RESUMO
Many high-value secondary metabolites are assembled by very large multifunctional polyketide synthases or non-ribosomal peptide synthetases encoded by giant genes, for instance, natamycin production in an industrial strain of Streptomyces gilvosporeus. In this study, a large operon reporter-based selection system has been developed using the selectable marker gene neo to report the expression both of the large polyketide synthase genes and of the entire gene cluster, thereby facilitating the selection of natamycin-overproducing mutants by iterative random mutagenesis breeding. In three successive rounds of mutagenesis and selection, the natamycin titer was increased by 110%, 230%, and 340%, respectively, and the expression of the whole biosynthetic gene cluster was correspondingly increased. An additional copy of the natamycin gene cluster was found in one overproducer. These findings support the large operon reporter-based selection system as a useful tool for the improvement of industrial strains utilized in the production of polyketides and non-ribosomal peptides.
Assuntos
Genes Reporter/genética , Melhoramento Genético/métodos , Engenharia Metabólica/métodos , Natamicina/biossíntese , Óperon/genética , Policetídeo Sintases/genética , Streptomyces/fisiologia , Vias Biossintéticas/genética , Perfilação da Expressão Gênica/métodos , Análise do Fluxo Metabólico/métodos , Redes e Vias Metabólicas/genética , Família Multigênica/genética , Natamicina/isolamento & purificação , Especificidade da Espécie , Streptomyces/classificaçãoRESUMO
The afsRS(cla) global regulatory genes from Streptomyces clavuligerus activate the production of two antibiotics in Streptomyces lividans. In this study, we gained an increase of 38% in the production of natamycin (3.56 g/L) in an industrial strain Streptomyces gilvosporeus TZ1401 through the integration of pHL851 that bears the afsRS(cla) global regulatory genes into its genome. We discovered by quantitive real-time reverse transcription PCR (qRT-PCR) that the expression of 6 genes of the natamycin biosynthetic gene cluster were improved from 1.9 to 2.7 times. This suggests that afsRS(cla) improve the production of natamycin through increased transcription. This study provides a good example for applying afsRS(cla) in high yield breeding of industrial antibiotic producers.