RESUMO
In vivo reconstitution of the TDP-l-megosamine pathway from the megalomicin gene cluster of Micromonospora megalomicea was accomplished by the heterologous expression of its biosynthetic genes in Escherichia coli. Mass spectrometric analysis of the TDP-sugar intermediates produced from operons containing different sets of genes showed that the production of TDP-l-megosamine from TDP-4-keto-6-deoxy-d-glucose requires only five biosynthetic steps, catalyzed by MegBVI, MegDII, MegDIII, MegDIV, and MegDV. Bioconversion studies demonstrated that the sugar transferase MegDI, along with the helper protein MegDVI, catalyzes the transfer of l-megosamine to either erythromycin C or erythromycin D, suggesting two possible routes for the production of megalomicin A. Analysis in vivo of the hydroxylation step by MegK indicated that erythromycin C is the intermediate of megalomicin A biosynthesis.
Assuntos
Aminoglicosídeos/biossíntese , Vias Biossintéticas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Micromonospora/genética , Família Multigênica , Aminoglicosídeos/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Eritromicina/metabolismo , Ordem dos Genes , Glucose/análogos & derivados , Glucose/metabolismo , Modelos Biológicos , Estrutura Molecular , Óperon , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Nucleotídeos de Timina/metabolismoRESUMO
Significant achievements in polyketide gene expression have made Escherichia coli one of the most promising hosts for the heterologous production of pharmacologically important polyketides. However, attempts to produce glycosylated polyketides, by the expression of heterologous sugar pathways, have been hampered until now by the low levels of glycosylated compounds produced by the recombinant hosts. By carrying out metabolic engineering of three endogenous pathways that lead to the synthesis of TDP sugars in E. coli, we have greatly improved the intracellular levels of the common deoxysugar intermediate TDP-4-keto-6-deoxyglucose resulting in increased production of the heterologous sugars TDP-L-mycarose and TDP-D-desosamine, both components of medically important polyketides. Bioconversion experiments carried out by feeding 6-deoxyerythronolide B (6-dEB) or 3-α-mycarosylerythronolide B (MEB) demonstrated that the genetically modified E. coli B strain was able to produce 60- and 25-fold more erythromycin D (EryD) than the original strain K207-3, respectively. Moreover, the additional knockout of the multidrug efflux pump AcrAB further improved the ability of the engineered strain to produce these glycosylated compounds. These results open the possibility of using E. coli as a generic host for the industrial scale production of glycosylated polyketides, and to combine the polyketide and deoxysugar combinatorial approaches with suitable glycosyltransferases to yield massive libraries of novel compounds with variations in both the aglycone and the tailoring sugars.
Assuntos
Produtos Biológicos/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia Genética , Macrolídeos/metabolismo , Produtos Biológicos/genética , Vias Biossintéticas , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , GlicosilaçãoRESUMO
Two putative C3-ketoreductases, MegBIIa and MegBIIb (formerly MegBII and MegDVII, respectively), homologues to members of the family 12 of aldo-keto reductase (AKR12) superfamily of enzymes, were identified in the megalomicin gene cluster from Micromonospora megalomicea. Proteins from this family are involved in the metabolism of TDP-sugars by actinomycetes. MegBIIa was originally proposed to be involved in the l-mycarose biosynthetic pathway, while MegBIIb in the l-megosamine biosynthetic pathway. In this work we have investigated the role of these proteins in the biosynthesis of dTDP-l-mycarose. In vivo analysis of the dTDP-sugar intermediates indicated that neither MegBIIa nor its homologue, MegBIIb, was a fully active enzyme by itself. Surprisingly, C3-ketoreductase activity was observed only in the presence of both MegBIIa and MegBIIb, suggesting the formation of an active complex. Copurification and size exclusion chromatography experiments confirmed that MegBIIa and MegBIIb interact forming a 1:1 heterodimeric complex. Finally, a mycarose operon containing megBIIa and megBIIb together with the other biosynthetic genes of the l-mycarose pathway was constructed and tested by bioconversion experiments in Escherichia coli. High levels of mycarosyl-erythronolide B were produced under the condition tested, confirming the role of these two proteins in this metabolic pathway.
Assuntos
Oxirredutases do Álcool/metabolismo , Hexoses/biossíntese , Micromonospora/metabolismo , Oxirredutases do Álcool/química , Oxirredutases do Álcool/isolamento & purificação , Aldeído Redutase , Aldo-Ceto Redutases , Sequência de Aminoácidos , Sequência de Bases , Cromatografia em Gel , Primers do DNA , Dimerização , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Espectrometria de Massas em TandemRESUMO
A new prenylated dibenzofuran, achyrofuran (2), was isolated from an extract of Achyrocline satureioides by bioassay-guided fractionation using the db/db mouse model for type 2 diabetes. Compound 2significantly lowered blood glucose levels in this model when administered orally at 20 mg/kg q.d.