RESUMO
PF1022A, a cyclooctadepsipeptide possessing strong anthelmintic properties and produced by the filamentous fungus Rosellinia sp. PF1022, consists of four alternating residues of N-methyl-L-leucine and four residues of D-lactate or D-phenyllactate. PF1022A derivatives obtained through modification of their benzene ring at the para-position with nitro or amino groups act as valuable starting materials for the synthesis of compounds with improved anthelmintic activities. Here we describe the production of such derivatives by fermentation through metabolic engineering of the PF1022A biosynthetic pathway in Rosellinia sp. PF1022. Three genes cloned from Streptomyces venezuelae, and required for the biosynthesis of p-aminophenylpyruvate from chorismate in the chloramphenicol biosynthetic pathway, were expressed in a chorismate mutase-deficient strain derived from Rosellinia sp. PF1022. Liquid chromatography-mass spectrometry and NMR analyses confirmed that this approach facilitated the production of PF1022A derivatives specifically modified at the para-position. This fermentation method is environmentally safe and can be used for the industrial scale production of PF1022A derivatives.
Assuntos
Anti-Helmínticos/química , Anti-Helmínticos/metabolismo , Cloranfenicol/biossíntese , Depsipeptídeos/química , Depsipeptídeos/metabolismo , Streptomyces/genética , Animais , Sequência de Bases , Biotransformação , Clonagem Molecular , Depsipeptídeos/biossíntese , Depsipeptídeos/genética , Fermentação , Engenharia Genética , Dados de Sequência Molecular , Streptomyces/metabolismoRESUMO
The avi2 gene encoding Avi2, which is a major cellulase produced by Humicola insolens FERM BP-5977, was cloned and sequenced. Avi2 showed high homology with other family 6 cellulases. The expression vector pNCE4 containing the avi2 gene was constructed, and this strain was transformed using a protoplast method. As a result, the pNCE4 transformant secreted 8-fold more Avi2 than the recipient strain.
Assuntos
Celulases/genética , Clonagem Molecular , Fungos Mitospóricos/genética , Celulases/biossíntese , Celulases/metabolismo , DNA Fúngico , Vetores Genéticos , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transformação GenéticaRESUMO
Three endoglucanase genes, designated the rce1, rce2, and rce3 genes, were isolated from Rhizopus oryzae as the first cellulase genes from the subdivision ZYGOMYCOTA: All the amino acid sequences deduced from the rce1, rce2, and rce3 genes consisted of three distinct domains: cellulose binding domains, linker domains, and catalytic domains belonging to glycosyl hydrolase family 45. The rce3 gene had two tandem repeated sequences of cellulose binding domains, while rce1 and rce2 had only one. rce1, rce2, and rce3 had various lengths of linker sequences.
