Polyoxin is a group of structurally-related peptidyl nucleoside antibiotics bearing C-5 modifications on the nucleoside skeleton. Although the structural diversity and bioactivity preference of polyoxin are, to some extent, affected by such modifications, the biosynthetic logic for their occurence remains obscure. Here we report the identification of PolB in polyoxin pathway as an unusual UMP C-5 methylase with thymidylate synthase activity which is responsible for the C-5 methylation of the nucleoside skeleton. To probe its molecular mechanism, we determined the crystal structures of PolB alone and in complexes with 5-Br UMP and 5-Br dUMP at 2.15 Å, 1.76 Å and 2.28 Å resolutions, respectively. Loop 1 (residues 117–131), Loop 2 (residues 192–201) and the substrate recognition peptide (residues 94–102) of PolB exhibit considerable conformational flexibility and adopt distinct structures upon binding to different substrate analogs. Consistent with the structural findings, a PolB homolog that harbors an identical function from Streptomyces viridochromogenes DSM 40736 was identified. The discovery of UMP C5-methylase opens the way to rational pathway engineering for polyoxin component optimization, and will also enrich the toolbox for natural nucleotide chemistry.

Xylitol is industrially synthesized by chemical reduction of d-xylose, which is more expensive than glucose. Thus, there is a growing interest in the production of xylitol from a readily available and much cheaper substrate, such as glucose. The commonly used yeast Pichia pastoris strain GS115 was shown to produce d-arabitol from glucose, and the derivative strain GS225 was obtained to produce twice amount of d-arabitol than GS115 by adaptive evolution during repetitive growth in hyperosmotic medium. We cloned the d-xylulose-forming d-arabitol dehydrogenase (DalD) gene from Klebsiella pneumoniae and the xylitol dehydrogenase (XDH) gene from Gluconobacter oxydans. Recombinant P. pastoris GS225 strains with the DalD gene only or with both DalD and XDH genes could produce xylitol from glucose in a single-fermentation process. Three-liter jar fermentation results showed that recombinant P. pastoris cells with both DalD and XDH converted glucose to xylitol with the highest yield of 0.078 g xylitol/g glucose and productivity of 0.29 g xylitol/L h. This was the first report to convert xylitol from glucose by the pathway of glucose–d-arabitol–d-xylulose–xylitol in a single process. The recombinant yeast could be used as a yeast cell factory and has the potential to produce xylitol from glucose.

Oligomycins are a group of 26 macrocyclic lactones that exhibit broad biological activities, including antifungal, anticancer and nematocidal activities. Analysis of the oligomycin biosynthetic gene cluster (olm) in S. avermitilis revealed 2 tandem LuxR-type regulators, OlmRI (931 aa) and OlmRII (941 aa), with shared identity of 38%. Gene replacement of olmRI or olmRII abolished oligomycin production, and this production could be partially restored in the disruptants by introducing cloned olmRI and olmRII with their native promoters, demonstrating the essential role of OlmRI and OlmRII for oligomycin biosynthesis. Quantitative real-time RT-PCR analysis revealed that transcription of 14 olm genes was differentially affected by the deletion of olmRI and olmRII. Unexpectedly, avermectin production in both mutants was enhanced at least 4-fold. The identification of the positive cluster-situated regulators, OlmRI and OlmRII, paves the way for the transcriptional analysis of oligomycin biosynthesis and for the enhancement of oligomycin and avermectin production through regulator engineering.

The nanchangmycin (NAN) produced by Streptomyces nanchangensis NS3226 is a polyether antibiotic resembling monensin in their gene clusters and the chemical structures. They can inhibit gram-positive bacteria and be a growth promoter for ruminants. Within the nanchangmycin gene cluster (nan), we identified that two SARP-family regulatory genes, nanR1 and nanR2, were both required to activate the transcription of all nan polyketide genes. Overexpression of NanR1 and NanR2 in wild-type increase NAN yields by at least three folds. Bioinformatic analysis of the immediate upstream DNA sequence of each nan gene and quantitative real-time RT-PCR analysis of the nan operons identified five putative SARP binding sites. Moreover, deletion of an AraC-family repressor gene nanR4 increased expression of NanR1 and R2 and led to a threefold increase in NAN production.

Muraymycin, a potent translocase I inhibitor with clinical potential, is produced by Streptomyces sp. NRRL 30471. The structure of muraymycin is highly unusual and contains the hexahydro-2-imino-4-pyrimidylglycyl moiety (epicapreomycidine) and an ureido bond. Here we report the identification of the muraymycin gene cluster from Streptomyces sp. NRRL 30471. Sequencing analysis of a 43.4-kb contiguous region revealed 33 ORFs, 26 of which were proposed to be involved in muraymycin biosynthesis. Independent targeted inactivation of mur16 and mur17 directly abolished muraymycin production, demonstrating the role of the genes essential for muraymycin biosynthesis. These data provide insights into the molecular mechanisms for muraymycin biosynthesis, and lay a foundation for the generation of muraymycin derivatives with enhanced bioactivity via the strategies of combinatorial biosynthesis.

Here we tell a 20-year long story. It began with an easily overlooked DNA degradation (Dnd) phenomenon during electrophoresis and eventually led to the discovery of an unprecedented DNA sulfur modification governed by five dnd genes. This unusual DNA modification, called phosphorothioation, is the first physiological modification identified on the DNA backbone, in which the nonbridging oxygen is replaced by sulfur in a sequence selective and stereo-specific manner. Homologous dnd gene clusters have been identified in diverse and distantly related bacteria and thus have drawn immediate attention of the entire microbial scientific community. Here, we summarize the progress in chemical, genetic, enzymatic, bioinformatical and analytical aspects of this novel postreplicative DNA modification. We also discuss perspectives on the physiological functions of the DNA phosphorothioate modification in bacteria and their implications.

Tunicamycin, a potent reversible translocase I inhibitor, is produced by several Actinomycetes species. The tunicamycin structure is highly unusual, and contains an 11-carbon dialdose sugar and an α, β-1″,11′-glycosidic linkage. Here we report the identification of a gene cluster essential for tunicamycin biosynthesis by high-throughput heterologous expression (HHE) strategy combined with a bioassay. Introduction of the genes into heterologous non-producing Streptomyces hosts results in production of tunicamycin by these strains, demonstrating the role of the genes for the biosynthesis of tunicamycins. Gene disruption experiments coupled with bioinformatic analysis revealed that the tunicamycin gene cluster is minimally composed of 12 genes (tunA-tunL). Amongst these is a putative radical SAM enzyme (Tun B) with a potentially unique role in biosynthetic carbon-carbon bond formation. Hence, a seven-step novel pathway is proposed for tunicamycin biosynthesis. Moreover, two gene clusters for the potential biosynthesis of tunicamycin-like antibiotics were also identified in Streptomyces clavuligerus ATCC 27064 and Actinosynnema mirums DSM 43827. These data provide clarification of the novel mechanisms for tunicamycin biosynthesis, and for the generation of new-designer tunicamycin analogs with selective/enhanced bioactivity via combinatorial biosynthesis strategies.

The complete DNA sequence of plasmid pHZ1358, a widely used vector for targeted gene disruption and replacement experiments in many Streptomyces hosts, has been determined. This has allowed a detailed analysis of the basis of its structural and segregational instability, compared to the high copy number plasmid pIJ101 of Streptomyces lividans 1326 from which it was derived. A 574-bp DNA region containing sti (strong incompatibility locus) was found to be a determinant for segregational instability in its original S. lividans 1326 host, while the structural instability was found to be related to the facile deletion of the entire Escherichia coli-derived part of pHZ1358, mediated by recombination between 36-bp direct repeats. A point mutation removing the BamHI site inside the rep gene encoding a replication protein (rep*) and/or a spontaneous deletion of the 694-bp region located between rep and sti including the uncharacterized ORF85 (orf85−) produced little or no effect on stability. A pHZ1358 derivative (pJTU412, sti−, rep*, orf85−) was then constructed which additionally lacked one of the 36-bp direct repeats. pJTU412 was demonstrated to be structurally stable but segregationally unstable and, in contrast to sti+ pHZ1358, allowed efficient targeted gene replacement in S. lividans 1326.

FR-008/candicidin is a heptaene macrolide with established antifungal activity, produced by Streptomyces sp. FR-008 as a complex mixture of compounds. Here, six components (FR-008-I to -VI) of the FR-008/candicidin complex were determined; III, V, and VI were confirmed as natural products, principally differing from each other at C-3 and C-9, while the other three were believed to originate from the respective conversions of the natural ones in vitro. Inactivation of KR21 and DH18, respectively, abolished production of V carrying a C-3 hydroxyl, and VI carrying a C-9 methylene. Combined inactivation created a mutant producing only III, with a C-3 ketone and a C-9 hydroxyl, and having antifungal activity superior to V and comparable to VI. Incomplete activities of KR21 and DH18 were, therefore, unambiguously identified as being involved in structural variations of FR-008 complex.

The polyketide backbone of the polyether ionophore antibiotic nanchangmycin (1) is assembled by a modular polyketide synthase in Streptomyces nanchangensis NS3226. The ACP-bound polyketide is thought to undergo a cascade of oxidative cyclizations to generate the characteristic polyether. Deletion of the glycosyl transferase gene nanG5 resulted in accumulation of the corresponding nanchangmycin aglycone (6). The discrete thioesterase NanE exhibited a nearly 17-fold preference for hydrolysis of 4, the N-acetylcysteamine (SNAC) thioester of nanchangmycin, over 7, the corresponding SNAC derivative of the aglycone, consistent with NanE-catalyzed hydrolysis of ACP-bound nanchangmycin being the final step in the biosynthetic pathway. Site-directed mutagenesis established that Ser96, His261, and Asp120, the proposed components of the NanE catalytic triad, were all essential for thioesterase activity, while Trp97 was shown to influence the preference for polyether over polyketide substrates.

Covering: 1994–2006

A 45 kb DNA sequencing analysis from Streptomyces hygroscopicus 5008 involved in validamycin A (VAL-A) biosynthesis revealed 16 structural genes, 2 regulatory genes, 5 genes related transport, transposition/integration or tellurium resistance; another 4 genes had no obvious identity. The VAL-A biosynthetic pathway was proposed, with assignment of the required genetic functions confined to the sequenced region. A cluster of eight reassembled genes was found to support VAL-A synthesis in a heterologous host, S. lividans 1326. In vivo inactivation of the putative glycosyltransferase gene (valG) abolished the final attachment of glucose for VAL production and resulted in accumulation of the VAL-A precursor, validoxylamine, while the normal production of VAL-A could be restored by complementation with valG. The role of valG in the glycosylation of validoxylamine to VAL-A was demonstrated in vitro by enzymatic assay.

The polyketide synthase (PKS) for the biosynthesis of the polyether nanchangmycin lacks an apparent thioesterase comparable to the type I thioesterase domains of the modular PKSs responsible for macrolide biosynthesis. Three candidate polyether chain-releasing factors were examined. Both the putative CR domain and the NanE protein appeared to be genetically relevant. Among the three heterologously expressed soluble proteins (recombinant CR domain, the ACP-CR didomain, and NanE) tested, only NanE hydrolyzed the polyether-SNAC. By contrast, recombinant DEBS TE from the erythromycin pathway, and the recombinant MonAX, a type II TE associated with the polyether monensin biosynthesis for which a homolog has not been detected in the nanchangmycin cluster, hydrolyzed a diketide-SNAC but not the polyether-SNAC. We could thus conclude that NanE is a dedicated thioesterase mediating the specific release of the polyether chain during nanchangmycin biosynthesis.

•A Y. lipolytica strain displaying sucrose isomerase was constructed.•The Y. lipolytica cells converted sucrose to isomaltulose at a yield of 93 ± 2%.•The sucrose isomerase displayed on yeast cell wall was more stable than its free form.•No trehalulose or glucose by-products were detected in the transformation.The gene encoding for sucrose isomerase from Pantoea dispersa (PdSIase) was successfully displayed on the cell surface of Yarrowia lipolytica, using the cell wall protein Pir1 as an anchor protein. The highest isomaltulose conversion yield of 93 ± 2% was obtained using the displayed PdSIase. The yeast strain displaying PdSIase was stable throughout broad ranges of pH values (4.5–7.0) and temperatures (20–40 °C). In addition, no trehalulose or glucose by-products were detected during the transformation process. The yeast cells remained highly viable in repeated batch operations for 12 cycles, with a high conversion yield of no less than 80%. The results in our study demonstrate that the PdSIase displayed by Y. lipolytica could continuously and efficiently convert sucrose to isomaltulose.

DNA phosphorothioation is widespread in many bacterial species. By homology analysis of the dnd gene cluster in Pseudomonas fluorescens Pf0-1, a spfBCDE gene cluster involved in DNA phosphorothioation was localized. Disruption of the spfD gene, a dndD homolog, caused the loss of the Dnd phenotype and demonstrated the involvement of spfD in DNA phosphorothioation in P. fluorescens Pf0-1. The ATPase activity of SpfD suggests that SpfD could hydrolyze ATP to provide the energy required in the DNA phosphorothioate modification process.