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221.
Yan X Probst K Linnenbrink A Arnold M Paululat T Zeeck A Bechthold A 《Chembiochem : a European journal of chemical biology》2012,13(2):224-230
Mensacarcin is a potent cytotoxic agent isolated from Streptomyces bottropensis. It possesses a high content of oxygen atoms and two epoxide groups, and shows cytostatic and cytotoxic activity comparable to that of doxorubicin, a widely used drug for antitumor therapy. Another natural compound, rishirilide A, was also isolated from the fermentation broth of S. bottropensis. Screening a cosmid library of S. bottropensis with minimal PKS-gene-specific primers revealed the presence of three different type II polyketide synthase (PKS) gene clusters in this strain: the msn cluster (mensacarcin biosynthesis), the rsl cluster (rishirilide biosynthesis), and the mec cluster (putative spore pigment biosynthesis). Interestingly, luciferase-like oxygenases, which are very rare in Streptomyces species, are enriched in both the msn cluster and the rsl cluster. Three cosmids, cos2 (containing the major part of the msn cluster), cos3 (harboring the mec cluster), and cos4 (spanning probably the whole rsl cluster) were introduced into the general heterologous host Streptomyces albus by intergeneric conjugation. Expression of cos2 and cos4 in S. albus led to the production of didesmethylmensacarcin (DDMM, a precursor of mensacarcin) and the production of rishirilide A and B (a precursor of rishirilide A), respectively. However, no product was detected from the expression of cos3. In addition, based on the results of isotope-feeding experiments in S. bottropensis, a putative biosynthesis pathway for mensacarcin is proposed. 相似文献
222.
223.
X Bian F Huang FA Stewart L Xia Y Zhang R Müller 《Chembiochem : a European journal of chemical biology》2012,13(13):1946-1952
The reconstruction of a natural product biosynthetic pathway from bacteria in a vector and subsequent heterologous expression in a technically amenable microbial system represents an efficient alternative to empirical traditional methods for functional discovery, yield improvement, and genetic engineering to produce "unnatural" derivatives. However, the traditional cloning procedure based on genomic library construction and screening are complicated due to the large size (>10 kb) of most biosynthetic pathways. Here, we describe the direct cloning of a partial syringolin biosynthetic gene cluster (sylCDE, 19 kb) from a digested genomic DNA mixture of Pseudomonas syringae into a plasmid in which sylCDE is under the control of an inducible promoter by one step linear-plus-linear homologous recombination (LLHR) in Escherichia coli. After expression in E. coli GB05-MtaA, two new syringolin derivatives were discovered. The complete syringolin gene cluster was assembled by addition of sylAB and exchange of a synthetic bidirectional promoter against the native promoter to drive sylB and sylC expression by using Red/ET recombineering. The varying production distribution of syringolin derivatives showed the different efficiencies of native and synthetic promoters in E. coli. The successful reconstitution and expression of the syringolin biosynthetic pathway shows that Red/ET recombineering is an efficient tool to clone and engineer secondary metabolite biosynthetic pathways. 相似文献
224.
Morgan A. Wyatt M. C. Y. Mok Prof. Dr. Murray Junop Prof. Dr. Nathan A. Magarvey 《Chembiochem : a European journal of chemical biology》2012,13(16):2408-2415
Through a number of strategies nonribosomal peptide assembly lines give rise to a metabolic diversity not possible by ribosomal synthesis. One distinction within nonribosomal assembly is that products are elaborated on an enzyme‐tethered substrate, and their release is enzyme catalysed. Reductive release by NAD(P)H‐dependent catalysts is one observed nonribosomal termination and release strategy. Here we probed the selectivity of a terminal reductase domain by using a full‐length heterologously expressed nonribosomal peptide synthetase for the dipeptide aureusimine and were able to generate 17 new analogues. Further, we generated an X‐ray structure of aureusimine terminal reductase to gain insight into the structural details associated with this enzymatic domain. 相似文献
225.
Noike M Liu C Ono Y Hamano Y Toyomasu T Sassa T Kato N Dairi T 《Chembiochem : a European journal of chemical biology》2012,13(4):566-573
Isoprenoids form the largest family of compounds found in nature. Isoprenoids are often attached to other moieties such as aromatic compounds, indoles/tryptophan, and flavonoids. These reactions are catalyzed by three phylogenetically distinct prenyltransferases: soluble aromatic prenyltransferases identified mainly in actinobacteria, soluble indole prenyltransferases mostly in fungi, and membrane‐bound prenyltransferases in various organisms. Fusicoccin A (FC A) is a diterpene glycoside produced by the plant‐pathogenic fungus Phomopsis amygdali and has a unique O‐prenylated glucose moiety. In this study, we identified for the first time, from a genome database of P. amygdali, a gene (papt) encoding a prenyltransferase that reversibly transfers dimethylallyl diphosphate (DMAPP) to the 6′‐hydroxy group of the glucose moiety of FC A to yield an O‐prenylated sugar. An in vitro assay with a recombinant enzyme was also developed. Detailed analyses with recombinant PAPT showed that the enzyme is likely to be a monomer and requires no divalent cations. The optimum pH and temperature were 8.0 and 50 °C, respectively. Km values were calculated as 0.49±0.037 μM for FC P (a plausible intermediate of FC A biosynthesis) and 8.3±0.63 μM for DMAPP, with a kcat of 55.3±3.3×10?3 s. The enzyme did not act on representative substrates of the above‐mentioned three types of prenyltransferase, but showed a weak transfer activity of geranyl diphosphate to FC P. 相似文献
226.
V Bučević-Popović M Sprung B Soldo M Pavela-Vrančič 《Chembiochem : a European journal of chemical biology》2012,13(13):1913-1920
The adenylation (A) domain in nonribosomal peptide synthetases catalyses a two-step reaction in which an amino acid is activated and then transferred to the neighbouring thiolation (T) domain. In this study, we investigated the role of the conserved A9 core sequence of the A-domain of tyrocidine synthetase 1, by analysis of single amino acid mutations in the A9 region. Mutation of an absolutely conserved proline (P490G) significantly reduced the conformational stability of the protein, as evidenced by increased susceptibility to proteolytic cleavage and denaturation. All mutant A-domains were capable of amino acid activation, but the activity in the overall reaction was reduced. Surprisingly, the S491R mutant (mutation at the first residue following the A9 motif) showed elevated overall activity compared to the wild-type protein. Our results suggest that the A9 core sequence plays a role in the second reaction step, in which it could serve as a "clip" for the proper positioning of residues important for the interaction with the T-domain, and/or stabilisation of the thioester-forming conformation. 相似文献
227.
Neumann CS Jiang W Heemstra JR Gontang EA Kolter R Walsh CT 《Chembiochem : a European journal of chemical biology》2012,13(7):972-976
Which came first? We have investigated the biosynthesis of the piperazic acid (Piz) building blocks in the kutzneride family of metabolites. The flavin-dependent oxygenase KtzI was shown to convert ornithine to N(5)-OH-Orn. LC-MS/MS showed (13)C(5)-labeled versions of these two amino acids to be direct precursors of piperazic acid in vivo. 相似文献
228.
Riclea R Aigle B Leblond P Schoenian I Spiteller D Dickschat JS 《Chembiochem : a European journal of chemical biology》2012,13(11):1635-1644
The volatiles released by several streptomycetes were collected by using a closed-loop stripping apparatus (CLSA) and analysed by GC-MS. The obtained headspace extracts of various species contained blastmycinone, a known degradation product of the fungicidal antibiotic, antimycin A(3b), and several unknown derivatives. The suggested structures of these compounds, based on their mass spectra and GC retention indices, were confirmed by comparison to synthetic reference samples. Additional compounds found in the headspace extracts were butenolides formed from the blastmycinones by elimination of the carboxylic acid moiety. Analysis of a gene knockout mutant in the antimycin biosynthetic gene cluster demonstrated that all blastmycinones and butenolides are formed via the antimycin biosynthetic pathway. The structural variation of the blastmycinones identified here is much larger than within the known antimycins, thus suggesting that several antimycin derivatives remain to be discovered. 相似文献
229.
Wu J Li L Deng Z Zabriskie TM He X 《Chembiochem : a European journal of chemical biology》2012,13(11):1613-1621
Mildiomycin (MIL) is a peptidyl-nucleoside antibiotic produced by Streptoverticillum remofaciens ZJU5119 that exhibits strong inhibitory activity against powdery mildew. The entire MIL biosynthesis gene cluster was cloned and expressed in Streptomyces lividans 1326. Systematic gene disruptions narrowed down the cluster to 16 functional ORFs and identified the boundaries of the gene cluster. A putative cytosylglucuronic acid (CGA) synthase gene, milC, was disrupted in Sv. remofaciens and heterologously expressed in E. coli. An in vitro assay revealed that purified MilC could utilize either cytosine or hydroxymethylcytosine as substrate to yield CGA or hydroxymethyl-CGA (HM-CGA), respectively. MilG is believed to be a key enzyme in the MIL biosynthesis pathway and contains the C(XXX)C(XX)C motif characteristic of members of the radical S-adenosyl methionine (SAM) superfamily. Disruption of milG leads to accumulation of HM-CGA. Labeling experiments with (13)C(6)-L-arginine indicated that decarboxylation at C5 of the pyranoside ring was coupled with the attachment of 5-guanidino-2,4-dihydroxyvalerate side chain through C-C bond formation. In contrast, exogenous (13)C(6)-labeled 4-hydroxy-L-arginine was not incorporated into the MIL structure. Comparative analysis of the 16 MIL ORFs with counterparts involved in the biosynthesis of the structurally similar compound blasticidin S, along with the results above, provide insight into the complete MIL biosynthetic pathway. 相似文献
230.
Michael Müller 《Advanced Synthesis \u0026amp; Catalysis》2012,354(17):3161-3174
Some of the recent developments in enzymatic asymmetric C C bond formation are described in this review. The close relationship of biocatalysis and biosynthesis is highlighted with a special emphasis on diversity and biogenesis. One focus of this review is the creation of tetrasubstituted carbon stereocenters. Members of the supposedly well‐known aldolase and hydroxynitrile lyase enzyme families possess the ability to catalyze the formation of tertiary alcohols. In the case of aldolases, this can occur through intramolecular cyclization or intermolecular asymmetric C C bond formation. Thiamine diphosphate‐dependent YerE has been identified as a potent catalyst for the acyloin condensation with ketones as acceptor substrates. C1 transformations such as methylation or carboxylation are catalyzed in an asymmetric manner by enzymes from different classes, for example S‐adenosylmethionine‐dependent (radical) enzymes or NADPH‐dependent oxidoreductases. Insights from biosynthetic and mechanistic studies of enzymatic reactions proceeding via radical intermediates give valuable hints towards possible applications in biocatalysis. Still, the oxygen sensitivity of many of these biocatalysts poses a considerable challenge for practical applications. 相似文献