Molecular basis for nuclear accumulation and targeting of the inhibitor of apoptosis BIRC2.

The inhibitor of apoptosis protein BIRC2 regulates fundamental cell death and survival signaling pathways. Here we show that BIRC2 accumulates in the nucleus via binding of its second and third BIR domains, BIRC2BIR2 and BIRC2BIR3, to the histone H3 tail and report the structure of the BIRC2BIR3-H3 complex. RNA-seq analysis reveals that the genes involved in interferon and defense response signaling and cell-cycle regulation are most affected by depletion of BIRC2. Overexpression of BIRC2 delays DNA damage repair and recovery of the cell-cycle progression. We describe the structural mechanism for targeting of BIRC2BIR3 by a potent but biochemically uncharacterized small molecule inhibitor LCL161 and demonstrate that LCL161 disrupts the association of endogenous BIRC2 with H3 and stimulates cell death in cancer cells. We further show that LCL161 mediates degradation of BIRC2 in human immunodeficiency virus type 1-infected human CD4+ T cells. Our findings provide mechanistic insights into the nuclear accumulation of and blocking BIRC2.

Template-mediated copper doped porous g-C3N4 for efficient photodegradation of antibiotic contaminants.

Graphite carbon nitride (g-C3N4) has great potential to treat antibiotic wastewater, but limited by small specific surface area, rapid recombination of photogenerated carriers and narrow visible light absorption range. In order to solve above problems, we designed a simple template-mediated approach by supramolecular self-assembly (Cu-melamine-cyanuric acid) to prepare copper doped porous graphitic carbon nitride (Cu-pCN) photocatalyst. The pre-organized template self-assembly driven by hydrogen bonds and electrostatic interaction, resulted in highly porous structure. The specific surface area of Cu-pCN increased to 142.8 m2/g from 11.37 m2/g of conventional bulk g-C3N4. In addition, the doping of Cu endowed them with better light absorption, higher separation and transfer rate of photogenerated carriers. Consequently, the obtained Cu-pCN displayed the superior photocatalytic degradation rate for tetracycline (TC) and high recycling stability.

Histone mimics: more tales to read.

Post-translational modifications (PTMs) on histone proteins are known as epigenetic marks that demarcate the status of chromatin. These modifications are 'read' by specific reader proteins, which in turn recruit additional factors to modulate chromatin accessibility and the activity of the underlying DNA. Accumulating evidence suggests that these modifications are not restricted solely to histones, many non-histone proteins may function in a similar way through mimicking the histones. In this commentary, we briefly discuss a systematic study of the discovery of histone H3 N-terminal mimicry proteins (H3TMs), and their implications in chromatin regulation and drug discoveries.

Nuclear condensates of p300 formed though the structured catalytic core can act as a storage pool of p300 with reduced HAT activity.

The transcriptional co-activator and acetyltransferase p300 is required for fundamental cellular processes, including differentiation and growth. Here, we report that p300 forms phase separated condensates in the cell nucleus. The phase separation ability of p300 is regulated by autoacetylation and relies on its catalytic core components, including the histone acetyltransferase (HAT) domain, the autoinhibition loop, and bromodomain. p300 condensates sequester chromatin components, such as histone H3 tail and DNA, and are amplified through binding of p300 to the nucleosome. The catalytic HAT activity of p300 is decreased due to occlusion of the active site in the phase separated droplets, a large portion of which co-localizes with chromatin regions enriched in H3K27me3. Our findings suggest a model in which p300 condensates can act as a storage pool of the protein with reduced HAT activity, allowing p300 to be compartmentalized and concentrated at poised or repressed chromatin regions.

ZZEF1 is a Histone Reader and Transcriptional Coregulator of Krüppel-Like Factors.

The ZZ-type zinc finger and EF-hand domain protein 1 (ZZEF1) is a multidomain-containing protein. Mutations of ZZEF1 has been implicated in several kinds of human diseases such as diabetes and cancers. However, the function of the ZZEF protein remains largely unknown. Here we show that ZZEF1 functions as a histone H3 reader. The second ZZ domain of ZZEF1 (ZZEF1ZZ2) binds to the N-terminus of histone H3 and is capable of accommodating common epigenetic marks on the H3 tail. The N-terminal amino acids, especially Ala1, of H3 and an acidic cavity of ZZEF1ZZ2 are critical for the ZZ-H3 interaction. RNA-seq analysis in human lung cancer cell line H1299 reveals that downregulated genes upon ZZEF1 depletion are specifically enriched in genes regulated by Krüppel-like factors. Indeed, ZZEF1 physically interacts with KLF9 and KLF6, and regulates a common set of target genes of these transcription factors. Together, our findings suggest a model in which ZZEF1 binds to histone H3 tail and promotes KLF9/6-mediated gene regulation.

Reassembly of the Biosynthetic Gene Cluster Enables High Epothilone Yield in Engineered Schlegelella brevitalea.

Epothilones, as a new class of microtubule-stabilizing anticancer drugs, exhibit strong bioactivity against taxane-resistant cells and show clinical activity for the treatment of advanced breast cancer. Additionally, they also show great potential for a central nervous system injury and Alzheimer's disease. However, due to the long fermentation period of the original producer and challenges of genetic engineering of nonribosomal peptide/polyketide (NRP/PK) megasynthase genes, the application of epothilones is severely limited. Here, we addressed these problems by reassembling a novel 56-kb epothilone biosynthetic gene cluster, optimizing the promoter of each gene based on RNA-seq profiling, and completing precursor synthetic pathways in engineered Schlegella brevitalea. Furthermore, we debottlenecked the cell autolysis by optimizing culture conditions. Finally, the yield of epothilones in shake flasks was improved to 82 mg/L in six-day fermentation. Overall, we not only constructed epothilone overproducers for further drug development but also provided a rational strategy for high-level NRP/PK compound production.

Reclassification of 'Polyangium brachysporum' DSM 7029 as Schlegelella brevitalea sp. nov.

Strain DSM 7029, isolated from a soil sample in Greece, can produce antitumour glidobactins, and has been found, as a heterologous host, to produce useful nonribosomal peptide synthetase-polyketide synthase hybrid molecules known as epothilones. This strain was originally named 'Polyangium brachysporum' of the family Polyangiaceae and the order Myxococcales. However, phylogenetic analysis of the 16S rRNA gene sequence of strain DSM 7029 indicated that it was clustered with members of Schlegelella. Significant growth occurred at 25-42 °C, pH 5.0-10.0 and in the presence of 0-0.2 % (w/v) NaCl. The predominant ubiquinone was Q-8. The major fatty acids were C16 : 1ω7c/C16 : 1ω6c, C16 : 0 and C18 : 1ω7c. The G+C content of genomic DNA was 67.51 mol%. The strain was clearly distinguishable from other neighbouring Schlegelella members and genera Caldimonas and Zhizhongheella, using phylogenetic analysis, fatty acid composition data and a range of physiological and biochemical characteristics and genome analysis. Therefore, strain DSM 7029 represents a novel species of the genus Schlegelella, for which the name Schlegelella brevitalea sp. nov. is proposed.

Identification of the streptothricin and tunicamycin biosynthetic gene clusters by genome mining in Streptomyces sp. strain fd1-xmd.

The genus Streptomyces have been highly regarded for their important source of natural products. Combined with the technology of genome sequencing and mining, we could identify the active ingredients from fermentation broth quickly. Here, we report on Streptomyces sp. strain fd1-xmd, which was isolated from a soil sample collected in Shanghai. Interestingly, the fermentation broth derived from this strain demonstrated broad-spectrum antimicrobial activity against gram-positive bacteria, gram-negative bacteria, and eukaryotes. To identify the antimicrobial substances and their biosynthetic gene clusters, we sequenced the fd1-xmd strain and obtained a genome 7,929,999 bp in length. The average GC content of the chromosome was 72.5 mol%. Knockout experiments demonstrated that out of eight biosynthetic gene clusters we could identify, two are responsible for the biosynthesis of the antibiotics streptothricin (ST) and tunicamycin (TM). The ST biosynthetic gene cluster from fd1-xmd was verified via successful heterologous expression in Streptomyces coelicolor M1146. ST production had a yield of up to 0.5 g/L after the optimization of culture conditions. This study describes a novel producer of ST and TM and outlines the complete process undertaken for Streptomyces sp. strain fd1-xmd genome mining.

Heterologous Production and Yield Improvement of Epothilones in Burkholderiales Strain DSM 7029.

The cloning of microbial natural product biosynthetic gene clusters and their heterologous expression in a suitable host have proven to be a feasible approach to improve the yield of valuable natural products and to begin mining cryptic natural products in microorganisms. Myxobacteria are a prolific source of novel bioactive natural products with only limited choices of heterologous hosts that have been exploited. Here, we describe the use of Burkholderiales strain DSM 7029 as a potential heterologous host for the functional expression of myxobacterial secondary metabolites. Using a newly established electroporation procedure, the 56 kb epothilone biosynthetic gene cluster from the myxobacterium Sorangium cellulosum was introduced into the chromosome of strain DSM 7029 by transposition. Production of epothilones A, B, C, and D was detected despite their yields being low. Optimization of the medium, introduction of the exogenous methylmalonyl-CoA biosynthetic pathway, and overexpression of rare tRNA genes resulted in an approximately 75-fold increase in the total yields of epothilones to 307 µg L-1. These results show that strain DSM 7029 has the potential to produce epothilones with reasonable titers and might be a broadly applicable host for the heterologous expression of other myxobacterial polyketide synthases and nonribosomal peptide synthetases, expediting the process of genome mining.

Streptomyces yangpuensis sp. nov., isolated from soil.

A novel Gram-stain-positive strain with sandy aerial mycelium and golden yellow substrate mycelium, designated fd2-tbT, was isolated from a soil sample collected in Shanghai, China, and its taxonomic status was established by phylogenetic analysis. 16S rRNA gene sequence analysis showed that strain fd2-tbT belonged to the genus Streptomyces and was related to Streptomyces amritsarensis JCM 19660T (99.9 % 16S rRNA gene sequence similarity), Streptomyces flavotricini NBRC 12770T (99.9 %), Streptomyces polychromogenes NBRC 13072T (99.8 %), Streptomyces racemochromogenes NRRL B-5430T (99.7 %), Streptomyces globosus LMG 19896T (99.5 %), Streptomyces toxytricini NBRC 12823T (99.5 %) and Streptomyces katrae NBRC 13447T (99.3 %). The cell wall of strain fd2-tbT contained ll-diaminopimelic acid, and whole-cell sugars were identified as glucose and ribose. The menaquinones MK-9(H4), MK-9(H6) and MK-9(H8) were also detected. In addition, the polar lipids diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, as well as five unidentified phospholipids, were detected. Major cellular fatty acids were identified as anteiso-C15 : 0, iso-C16 : 0, anteiso-C17 : 0, iso-C15 : 0 and C16 : 0. DNA-DNA hybridization experiments showed that strain fd2-tbT exhibited 36.5 ± 0.6 %, 43.5 ± 2.0 %, 11.1 ± 1.3 %, 10.3 ± 3.1 %, 9.8 ± 1.9 %, 48.9 ± 3.9 % and 16.3 ± 1.7 % relatedness to S. amritsarensis JCM 40119660T, S. flavotricini NBRC 12770T, S. polychromogenes NBRC 13072T, S. racemochromogenes NRRL B-5430T, S. globosus LMG 19896T, S. toxytricini NBRC 12823T and S. katrae NBRC 13447T, respectively. Based on these analyses as well as some phenotypic differences, strain fd2-tbT is considered to represent a novel species of the genus Streptomyces, for which the name Streptomyces yangpuensis sp. nov. is proposed. The type strain is fd2-tbT ( = DSM 100336T = CGMCC 4.7256T).

A systematic study of the whole genome sequence of Amycolatopsis methanolica strain 239T provides an insight into its physiological and taxonomic properties which correlate with its position in the genus.

The complete genome of methanol-utilizing Amycolatopsis methanolica strain 239T was generated, revealing a single 7,237,391 nucleotide circular chromosome with 7074 annotated protein-coding sequences (CDSs). Comparative analyses against the complete genome sequences of Amycolatopsis japonica strain MG417-CF17T, Amycolatopsis mediterranei strain U32 and Amycolatopsis orientalis strain HCCB10007 revealed a broad spectrum of genomic structures, including various genome sizes, core/quasi-core/non-core configurations and different kinds of episomes. Although polyketide synthase gene clusters were absent from the A. methanolica genome, 12 gene clusters related to the biosynthesis of other specialized (secondary) metabolites were identified. Complete pathways attributable to the facultative methylotrophic physiology of A. methanolica strain 239T, including both the mdo/mscR encoded methanol oxidation and the hps/hpi encoded formaldehyde assimilation via the ribulose monophosphate cycle, were identified together with evidence that the latter might be the result of horizontal gene transfer. Phylogenetic analyses based on 16S rDNA or orthologues of AMETH_3452, a novel actinobacterial class-specific conserved gene against 62 or 18 Amycolatopsis type strains, respectively, revealed three major phyletic lineages, namely the mesophilic or moderately thermophilic A. orientalis subclade (AOS), the mesophilic Amycolatopsis taiwanensis subclade (ATS) and the thermophilic A. methanolica subclade (AMS). The distinct growth temperatures of members of the subclades correlated with corresponding genetic variations in their encoded compatible solutes. This study shows the value of integrating conventional taxonomic with whole genome sequence data.

Draft Genome Sequence of the Streptothricin-Producing Strain Streptomyces sp. fd2-tb.

Streptomyces sp. fd2-tb can produce streptothricin class antibiotics with broad antimicrobial spectra. To better understand the mechanism of streptothricin biosynthesis and to assess the capacity of this strain in secondary metabolism, we report the draft genome sequence of Streptomyces sp. strain fd2-tb.

Complete genome sequence of the glidobactin producing strain [Polyangium] brachysporum DSM 7029.

[Polyangium] brachysporum DSM7029 can produce glidobactin, which is a peptide-based proteasome inhibitor that shows great potential as an anticancer drug. To better understand the glidobactin biosynthesis mechanism and to aid further studies of this strain, we report the annotated complete genome sequence of DSM7029, which is 6,476,147 bp in length.