Determination of the Protein-Protein Interactions within Acyl Carrier Protein (MmcB)-Dependent Modifications in the Biosynthesis of Mitomycin.

Mitomycin has a unique chemical structure and contains densely assembled functionalities with extraordinary antitumor activity. The previously proposed mitomycin C biosynthetic pathway has caused great attention to decipher the enzymatic mechanisms for assembling the pharmaceutically unprecedented chemical scaffold. Herein, we focused on the determination of acyl carrier protein (ACP)-dependent modification steps and identification of the protein-protein interactions between MmcB (ACP) with the partners in the early-stage biosynthesis of mitomycin C. Based on the initial genetic manipulation consisting of gene disruption and complementation experiments, genes mitE, mmcB, mitB, and mitF were identified as the essential functional genes in the mitomycin C biosynthesis, respectively. Further integration of biochemical analysis elucidated that MitE catalyzed CoA ligation of 3-amino-5-hydroxy-bezonic acid (AHBA), MmcB-tethered AHBA triggered the biosynthesis of mitomycin C, and both MitB and MitF were MmcB-dependent tailoring enzymes involved in the assembly of mitosane. Aiming at understanding the poorly characterized protein-protein interactions, the in vitro pull-down assay was carried out by monitoring MmcB individually with MitB and MitF. The observed results displayed the clear interactions between MmcB and MitB and MitF. The surface plasmon resonance (SPR) biosensor analysis further confirmed the protein-protein interactions of MmcB with MitB and MitF, respectively. Taken together, the current genetic and biochemical analysis will facilitate the investigations of the unusual enzymatic mechanisms for the structurally unique compound assembly and inspire attempts to modify the chemical scaffold of mitomycin family antibiotics.

Nonomuraea nitratireducens sp. nov., a new actinobacterium isolated from Suaeda australis Moq. rhizosphere.

A novel actinomycete, designated WYY166T, was isolated from the rhizosphere of Suaeda australis Moq. collected in Dongfang, PR China. The taxonomic position of this strain was investigated using a polyphasic approach. Phylogenetic analysis based on its 16S rRNA gene referred strain WYY166T to the genus Nonomuraea, and it was most closely related to the type strains Nonomuraea candida HMC10T, Nonomuraea turkmeniaca DSM 43926T, Nonomuraea maritima NBRC 106687T and Nonomuraea polychroma DSM 43925T (98.35, 97.60, 97.36 and 97.30% sequence similarity, respectively). Genome sequencing revealed a genome size of 11.27 Mbp and a G+C content of 71.10 mol%. The genome average nucleotide identity (ANI) values and the digital DNA - DNA hybridization (dDDH) values between strain WYY166T and the other species of the genus were found to be low (ANI 81.63~85.23 %, dDDH 23.6~31.6 %), suggesting that it represented a new species. The physiological evaluation showed that it had remarkable nitrate reduction activity. The whole-cell hydrolysates contained meso-diaminopimelic acid and madurose. The N-acyl type of muramic acid was acetyl. The major menaquinones were MK-9 (H4) (86.9 %) and MK-9 (H2) (13.1 %). The predominant fatty acids were iso-C16 : 0 (53.2 %), 10-methyl C17 : 0 (10.7 %), C17 : 1 ω6c (8.3 %) and iso-C16 : 1 h (7.3 %). These physiological, biochemical and chemotaxonomic data suggested that strain WYY166T should be classified as representing a novel species of the genus Nonomuraea, for which the name Nonomuraea nitratireducens sp. nov. is proposed. The type strain is WYY166T (=MCCC 1K03779T=KCTC 49343T).

Generation of tetramycin B derivative with improved pharmacological property based on pathway engineering.

Polyene antibiotics, including amphotericin, nystatin, pimaricin, and tetramycin, are important antifungal agents. Increasing the production of polyenes and generation of their improved analogues based on the biosynthetic pathway engineering has aroused wide concern in application researches. Herein, tetramycin and nystatin, both of which share most of acyl-CoA precursors, are produced by Streptomyces hygrospinosus var. beijingensis CGMCC 4.1123. Thus, the intracellular malonyl-CoA is found to be insufficient for PKSs (polyketide synthases) extension of tetramycin by quantitative analysis in this wild-type strain. To circumvent this problem and increase tetramycin titer, the acyl-CoA competing biosynthetic gene cluster (BGC) of nystatin was disrupted, and the biosynthetic genes of malonyl-CoA from S. coelicolor M145 were integrated and overexpressed in nys-disruption mutant strain (SY02). Moreover, in order to specifically accumulate tetramycin B from A, two copies of tetrK and a copy of tetrF were introduced, resulting in elevating tetramycin B fermentration titer by 122% to 865 ± 8 mg/L than the wild type. In this optimized strain, a new tetramycin derivative, 12-decarboxy-12-methyl tetramycin B, was generated with a titer of 371 ± 26 mg/L through inactivation of a P450 monooxygenase gene tetrG. Compared with tetramycin B, the new compound exhibited higher antifungal activity against Saccharomyces cerevisiae and Rhodotorula glutinis, but lower hemolytic toxicity to erythrocyte. This research provided a good example of employing biosynthetic engineering strategies for fermentation titer improvement of polyene and development of the derivatives for medicinal applications.

p-Aminophenylalanine Involved in the Biosynthesis of Antitumor Dnacin B1 for Quinone Moiety Formation.

Actinosynnema species produce diverse natural products with important biological activities, which represent an important resource of antibiotic discovery. Advances in genome sequencing and bioinformatics tools have accelerated the exploration of the biosynthetic gene clusters (BGCs) encoding natural products. Herein, the completed BGCs of dnacin B1 were first discovered in two Actinosynnema pretiosum subsp. auranticum strains DSM 44131T (hereafter abbreviated as strain DSM 44131T) and X47 by comparative genome mining strategy. The BGC for dnacin B1 contains 41 ORFs and spans a 66.9 kb DNA region in strain DSM 44131T. Its involvement in dnacin B1 biosynthesis was identified through the deletion of a 9.7 kb region. Based on the functional gene analysis, we proposed the biosynthetic pathway for dnacin B1. Moreover, p-amino-phenylalanine (PAPA) unit was found to be the dnacin B1 precursor for the quinone moiety formation, and this was confirmed by heterologous expression of dinV, dinE and dinF in Escherichia coli. Furthermore, nine potential PAPA aminotransferases (APAT) from the genome of strain DSM 44131T were explored and expressed. Biochemical evaluation of their amino group transformation ability was carried out with p-amino-phenylpyruvic acid (PAPP) or PAPA as the substrate for the final product formation. Two of those, APAT4 and APAT9, displayed intriguing aminotransferase ability for the formation of PAPA. The proposed dnacin B1 biosynthetic machinery and PAPA biosynthetic investigations not only enriched the knowledge of tetrahydroisoquinoline (THIQ) biosynthesis, but also provided PAPA building blocks to generate their structurally unique homologues.

Streptomyces-based whole-cell biosensors for detecting diverse cell envelope-targeting antibiotics.

Cell envelope-targeting antibiotics are potent therapeutic agents against various bacterial infections. The emergence of multiple antibiotic-resistant strains underscores the significance of identifying potent antimicrobials specifically targeting the cell envelope. However, current drug screening approaches are tedious and lack sufficient specificity and sensitivity, warranting the development of more efficient methods. Genetic circuit-based whole-cell biosensors hold great promise for targeted drug discovery from natural products. Here, we performed comparative transcriptomic analysis of Streptomyces coelicolor M1146 exposed to diverse cell envelope-targeting antibiotics, aiming to identify regulatory elements involved in perceiving and responding to these compounds. Differential gene expression analysis revealed significant activation of VanS/R two-component system in response to the glycopeptide class of cell envelope-acting antibiotics. Therefore, we engineered a pair of VanS/R-based biosensors that exhibit functional complementarity and possess exceptional sensitivity and specificity for glycopeptides detection. Additionally, through promoter screening and characterization, we expanded the biosensor's detection range to include various cell envelope-acting antibiotics beyond glycopeptides. Our genetically engineered biosensor exhibits superior performance, including a dynamic range of up to 887-fold for detecting subtle antibiotic concentration changes in a rapid 2-h response time, enabling high-throughput screening of natural product libraries for antimicrobial agents targeting the bacterial cell envelope.

Insertion sequence transposition inactivates CRISPR-Cas immunity.

CRISPR-Cas immunity systems safeguard prokaryotic genomes by inhibiting the invasion of mobile genetic elements. Here, we screened prokaryotic genomic sequences and identified multiple natural transpositions of insertion sequences (ISs) into cas genes, thus inactivating CRISPR-Cas defenses. We then generated an IS-trapping system, using Escherichia coli strains with various ISs and an inducible cas nuclease, to monitor IS insertions into cas genes following the induction of double-strand DNA breakage as a physiological host stress. We identified multiple events mediated by different ISs, especially IS1 and IS10, displaying substantial relaxed target specificity. IS transposition into cas was maintained in the presence of DNA repair machinery, and transposition into other host defense systems was also detected. Our findings highlight the potential of ISs to counter CRISPR activity, thus increasing bacterial susceptibility to foreign DNA invasion.

Exploring Diversity through Dimerization in Natural Products by a Rational Tandem Mass-Based Molecular Network Strategy.

The step- and atom-efficient dimerization strategy is frequently used in nature to build structural complexity and diversity. We propose the rationale and structural features of the versatile monomers that are responsible for "diversity through dimerization". Using 5-FAM-maleimide combined with a UHPLC-MS/MS-FBMN workflow, we successfully identified a diverse set of dimeric natural products from fungus Panus rudis F01315, in which all four complex 4'5-ring scaffolds are derived from one monomeric epoxyquinol and endowed with functional diversity.

Guanacastane-type diterpenoids with cytotoxic activity from Coprinus plicatilis.

Four new guanacastane-type diterpenoids (1-4), together with the known compound, guanacastepene E (5), were isolated from a basidiomycete of the macro-fungi, Coprinus plicatilis 82. Their structures were elucidated on the basis of extensive spectroscopic analyses, including FT-ICR-MS, UV, IR and 1D and 2D NMR experiments. The in vitro cytotoxic activities of all compounds against the human cancer cell lines HepG2, HeLa, MDA-MB-231, BGC-823, HCT 116, and U2OS were evaluated, only compound 1 exhibited significant cytotoxicities with IC(50) values ranging from 1.2 to 6.0 µM.

Adaptive Optimization Boosted the Production of Moenomycin A in the Microbial Chassis Streptomyces albus J1074.

Great efforts have been made to improve Streptomyces chassis for efficient production of targeted natural products. Moenomycin family antibiotics, represented by moenomycin (Moe) and nosokomycin, are phosphoglycolipid antibiotics that display extraordinary inhibition against Gram-positive bacteria. Herein, we assembled a completed 34 kb hybrid biosynthetic gene cluster (BGC) of moenomycin A (moe-BGC) based on a 24 kb nosokomycin analogue biosynthetic gene cluster (noso-BGC). The heterologous expression of the hybrid moe-BGC in Streptomyces albus J1074 achieved the production of moenomycin A in the recombinant strain LX01 with a yield of 12.1 ± 2 mg/L. Further strong promoter refactoring to improve the transcriptional levels of all of the functional genes in strain LX02 enhanced the production of moenomycin A by 58%. However, the yield improvement of moenomycin A resulted in a dramatic 38% decrease in the chassis biomass compared with the control strain. To improve the weak physiological tolerance to moenomycin A of the chassis, another copy of the gene salb-PBP2 (P238N&F200D), encoding peptidoglycan biosynthetic protein PBP2, was introduced into the chassis strain, producing strain LX03. Cell growth was restored, and the fermentation titer of moenomycin A was 130% higher than that of LX01. Additionally, the production of moenomycin A in strain LX03 was further elevated by 45% to 40.0 ± 3 mg/L after media optimization. These results suggested that the adaptive optimization strategy of strong promoter refactoring in the BGC plus physiological tolerance in the chassis was an efficient approach for obtaining the desired natural products with high titers.

Optimization of the culture condition of Bacillus mucilaginous using Agaricus bisporus industrial wastewater by Plackett-Burman combined with Box-Behnken response surface method.

In the present study, conditions for Bacillus mucilaginous fermentation using Agaricus bisporus wastewater as culture medium were optimized. We analyzed the total number of living B. mucilaginous in the fermentation broth using multispectral imaging flow cytometry. Single-factor experiments were carried out, where a Plackett-Burman design was used to screen out three factors from the original six factors of processing wastewater solubility, initial pH, inoculum size, liquid volume, culture temperature, and rotation speed that affected the total number of viable B. mucilaginous. The Box-Behnken response surface method was used to optimize interactions between the three main factors and predict optimal fermentation conditions. Factors significantly affecting the total number of viable B. mucilaginous, including shaking speed, culturing temperature, and initial pH, were investigated. The optimum conditions for B. mucilaginous fermentation in A. bisporus wastewater were a rotational speed of 195 rpm, culture temperature of 29 °C, initial pH of 6.5, solubility of 0.5%, 8% inoculation volume, and 90 mL liquid volume in a 250 mL flask, culture time of 48 h. Under these conditions, the concentration of total viable bacteria reached 2.16 ± 0.02 × 108 Obj/mL, which meets the national standard. A. bisporus wastewater can be used for the cultivation of B. mucilaginous.

Optimization of Cultural Conditions for Bacillus megaterium Cultured in Agaricus bisporus Industrial Wastewater.

The aim of this study was to optimize the cultural conditions for Bacillus megaterium using Agaricus bisporus industrial wastewater as nature culture through response surface methodology. In our present study, we analyzed the total number of living B. megaterium in the fermentation broth using multispectral imaging flow cytometry. Plackett-Burman design was applied to evaluate the effects of six variables, namely, initial pH, industrial wastewater solubility, rotating speed, culture temperature, inoculum size, and loading volume. Loading volume, initial pH, and culture temperature were found to influence the biomass of B. megaterium significantly and were further optimized by Box-Behnken design. After verification test, the optimum fermentation conditions of B. megaterium using the A. bisporus processing wastewater as nature culture media were obtained as follows: initial pH of 7.4, culture temperature of 25°C, loading volume of 40 mL/250 mL, culture time of 24 h, industrial wastewater solubility of 1%, rotating speed of 200 rpm, and inoculum size of 8%. The predicted optimum model's value was 8.88 × 108 Obj/mL and the average experimental value was 9.03 ± 0.02 × 108 Obj/mL, which met the national microbial fertilizers' standard. Furthermore, the field experiment results showed that the fermentation broth of B. megaterium could significantly improve the yield of Spinacia oleracea L.

Three new 2,5-diketopiperazines from the fish intestinal Streptomyces sp. MNU FJ-36.

The gut actinobacteria of marine-inhabited fish is one of the most important reservoirs of novel natural products. Currently, the Streptomyces sp. MNU FJ-36 was isolated from the intestinal fabric of Katsuwonus sp. and determined by 16S rRNA analysis. From the cultures of the S. sp. MNU FJ-36, three new 2,5-diketopiperazines (2,5-DKPs) were discovered and identified as 3-(3-hydroxy-4-methoxybenzyl)-6-isobutyl-2,5-diketopiperazine (1), 3-(1,3-benzodioxol-5-ylmethyl)-6-isobutyl-2,5-diketopiperazine (2) and 3-(1,3-benzodioxol-5-ylmethyl)-6-isopropyl-2,5-diketopiperazine (3). Their structures were elucidated on the basis of spectroscopic data analysis. All the compounds were also evaluated for their inhibitory activity against P388, A-549 and HCT-116 cell lines with the MTT assay.

Hepatoprotective effects of polysaccharide isolated from Agaricus bisporus industrial wastewater against CCl4-induced hepatic injury in mice.

During the industrial production of canned mushroom (Agaricus bisporus), a large quantity of wastewater is produced. In this study, the wastewater generated during the canning of mushroom was analyzed. From this wastewater, four polysaccharide components (Abnp1001, Abnp1002, Abap1001, and Abap1002) with hepatic-protective activity were isolated by ultrafiltration, DEAE cellulose-52 chromatography and Sephadex G-200 size-exclusion chromatography. Results of ultraviolet spectra analysis and molecular weight determination showed that Abnp1001, Abnp1002, Abap1001 and Abap1002 were uniform with average molecular weights of 336, 12.8, 330 and 15.8kDa, respectively. The monosaccharide composition analysis using gas chromatography (GC) showed that the four fractions were heteropolysaccharides and mainly composed of glucose. Fourier transform-infrared (FT-IR) analysis showed that the isolated fractions were all composed of ß-glycoside linkages. Additionally, the potential hepatoprotective activities of these polysaccharides against CCl4-induced hepatic injury in mice were studied. Notably, Abnp1002 and Abap1002 could lower the alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations in serum in a dose dependent manner and reduce the hepatocellular degeneration and necrosis, as well as inflammatory infiltration. These results indicate that these two polysaccharides had protective effects on acute hepatic injury induced by CCl4 in mice and suggest that the polysaccharides extracted from A. bisporus industrial wastewater might have potential in therapeutics of acute hepatic injury.

Guanacastane-type diterpenoids from Coprinus radians.

Thirteen diterpenoids, named radianspenes A-M (1-13), including three lactams radianspenes J (10), K (11) and L (12) and one dimer radianspene M (13), were isolated from fermentation products of the higher fungal strain Coprinus radians M65. All these compounds possessing guanacastane skeleton were evaluated for antitumor activity using MDA-MB-435 cell line. Radianspene C exhibited inhibitory activity with IC(50) of 0.91 µM.