In a survey of microbial systems capable of generating unusual metabolite structural variability, Streptomyces venezuelae ATCC 15439 is notable in its ability to produce two distinct groups of macrolide antibiotics. Methymycin and neomethymycin are derived from the 12-membered ring macrolactone 10-deoxymethynolide, whereas narbomycin and pikromycin are derived from the 14-membered ring macrolactone, narbonolide. This report describes the cloning and characterization of the biosynthetic gene cluster for these antibiotics. Central to the cluster is a polyketide synthase locus (pikA) that encodes a six-module system comprised of four multifunctional proteins, in addition to a type II thioesterase (TEII). Immediately downstream is a set of genes for desosamine biosynthesis (des) and macrolide ring hydroxylation. The study suggests that Pik TEII plays a role in forming a metabolic branch through which polyketides of different chain length are generated, and the glycosyl transferase (encoded by desVII) has the ability to catalyze glycosylation of both the 12-and 14-membered ring macrolactones. Moreover, the pikC-encoded P450 hydroxylase provides yet another layer of structural variability by introducing regiochemical diversity into the macrolide ring systems. The data support the notion that the architecture of the pik gene cluster as well as the unusual substrate specificity of particular enzymes contributes to its ability to generate four macrolide antibiotics.

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Purpose: NOTCH signaling pathway is essential in T-cell development and NOTCH1 mutations are frequently present inT-cell acute lymphoblastic leukemia (T-ALL). To gain insight into its clinical significance, NOTCH1 mutation was investigated in 77 patients withT-ALL. Experimental Design: Detection of NOTCH1 mutation was done using reverse transcription-PCR amplification and direct sequencing, and thereby compared according to the clinical/ biological data of the patients. Results: Thirty-two mutations were identified in 29 patients (with dual mutations in 3 cases), involving not only the heterodimerization and proline/glutamic acid/serine/threonine domains as previously reported but also the transcription activation and ankyrin repeat domains revealed for the first time. These mutations were significantly associated with elevated WBC count at diagnosis and independently linked to short survival time. Interestingly, the statistically significant difference of survival according to NOTCH1 mutations was only observed in adult patients (>18 years) but not in pediatric patients (V18 years), possibly due to the relatively good overall response of childhoodT-ALL to the current chemotherapy. NOTCH1 mutations could coexist with HOX11, HOX11L2, or SIL-TAL1 expression. The negative effect of NOTCH1 mutation on prognosis was potentiated by HOX11L2 but was attenuated by HOX11. Conclusion: NOTCH1 mutation is an important prognostic marker in T-ALL and its predictive value could be even further increased if coevaluated with other T-cell-related regulatory genes. NOTCH pathway thus acts combinatorially with oncogenic transcriptional factors on T-ALL pathogenesis.

Modular polyketide synthases are giant multifunctional enzymes that catalyse the condensation of small carboxylic acids such as acetate and propionate into structurally diverse polyketides that possess a spectrum of biological activities. In a modular polyketide synthase, an enzymatic domain catalyses a specific reaction, and three to six enzymatic domains involved in a condensation-processing cycle are organized into a module. A fundamental aspect of a modular polyketide synthase is that its module arrangement linearly specifies the structure of its polyketide product. Here we report a natural example in which alternative expression of the pikromycin polyketide synthase results in the generation of two macrolactone structures. Expression of the full-length modular polyketide synthase PikAIV in Streptomyces venezuelae generates the 14-membered ring macrolactone narbonolide, whereas expression of the amino-terminal truncated form of PikAIV leads to 'skipping' of the final condensation cycle in polyketide biosynthesis to generate the 12-membered ring macrolactone 10-deoxymethynolide. Our findings provide insight into the structure and function of modular polyketide synthases, as well as a new set of tools to generate structural diversity in polyketide natural products.

The Streptomyces venezuelae pikD gene from the pikromycin biosynthetic cluster was analyzed, and its deduced product (PikD) was found to have amino acid sequence homology with a small family of bacterial regulatory proteins. Database comparisons revealed two hypothetical domains, including an N-terminal triphosphate-binding domain and a C-terminal helix-turn-helix DNA-binding motif. Analysis of PikD was initiated by deletion of the corresponding gene (pikD) from the chromosome of S. venezuelae, resulting in complete loss of antibiotic production. Complementation by a plasmid carrying pikD restored macrolide biosynthesis, demonstrating that PikD is a positive regulator. Mutations were made in the predicted nucleotide triphosphate-binding domain, confirming the active-site amino acid residues of the Walker A and B motifs. Feeding of macrolide intermediates was carried out to gauge the points of operon control by PikD. Although the pikD mutant strain was unable to convert macrolactones (10-deoxymethynolide and narbonolide) to glycosylated products, macrolide intermediates (YC-17 and narbomycin) were hydroxylated with high efficiency. To study further the control of biosynthesis, presumed promoter regions from pik cluster loci were linked to the xylE reporter and placed in S. venezuelae wild-type and pikD mutant strains. This analysis demonstrated that PikD-mediated transcriptional regulation occurs at promoters controlling expression of pikRII, pikAI, and desI but not those controlling pikRI or pikC.The soil bacteria belonging to the genus Streptomyces have been of great interest due to their well-known capacity to produce a diverse range of antibiotics and other secondary metabolites (4, 24). Production typically occurs according to a growth phase-dependent profile (11,12) and is often accompanied by the development of spore-bearing aerial mycelia (9).The regulatory elements involved in generating antibiotics are of significant interest. An entire family of regulatory genes called SARPs (Streptomyces antibiotic regulatory proteins) has been identified based on sequence and motif homology, as well as by complementation studies (2,8). Sequence analysis links the SARP family together by the presence of OmpR-like DNA-binding domains (25). Members of this family include the positive regulators ActII-ORF4 of the actinorhodin biosynthetic cluster (2), RedD of the undecylprodigiosin gene cluster (26, 36), DnrI of the daunorubicin biosynthetic system (33, 36), and CcaR, which regulates both the cephamycin and clavulanic acid pathways (28, 36). Other genes have been identified that encode transcriptional activators of specific SARPs. These include redZ and dnrN, which activate redD and dnrI, respectively (15, 35).A general network or system of regulatory elements involved in the control of secondary metabolite pathways has not yet emerged. For example, the srmR gene of Streptomyces ambofaciens, which regulates spiramycin production, shows no sequence homology to other regulatory proteins (16). The tylosin biosynthetic pathw...

. These results demonstrate that PikC is responsible for the conversion of YC-17 to methymycin and neomethymycin, and narbomycin to pikromycin in S. venezuelae. This substrate flexibility is unique and represents the first example of a P450 hydroxylase that can accept 12- and 14-membered ring macrolides as substrates, as well as functionalize at two positions on the macrolactone system. The broad substrate specificity of PikC provides a potentially valuable entry into the construction of novel macrolide- and ketolide-based antibiotics.

Nature continues to be the inspiration for most pharmaceutical drug leads, and given the synthetic challenge posed by many complex secondary metabolites, the emerging field of combinatorial biosynthesis has become a rich new source for modified non-natural scaffolds. 1 Yet, many naturally occurring bioactive secondary metabolites possess unusual carbohydrate ligands, which serve as molecular recognition elements critical for biological activity. 2 Without these essential sugar attachments, the biological activities of most clinically important secondary metabolites are either completely abolished or dramatically decreased. We and others have demonstrated that glycosyltransferases, responsible for the final glycosylation of certain secondary metabolites, show a high degree of promiscuity toward the nucleotide sugar donor. 3,4 These discoveries have opened the door to the possibility of manipulating the corresponding biosynthetic pathways for modifying the crucial glycosylation pattern of natural, or non-natural, secondary metabolite scaffolds in a combinatorial fashion. To date, the genetic manipulation of the carbohydrate appendage for any given metabolite has been limited to alterations and/or knock-outs of the small subset of genes required to construct and attach each desired carbohydrate moiety. However, a significant expansion of the saccharide structure diversity obtained by these methods might be accomplished via the recruitment and collaborative action of sugar genes from a variety of biosynthetic pathways to construct composite clusters with the potential to make and attach non-natural sugars.To test this possibility, we selected the Streptomyces Venezuelae methymycin/pikromycin gene cluster as the parent system and a gene from the calicheamicin biosynthetic gene cluster (from Micromonospora echinospora spp. Calichensis) as the foreign collaborator gene. The parent cluster encodes the biosynthetic enzymes for methymycin (1), neomethymycin (2), pikromycin (3), and narbomycin (4), all of which are macrolides containing desosamine (5) as the sole sugar component crucial for antibiotic activity. 5 Eight open reading frames (desI-desVIII) in this cluster have been assigned as genes involved in desosamine biosynthesis (Scheme 1). The antitumor agent calicheamicin (6) contains four unique sugars crucial to tight DNA binding (K a ≈ 10 6 -10 8 ), one of which (9) is derived from 4-amino-4,6-dideoxyglucose (8) and is part of the unusually restricted N-O connection between sugars A and B (Scheme 2). 6 Compound 8 is anticipated to be derived from the corresponding 4-ketosugar 7 via a transamination reaction, and recent work has led to the assignment of a gene (calH) as encoding the desired C-4 aminotransferase (Scheme 2). 7 Interestingly, the proposed substrate for CalH, 7, is also an intermediate in the desosamine pathway and is expected to exist Thamchaipenet, A.; Gustafsson, C.; Fu, H.; Betlach, M.; Betlach, M.; Ashley, G. Targeted deletion/disruption of the desVI, desV, or desI genes in the methymycin/pikromycin...

The online version of this article has a Supplementary Appendix. BackgroundMutations in the PHF6 gene were recently described in patients with T-cell acute lymphoblastic leukemia and in those with acute myeloid leukemia. The present study was designed to determine the prevalence of PHF6 gene alterations in T-cell acute lymphoblastic leukemia. Design and MethodsWe analyzed the incidence and prognostic value of PHF6 mutations in 96 Chinese patients with T-cell acute lymphoblastic leukemia. PHF6 deletions were screened by real-time quantitative polymerase chain reaction and array-based comparative genomic hybridization. Patients were also investigated for NOTCH1, FBXW7, WT1, and JAK1 mutations together with CALM-AF10, SET-NUP214, and SIL-TAL1 gene rearrangements. ResultsPHF6 mutations were identified in 11/59 (18.6%) adult and 2/37 (5.4%) pediatric cases of Tcell acute lymphoblastic leukemia, these incidences being significantly lower than those recently reported. Although PHF6 is X-linked and mutations have been reported to occur almost exclusively in male patients, we found no sex difference in the incidences of PHF6 mutations in Chinese patients with T-cell acute lymphoblastic leukemia. PHF6 deletions were detected in 2/79 (2.5%) patients analyzed. NOTCH1 mutations, FBXW7 mutations, WT1 mutations, JAK1 mutations, SIL-TAL1 fusions, SET-NUP214 fusions and CALM-AF10 fusions were present in 44/96 (45.8%), 9/96 (9.4%), 4/96 (4.1%), 3/49 (6.1%), 9/48 (18.8%), 3/48 (6.3%) and 0/48 (0%) of patients, respectively. The molecular genetic markers most frequently associated with PHF6 mutations were NOTCH1 mutations (P=0.003), SET-NUP214 rearrangements (P=0.002), and JAK1 mutations (P=0.005). No differences in disease-free survival and overall survival between T-cell acute lymphoblastic leukemia patients with and without PHF6 mutations were observed in a short-term follow-up. ConclusionsOverall, these results indicate that, in T-cell acute lymphoblastic leukemia, PHF6 mutations are a recurrent genetic abnormality associated with mutations of NOTCH1, JAK1 and rearrangement of SET-NUP214.