RESUMO
As glycosyltransferases found in nature often show distinct substrate specificity, glycosyltransferase engineering is an important research field. In this work, we were able to introduce an activity into a glycosyltransferase involved in natural product (landomycin E) biosynthesis. This was achieved by recognizing hot spot amino acids in glycosyltransferases which are strongly involved in determining substrate specificity.
Assuntos
Glicosiltransferases/genética , Sequência de Aminoácidos , Aminoglicosídeos/biossíntese , Glicosiltransferases/metabolismo , Dados de Sequência Molecular , Mutação , Engenharia de Proteínas , Homologia de Sequência de Aminoácidos , Especificidade por SubstratoRESUMO
We report here a system for the functional expression of the Flp recombinase in several actinomycetes: Streptomyces coelicolor, S. lividans, and Saccharotrix espanaensis. We have constructed a synthetic gene encoding the Flp recombinase with a GC content of 60.6% optimized for expression in high-GC bacteria. Using the synthetic flp(a) gene, we have removed an apramycin resistance gene flanked by FRT sites from the chromosome of actinomycetes with an efficiency of 40%. Sequencing the region of chromosome showed that excision of the apramycin cassette by Flp recombinase was specific.
Assuntos
Actinomycetales/genética , DNA Nucleotidiltransferases/metabolismo , Resistência Microbiana a Medicamentos/genética , Engenharia Genética/métodos , Cromossomos Fúngicos , DNA Nucleotidiltransferases/genética , Expressão Gênica , Genes Sintéticos , Marcadores Genéticos , Genoma Fúngico , Nebramicina/análogos & derivados , Nebramicina/farmacologia , Recombinação Genética , Streptomyces coelicolor/genética , Streptomyces lividans/genéticaRESUMO
Site-specific recombinases revolutionized "in vivo" genetic engineering because they can catalyze precise excisions, integrations, inversions, or translocations of DNA between their distinct recognition target sites. We have constructed a synthetic gene encoding Cre recombinase with the GC content 67.7% optimized for expression in high-GC bacteria and demonstrated this gene to be functional in Streptomyces lividans. Using the synthetic cre(a) gene, we have removed an apramycin resistance gene flanked by loxP sites from the chromosome of S. lividans with 100% efficiency. Sequencing of the chromosomal DNA part showed that excision of the apramycin cassette by Cre recombinase was specific.
Assuntos
Expressão Gênica , Engenharia Genética/métodos , Integrases/genética , Streptomyces lividans/genética , Clonagem Molecular , Integrases/metabolismo , Plasmídeos/genética , Streptomyces lividans/metabolismo , Proteínas Virais/genética , Proteínas Virais/metabolismoRESUMO
Detailed studies on the biosynthesis of the hexasaccharide side chain of landomycin A, produced by S. cyanogenus S136, revealed the function of each glycosyltransferase gene of the biosynthetic gene cluster. Analyses of generated mutants as well as feeding experiments allowed us to determine that LanGT2 and LanGT3 catalyze the attachment of one sugar, whereas LanGT1 and LanGT4 attach two sugars during landomycin A biosynthesis. The generation of a lanZ2 deletion mutant provided evidence that LanZ2 is controlling the elongation of the saccharide side chain.
Assuntos
Aminoglicosídeos/biossíntese , Antibacterianos/biossíntese , Glicosiltransferases/metabolismo , Polissacarídeos/biossíntese , Aminoglicosídeos/química , Catálise , Cromatografia Gasosa-Espectrometria de Massas , Glicosiltransferases/genética , Mutação , Polissacarídeos/química , Streptomyces/genética , Streptomyces/metabolismoRESUMO
The glycosyltransferase LanGT2 is involved in the biosynthesis of the hexasaccharide side chain of the angucyclic antibiotic landomycin A. Its function was elucidated by targeted gene inactivation of lanGT2. The main metabolite of the obtained mutant was identified as tetrangulol (4), the progenitor of the landomycin aglycon (7). The lack of the sugar side chain indicates that LanGT2 catalyzes the priming glycosyl transfer in the hexasaccharide biosynthesis: the attachment of a D-olivose to O-8 of the polyketide backbone. Heterologous expression of urdGT2 from S. fradiae Tü2717 in this mutant resulted in the production of a novel C-glycosylated angucycline (6).
Assuntos
Aminoglicosídeos/biossíntese , Glicosiltransferases/metabolismo , Benzo(a)Antracenos/química , Benzo(a)Antracenos/metabolismo , Catálise , Desoxiaçúcares/química , Desoxiaçúcares/metabolismo , Ativação Enzimática , Glicosilação , Glicosiltransferases/genética , Estrutura Molecular , Mutação , Streptomyces/enzimologiaRESUMO
Two genes from Streptomyces cyanogenous S136 that encode the reductase LanZ4 and the hydroxylase LanZ5, which are involved in landomycin A biosynthesis, were characterized by targeted gene inactivation. Analyses of the corresponding mutants as well as complementation experiments have allowed us to show that LanZ4 and LanZ5 are responsible for the unique C-11-hydroxylation that occurs during landomycin biosynthesis. Compounds accumulated by the lanZ4/Z5 mutants are the previously described landomycin F and the new landomycins M and O.
Assuntos
Aminoglicosídeos/biossíntese , Dissacarídeos/biossíntese , Marcação de Genes , Oligossacarídeos/biossíntese , Streptomyces/genética , Aminoglicosídeos/farmacologia , Antibióticos Antineoplásicos/biossíntese , Antibióticos Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Dissacarídeos/farmacologia , Humanos , Concentração Inibidora 50 , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Naftoquinonas/química , Naftoquinonas/farmacologia , Oligossacarídeos/química , Oligossacarídeos/farmacologia , Oxigenases/genética , Streptomyces/metabolismoRESUMO
The glycosyltransferase gene lanGT3, involved in the biosynthesis of the angucyclic antibiotic landomycin A, has been characterised by targeted gene deletion. A lanGT3 mutant was shown to produce landomycin E, which consists of a trisaccharide side chain attached to the polyketide moiety. Expression of lanGT3 in the mutant restored landomycin A production. Our results indicate that LanGT3 is responsible for the transfer of the fourth sugar during landomycin A biosynthesis.