RESUMO
AIMS: Production of minor asukamycin congeners and its new derivatives by combination of targeted genetic manipulations with specific precursor feeding in the producer of asukamycin, Streptomyces nodosus ssp. asukaensis. METHODS AND RESULTS: Structural variations of manumycins lie only in the diverse initiation of the 'upper' polyketide chain. Inactivation of the gene involved in the biosynthesis of cyclohexanecarboxylic acid (CHC) turned off the production of asukamycin in the mutant strain and allowed an increased production of other manumycins with the branched end of the upper chain. The ratio of produced metabolites was further affected by specific precursor feeding. Precursor-directed biosynthesis of a new asukamycin analogue (asukamycin I, 28%) with linear initiation of the upper chain was achieved by feeding norleucine to the mutant strain. Another asukamycin analogue with the unbranched upper chain (asukamycin H, 14%) was formed by the CHC-deficient strain expressing a heterologous gene putatively involved in the formation of the n-butyryl-CoA starter unit of manumycin A. CONCLUSIONS: Combination of the described techniques proved to be an efficient tool for the biosynthesis of minor or novel manumycins. SIGNIFICANCE AND IMPACT OF THE STUDY: Production of two novel asukamycin derivatives, asukamycins H and I, was achieved. Variations appeared in the upper polyketide chain, the major determinant of enzyme-inhibitory features of manumycins, affecting their cancerostatic or anti-inflammatory features.
Assuntos
Antibacterianos/biossíntese , Polienos/metabolismo , Alcamidas Poli-Insaturadas/metabolismo , Streptomyces/metabolismo , Acil Coenzima A/metabolismo , Aminoácidos/metabolismo , Meios de Cultura , Ácidos Cicloexanocarboxílicos/metabolismo , Engenharia Genética , Mutagênese Insercional , Mutação , Streptomyces/genéticaAssuntos
Legionella/isolamento & purificação , Doença dos Legionários/microbiologia , Orofaringe/microbiologia , Anti-Infecciosos/administração & dosagem , Proteínas de Bactérias/genética , Ciprofloxacina/administração & dosagem , Primers do DNA/química , Humanos , Terapia de Imunossupressão/efeitos adversos , Transplante de Rim , Doença dos Legionários/diagnóstico , Doença dos Legionários/tratamento farmacológico , Transplante de Fígado , Transplante de Pâncreas , Peptidilprolil Isomerase/genética , Reação em Cadeia da Polimerase/métodosRESUMO
A gene pknA, coding for an eukaryotic-type protein Ser/Thr kinase, was cloned from the Streptomyces coelicolor A3(2) chromosome. The PknA protein kinase, containing the C-terminal eukaryotic-type kinase domain with an N-terminal extension, was expressed in Escherichia coli and Streptomyces lividans. The affinity purified MBP-PknA fusion protein was assayed for kinase activity that showed its ability to autophosphorylate in vitro in the presence of [gamma-32P]ATP. The activity was Mn2+ dependent. The preautophosphorylated kinase phosphorylated at least two proteins (sizes 30 and 32 kDa) in the S. coelicolor J1501 cell-free extracts of all developmental stages. The larger of them was also phosphorylated in vitro by an endogenous protein kinase in late stages extracts, but not earlier. Although Mn2+ dependent protein phosphorylation has previously been described in Streptomyces, this is the first report of a gene encoding such an enzyme in this genus.
