Gene Replacement for the Generation of Designed Novel Avermectin Derivatives with Enhanced Acaricidal and Nematicidal Activities

Abstract

c Avermectin (AVM) and ivermectin (IVM) are potent pesticides and acaricides which have been widely used during the past 30 years. As insect resistance to AVM and IVM is greatly increasing, alternatives are urgently needed. Here, we report two novel AVM derivatives, tenvermectin A (TVM A) and TVM B, which are considered a potential new generation of agricultural and veterinary drugs. The molecules of the TVMs were designed based on structure and pharmacological property comparisons among AVM, IVM, and milbemycin (MBM). To produce TVMs, a genetically engineered strain, MHJ1011, was constructed from Streptomyces avermitilis G8-17, an AVM industrial strain. In MHJ1011, the native aveA1 gene was seamlessly replaced with milA1 from Streptomyces hygroscopicus. The total titer of the two TVMs produced by MHJ1011 reached 3,400 mg/liter. Insecticidal tests proved that TVM had enhanced activities against Tetranychus cinnabarinus and Bursaphelenchus xylophilus, as desired. This study provides a typical example of exploration for novel active compounds through a new method of polyketide synthase (PKS) reassembly for gene replacement. The results of the insecticidal tests may be of use in elucidating the structure-activity relationship of AVMs and MBMs.A vermectins (AVMs) are a series of 16-membered macrocyclic lactone derivatives with potent anthelmintic and insecticidal properties (1, 2). Ivermectin (IVM), a hydrogenated product of AVM B1, has been one of the best-selling antiparasitics since 1981 (3). However, as insect resistance to AVM (4) and IVM (5-7) is on the rise, alternatives or new products with enhanced potency and expanded spectra of activity are urgently needed.Milbemycins (MBMs) are another group of 16-membered macrolides that share similar structures with AVMs. The insecticidal activity of milbemectin (a mixture of MBMs A3 and A4) against some parasites is higher than those of AVM and IVM, while the toxicity is significantly lower (8).The structural differences among AVM, IVM, and MBM are in C-25, C-22-23, and C-13 ( Fig. 1) (9). IVM differs from AVM in C-22-23 (the former has a saturated bond, whereas the latter has a double bond in that position), but its toxicity is lower than that of AVM (10), implying that the single bond in C-22-23 should be the better structure in terms of safety. The major structural difference between IVM and MBM is a bisoleandrosyloxy substituent attached at C-13 of IVM, whereas that position is unsubstituted in MBM. Also, there are different alkyl substituents at C-25: in IVM, the substituent can be isopropyl or secondary butyl, while in MBM, it can be methyl or ethyl. However, MBM is more potent against some parasites and is safer than IVM, suggesting that one or both of the moieties in C-25 and C-13 of MBM is (are) the better structure(s) in these ways than that (those) of IVM. Another conclusion is that the two oleandroses in C-13 are important to the antiparasitic activity of IVM, since their removal will reduce the activity significantly, about 30-fold (10). Based on the curr...

New 5-oxomilbemycins from a Genetically Engineered Strain Streptomyces bingchenggensis BCJ60

“…The residual oily substance was subjected to a silica gel column chromatography with a gradient petroleum ether/acetone solvent system (from 20:1 to 3:2) to give two fractions (I and II). 12 (1). White …”

“…In addition, the C-6 oxygenated methine in 5-oxomilbemycin A3 was replaced by a methylene group in 1. The differences between compound 1 and milbemycin β 12 were that an aldehyde group and a carbonyl group were situated at C-27 and C-5, respectively, in 1. The observed HMBC correlations from H-9 (δ H 7.39) to C-27 (δ C 189.3), from H-27 (10.22) to C-8 (δ C 135.4), from H-6 (δ H 2.40, 3.38) to C-2 (δ C 48.6), C-4 (δ C 136.7), and from H 3 -26 (δ H 1.86) to C-3 (δ C 136.2), C-4 (δ C 136.7) and C-5 (δ C 196.6) supported the structural assignment of compound 1.…”

“…The observed HMBC correlations from H-9 (δ H 7.39) to C-27 (δ C 189.3), from H-27 (10.22) to C-8 (δ C 135.4), from H-6 (δ H 2.40, 3.38) to C-2 (δ C 48.6), C-4 (δ C 136.7), and from H 3 -26 (δ H 1.86) to C-3 (δ C 136.2), C-4 (δ C 136.7) and C-5 (δ C 196.6) supported the structural assignment of compound 1. Therefore, the planar structure of compound 1 was elucidated and named 27-aldehyde-5-oxomilbemycin β 12 . The relative configuration of 1 was assigned as occurring with 5-oxomilbemycins A3/A4.…”

“…The acaricidal and nematocidal capacities of compounds 1 and 2 were evaluated against Tetranychus cinnabarinus and Bursaphelenchus xylophilus, respectively [12][13]. The mixture of commercial acaricide and nematocide milbemycins A3/A4 was used as a positive control.…”

“…Previously, we have reported the isolation and structural elucidation of two new milbemycin derivatives from the genetically engineered strain S. bingchenggensis BCJ60 [6]. To further exploit the active constituents produced by this strain, the detailed fractionation of the crude extract was conducted and two new 5-oxomilbemycins, 27-aldehyde-5-oxomilbemycin β 12 (1) and 2-hydroxymilbemycin K (2) (Figure 1) were subsequently obtained. In this paper, we describe the isolation, structural elucidation, acaricidal and nematocidal activities of compounds 1 and 2.…”