RESUMO
The glycosylation of natural product scaffolds with highly modified deoxysugars is often essential for their biological activity, being responsible for specific contacts to molecular targets and significantly affecting their pharmacokinetic properties. In order to provide tools for the targeted alteration of natural product glycosylation patterns, significant strides have been made to understand the biosynthesis of activated deoxysugars and their transfer. We report here efforts towards the production of plasmid-borne biosynthetic gene cassettes capable of producing TDP-activated forms of D-mycaminose, D-angolosamine and D-desosamine. We additionally describe the transfer of these deoxysugars to macrolide aglycones using the glycosyl transferases EryCIII, TylMII and AngMII, which display usefully broad substrate tolerance.
Assuntos
Glucosamina/análogos & derivados , Macrolídeos/química , Macrolídeos/metabolismo , Clonagem Molecular , Engenharia Genética , Glucosamina/química , Glucosamina/metabolismo , Estrutura Molecular , Família Multigênica/genética , Análise de Sequência , Streptomyces/química , Streptomyces/genética , Streptomyces/metabolismoRESUMO
The spinosyns are a family of potent and highly selective insect control agents that display a favorable environmental profile. As some regions of the spinosyn molecule are recalcitrant to chemical modification, a targeted genetic approach was carried out to generate new analogues. The polyketide synthase (PKS) loading modules from the avermectin PKS of Streptomyces avermitilis and the erythromcyin PKS of Saccharopolyspora erythraea were each used to replace the spinosyn PKS loading module. Both of the resulting strains containing hybrid PKS pathways produced the anticipated spinosyn analogues. Supplementation of the culture media with a range of exogenous carboxylic acids led to the successful incorporation of these novel elements to yield further novel spinosyn molecules, some of which demonstrated potent and new insecticidal activities. Furthermore, it has been demonstrated that semisynthesis of such novel metabolites can then be used to generate active analogues, demonstrating the effectiveness of utilizing these complementary methods to search the chemical space around this template.
Assuntos
DNA/química , Inseticidas/química , Macrolídeos/química , Policetídeo Sintases/química , Tetranychidae/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Sequência de Bases , Eritromicina/química , Escherichia coli/metabolismo , Ivermectina/análogos & derivados , Ivermectina/química , Modelos Moleculares , Engenharia de Proteínas , Saccharopolyspora/enzimologia , Saccharopolyspora/metabolismo , Streptomyces/enzimologia , Streptomyces/metabolismoRESUMO
The function of gene products involved in the biosynthesis of the clinically important polyketide rapamycin were elucidated by biotransformation and gene complementation.
Assuntos
Genes , Sirolimo/metabolismo , Teste de Complementação Genética , Espectrometria de Massas , Sirolimo/análogos & derivados , Sirolimo/químicaRESUMO
Ivermectin, a mixture of 22,23-dihydroavermectin B1a9 with minor amounts of 22,23-dihydroavermectin B1b 10, is one of the most successful veterinary antiparasitic drugs ever produced. In humans, ivermectin has been used for the treatment of African river blindness (onchocerciasis) resulting in an encouraging decrease in the prevalence of skin and eye diseases linked to this infection. The components of ivermectin are currently synthesized by chemical hydrogenation of a specific double bond at C22-C23 in the polyketide macrolides avermectins B1a 5 and B1b 6, broad-spectrum antiparasitic agents isolated from the soil bacterium Streptomyces avermitilis. We describe here the production of such compounds (22,23-dihydroavermectins B1a 9 and A1a 11) by direct fermentation of a recombinant strain of S. avermitilis containing an appropriately-engineered polyketide synthase (PKS). This suggests the feasibility of a direct biological route to this valuable drug.