Avermectin biosynthesis

The proposed pathway for the biosynthesis of the avermectins (Fig. 3) has been described in a review (23). Some of the details are yet to be elucidated, although the steps, in general, are based on firm evidence from four types of studies incorporation of labeled precursors, conversion of putative intermediates by producing strains and blocked mutants, in vitro measurement of biosynthetic enzymes, and studies with enzyme inhibitors. The biosynthesis of the oleandrose units was elucidated from studies using and labeled glucose, which indicated a direct conversion of glucose to... 

In summary, a novel cyclohexyl avermectin analog B1 with enhanced antiparasitic activity was discovered and produced with high selectivity and excellent fermentation titer through deciphering biosynthesis, pathway engineering, and directed evolution. The new product, doramectin, is sold commercially as Dectomax. 

Ikeda, H. and Omura, S. (1995) Control of avermectin biosynthesis in Streptomyces avermitilis for the selective production of a useful component. Journal of Antibiotics (Tokyo), 48, 549-562. 

CD Denoya, RW Fedechko, EW Hafner, HAI McArthur, MR Morgenstern, DD Skinner, K Stutzman-Engwall, RG Wax, WC Wemau. A second branched-chain OC-keto acid dehydrogenase gene cluster (bkdFGH) from Streptomyces avermitilis. its relationship to avermectin biosynthesis and the construction of a bkdF mutant suitable for the production of novel antiparasitic avermectins. J Bacteriol 177 3504-3511, 1995. 

As discussed earlier, the avermectin polyketide backbone is derived from seven acetate and five propionate extender units added to an a branched-chain fatty acid starter, which is either (S( I )-a-mcthylbutyric acid or isobutyric acid. The C25 position of naturally occurring avermectins has two possible substituents a. sec-butyl residue derived from the incorporation of S(+)-a-methy lbutyry 1-CoA ( a avermectins), or an isopropyl residue derived from the incorporation of isobutyiyl-CoA ( b avermectins). These a branched-chain fatty acids, which act as starter units in the biosynthesis of the polyketide ring, are derived from the a branched-chain amino acids isoleucine and valine through a branched-chain amino acid transaminase reaction followed by a branched-chain a-keto acid dehydrogenase (BCDH) reaction (Fig. 5) [23]. 

Bu Lock et al. had shown initially [24] that supplementation of S. avermitilis fermentations with a range of fatty acids resulted in their uptake and incorporation to generate novel avermectins modified at the C25 side chain of the molecule. This approach, described as precursor-directed biosynthesis, has been employed to produce many new antibiotics [25], but the co-expression of the parent molecule interferes with the detection and isolation of the novel analogs. To circumvent these difficulties, the elegant technique of mutational biosynthesis [26] or mutasynthesis [27,28] was developed. In this approach, a mutant of an organism, deficient in the production of an essential precursor for the secondary metabolite of choice, is isolated, and precursor-directed biosynthesis is then employed to generate only the novel analogs. 

In applying mutasynthesis to the production of novel avermectins, the elimination of branched-chain a-keto acid dehydrogenase (BCDH) was targeted. This multienzyme complex is responsible for supplying the 2-methylbutyryl- and iso-butyryl-CoA starter units that initiate natural avermectin biosynthesis [21,29],... 

Figure 5 Pathways of valine and isoleucine catabolism and their postulated relationship to avermectin biosynthesis.

These results confirmed that branched-chain amino acid catabolism via the BCDH reaction provides the fatty acid precursors for natural avermectin biosynthesis in S. avermitilis. In contrast, B. subtilis appears to possess two mechanisms for branched-chain precursor supply. The dual substrate pyruvate/branched-chain a-keto acid dehydrogenase (aceA) and an a-keto acid dehydrogenase (bfmB), which also has some ability to metabolize pyruvate, appears to be primarily involved in supplying the branched-chain initiators of long, branched-chain fatty acid biosynthesis [32,42], Two mutations are therefore required to generate the bkd phenotype in B. subtilis [31,42],... 

S. avermitilis and the biosynthesis of avermectins constitute an interesting example where traditional techniques such as chemical mutagenesis and protoplast fusion combined with recombinant DNA technology have been successfully applied in mutant isolation and strain improvement. In addition, this system offered the first opportunity to apply mutasynthesis to the production of better analogs, an application that had never before been exploited commercially. An intense doramectin development effort was therefore initiated with the Mrf-deficient mutants of S. avermitilis. The first step in this process involved the isolation of mutants deficient in 5-Omethyltransferase activity to maximize levels of the more bioactive class B avermectins [13], Thereafter, a combination of strain im-... 

TS Chen, BH Arison, VP Gullo, ES Inamine. Further studies on the biosynthesis of the avermectins. J Ind Microbiol 4 231-238, 1989. 

TS Chen, OD Hensens, MD Schulman. Biosynthesis. In WC Campbell, ed. Ivermectin and Avermectin. New York Springer-Verlag, pp 55-72, 1989. 

CJ Dutton, SP Gibson, AC Goudie, KS Holdom, MS Pacey, JC Ruddock, JD Bu Lock, MS Richards. Novel avermectins produced by mutational biosynthesis. J Antibiot 44 357-365, 1991. 

Figure 19 Altering the starter unit specificity of DEBS 1-TE. A hybrid PKS was constructed by replacing the erythromycin loading domains with those of the avermectin PKS. The resulting mini-PKS produced lactones incorporating the natural starter units of erythromycin biosynthesis, acetate and propionate, as well as those characteristic of avermectin, isobutyrate, and 2-methylbutyrate.

CJ Dutton, AM Hooper, PF Leadlay, J Staunton. Avermectin biosynthesis. Intact incorporation of a diketide chain-assembly intermediate into the polyketide mac-rocylic ring. Tetrahedron Lett 35 327-330, 1994. 

A final example of metabolic pathway engineering is based on polyketide and nonribosomal peptide biosynthesis. Polyketides and nonribosomal peptides are complex natural products with numerous chiral centers, which are of substantial economic benefit as pharmaceuticals. These natural products function as antibiotics [erythromycin A (65), vancomycin (66)], antifungals (rapamycin, amphotericin B), antiparasitics [avermectin Ala (67)], antitumor agents [epothiolone A (68), calicheamicin yj, and immunosuppressants [FK506 (69), cyclosporin A], Because this exponentially growing and intensely researched field has developed, the reader is directed to review articles for additional details.347-359 Also with the potential economic benefit to develop the next blockbuster pharmaceutical, a number of patents and patent applications have been published.360-366... 

In the first part of this chapter, we deal with insecticides including miticides and nematocides, which include very useful compounds such as avermectins and milbemycins, produced by bacteria and fungi. We list out microbial insecticides of importance and review the works mainly on the mode of action and biosynthesis of each metabolite. In the next part, major mycotoxins are listed and recent topics on them, especially on their biosynthesis, are described. Since contamination of two major mycotoxin groups, aflatoxins (AFs) and trichothecenes, in food and feed is a worldwide problem, they are treated in detail in the last part of this chapter. Recent studies on their biosynthesis, regulatory mechanism for their production, and inhibitors of their production are described. 

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