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

Zhang et al. Microb Cell Fact (2015) 14:152 DOI 10.1186/s12934-015-0337-y Open Access RESEARCH Designed biosynthesis of 25‑methyl and 25‑ethyl ivermectin with enhanced insecticidal activity by domain swap of avermectin polyketide synthase Ji Zhang1, Yi‑Jun Yan1,3, Jing An1, Sheng‑Xiong Huang3, Xiang‑Jing Wang1* and Wen‑Sheng Xiang1,2* 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 aver‑ mectin 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 avermec‑ tin-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 poten‑ tial 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. Keywords: 25-Ethyl ivermectin, 25-Methyl ivermectin, Domain swap, Insecticidal activity Background Avermectins and milbemycins (Fig. 1), the 16-membered macrolide antibiotics with potent anthelmintic and *Correspondence: ; xiangwensheng@neau. edu.cn 1 School of Life Science, Northeast Agricultural University, No. 59 Mucai Street, Xiangfang District, Harbin 150030, China Full list of author information is available at the end of the article insecticidal activity, have been widely used for broadspectrum parasite control in agricultural, medical, and veterinary fields [1–3]. They are structurally related compounds with structural differences at C25, C22–C23, and C13, leading to their own unique ‘spectral fingerprint’ with various strengths and dosage-limiting species. The subsequent chemically modification of avermectins and milbemycins resulted in series of analogs, some of © 2015 Zhang et al. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhang et al. Microb Cell Fact (2015) 14:152 Page 2 of 12 Fig. 1 The structures of avermectins, milbemycins, and 25-methyl/25-ethyl ivermectin (1 and 2) which are commercially developed as anthelmintics and insecticides, such as ivermectin, selamectin, abamectin, emamectin, doramectin, milbemycin oxime, lepimectin, and latidectin [2]. Among these analogs, ivermectin (22,23-dihydroavermectin B1), showing the same effective antiparasitic activity and lesser toxic side effect than avermectins B1, has been worldwide used as an anthelmintic for livestock and companion animals and as an agricultural insecticide. Moreover, ivermectin has also been applied in human medicine, particularly treatment of onchocerciasis and lymphatic filariasis [4]. In the case of milbemycins, moxidectin is currently undergoing a phase III clinical trial to compare its efficacy with ivermectin in subjects with Onchocerca volvulus infection [1]; milbemycin oxime has been used against intestinal nematodes in dogs and cats, against adult heartworm in dogs, and against ectoparasites in companion animals [5]. Recently, it has been reported that ivermectin, selamectin and moxidectin demonstrated antibacterial activity against Mycobacterium tuberculosis, especially the multidrug-resistant and extensively drug-resistant clinical strains [1]. The approval for clinical and veterinary uses as well as the documented pharmacokinetic and safety profiles of these compounds make them potential therapeutic options for treating M. tuberculosis. The outstanding activities of avermectins and milbemycins together with the potential uses in the filed of human medicine and agriculture stimulate the semisynthetic derivatives of avermectins and milbemycins. On the other hand, the wide use of avermectins in agriculture has inevitably caused serious drug resistance [2], it is therefore imperative to develop novel avermectins. The previously established industrial process for preparing the analogs of avermectins and milbemycins involves extracting avermectins or milbemycins from the fermentation broth and the subsequently chemical modification. However, this process suffers several drawbacks, such as the expensive cost, the heavy metal pollution, and low efficiency [3, 6–8]. Fortunately, the combinatorial biosynthesis and genetic manipulation provide alternative and efficient strategies to generate the known semi-biosynthetic polyketides and new hybrid compounds according to the module polyketide synthase (PKS) assembly line [3, 9, 10]. For example, on the basis of understanding the biosynthetic mechanism of avermectin, 22,23-dihydroavermectins including ivermectin were successfully produced by direct fermentation of engineered S. avermitilis, in which the DNA Zhang et al. Microb Cell Fact (2015) 14:152 region encoding the dehydratase (DH) and ketoreductase (KR) domains of module 2 from the avermecti (...truncated)