Abstract
BackgroundMilbemycins, produced from Streptomyces hygroscopicus subsp. aureolacrimosus and Streptomyces bingchenggensis, are 16-membered macrolides that share structural similarity with avermectin produced from Streptomyces avermitilis. Milbemycins possess strong acaricidal, insecticidal, and anthelmintic activities but low toxicity. Due to the high commercial value of the milbemycins and increasing resistance to the avermectins and their derivatives, it is imperative to develop an efficient combinatorial biosynthesis system exploiting an overproduction host strain to produce the milbemycins and novel analogs in large quantities.ResultsThe respective replacement of AveA1 and AveA3 (or module 7 in AveA3) of the avermectin polyketide synthase (PKS) in the avermectin high-producing strain S. avermitilis SA-01 with MilA1 and MilA3 (or module 7 in MilA3) of the milbemycin PKS resulted in the production of milbemycins A3, A4, and D in small amounts and their respective C5-O-methylated congener milbemycins B2, B3, and G as major products with total titers of approximately 292 mg/l. Subsequent inactivation of the C5-O-methyltransferase AveD led to a production of milbemycins A3/A4 (the main components of the commercial product milbemectin) in approximately 225 and 377 mg/l in the flask and 5 l fermenter culture, respectively, along with trace amounts of milbemycin D.ConclusionsWe demonstrated that milbemycin biosynthesis can be engineered in the avermectin-producing S. avermitilis by combinatorial biosynthesis with only a slight decrease in its production level. Application of a similar strategy utilizing higher producing industrial strains will provide a more efficient combinatorial biosynthesis system based on S. avermitilis for further enhanced production of the milbemycins and their novel analogs with improved insecticidal potential.
Highlights
Milbemycins, produced from Streptomyces hygroscopicus subsp. aureolacrimosus and Streptomyces bingchenggensis, are 16-membered macrolides that share structural similarity with avermectin produced from Streptomyces avermitilis
The difference lies in the fact that the milA1 gene encoding the loading module and two extension modules is located 55- and 62-kb apart from the other three polyketide synthase (PKS) genes in the milbemycin gene cluster of S. nanchangensis and S. bingchenggensis, respectively, and there are several significant differences between the two biosynthetic genes at the domain level which directs the structural diversity of these molecules (Fig. 2)
In order to reduce the number of time-consuming double crossover processes, we decided to replace the AveA1 PKS on the chromosome in S. avermitilis SA-01, the previously optimized avermectin high-producing mutant strain, with MilA1 from S. hygroscopicus subsp. aureolacrimosus NRRL 5739 rather than individually replace the corresponding domain/modules for changing the structures at C25 and C22–C23 of avermectins
Summary
Milbemycins, produced from Streptomyces hygroscopicus subsp. aureolacrimosus and Streptomyces bingchenggensis, are 16-membered macrolides that share structural similarity with avermectin produced from Streptomyces avermitilis. The milbemycins and avermectins are structurally related 16-membered macrolides with excellent anthelmintic and insecticidal activities (Fig. 1). The biosynthetic gene clusters of the milbemycins in Streptomyces nanchangensis NS3226 (milbemycins α11, α13, α14, β1, and β9 are identical to meilingmycins A to E, respectively, produced by S. nanchangensis) [14] and S. bingchenggensis [15] (Fig. 2a) as well as the avermectins in S. avermitilis [16] (Fig. 2b) were characterized. The difference lies in the fact that the milA1 gene encoding the loading module and two extension modules is located 55- and 62-kb apart from the other three PKS genes in the milbemycin gene cluster of S. nanchangensis and S. bingchenggensis, respectively, and there are several significant differences between the two biosynthetic genes at the domain level which directs the structural diversity of these molecules (Fig. 2). Incorporation of 2-methylbutyryl-CoA or isobutyryl-CoA as a starter unit results
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