Over-expression of the pathway specific positive regulator gene is an effective way to activate silent gene cluster. In the curret study, the SARP family regulatory gene, vasR2, was over-expressed in strain Verrucosispora sp. NS0172 and the cryptic gene cluster responsible for the biosynthesis of pentaketide ansamycin was partially activated. Two tetraketides (1 and 2) and a triketide (3) ansamycins, together with five known compounds (4-8), were isolated and elucidated from strain NS0172OEvasR2. Their NMR data were completely assigned by analysis of their HR-ESI-MS and
Micromonosporaceae/metabolism* ; Multigene Family ; Polyketides/metabolism* ; Rifabutin/metabolism*

Chuangxinmycin (CM) from Actinoplanes tsinanensis was an antibiotic discovered by Chinese scientists about 40 years ago. It contains a new heterocyclic system of indole fused with dihydrothiopyran, whose biosynthetic mechanism remains unclear. CM is used as an oral medicine in the treatment of bacterial infections in China. The simple structure makes CM as an attractive candidate of structure modification for improvement of antibacterial activity. Recently, we analyzed the secondary metabolites of Actinoplanes tsinanensis CPCC 200056, a CM producing strain, as a natural CM analogue. We discovered the first natural CM analogue 3-demethylchuangxinmycin (DCM) as a new natural product. Compared to CM, DCM exhibited a much weaker activity in the inhibition of the bacterial strains tested. The finding provides valuable information for the structure-activity relationship in the biosynthesis of CM.
Anti-Bacterial Agents ; chemistry ; isolation & purification ; China ; Indoles ; chemistry ; isolation & purification ; Micromonosporaceae ; chemistry ; Structure-Activity Relationship

The present study was designed to identify the difference between two rapamycin biosynthetic gene clusters from Streptomyces hygroscopicus ATCC29253 and Actinoplanes sp. N902-109 by comparing the sequence and organization of the gene clusters. The biosynthetic gene cluster for rapamycin in Streptomyces hygroscopicus ATCC29253 was reported in 1995. The second rapamycin producer, Actinoplanes sp. N902-109, which was isolated in 1995, could produce more rapamycin than Streptomyces hygroscopicus ATCC29253. The genomic map of Actinoplanes sp. N902-109 has been elucidated in our laboratory. Two gene clusters were compared using the online software anti-SMASH, Glimmer 3.02 and Subsystem Technology (RAST). Comparative analysis revealed that the organization of the multifunctional polyketide synthases (PKS) genes: RapA, RapB, RapC, and NRPS-like RapP were identical in the two clusters. The genes responsible for precursor synthesis and macrolactone modification flanked the PKS core region in N902-109, while the homologs of those genes located downstream of the PKS core region in ATCC29253. Besides, no homolog of the gene encoding a putative type II thioesterase that may serve as a PKS "editing" enzyme accounted for over-production of rapamycin in N902-109, was found in ATCC29253. Furthermore, no homologs of genes rapQ (encoding a methyltransferase) and rapG in N902-109 were found in ATCC29253, however, an extra rapM gene encoding methyltransferase was discovered in ATCC29253. Two rapamycin biosynthetic gene clusters displayed overall high homology as well as some differences in gene organization and functions.
Amino Acid Sequence ; Bacterial Proteins ; chemistry ; genetics ; metabolism ; Biosynthetic Pathways ; Micromonosporaceae ; chemistry ; genetics ; metabolism ; Molecular Sequence Data ; Multigene Family ; Sequence Alignment ; Sirolimus ; metabolism ; Streptomyces ; chemistry ; genetics ; metabolism

In the present study, we introduced point mutations into Ac_rapA which encodes a polyketide synthase responsible for rapamycin biosynthesis in Actinoplanes sp. N902-109, in order to construct a mutant with an inactivated enoylreductase (ER) domain, which was able to synthesize a new rapamycin analog. Based on the homologous recombination induced by double-strand breaks in chromosome mediated by endonuclease I-SceI, the site-directed mutation in the first ER domain of Ac_rapA was introduced using non-replicating plasmid pLYERIA combined with an I-SceI expression plasmid. Three amino acid residues of the active center, Ala-Gly-Gly, were converted to Ala-Ser-Pro. The broth of the mutant strain SIPI-027 was analyzed by HPLC and a new peak with the similar UV spectrum to that of rapamycin was found. The sample of the new peak was prepared by solvent extraction, column chromatography, and crystallization methods. The structure of new compound, named as SIPI-rapxin, was elucidated by determining and analyzing its MS and NMR spectra and its biological activity was assessed using mixed lymphocyte reaction (MLR). An ER domain-deficient mutant of Actinoplanes sp. N902-109, named as SIPI-027, was constructed, which produced a novel rapamycin analog SIPI-rapxin and its structure was elucidated to be 35, 36-didehydro-27-O-demethylrapamycin. The biological activity of SIPI-rapxin was better than that of rapamycin. In conclusion, inactivation of the first ER domain of rapA, one of the modular polyketide synthase responsible for macro-lactone synthesis of rapamycin, gave rise to a mutant capable of producing a novel rapamycin analog, 35, 36-didehydro-27-O-demethylrapamycin, demonstrating that the enoylreductase domain was responsible for the reduction of the double bond between C-35 and C-36 during rapamycin synthesis.
Anti-Bacterial Agents ; chemistry ; metabolism ; Bacterial Proteins ; chemistry ; genetics ; metabolism ; Genetic Engineering ; Micromonosporaceae ; chemistry ; enzymology ; genetics ; metabolism ; Mutation ; Polyketide Synthases ; chemistry ; genetics ; metabolism ; Protein Domains ; Sirolimus ; analogs & derivatives ; metabolism

A new sisomicin resistance gene sisR was cloned from sisomicin-producing Micromonospora inyoensis. The sisR fragment was obtained by PCR amplification. The primer pairs were designed based on grm gene sequence from gentamicin-producing Micromonospora purpurea. The template DNA was isolated from Micromonospora inyoensis. A series of different DNA fragments were amplified by PCR, which were sub-cloned to vector pUC19 for further identification. It was found that five specific transformants containing target DNA fragments could resist high concentrations of sisomicin (over 1000 microg/mL sisomicin). One of them designated as sisR, was then sequenced and the alignment among sisR and other related genes showed that sisR gene differs from any known genes. It was concluded that sisR gene is a sequence that has not been reported so far.
Anti-Bacterial Agents ; pharmacology ; Bacterial Proteins ; genetics ; Base Sequence ; Cloning, Molecular ; Drug Resistance, Microbial ; genetics ; Genes, Bacterial ; Micromonospora ; genetics ; Molecular Sequence Data ; Sisomicin ; pharmacology