Harnessing the Potential of Halogenated Natural Product Biosynthesis by Mangrove-Derived Actinomycetes

Mangrove-derived actinomycetes are promising sources of bioactive natural products. In this study, using homologous screening of the biosynthetic genes and anti-microorganism/tumor assaying, 163 strains of actinomycetes isolated from mangrove sediments were investigated for their potential to produce halogenated metabolites. The FADH₂-dependent halogenase genes, identified in PCR-screening, were clustered in distinct clades in the phylogenetic analysis. The coexistence of either polyketide synthase (PKS) or nonribosomal peptide synthetase (NRPS) as the backbone synthetases in the strains harboring the halogenase indicated that these strains had the potential to produce structurally diversified antibiotics. As a validation, a new enduracidin producer, Streptomyces atrovirens MGR140, was identified and confirmed by gene disruption and HPLC analysis. Moreover, a putative ansamycin biosynthesis gene cluster was detected in Streptomyces albogriseolus MGR072. Our results highlight that combined genome mining is an efficient technique to tap promising sources of halogenated natural products synthesized by mangrove-derived actinomycetes.     

LC-MS-Based Metabolomics Study of Marine Bacterial Secondary Metabolite and Antibiotic Production in Salinispora arenicola

An LC-MS-based metabolomics approach was used to characterise the variation in secondary metabolite production due to changes in the salt content of the growth media as well as across different growth periods (incubation times). We used metabolomics as a tool to investigate the production of rifamycins (antibiotics) and other secondary metabolites in the obligate marine actinobacterial species Salinispora arenicola, isolated from Great Barrier Reef (GBR) sponges, at two defined salt concentrations and over three different incubation periods. The results indicated that a 14 day incubation period is optimal for the maximum production of rifamycin B, whereas rifamycin S and W achieve their maximum concentration at 29 days. A “chemical profile” link between the days of incubation and the salt concentration of the growth medium was shown to exist and reliably represents a critical point for selection of growth medium and harvest time.     

Complete Genome Sequence of Two Deep-Sea Streptomyces Isolates from Madeira Archipelago and Evaluation of Their Biosynthetic Potential

The deep-sea constitutes a true unexplored frontier and a potential source of innovative drug scaffolds. Here, we present the genome sequence of two novel marine actinobacterial strains, MA3_2.13 and S07_1.15, isolated from deep-sea samples (sediments and sponge) and collected at Madeira archipelago (NE Atlantic Ocean; Portugal). The de novo assembly of both genomes was achieved using a hybrid strategy that combines short-reads (Illumina) and long-reads (PacBio) sequencing data. Phylogenetic analyses showed that strain MA3_2.13 is a new species of the Streptomyces genus, whereas strain S07_1.15 is closely related to the type strain of Streptomyces xinghaiensis. In silico analysis revealed that the total length of predicted biosynthetic gene clusters (BGCs) accounted for a high percentage of the MA3_2.13 genome, with several potential new metabolites identified. Strain S07_1.15 had, with a few exceptions, a predicted metabolic profile similar to S. xinghaiensis. In this work, we implemented a straightforward approach for generating high-quality genomes of new bacterial isolates and analyse in silico their potential to produce novel NPs. The inclusion of these in silico dereplication steps allows to minimize the rediscovery rates of traditional natural products screening methodologies and expedite the drug discovery process.     

Metabolomic Tools for Secondary Metabolite Discovery from Marine Microbial Symbionts

Marine invertebrate-associated symbiotic bacteria produce a plethora of novel secondary metabolites which may be structurally unique with interesting pharmacological properties. Selection of strains usually relies on literature searching, genetic screening and bioactivity results, often without considering the chemical novelty and abundance of secondary metabolites being produced by the microorganism until the time-consuming bioassay-guided isolation stages. To fast track the selection process, metabolomic tools were used to aid strain selection by investigating differences in the chemical profiles of 77 bacterial extracts isolated from cold water marine invertebrates from Orkney, Scotland using liquid chromatography-high resolution mass spectrometry (LC-HRMS) and nuclear magnetic resonance (NMR) spectroscopy. Following mass spectrometric analysis and dereplication using an Excel macro developed in-house, principal component analysis (PCA) was employed to differentiate the bacterial strains based on their chemical profiles. NMR ¹H and correlation spectroscopy (COSY) were also employed to obtain a chemical fingerprint of each bacterial strain and to confirm the presence of functional groups and spin systems. These results were then combined with taxonomic identification and bioassay screening data to identify three bacterial strains, namely Bacillus sp. 4117, Rhodococcus sp. ZS402 and Vibrio splendidus strain LGP32, to prioritize for scale-up based on their chemically interesting secondary metabolomes, established through dereplication and interesting bioactivities, determined from bioassay screening.     

紫黑链霉菌进化枝菌株Streptomyces solisilvae HNM0141T的全基因组分析

链霉菌属放线菌是最重要的抗生素产生菌,也是包含放线菌种类最多的放线菌属.链霉菌属的有效发表种的模式菌株在16S rRNA基因序列系统发育树上分散归簇于不同的进化分枝.紫黑链霉菌16S rRNA基因进化枝(Streptomyces violaceusniger 16S rRNA gene clade)是其中的一个分枝.该...     

Secondary Metabolites and Biosynthetic Gene Clusters Analysis of Deep-Sea Hydrothermal Vent-Derived Streptomyces sp. SCSIO ZS0520

Streptomyces sp. SCSIO ZS0520 is a deep-sea hydrothermal vent-derived actinomycete. Our previous metabolism investigation showed that Streptomyces sp. SCSIO ZS0520 is a producer of cytotoxic actinopyrones. Here, another four types of secondary metabolites were identified, including six salinomycin isomers (2–7), the macrolide elaiophylin (8), the triterpene N-acetyl-aminobacteriohopanetriol (9), and the pyrone minipyrone (10). Among them, compounds 2–6 and 10 are new compounds. To understand the biosynthetic pathway of these compounds, a bioinformatic analysis of the whole genome was carried out, which identified 34 secondary metabolite biosynthetic gene clusters. Next, the biosynthetic pathways responsive to four types of products were deduced on the basis of gene function predictions and structure information. Taken together, these findings prove the metabolite potential of ZS0520 and lay the foundations to solve the remaining biosynthetic issues in four types of marine natural products.     

Comprehensive Genomic Analysis of Marine Strain Streptomyces sp. 891, an Excellent Producer of Chrysomycin A with Therapeutic Potential

Chrysomycin A is one of the most promising therapeutic candidates for treating infections caused by multidrug-resistant Gram-positive bacteria. By hybridizing next-step generation (Illumina) and third-generation (PacBio) sequencing technologies, a high-quality chromosome-level genome together with a plasmid was firstly assembled for chrysomycin A-producing marine strain 891. Phylogenetic analysis of the 16S rRNA gene and genome sequences revealed that this strain unambiguously belonged to the genus Streptomyces, and its genomic features and functional genes were comprehensively analyzed and annotated. AntiSMASH analysis of this strain unveiled one key biosynthetic gene cluster, T2PKS, responsible for the biosynthesis of chrysomycin, the biosynthesis pathway of which was putatively proposed. These findings definitely shed light on further investigation for construction of a robust industrial strain with high-yield chrysomycin A production using genetic engineering techniques and combinatorial biology approaches.     

生防菌株Bacillus velezensis Z全基因组测序分析

生防菌株Bacillus velezensis Z对胡椒瘟病等多种植物病害具有良好的生防效果;全基因组测序能够为其分子机理研究和开发应用提供信息基础。本研究开展该菌株全基因组测序,并进行比较基因组学和抑菌次生代谢产物合成基因簇预测研究。结果表明:B.velezensisZ菌株的基因组中含有1条4054780bp大小的环形染色体DNA和1个17 122 bp大小的环形质粒,总基因组的GC含量为46.24%,共编码基因4173个;包含27个rRNA,86个tRNA基因,34个sRNA;含有串联重复序列179个,其中13个微卫星DNA,138个小卫星DNA;通过比较基因组学分析,结果发现该菌株与贝莱斯芽孢杆菌模式菌株FZB42同源性极高,与枯草芽孢杆菌模式菌株168之间具有一定的遗传距离;同时发现B. velezensis Z基因组中共编码次生代谢产物合成基因簇13个,其中8个与表面活性素(surfactin)、泛革素(fengycin)、溶杆菌素(bacilysin)、macrolactinH、bacillaene、difficidin、plantazolicin、amylocyclici...     

胡椒瘟病生防菌 Bacillus subtilis VD18R19 全基因组测序及 比较基因组学分析

Bacillus subtilis VD18R19 具有促进胡椒生长和防治胡椒瘟病的效果,全基因组测序是其分子机理研究和开发 利用的重要基础。本研究采用第二代 Illumina 平台与第三代 PacBio 平台相结合的测序技术,对生防菌 VD18R19 进行 全基因组测序,并进行比较基因组学分析。结果发现,VD18R19 全基因组大小为 4 123 380 bp,GC 含量为 43.80%, 编码基因 4 245 个;含有 tRNA 85 个、rRNA 30 个、sRNA 33 个;含有串联重复序列 60 个,其中小卫星 DNA 43 个、 微卫星 DNA 1 个;共线性分析、core-pan 基因分析及基因家族分析结果均显示 VD18R19 与模式菌株 B. subtilis 168 具 有高度的同源性;antiSMASH 软件预测及同源序列比对结果显示,VD18R19 菌株中含有 6 个抑菌次生代谢产物合成基 因簇,编码 surfactin、plipastatin、bacillibactin、bacilysin、bacillaene、subtilosin A 等抑菌物质,其合成途径涵盖了核 糖体途径、非核糖体途径和聚酮合酶途径。本研究为深入研究生防菌 VD18R19 的分子机理奠定生物信息学基础,有利 于生防菌株及其抑菌次生代谢产物的开发和利用。     

热带植物基因组研究进展

基于全基因组测序结果,探讨了包括热带水果香蕉（Musa spp.）、龙眼（Dimocarpus longan）、番木瓜（Carica papaya）、菠萝（Ananas comosus）、椰子（Cocos nucifera）、榴莲（Durio zibethinus）,经济作物橡胶（Hevea brasiliensis）、木薯（Manihot esculenta）、枣椰（Phoenix dactylifera）、可可（Theobroma cacao）、油棕（Elaeis guineensis）、咖啡（Coffea canephora）以及药用植物铁皮石斛（Dendrobium officinale）在内的13种热带植物的全基因组测序的发展历程,并对热带植物基因组研究进行了概述。     

Activation of the Silent Secondary Metabolite Production by Introducing Neomycin-Resistance in a Marine-Derived Penicillium purpurogenum G59

Introduction of neomycin-resistance into a marine-derived, wild-type Penicillium purpurogenum G59 resulted in activation of silent biosynthetic pathways for the secondary metabolite production. Upon treatment of G59 spores with neomycin and dimethyl sulfoxide (DMSO), a total of 56 mutants were obtained by single colony isolation. The acquired resistance of mutants to neomycin was testified by the resistance test. In contrast to the G59 strain, the EtOAc extracts of 28 mutants inhibited the human cancer K562 cells, indicating that the 28 mutants have acquired the capability to produce bioactive metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses further indicated that diverse secondary metabolites have been newly produced in the bioactive mutant extracts. Followed isolation and characterization demonstrated that five bioactive secondary metabolites, curvularin (1), citrinin (2), penicitrinone A (3), erythro-23-O-methylneocyclocitrinol (4) and 22E-7α-methoxy-5α,6α-epoxyergosta-8(14),22-dien-3β-ol (5), were newly produced by a mutant, 4-30, compared to the G59 strain. All 1–5 were also not yet found in the secondary metabolites of other wild type P. purpurogenum strains. Compounds 1–5 inhibited human cancer K562, HL-60, HeLa and BGC-823 cells to varying extents. Both present bioassays and chemical investigations demonstrated that the introduction of neomycin-resistance into the marine-derived fungal G59 strain could activate silent secondary metabolite production. The present work not only extended the previous DMSO-mediated method for introducing drug-resistance in fungi both in DMSO concentrations and antibiotics, but also additionally exemplified effectiveness of this method for activating silent fungal secondary metabolites. This method could be applied to other fungal isolates to elicit their metabolic potentials to investigate secondary metabolites from silent biosynthetic pathways.     

橡胶树产胶生物学研究进展

天然橡胶（顺式-1,4-聚异戊二烯）是一种重要的工业原料,主要来自橡胶树。以天然橡胶的生物合成与产量形成为主要内容的产胶生物学研究为橡胶树高产遗传改良提供理论指导,近10年取得了重要进展。本文从橡胶树基因组测序、橡胶转移酶、转基因、以及转录组和蛋白质组等4个方面介绍产胶生物学研究主要进展,并讨论了相关领域研究存在的问题,对未来5~10年需重点关注的研究内容提出了建议。在介绍橡胶转移酶时,同时概述其他几种产胶植物的相关研究进展。     

Activation of Dormant Secondary Metabolite Production by Introducing Neomycin Resistance into the Deep-Sea Fungus,Aspergillus versicolor ZBY-3

A new ultrasound-mediated approach has been developed to introduce neomycin-resistance to activate silent pathways for secondary metabolite production in a bio-inactive, deep-sea fungus, Aspergillus versicolor ZBY-3. Upon treatment of the ZBY-3 spores with a high concentration of neomycin by proper ultrasound irradiation, a total of 30 mutants were obtained by single colony isolation. The acquired resistance of the mutants to neomycin was confirmed by a resistance test. In contrast to the ZBY-3 strain, the EtOAc extracts of 22 of the 30 mutants inhibited the human cancer K562 cells, indicating that these mutants acquired a capability to produce antitumor metabolites. HPLC-photodiode array detector (PDAD)-UV and HPLC-electron spray ionization (ESI)-MS analyses of the EtOAc extracts of seven bioactive mutants and the ZBY-3 strain indicated that diverse secondary metabolites have been newly produced in the mutant extracts in contrast to the ZBY-3 extract. The followed isolation and characterization demonstrated that six metabolites, cyclo(d-Pro-d-Phe) (1), cyclo(d-Tyr-d-Pro) (2), phenethyl 5-oxo-l-prolinate (3), cyclo(l-Ile-l-Pro) (4), cyclo(l-Leu-l-Pro) (5) and 3β,5α,9α-trihydroxy-(22E,24R)-ergosta-7,22-dien-6-one (6), were newly produced by the mutant u2n2h3-3 compared to the parent ZBY-3 strain. Compound 3 was a new compound; 2 was isolated from a natural source for the first time, and all of these compounds were also not yet found in the metabolites of other A. versicolor strains. Compounds 1–6 inhibited the K562 cells, with inhibition rates of 54.6% (1), 72.9% (2), 23.5% (3), 29.6% (4), 30.9% (5) and 51.1% (6) at 100 μg/mL, and inhibited also other human cancer HL-60, BGC-823 and HeLa cells, to some extent. The present study demonstrated the effectiveness of the ultrasound-mediated approach to activate silent metabolite production in fungi by introducing acquired resistance to aminoglycosides and its potential for discovering new compounds from silent fungal metabolic pathways. This approach could be applied to elicit the metabolic potentials of other fungal isolates to discover new compounds from cryptic secondary metabolites.     

Rapid Evaporative Ionization Mass Spectrometry (REIMS): a Potential and Rapid Tool for the Identification of Insecticide Resistance in Mosquito Larvae

Insecticide resistance is a significant challenge facing the successful control of mosquito vectors globally. Bioassays are currently the only method for phenotyping resistance. They require large numbers of mosquitoes for testing, the availability of a susceptible comparator strain, and often insectary facilities. This study aimed to trial the novel use of rapid evaporative ionization mass spectrometry (REIMS) for the identification of insecticide resistance in mosquitoes. No sample preparation is required for REIMS and analysis can be rapidly conducted within hours. Temephos resistant Aedes aegypti (Linnaeus) larvae from Cúcuta, Colombia and temephos susceptible larvae from two origins (Bello, Colombia, and the lab reference strain New Orleans) were analyzed using REIMS. We tested the ability of REIMS to differentiate three relevant variants: population source, lab versus field origin, and response to insecticide. The classification of these data was undertaken using linear discriminant analysis (LDA) and random forest. Classification models built using REIMS data were able to differentiate between Ae. aegypti larvae from different populations with 82% (±0.01) accuracy, between mosquitoes of field and lab origin with 89% (±0.01) accuracy and between susceptible and resistant larvae with 85% (±0.01) accuracy. LDA classifiers had higher efficiency than random forest with this data set. The high accuracy observed here identifies REIMS as a potential new tool for rapid identification of resistance in mosquitoes. We argue that REIMS and similar modern phenotyping alternatives should complement existing insecticide resistance management tools.     

Vertical Distribution of Sodium in Roots of Rice Plants Exposed to Salinity as Analyzed by Cryo Time-of-Flight Secondary Ion Mass Spectrometry

Abstract

Distribution of Na⁺ along the root axis under salinity stress was analyzed in two rice (Oryza sativa L.) cultivars with different salt resistance (salt-sensitive IR 24 and salt-resistant Pokkali). Rice plants were grown hydroponically and NaCl was applied with nutrient solution at concentrations of 0, 25 and 50 mM for 7 d after germination. The distribution of Na⁺ in roots under salinity was analyzed by the cryo time-of-flight secondary ion mass spectrometry (cryo TOF-SIMS). The Na⁺ content in the root was higher in salt-sensitive IR 24 than in salt-resistant Pokkali under NaCl stress. The content was highest at the root tip and was decreased basipetally along the root axis. The difference in Na⁺ content between the cultivars was apparent in all regions from the root tip.