 5.7-fold higher than that of milbemycin A3/A4 against C. elegans and the second-instar larva of Mythimna separate, respectively.

Conclusions: Two new avermectin derivatives 25-methyl and 25-ethyl ivermectin were generated by the domain swap of avermectin PKS. The enhanced insecticidal activity of 25-methyl and 25-ethyl ivermectin implied the potential use as insecticide in agriculture. Furthermore, the high yield and genetic stability of the engineered strains S. avermitilis AVE-T27 and AVE-H39 suggested the enormous potential in industrial production of the commercial insecticide ivermectin and 25-methyl/25-ethyl ivermectins, respectively.

No MeSH data available.


Related in: MedlinePlus

HPLC analysis of the mycelial extracts from the parental and mutant strains. aS. avermitilis NA-108; bS. avermitlis AVE-T27; c standard sample ivermectin B1a; dS. avermitilis AVE-H39. B1a avermectin B1a, B2a avermectin B2a, B1b avermectin B1b, B2b avermectin B2b, Ive B1a ivermectin B1a, 1 compound 1, 2 compound 2
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Fig4: HPLC analysis of the mycelial extracts from the parental and mutant strains. aS. avermitilis NA-108; bS. avermitlis AVE-T27; c standard sample ivermectin B1a; dS. avermitilis AVE-H39. B1a avermectin B1a, B2a avermectin B2a, B1b avermectin B1b, B2b avermectin B2b, Ive B1a ivermectin B1a, 1 compound 1, 2 compound 2

Mentions: On the basis of understanding the biosynthetic pathways of avermectin and milbemycin, we attempted to construct an ivermectin-producing strain by replacing the aveDH2-KR2 of avermectin biosynthetic gene cluster in S. avermitilis NA-108 with milDH2-ER2-KR2 of milbemycin biosynthetic gene cluster from S. bingchenggensis (Fig. 2). PCR verification using the primers E1 and E2 (Additional file 2: Table S1) demonstrated that the expected 2.6-kb DNA fragment encoding MilDH2-ER2-KR2 was obtained from the genomic DNA of S. bingchenggensis and the double-crossover mutant S. avermitilis AVE-T27, whereas no PCR product was detected from the genomic DNA of S. avermitilis NA-108 (Fig. 3a, b). The PCR product was then sequenced, and the results further confirmed that aveDH2-KR2 in avermectin biosynthetic gene cluster was successfully replaced with milDH2-ER2-KR2 by double crossover. Compared to the parental strain S. avermitilis NA-108, strain AVE-T27 demonstrated a different metabolic profile. As shown in Fig. 4, in addition to the disappearance of avermectin “a” components and the remarkable decrease of avermectin “b” components, a new compound, which showed identical retention time and molecular mass (m/z = 897, [M+Na]+) to those of the authentic sample ivermectin B1a, was detected in culture extracts of S. avermitilis AVE-T27 by HPLC analysis. Therefore, the compound was considered to be ivermectin B1a, which was consistent with the designed biosynthetic strategy (Fig. 2).Fig. 2


Designed biosynthesis of 25-methyl and 25-ethyl ivermectin with enhanced insecticidal activity by domain swap of avermectin polyketide synthase.

Zhang J, Yan YJ, An J, Huang SX, Wang XJ, Xiang WS - Microb. Cell Fact. (2015)

HPLC analysis of the mycelial extracts from the parental and mutant strains. aS. avermitilis NA-108; bS. avermitlis AVE-T27; c standard sample ivermectin B1a; dS. avermitilis AVE-H39. B1a avermectin B1a, B2a avermectin B2a, B1b avermectin B1b, B2b avermectin B2b, Ive B1a ivermectin B1a, 1 compound 1, 2 compound 2
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4581413&req=5

Fig4: HPLC analysis of the mycelial extracts from the parental and mutant strains. aS. avermitilis NA-108; bS. avermitlis AVE-T27; c standard sample ivermectin B1a; dS. avermitilis AVE-H39. B1a avermectin B1a, B2a avermectin B2a, B1b avermectin B1b, B2b avermectin B2b, Ive B1a ivermectin B1a, 1 compound 1, 2 compound 2
Mentions: On the basis of understanding the biosynthetic pathways of avermectin and milbemycin, we attempted to construct an ivermectin-producing strain by replacing the aveDH2-KR2 of avermectin biosynthetic gene cluster in S. avermitilis NA-108 with milDH2-ER2-KR2 of milbemycin biosynthetic gene cluster from S. bingchenggensis (Fig. 2). PCR verification using the primers E1 and E2 (Additional file 2: Table S1) demonstrated that the expected 2.6-kb DNA fragment encoding MilDH2-ER2-KR2 was obtained from the genomic DNA of S. bingchenggensis and the double-crossover mutant S. avermitilis AVE-T27, whereas no PCR product was detected from the genomic DNA of S. avermitilis NA-108 (Fig. 3a, b). The PCR product was then sequenced, and the results further confirmed that aveDH2-KR2 in avermectin biosynthetic gene cluster was successfully replaced with milDH2-ER2-KR2 by double crossover. Compared to the parental strain S. avermitilis NA-108, strain AVE-T27 demonstrated a different metabolic profile. As shown in Fig. 4, in addition to the disappearance of avermectin “a” components and the remarkable decrease of avermectin “b” components, a new compound, which showed identical retention time and molecular mass (m/z = 897, [M+Na]+) to those of the authentic sample ivermectin B1a, was detected in culture extracts of S. avermitilis AVE-T27 by HPLC analysis. Therefore, the compound was considered to be ivermectin B1a, which was consistent with the designed biosynthetic strategy (Fig. 2).Fig. 2

Bottom Line: Moreover, the insecticidal activity of the mixture of 25-methyl and 25-ethyl ivermectin was 2.5-fold and 5.7-fold higher than that of milbemycin A3/A4 against C. elegans and the second-instar larva of Mythimna separate, respectively.Two new avermectin derivatives 25-methyl and 25-ethyl ivermectin were generated by the domain swap of avermectin PKS.The enhanced insecticidal activity of 25-methyl and 25-ethyl ivermectin implied the potential use as insecticide in agriculture.

View Article: PubMed Central - PubMed

Affiliation: School of Life Science, Northeast Agricultural University, No. 59 Mucai Street, Xiangfang District, Harbin, 150030, China. zhangji@neau.edu.cn.

ABSTRACT

Background: Avermectin and milbemycin are important 16-membered macrolides that have been widely used as pesticides in agriculture. However, the wide use of these pesticides inevitably causes serious drug resistance, it is therefore imperative to develop new avermectin and milbemycin analogs. The biosynthetic gene clusters of avermectin and milbemycin have been identified and the biosynthetic pathways have been elucidated. Combinatorial biosynthesis by domain swap provides an efficient strategy to generate chemical diversity according to the module polyketide synthase (PKS) assembly line.

Results: The substitution of aveDH2-KR2 located in avermectin biosynthetic gene cluster in the industrial avermectin-producing strain Streptomyces avermitilis NA-108 with the DNA regions milDH2-ER2-KR2 located in milbemycin biosynthetic gene cluster in Streptomyces bingchenggensis led to S. avermitilis AVE-T27, which produced ivermectin B1a with high yield of 3450 ± 65 μg/ml. The subsequent replacement of aveLAT-ACP encoding the loading module of avermectin PKS with milLAT-ACP encoding the loading module of milbemycin PKS led to strain S. avermitilis AVE-H39, which produced two new avermectin derivatives 25-ethyl and 25-methyl ivermectin (1 and 2) with yields of 951 ± 46 and 2093 ± 61 μg/ml, respectively. Compared to commercial insecticide ivermectin, the mixture of 25-methyl and 25-ethyl ivermectin (2:1 = 3:7) exhibited 4.6-fold increase in insecticidal activity against Caenorhabditis elegans. Moreover, the insecticidal activity of the mixture of 25-methyl and 25-ethyl ivermectin was 2.5-fold and 5.7-fold higher than that of milbemycin A3/A4 against C. elegans and the second-instar larva of Mythimna separate, respectively.

Conclusions: Two new avermectin derivatives 25-methyl and 25-ethyl ivermectin were generated by the domain swap of avermectin PKS. The enhanced insecticidal activity of 25-methyl and 25-ethyl ivermectin implied the potential use as insecticide in agriculture. Furthermore, the high yield and genetic stability of the engineered strains S. avermitilis AVE-T27 and AVE-H39 suggested the enormous potential in industrial production of the commercial insecticide ivermectin and 25-methyl/25-ethyl ivermectins, respectively.

No MeSH data available.


Related in: MedlinePlus