The targeted inactivation of polyketide synthasemycAVin the mycinamicin producer,Micromonospora griseorubida, and a complementation study

Abstract

Mycinamicin is a 16-membered macrolide antibiotic produced by Micromonospora griseorubida A11725, which shows strong antimicrobial activity against gram-positive bacteria. Recently, the nucleotide sequences of the mycinamicn biosynthetic gene cluster in M. griseorubida have been completely determined. Mycinamicin non-producer M7A21 was isolated by mycAV inactivation, which encodes the module 7 of mycinamicin polyketide synthase (PKS) required for the biosynthesis of the mycinamicin biosynthetic intermediate protomycinolide-IV (PML-IV). When the bioconversion to mycinamicin II (M-II) from PML-IV was performed using M7A21 and the feeding culture method, the productivity of M-II was the same as that of M-II in wild-type strain A11725. p446M7 containing mycAV was constructed using the Escherichia coli-Streptomyces shuttle vector pGM446. The mycinamicin productivity of M7A21 was restored by the introduction of p446M7 into the M7A21 cell, but almost all p446M7 was integrated into the chromosome of M7A21 because the plasmid was unstable in M7A21. The feeding culture and the introduction of the complement gene for M7A21 would be powerful tools to perform combinatorial biosynthesis for the production of new macrolide antibiotics.

Gene targeting forO-methyltransferase genes,mycEandmycF, on the chromosome ofMicromonospora griseorubidaproducing mycinamicin with a disruption cassette containing the bacteriophage φC31attBattachment site

“…Plasmid and genomic DNA amplification, restriction enzyme digestion, fragment isolation, cloning, and DNA fragment amplification were performed according to standard procedures. Southern blot analysis was performed according to our previous procedure (Anzai et al , 2004a).…”

“…The intergeneric conjugation from E. coli S17‐1 to M. griseorubida was performed using a modified protocol of our previous procedure (Anzai et al , 2004a). A mixture of the E. coli donor cells and the M. griseorubida recipient cells was spread on MR0.1S plates or AS‐1 agar plates (Alexander et al , 2003).…”

“…The strains used in this study are shown in Table 1. The culture conditions of M. griseorubida and E. coli were according to our previous report (Anzai et al , 2004a). FMM broth containing 7% dextrin, 0.5% glucose, 0.5% yeast extract, 0.5% soybean meal (Ajinomoto, Japan), 0.5% CaCO 3 , 0.1% K 2 HPO 4 , 0.4% MgSO 4 ·7H 2 O, and 0.0002% CoCl 2 ·6H 2 O was used for fermentation of M. griseorubida .…”

Function of Cytochrome P450 Enzymes MycCI and MycG in Micromonospora griseorubida, a Producer of the Macrolide Antibiotic Mycinamicin

“…Intermediate feeding into the M. griseorubida culture plate and sample preparation for high-performance liquid chromatography (HPLC) analysis were performed using a protocol modified from our previous study (3). A seed culture was grown in 5 ml of MR0.1S broth for 5 days, and 150 l of this seed culture was then spread on agar plates containing 15 ml of MR0.1S.…”

Production of rosamicin derivatives in Micromonospora rosaria by introduction of d-mycinose biosynthetic gene with ΦC31-derived integration vector pSET152

“…All the ampliWed DNA fragments were cloned into pDrive by using the TA cloning system, and the sequences of the cloned DNA fragments were determined. The intergeneric conjugation from E. coli ET12567/ pUZ8002 to M. rosaria IFO13697 was performed by using a protocol similar to our previous procedure [1]. An overnight culture of the E. coli donor strain was diluted in fresh medium and incubated for 3-5 h. The cells were harvested, washed twice, and concentrated tenfold in TSB.…”