Development of a physical map for three Frankia strains and a partial genetic map for Frankia EuI1c

Physical maps for Frankia strains CcI3, EAN1pec and EuI1c chromosomes were constructed by the use of macrorestriction analysis and pulsed-field gel electrophoresis (PFGE). The restriction enzymes Ase I, Pme I, Swa I and Ssp I were used to cut the Frankia chromosome into a limited number of large fragments and for double digestions. The genomes sizes, as determined by the addition of the estimated fragment sizes, were 5430 ± 35 kb, 9101 ± 109 kb and 8105 ± 842 kb for strains CcI3, EAN1pec and EuI1c, respectively. A complete physical map was achieved by the analysis of PFGE for the single and double digestions and by two-dimensional PFGE to determine doublets and overlapping fragments. For strain EuI1c, a partial genetic map was also constructed by positioning the 16S rRNA, gln II, gln A and hbo O genes on the physical map. PFGE analysis of DNA with and without proteinase K treatment together with the other results suggested a circular genome.     

Expanding our Understanding of Sequence-Function Relationships of Type II Polyketide Biosynthetic Gene Clusters: Bioinformatics-Guided Identification of Frankiamicin A from Frankia sp. EAN1pec

A large and rapidly increasing number of unstudied “orphan” natural product biosynthetic gene clusters are being uncovered in sequenced microbial genomes. An important goal of modern natural products research is to be able to accurately predict natural product structures and biosynthetic pathways from these gene cluster sequences. This requires both development of bioinformatic methods for global analysis of these gene clusters and experimental characterization of select products produced by gene clusters with divergent sequence characteristics. Here, we conduct global bioinformatic analysis of all available type II polyketide gene cluster sequences and identify a conserved set of gene clusters with unique ketosynthase α/β sequence characteristics in the genomes of Frankia species, a group of Actinobacteria with underexploited natural product biosynthetic potential. Through LC-MS profiling of extracts from several Frankia species grown under various conditions, we identified Frankia sp. EAN1pec as producing a compound with spectral characteristics consistent with the type II polyketide produced by this gene cluster. We isolated the compound, a pentangular polyketide which we named frankiamicin A, and elucidated its structure by NMR and labeled precursor feeding. We also propose biosynthetic and regulatory pathways for frankiamicin A based on comparative genomic analysis and literature precedent, and conduct bioactivity assays of the compound. Our findings provide new information linking this set of Frankia gene clusters with the compound they produce, and our approach has implications for accurate functional prediction of the many other type II polyketide clusters present in bacterial genomes.     

Gene probes for the detection of 6-deoxyhexose metabolism in secondary metabolite-producing streptomycetes

DNA probes were designed from the streptomycin production genes strDELM of Streptomyces griseus involved in the biosynthesis of the 6-deoxyhexose (6DOH) dihydrostreptose which could detect the genomic fragments coding for 6DOH formation in other actinomycetes strains. In about 70% of the 43 strains tested at least one signal could be detected with strD-, strE- or strLM-specific probes. Evidence is presented that the hybridizing genes are mostly clustered and probably engaged in the formation of secondary metabolites. Because of the wide-spread use of 6DOH constituents in natural products these probes should allow to detect a vast array of different secondary metabolic gene clusters in actinomycetes.     

专性海洋放线菌盐孢菌的研究进展

盐孢菌属(Salinispora)作为首个被报道的专性海洋放线菌,主要分布于热带和亚热带海洋沉积环境中,在海绵、海鞘中也有发现。与其他大多数放线菌一样,盐孢菌属的菌株可以产生大量具有抗细菌、抗病毒、抗肿瘤细胞活性、结构新颖的次级代谢产物且表现出物种特异性。全基因组序列分析显示,盐孢菌属菌株基因组中超过10%的基因序列与次级代谢产物合成相关,但绝大多数生物合成基因簇编码的产物未被发现,表明盐孢菌属还存在巨大的生物合成潜能,有待深入发掘。目前新的培养方法、测序技术及生物信息学、基因组发掘技术、合成生物学技术的发展对提升盐孢菌属菌株新型药物的生产潜力发挥重要作用。本文对盐孢菌属的物种多样性、系统分类与化合物发现等方面的研究进行了系统综述。     

A proteomic survey of nonribosomal peptide and polyketide biosynthesis in actinobacteria

Actinobacteria such as streptomycetes are renowned for their ability to produce bioactive natural products including nonribosomal peptides (NRPs) and polyketides (PKs). The advent of genome sequencing has revealed an even larger genetic repertoire for secondary metabolism with most of the small molecule products of these gene clusters still unknown. Here, we employed a "protein-first" method called PrISM (Proteomic Investigation of Secondary Metabolism) to screen 26 unsequenced actinomycetes using mass spectrometry-based proteomics for the targeted detection of expressed nonribosomal peptide synthetases or polyketide synthases. Improvements to the original PrISM screening approach (Nat. Biotechnol. 2009, 27, 951-956), for example, improved de novo peptide sequencing, have enabled the discovery of 10 NRPS/PKS gene clusters from 6 strains. Taking advantage of the concurrence of biosynthetic enzymes and the secondary metabolites they generate, two natural products were associated with their previously "orphan" gene clusters. This work has demonstrated the feasibility of a proteomics-based strategy for use in screening for NRP/PK production in actinomycetes (often >8 Mbp, high GC genomes) versus the bacilli (2-4 Mbp genomes) used previously.     

西沙海藻共附生放线菌的分离鉴定及其抗菌活性评价

【目的】为了探究南海海藻共附生放线菌资源的多样性及潜在的应用价值,对中国西沙群岛来源的海藻进行共附生放线菌的分离鉴定与抗菌活性筛选。【方法】利用稀释涂布平板法,采用2种不同分离培养基对不同采样位点的6种海藻进行放线菌分离;通过16S rRNA基因序列分析、构建系统发育树对分离的放线菌进行鉴定;用打孔法对无乳链球菌(Streptococcus agalactiae)等10种敏感细菌进行抗菌活性筛选;对筛选得到的目标活性菌株HZ014进行全基因组测序,通过AntiSMASH在线工具分析其次级代谢产物生物合成基因簇,预测其产生新型活性物质的潜力。【结果】从6种海藻中分离得到36株共附生放线菌,基于16S rRNA基因序列比对和系统发育分析,鉴定结果为链霉菌属(Streptomyces) 2株、红球菌属(Rhodococcus) 2株、诺卡氏菌属(Nocardia)3株、小单孢菌属(Micromonospora) 5株和盐孢菌属(Salinispora) 24株;抗菌活性筛选结果表明,36株共附生放线菌发酵粗提物对至少1种敏感细菌表现出一定的抑制作用,不同菌株发酵粗提物的抗菌活性存在明显差异,...     

Heterologous expression of natural product biosynthetic gene clusters in Streptomyces coelicolor: from genome mining to manipulation of biosynthetic pathways

Heterologous gene expression is one of the main strategies used to access the full biosynthetic potential of actinomycetes, as well as to study the metabolic pathways of natural product biosynthesis and to create unnatural pathways. Streptomyces coelicolor A3(2) is the most studied member of the actinomycetes, bacteria renowned for their prolific capacity to synthesize a wide range of biologically active specialized metabolites. We review here the use of strains of this species for the heterologous production of structurally diverse actinomycete natural products.     

Biosynthesis pathways for deoxysugars in antibiotic-producing actinomycetes: isolation, characterization and generation of novel glycosylated derivatives

Many bioactive natural products synthesized by actinomycetes are glycosylated compounds in which the appended sugars contribute to specific interactions with their biological target. Most of these sugars are 6-deoxyhexoses, of which more than 70 different forms have been identified, and an increasing number of gene clusters involved in 6-deoxyhexoses biosynthesis are being characterized from antibiotic-producing actinomycetes. Novel glycosylated compounds have been generated by modifying natural deoxysugar biosynthesis pathways in the producer organisms, and/or the simultaneous expression in these strains of selected deoxysugar biosynthesis genes from other strains. Non-producing strains endowed with the capacity to synthesize novel deoxysugars through the expression of engineered deoxysugar biosynthesis clusters can also be used as alternative hosts. Transfer of these deoxysugars to a multiplicity of aglycones relies upon the existence of glycosyltransferases with an inherent degree of 'relaxed substrate specificity'. In this review, we analyze how the knowledge coming out from isolation and characterization of deoxysugar biosynthesis pathways from actinomycetes is being used to produce novel glycosylated derivatives of natural products.     

Nature’s combinatorial biosynthesis and recently engineered production of nucleoside antibiotics in <Emphasis Type="Italic">Streptomyces</Emphasis>

Modified nucleosides produced by Streptomyces and related actinomycetes are widely used in agriculture and medicine as antibacterial, antifungal, anticancer and antiviral agents. These specialized small-molecule metabolites are biosynthesized by complex enzymatic machineries encoded within gene clusters in the genome. The past decade has witnessed a burst of reports defining the key metabolic processes involved in the biosynthesis of several distinct families of nucleoside antibiotics. Furthermore, genome sequencing of various Streptomyces species has dramatically increased over recent years. Potential biosynthetic gene clusters for novel nucleoside antibiotics are now apparent by analysis of these genomes. Here we revisit strategies for production improvement of nucleoside antibiotics that have defined mechanisms of action, and are in clinical or agricultural use. We summarize the progress for genetically manipulating biosynthetic pathways for structural diversification of nucleoside antibiotics. Microorganism-based biosynthetic examples are provided and organized under genetic principles and metabolic engineering guidelines. We show perspectives on the future of combinatorial biosynthesis, and present a working model for discovery of novel nucleoside natural products in Streptomyces.     

西藏仲巴五彩沙漠放线菌资源勘探及生物活性筛选

【背景】细菌耐药性问题日益严峻,新抗生素的研发速度远远落后于临床需要,从特殊生境中挖掘微生物药物资源有望解决以上问题。【目的】勘探西藏仲巴五彩沙漠土壤放线菌多样性并进行生物活性筛选,为发现药用放线菌资源、开发新型抗生素奠定基础。【方法】采用8种分离培养基,通过平板稀释涂布法分离放线菌;根据分离菌株的16S r RNA基因序列同源性分析放线菌多样性;采用PCR技术对分离的放线菌菌株进行II型聚酮合酶(PKS-II)酮缩酶结构域KS、非核糖体多肽合成酶(NRPS)腺苷酸化结构域A、安莎类抗生素生物合成前体3-氨基-5-羟基-苯甲酸合酶(AHBA)保守区、黄素腺嘌呤二核苷酸卤化酶(Halo)保守区抗生素生物合成基因检测;对生物合成基因检测阳性的菌株进行液体发酵,发酵液经乙酸乙酯萃取、菌体经丙酮浸提,获得提取浓缩物样品进行抑菌活性和抗氧化活性筛选。【结果】从4份土样中分离纯化到231株放线菌,分布于7个属,其中链霉菌为优势菌属。68株放线菌的生物合成基因分析显示至少具有1种生物合成基因簇,其中6株同时具有4种生物合成基因簇;进一步的抑菌活性检测显示所有检测的菌株至少表现为对1株检定菌具有抑菌活性,其中8株具有广谱抗菌活性;抗氧化活性筛选结果为13株显示总抗氧化能力阳性,10株具有较好的羟自由基清除能力,3株显示较强的氧自由基清除能力。【结论】西藏仲巴五彩沙漠土壤中含有较丰富的放线菌药用资源,具有从中发现放线菌新菌种和开发新抗生素的潜力。     

A genomics-guided approach for discovering and expressing cryptic metabolic pathways

Genome analysis of actinomycetes has revealed the presence of numerous cryptic gene clusters encoding putative natural products. These loci remain dormant until appropriate chemical or physical signals induce their expression. Here we demonstrate the use of a high-throughput genome scanning method to detect and analyze gene clusters involved in natural-product biosynthesis. This method was applied to uncover biosynthetic pathways encoding enediyne antitumor antibiotics in a variety of actinomycetes. Comparative analysis of five biosynthetic loci representative of the major structural classes of enediynes reveals the presence of a conserved cassette of five genes that includes a novel family of polyketide synthase (PKS). The enediyne PKS (PKSE) is proposed to be involved in the formation of the highly reactive chromophore ring structure (or "warhead") found in all enediynes. Genome scanning analysis indicates that the enediyne warhead cassette is widely dispersed among actinomycetes. We show that selective growth conditions can induce the expression of these loci, suggesting that the range of enediyne natural products may be much greater than previously thought. This technology can be used to increase the scope and diversity of natural-product discovery.     

链霉菌Streptomyces antibioticus NRRL 8167中Naphthgeranine A生物合成基因簇的分析鉴定

【背景】微生物来源的天然产物是小分子药物或药物先导物的重要来源。对链霉菌Streptomyces antibioticus NRRL 8167的基因组分析显示,其包含多个次级代谢产物的生物合成基因簇,具有产生多种新化合物的潜力。【目的】对链霉菌S. antibioticus NRRL 8167中次级代谢产物进行研究,以期发现结构新颖或生物活性独特的化合物,并对相应产物的生物合成基因簇和生物合成途径进行解析。【方法】利用HPLC图谱结合特征性紫外吸收和LC-MS方法,排除S. antibioticus NRRL 8167产生的已知化合物,确定具有特殊紫外吸收的化合物作为挖掘对象,然后利用正、反相硅胶柱色谱、高效液相色谱等技术对次级代谢产物进行分离纯化,分离化合物。利用质谱及核磁共振光谱技术对化合物结构进行解析和鉴定;提取链霉菌S. antibioticus NRRL 8167基因组DNA,利用PacBio测序平台进行基因组测序;利用生物信息学对基因组进行注释,并对合成该化合物的基因簇进行定位分析,推导其生物合成途径。【结果】确定这个化合物是NaphthgeranineA,属于聚酮类化合物。全基因组序列分析发现S.antibioticusNRRL8167基因组含有28个次级代谢产物生物合成基因簇,其中基因簇20可能负责Naphthgeranine A的生物合成,并对其生物合成途径进行了推导。【结论】基于紫外吸收光谱和质谱特征,从S. antibioticus NRRL 8167菌株的发酵提取物中分离鉴定了一个聚酮类化合物Naphthgeranine A。该菌株的全基因组测序为其生物合成基因簇的鉴定提供了前提,对Naphthgeranine A生物合成基因簇和生物合成途径的推测为进一步研究这个化合物的生物合成机制奠定了基础。     

Sponge-associated sp. RM66 metabolome induction with N-acetylglucosamine: Antibacterial,antifungal and anti-trypanosomal activities

The marine sponge Amphimedon sp., collected from Hurghada (Egypt) was investigated for its sponge-derived actinomycetes diversity. Nineteen actinomycetes were cultivated and phylogenetically identified using 16S rDNA gene sequencing were carried out. The strains belong to genera Kocuria, Dietzia, Micrococcus, Microbacterium and Streptomyces. Many silent biosynthetic genes clusters were investigated using genome sequencing of actinomycete strains and has revealed in particular the genus Streptomyces that has indicated their exceptional capacity for the secondary metabolites production that not observed under classical cultivation conditions. In this study, the effect of N-acetylglucosamine on the metabolome of Streptomyces sp. RM66 was investigated using three actinomycetes media (ISP2, M1 and MA). In total, twelve extracts were produced using solid and liquid fermentation approaches. Liquid chromatography-high resolution tandem mass spectrometry (LC-HRMS/MS) data were analysed using metabolomics tools to compare natural product production across all crude extracts. Our study highlighted the elicitation effect of N-acetylglucosamine on the secondary metabolite profiles of Streptomyces sp. RM66. These results highlight the of N-acetylglucosamine application as an elicitor to induce the cryptic metabolites and for increasing the chemical diversity. All the twelve extracts were tested for their antibacterial activity was tested against Staphylococcus aureus NCTC 8325, antifungal activity against Candida albicans 5314 (ATCC 90028) and anti-trypanosomal activity against Trypanosoma brucei brucei. Extract St1 showed the most potent one with activities 2.3, 3.2 and 4.7 ug/ml as antibacterial, antifungal and anti-trypanosomal, respectively.     

Biosynthesis of a natural polyketide-isoprenoid hybrid compound, furaquinocin A: identification and heterologous expression of the gene cluster

Furaquinocin (FQ) A, produced by Streptomyces sp. strain KO-3988, is a natural polyketide-isoprenoid hybrid compound that exhibits a potent antitumor activity. As a first step toward understanding the biosynthetic machinery of this unique and pharmaceutically useful compound, we have cloned an FQ A biosynthetic gene cluster by taking advantage of the fact that an isoprenoid biosynthetic gene cluster generally exists in flanking regions of the mevalonate (MV) pathway gene cluster in actinomycetes. Interestingly, Streptomyces sp. strain KO-3988 was the first example of a microorganism equipped with two distinct mevalonate pathway gene clusters. We were able to localize a 25-kb DNA region that harbored FQ A biosynthetic genes (fur genes) in both the upstream and downstream regions of one of the MV pathway gene clusters (MV2) by using heterologous expression in Streptomyces lividans TK23. This was the first example of a gene cluster responsible for the biosynthesis of a polyketide-isoprenoid hybrid compound. We have also confirmed that four genes responsible for viguiepinol [3-hydroxypimara-9(11),15-diene] biosynthesis exist in the upstream region of the other MV pathway gene cluster (MV1), which had previously been cloned from strain KO-3988. This was the first example of prokaryotic enzymes with these biosynthetic functions. By phylogenetic analysis, these two MV pathway clusters were identified as probably being independently distributed in strain KO-3988 (orthologs), rather than one cluster being generated by the duplication of the other cluster (paralogs).     

基因簇大片段克隆技术研究进展及挑战

天然产物结构复杂、活性多样,是新药开发的重要来源,对天然产物生物合成途径的研究,有利于探索酶催化的合成机制,促进复杂天然产物的应用。天然产物的生物合成由其对应的基因簇调控,其中大量天然产物生物合成基因簇(biosynthetic gene clusters,BGCs)在野生型菌株中无法表达或表达量低。对这些基因簇的研究,需要进行克隆表达,而如何克隆大片段基因簇并使其表达,从而发现新型天然产物是一个具有挑战性的问题。其中构建基因组文库、转化关联重组(transformation-associated recombination,TAR)、Red/ET重组等是克隆大片段基因簇的重要技术。本文从克隆技术的策略和应用两个方面,总结了这3种克隆技术目前的研究进展,讨论了目前大片段基因簇克隆技术面临的挑战,为研究大片段基因簇提供方法学借鉴。     

西沙石珊瑚共附生放线菌的分离培养及抗菌活性评价

【背景】珊瑚礁生态系统是海洋中一类极其重要的生态系统,健康珊瑚礁中丰富的共附生放线菌群体是珊瑚抵御各种致病菌的重要防线,因此,这类放线菌是寻找抗菌活性分子的重要资源,其药用潜力巨大。【目的】从西沙石珊瑚样品中分离共附生放线菌,并从中筛选具有良好抗菌活性的菌株。【方法】通过稀释涂布法分离珊瑚共附生放线菌,并根据16S rRNA基因序列构建系统发育树进行菌种鉴定;通过平板对峙法对放线菌进行抗菌活性筛选并确定目标菌株;将目标菌株涂布于不同氯化钠浓度的ISP2固体培养基上培养,测试其盐度耐受能力;通过平板对峙法对该菌株发酵产物的热稳定性和光稳定性进行测试;采用NanoPore和Illumina方法完成目标活性放线菌全基因组测序,并通过antiSMASH在线分析预测其次级代谢产物生物合成基因簇及其结构类型。【结果】从6份西沙石珊瑚样品中分离得到104株可培养放线菌,根据菌落形态和分离来源去重后对其中27株放线菌进行16S rRNA基因序列测序,通过序列比对和系统发育树分析将菌株初步鉴定为盐孢菌属(Salinispora)(25株)、链霉菌属(Streptomyces)(1株)和戈登菌属(Gord...     

PCR screening reveals considerable unexploited biosynthetic potential of ansamycins and a mysterious family of AHBA‐containing natural products in actinomycetes

Aims

Ansamycins are a family of macrolactams that are synthesized by type I polyketide synthase (PKS) using 3‐amino‐5‐hydroxybenzoic acid (AHBA) as the starter unit. Most members of the family have strong antimicrobial, antifungal, anticancer and/or antiviral activities. We aimed to discover new ansamycins and/or other AHBA‐containing natural products from actinobacteria.

Methods and Results

Through PCR screening of AHBA synthase gene, we identified 26 AHBA synthase gene–positive strains from 206 plant‐associated actinomycetes (five positives) and 688 marine‐derived actinomycetes (21 positives), representing a positive ratio of 2·4–3·1%. Twenty‐five ansamycins, including eight new compounds, were isolated from six AHBA synthase gene–positive strains through TLC‐guided fractionations followed by repeated column chromatography. To gain information about those potential ansamycin gene clusters whose products were unknown, seven strains with phylogenetically divergent AHBA synthase genes were subjected to fosmid library construction. Of the seven gene clusters we obtained, three show characteristics for typical ansamycin gene clusters, and other four, from Micromonospora spp., appear to lack the amide synthase gene, which is unusual for ansamycin biosynthesis. The gene composition of these four gene clusters suggests that they are involved in the biosynthesis of a new family of hybrid PK‐NRP compounds containing AHBA substructure.

Conclusions

PCR screening of AHBA synthase is an efficient approach to discover novel ansamycins and other AHBA‐containing natural products.

Significance and Impact of the Study

This work demonstrates that the AHBA‐based screening method is a useful approach for discovering novel ansamycins and other AHBA‐containing natural products from new microbial resources.     

Synthetic biology and metabolic engineering of actinomycetes for natural product discovery

Actinomycetes are one of the most valuable sources of natural products with industrial and medicinal importance. After more than half a century of exploitation, it has become increasingly challenging to find novel natural products with useful properties as the same known compounds are often repeatedly re-discovered when using traditional approaches. Modern genome mining approaches have led to the discovery of new biosynthetic gene clusters, thus indicating that actinomycetes still harbor a huge unexploited potential to produce novel natural products. In recent years, innovative synthetic biology and metabolic engineering tools have greatly accelerated the discovery of new natural products and the engineering of actinomycetes. In the first part of this review, we outline the successful application of metabolic engineering to optimize natural product production, focusing on the use of multi-omics data, genome-scale metabolic models, rational approaches to balance precursor pools, and the engineering of regulatory genes and regulatory elements. In the second part, we summarize the recent advances of synthetic biology for actinomycetal metabolic engineering including cluster assembly, cloning and expression, CRISPR/Cas9 technologies, and chassis strain development for natural product overproduction and discovery. Finally, we describe new advances in reprogramming biosynthetic pathways through polyketide synthase and non-ribosomal peptide synthetase engineering. These new developments are expected to revitalize discovery and development of new natural products with medicinal and other industrial applications.