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Genome sequencing and molecular networking analysis of the wild fungus Anthostomella pinea reveal its ability to produce a diverse range of secondary metabolites. 对野生真菌 Anthostomella pinea 的基因组测序和分子网络分析揭示了其产生多种次级代谢产物的能力。
Q1 Agricultural and Biological Sciences Pub Date : 2024-01-03 DOI: 10.1186/s40694-023-00170-1
R Iacovelli, T He, J L Allen, T Hackl, K Haslinger

Background: Filamentous fungi are prolific producers of bioactive molecules and enzymes with important applications in industry. Yet, the vast majority of fungal species remain undiscovered or uncharacterized. Here we focus our attention to a wild fungal isolate that we identified as Anthostomella pinea. The fungus belongs to a complex polyphyletic genus in the family of Xylariaceae, which is known to comprise endophytic and pathogenic fungi that produce a plethora of interesting secondary metabolites. Despite that, Anthostomella is largely understudied and only two species have been fully sequenced and characterized at a genomic level.

Results: In this work, we used long-read sequencing to obtain the complete 53.7 Mb genome sequence including the full mitochondrial DNA. We performed extensive structural and functional annotation of coding sequences, including genes encoding enzymes with potential applications in biotechnology. Among others, we found that the genome of A. pinea encodes 91 biosynthetic gene clusters, more than 600 CAZymes, and 164 P450s. Furthermore, untargeted metabolomics and molecular networking analysis of the cultivation extracts revealed a rich secondary metabolism, and in particular an abundance of sesquiterpenoids and sesquiterpene lactones. We also identified the polyketide antibiotic xanthoepocin, to which we attribute the anti-Gram-positive effect of the extracts that we observed in antibacterial plate assays.

Conclusions: Taken together, our results provide a first glimpse into the potential of Anthstomella pinea to provide new bioactive molecules and biocatalysts and will facilitate future research into these valuable metabolites.

背景:丝状真菌是生物活性分子和酶的大量生产者,在工业中有着重要的应用。然而,绝大多数真菌物种仍未被发现或定性。在此,我们重点关注一种野生真菌分离物,并将其鉴定为Anthostomella pinea。该真菌属于木犀科的一个复杂的多单胞属,众所周知,木犀科由内生真菌和致病真菌组成,能产生大量有趣的次级代谢产物。尽管如此,Anthostomella 在很大程度上仍未得到充分研究,只有两个物种在基因组水平上得到了完整测序和特征描述:在这项工作中,我们利用长线程测序技术获得了 53.7 Mb 的完整基因组序列,其中包括完整的线粒体 DNA。我们对编码序列进行了广泛的结构和功能注释,包括可能应用于生物技术的酶编码基因。其中,我们发现 A. pinea 的基因组编码 91 个生物合成基因簇、600 多个 CAZymes 和 164 个 P450s。此外,通过对栽培提取物进行非靶向代谢组学和分子网络分析,我们发现了丰富的次生代谢,尤其是大量的倍半萜和倍半萜内酯。我们还发现了多酮类抗生素黄腐素,我们在抗菌平板试验中观察到提取物具有抗革兰氏阳性菌的作用:综上所述,我们的研究结果让人们首次看到了茴芹提供新生物活性分子和生物催化剂的潜力,并将促进未来对这些宝贵代谢物的研究。
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引用次数: 0
Mechanical, physical and thermal properties of composite materials produced with the basidiomycete Fomes fomentarius. 由担子菌发酵而成的复合材料的机械、物理和热性能。
Q1 Agricultural and Biological Sciences Pub Date : 2023-12-04 DOI: 10.1186/s40694-023-00169-8
Bertram Schmidt, Carsten Freidank-Pohl, Justus Zillessen, Lisa Stelzer, Tamara Núñez Guitar, Carsten Lühr, Henri Müller, Fangxing Zhang, Jörg U Hammel, Heiko Briesen, Sascha Jung, Hans-Jörg Gusovius, Vera Meyer

Background: To achieve climate neutrality, fundamentally new concepts of circularity need to be implemented by the building sector as it contributes to 40% of anthropogenic CO2 emission. Fungal biotechnology can make a significant contribution here and help eliminate fossil dependency for building material production. Recently, we have shown that the medicinal polypore Fomes fomentarius feeds well on renewable lignocellulosic biomass and produces composite materials that could potentially replace fossil fuel-based expanded polystyrene as insulation material.

Results: In this study, we explored the mechanical, physical, and thermal properties of F. fomentarius-based composite materials in more detail and determined key performance parameters that are important to evaluate the usability of F. fomentarius-based composite materials in the construction sector. These parameters were determined according to European standards and included compressive strength, modulus of elasticity, thermal conductivity, water vapour permeability, and flammability of uncompressed composites as well as flexural strength, transverse tensile strength, and water absorption capacity of heat-pressed composites, among others. We could show that uncompressed composites obtained from F. fomentarius and hemp shives display a thermal conductivity of 0.044 W (m K)-1 which is in the range of natural organic fibres. A water vapour permeability of 1.72 and classification into flammability class B1 clearly surpasses fossil-based insulation materials including expanded polystyrene and polyurethane. We could furthermore show that heat-pressing can be used to reliably generate stiff and firm particleboards that have the potential to replace current wood-based particleboards that contain synthetic additives. X-ray microcomputed tomography finally visualized for the first time the growth of hyphae of F. fomentarius on and into the hemp shive substrates and generated high-resolution images of the microstructure of F. fomentarius-based composites.

Conclusion: This study demonstrates that fungal-based composites produced with F. fomentarius partially meet or even exceed key performance parameters of currently used fossil fuel-based insulation materials and can also be used to replace particleboards.

背景:为了实现气候中和,建筑行业需要从根本上实施新的循环概念,因为它贡献了40%的人为二氧化碳排放。真菌生物技术可以在这方面做出重大贡献,并有助于消除建筑材料生产对化石的依赖。最近,我们已经证明,药用多孔Fomes fomentarius很好地以可再生木质纤维素生物质为食,并生产复合材料,有可能取代化石燃料为基础的膨胀聚苯乙烯作为绝缘材料。结果:在本研究中,我们更详细地探讨了F. fomentarius基复合材料的机械、物理和热性能,并确定了评估F. fomentarius基复合材料在建筑领域可用性的关键性能参数。这些参数是根据欧洲标准确定的,包括未压缩复合材料的抗压强度、弹性模量、导热系数、水蒸气渗透性和可燃性,以及热压复合材料的抗弯强度、横向抗拉强度和吸水能力等。我们可以证明,从F. fomentarius和hemp shives中获得的未压缩复合材料的导热系数为0.044 W (m K)-1,在天然有机纤维的范围内。水蒸汽渗透性为1.72,可燃性为B1级,明显超过化石基绝缘材料,包括膨胀聚苯乙烯和聚氨酯。我们还可以进一步证明,热压可以可靠地生产出坚硬的刨花板,这种刨花板有可能取代目前含有合成添加剂的木质刨花板。最终,x射线显微计算机断层扫描首次显示了麻屑基质上和基质中fomentarius菌丝的生长情况,并生成了以fomentarius为基础的复合材料微观结构的高分辨率图像。结论:本研究表明,以真菌为原料制备的真菌基复合材料部分达到甚至超过了目前使用的化石燃料基绝热材料的关键性能参数,也可用于替代刨花板。
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引用次数: 0
Compatible solutes determine the heat resistance of conidia. 相容溶质决定分生孢子的耐热性。
Q1 Agricultural and Biological Sciences Pub Date : 2023-11-13 DOI: 10.1186/s40694-023-00168-9
Sjoerd J Seekles, Tom van den Brule, Maarten Punt, Jan Dijksterhuis, Mark Arentshorst, Maryam Ijadpanahsaravi, Winfried Roseboom, Gwendolin Meuken, Véronique Ongenae, Jordy Zwerus, Robin A Ohm, Gertjan Kramer, Han A B Wösten, Johannes H de Winde, Arthur F J Ram

Background: Asexually developed fungal spores (conidia) are key for the massive proliferation and dispersal of filamentous fungi. Germination of conidia and subsequent formation of a mycelium network give rise to many societal problems related to human and animal fungal diseases, post-harvest food spoilage, loss of harvest caused by plant-pathogenic fungi and moulding of buildings. Conidia are highly stress resistant compared to the vegetative mycelium and therefore even more difficult to tackle.

Results: In this study, complementary approaches are used to show that accumulation of mannitol and trehalose as the main compatible solutes during spore maturation is a key factor for heat resistance of conidia. Compatible solute concentrations increase during conidia maturation, correlating with increased heat resistance of mature conidia. This maturation only occurs when conidia are attached to the conidiophore. Moreover, conidia of a mutant Aspergillus niger strain, constructed by deleting genes involved in mannitol and trehalose synthesis and consequently containing low concentrations of these compatible solutes, exhibit a sixteen orders of magnitude more sensitive heat shock phenotype compared to wild-type conidia. Cultivation at elevated temperature results in adaptation of conidia with increased heat resistance. Transcriptomic and proteomic analyses revealed two putative heat shock proteins to be upregulated under these conditions. However, conidia of knock-out strains lacking these putative heat shock proteins did not show a reduced heat resistance.

Conclusions: Heat stress resistance of fungal conidia is mainly determined by the compatible solute composition established during conidia maturation. To prevent heat resistant fungal spore contaminants, food processing protocols should consider environmental conditions stimulating compatible solute accumulation and potentially use compatible solute biosynthesis as a novel food preservation target.

背景:无性发育的真菌孢子(分生孢子)是丝状真菌大量繁殖和扩散的关键。分生孢子的萌发和随后菌丝体网络的形成引起了许多社会问题,这些问题与人类和动物的真菌疾病、收获后的食物腐败、植物病原真菌引起的收获损失和建筑物的霉变有关。与营养菌丝相比,分生孢子具有很强的抗逆性,因此更难处理。结果:本研究采用互补方法表明,甘露醇和海藻糖作为主要相容溶质在孢子成熟过程中的积累是分生孢子耐热性的关键因素。相容溶质浓度在分生孢子成熟过程中增加,与成熟分生孢子耐热性增加有关。只有当分生孢子附着在分生孢子上时,这种成熟才会发生。此外,通过删除甘露醇和海藻糖合成相关基因构建的黑曲霉突变菌株的分生孢子含有低浓度的这些相容溶质,与野生型分生孢子相比,表现出16个数量级的热休克敏感表型。高温培养使分生孢子耐热性增强。转录组学和蛋白质组学分析揭示了两种假定的热休克蛋白在这些条件下上调。然而,敲除菌株缺乏这些假定的热休克蛋白的分生孢子没有表现出降低的耐热性。结论:真菌分生孢子的耐热性主要取决于分生孢子成熟过程中形成的相容溶质组成。为了防止耐热真菌孢子污染,食品加工方案应考虑刺激相容溶质积累的环境条件,并可能使用相容溶质生物合成作为新的食品保存目标。
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引用次数: 0
Identification and evaluation of suitable reference genes for RT-qPCR analyses in Trichoderma atroviride under varying light conditions. 在不同光照条件下鉴定和评价用于RT-qPCR分析的合适参考基因。
Q1 Agricultural and Biological Sciences Pub Date : 2023-10-03 DOI: 10.1186/s40694-023-00167-w
Daniel Flatschacher, Alexander Eschlböck, Susanne Zeilinger

Background: Trichoderma atroviride is a competitive soil-borne mycoparasitic fungus with extensive applications as a biocontrol agent in plant protection. Despite its importance and application potential, reference genes for RT-qPCR analysis in T. atroviride have not been evaluated. Light exerts profound effects on physiology, such as growth, conidiation, secondary metabolism, and stress response in T. atroviride, as well as in other fungi. In this study, we aimed to address this gap by identifying stable reference genes for RT-qPCR experiments in T. atroviride under different light conditions, thereby enhancing accurate and reliable gene expression analysis in this model mycoparasite. We measured and compared candidate reference genes using commonly applied statistical algorithms.

Results: Under cyclic light-dark cultivation conditions, tbp and rho were identified as the most stably expressed genes, while act1, fis1, btl, and sar1 were found to be the least stable. Similar stability rankings were obtained for cultures grown under complete darkness, with tef1 and vma1 emerging as the most stable genes and act1, rho, fis1, and btl as the least stable genes. Combining the data from both cultivation conditions, gapdh and vma1 were identified as the most stable reference genes, while sar1 and fis1 were the least stable. The selection of different reference genes had a significant impact on the calculation of relative gene expression, as demonstrated by the expression patterns of target genes pks4 and lox1.

Conclusion: The data emphasize the importance of validating reference genes for different cultivation conditions in fungi to ensure accurate interpretation of gene expression data.

背景:萎缩木霉是一种具有竞争力的土传真菌寄生菌,在植物保护中具有广泛的生物防治作用。尽管其重要性和应用潜力很大,但用于逆转录-qPCR分析的参考基因尚未得到评估。光对T.atroviride和其他真菌的生长、分生孢子、次生代谢和应激反应等生理学产生了深远的影响。在这项研究中,我们旨在通过在不同光照条件下在曲韦里进行RT-qPCR实验来确定稳定的参考基因来解决这一差距,从而增强该模型分枝杆菌中准确可靠的基因表达分析。我们使用常用的统计算法测量和比较候选参考基因。结果:在循环明暗培养条件下,tbp和rho被鉴定为最稳定表达的基因,而act1、fis1、btl和sar1被发现是最不稳定的。在完全黑暗下生长的培养物也获得了类似的稳定性排名,其中tef1和vma1是最稳定的基因,act1、rho、fis1和btl是最不稳定的基因。结合两种培养条件的数据,gapdh和vma1被确定为最稳定的参考基因,而sar1和fis1是最不稳定的。目标基因pks4和lox1的表达模式表明,不同参考基因的选择对相对基因表达的计算有显著影响。结论:这些数据强调了在真菌的不同培养条件下验证参考基因的重要性,以确保基因表达数据的准确解释。
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引用次数: 0
Non-canonical two-step biosynthesis of anti-oomycete indole alkaloids in Kickxellales. 木螺中抗卵霉菌吲哚类生物碱的非典型两步生物合成。
Q1 Agricultural and Biological Sciences Pub Date : 2023-09-05 DOI: 10.1186/s40694-023-00166-x
Johannes Rassbach, Nathalie Hilsberg, Veit G Haensch, Sebastian Dörner, Julia Gressler, Robin Sonnabend, Caroline Semm, Kerstin Voigt, Christian Hertweck, Markus Gressler

Background: Fungi are prolific producers of bioactive small molecules of pharmaceutical or agricultural interest. The secondary metabolism of higher fungi (Dikarya) has been well-investigated which led to > 39,000 described compounds. However, natural product researchers scarcely drew attention to early-diverging fungi (Mucoro- and Zoopagomycota) as they are considered to rarely produce secondary metabolites. Indeed, only 15 compounds have as yet been isolated from the entire phylum of the Zoopagomycota.

Results: Here, we showcase eight species of the order Kickxellales (phylum Zoopagomycota) as potent producers of the indole-3-acetic acid (IAA)-derived compounds lindolins A and B. The compounds are produced both under laboratory conditions and in the natural soil habitat suggesting a specialized ecological function. Indeed, lindolin A is a selective agent against plant-pathogenic oomycetes such as Phytophthora sp. Lindolin biosynthesis was reconstituted in vitro and relies on the activity of two enzymes of dissimilar evolutionary origin: Whilst the IAA-CoA ligase LinA has evolved from fungal 4-coumaryl-CoA synthetases, the subsequently acting IAA-CoA:anthranilate N-indole-3-acetyltransferase LinB is a unique enzyme across all kingdoms of life.

Conclusions: This is the first report on bioactive secondary metabolites in the subphylum Kickxellomycotina and the first evidence for a non-clustered, two-step biosynthetic route of secondary metabolites in early-diverging fungi. Thus, the generally accepted "gene cluster hypothesis" for natural products needs to be reconsidered for early diverging fungi.

背景:真菌是多产的具有生物活性的小分子药物或农业利益的生产者。高等真菌(Dikarya)的次级代谢已经得到了很好的研究,导致了大约39,000种已描述的化合物。然而,天然产物研究人员很少注意到早期分化真菌(Mucoro-和Zoopagomycota),因为它们被认为很少产生次生代谢物。事实上,迄今为止,只有15种化合物从整个动物菌门中分离出来。结果:在这里,我们展示了八种Kickxellales (Zoopagomycota门)作为吲哚-3-乙酸(IAA)衍生化合物lindolins A和lindolins b的强有力的产生者,这些化合物在实验室条件下和自然土壤栖息地都能产生,表明一种特殊的生态功能。事实上,lindolin A是一种针对植物致病性卵菌(如疫霉菌)的选择性制剂。lindolin的生物合成在体外重建,依赖于两种不同进化起源的酶的活性:虽然IAA-CoA连接酶LinA是从真菌的4-香豆烯- coa合成酶进化而来,但随后作用的IAA-CoA: n -吲哚-3-乙酰转移酶LinB是所有生命领域中独特的酶。结论:本文首次报道了Kickxellomycotina亚门次生代谢物的生物活性,首次证实了早期分化真菌次生代谢物的非聚类、两步生物合成途径。因此,普遍接受的天然产物“基因簇假说”需要重新考虑早期分化真菌。
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引用次数: 1
CRISPR/Cas9 mediated gene editing of transcription factor ACE1 for enhanced cellulase production in thermophilic fungus Rasamsonia emersonii. CRISPR/Cas9介导的转录因子ACE1基因编辑增强了嗜热真菌拉森索尼的纤维素酶生产。
Q1 Agricultural and Biological Sciences Pub Date : 2023-09-01 DOI: 10.1186/s40694-023-00165-y
Varinder Singh, Yashika Raheja, Neha Basotra, Gaurav Sharma, Adrian Tsang, Bhupinder Singh Chadha

Background: The filamentous fungus Rasamsonia emersonii has immense potential to produce biorefinery relevant thermostable cellulase and hemicellulase enzymes using lignocellulosic biomass. Previously in our lab, a hyper-cellulase producing strain of R. emersonii was developed through classical breeding and system biology approaches. ACE1, a pivotal transcription factor in fungi, plays a crucial role in negatively regulating the expression of cellulase genes. In order to identify the role of ACE1 in cellulase production and to further improve the lignocellulolytic enzyme production in R. emersonii, CRISPR/Cas9 mediated disruption of ACE1 gene was employed.

Results: A gene-edited ∆ACE1 strain (GN11) was created, that showed 21.97, 20.70 and 24.63, 9.42, 18.12%, improved endoglucanase, cellobiohydrolase (CBHI), β-glucosidase, FPase, and xylanase, activities, respectively, as compared to parental strain M36. The transcriptional profiling showed that the expression of global regulator (XlnR) and different CAZymes genes including endoglucanases, cellobiohydrolase, β-xylosidase, xylanase, β-glucosidase and lytic polysaccharide mono-oxygenases (LPMOs) were significantly enhanced, suggesting critical roles of ACE1 in negatively regulating the expression of various key genes associated with cellulase production in R. emersonii. Whereas, the disruption of ACE1 significantly down-regulated the expression of CreA repressor gene as also evidenced by 2-deoxyglucose (2-DG) resistance phenotype exhibited by edited strain GN11 as well as appreciably higher constitutive production of cellulases in the presence of glucose and mixture of glucose and disaccharide (MGDs) both in batch and flask fed batch mode of culturing. Furthermore, ∆ACE1 strains were evaluated for the hydrolysis of biorefinery relevant steam/acid pretreated unwashed rice straw slurry (Praj Industries Ltd; 15% substrate loading rate) and were found to be significantly superior when compared to the benchmark enzymes produced by parent strain M36 and Cellic Ctec3.

Conclusions: Current work uncovers the crucial role of ACE1 in regulating the expression of the various cellulase genes and carbon catabolite repression mechanism in R. emersonii. This study represents the first successful report of utilizing CRISPR/Cas9 genome editing technology to disrupt the ACE1 gene in the thermophlic fungus R. emersonii. The improved methodologies presented in this work might be applied to other commercially important fungal strains for which genetic manipulation tools are limited.

背景:丝状真菌拉斯穆森(Rasamsonia emersonii)利用木质纤维素生物质生产生物精炼相关的耐热纤维素酶和半纤维素酶具有巨大的潜力。在我们实验室之前,通过经典育种和系统生物学方法开发了一种产生超纤维素酶的爱默生氏弧菌菌株。ACE1是真菌中的关键转录因子,在负向调控纤维素酶基因的表达中起着至关重要的作用。为了确定ACE1在纤维素酶生产中的作用,进一步提高爱默生氏弧菌的木质纤维素酶生产,采用CRISPR/Cas9介导的ACE1基因的破坏。结果:获得基因编辑的∆ACE1菌株(GN11),与亲本菌株M36相比,其内切葡聚糖酶、纤维生物水解酶(chi)、β-葡萄糖苷酶、FPase和木聚糖酶活性分别提高了21.97、20.70和24.63、9.42、18.12%。转录谱分析结果显示,acei - 1与内源性葡聚糖酶、纤维素生物水解酶、β-木糖苷酶、木聚糖酶、β-葡萄糖苷酶和水解多糖单加氧酶等CAZymes基因的表达显著增强,表明acei - 1在负调控与纤维素酶生产相关的多种关键基因的表达中起着关键作用。然而,ACE1的破坏显著下调了CreA抑制基因的表达,这也证明了编辑菌株GN11表现出的2-脱氧葡萄糖(2-DG)抗性表型,以及在葡萄糖和葡萄糖-双糖混合物(MGDs)存在下的纤维素酶的组成产量明显提高,无论是在批量培养还是瓶喂批量培养模式下。此外,测定了∆ACE1菌株对生物炼制相关蒸汽/酸预处理的未水洗稻草浆的水解作用(Praj Industries Ltd;15%底物负载率),与亲本菌株M36和Cellic Ctec3产生的基准酶相比,具有显著的优势。结论:目前的工作揭示了ACE1在调节爱默生氏弧菌各种纤维素酶基因表达和碳分解代谢抑制机制中的重要作用。本研究首次成功报道了利用CRISPR/Cas9基因组编辑技术破坏嗜热真菌爱默生弧菌(R. emersonii)中的ACE1基因。在这项工作中提出的改进方法可能应用于其他商业上重要的真菌菌株,其中遗传操作工具是有限的。
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引用次数: 0
Basidiomycete non-reducing polyketide synthases function independently of SAT domains. 担子菌非还原性多酮合成酶独立于SAT结构域起作用。
Q1 Agricultural and Biological Sciences Pub Date : 2023-08-04 DOI: 10.1186/s40694-023-00164-z
Nikolai A Löhr, Malik Rakhmanov, Jacob M Wurlitzer, Gerald Lackner, Markus Gressler, Dirk Hoffmeister

Background: Non-reducing polyketide synthases (NR-PKSs) account for a major share of natural product diversity produced by both Asco- and Basidiomycota. The present evolutionary diversification into eleven clades further underscores the relevance of these multi-domain enzymes. Following current knowledge, NR-PKSs initiate polyketide assembly by an N-terminal starter unit:acyl transferase (SAT) domain that catalyzes the transfer of an acetyl starter from the acetyl-CoA thioester onto the acyl carrier protein (ACP).

Results: A comprehensive phylogenetic analysis of NR-PKSs established a twelfth clade from which three representatives, enzymes CrPKS1-3 of the webcap mushroom Cortinarius rufoolivaceus, were biochemically characterized. These basidiomycete synthases lack a SAT domain yet are fully functional hepta- and octaketide synthases in vivo. Three members of the other clade of basidiomycete NR-PKSs (clade VIII) were produced as SAT-domainless versions and analyzed in vivo and in vitro. They retained full activity, thus corroborating the notion that the SAT domain is dispensable for many basidiomycete NR-PKSs. For comparison, the ascomycete octaketide synthase atrochrysone carboxylic acid synthase (ACAS) was produced as a SAT-domainless enzyme as well, but turned out completely inactive. However, a literature survey revealed that some NR-PKSs of ascomycetes carry mutations within the catalytic motif of the SAT domain. In these cases, the role of the domain and the origin of the formal acetate unit remains open.

Conclusions: The role of SAT domains differs between asco- and basidiomycete NR-PKSs. For the latter, it is not part of the minimal set of NR-PKS domains and not required for function. This knowledge may help engineer compact NR-PKSs for more resource-efficient routes. From the genomic standpoint, seemingly incomplete or corrupted genes encoding SAT-domainless NR-PKSs should not automatically be dismissed as non-functional pseudogenes, but considered during genome analysis to decipher the potential arsenal of natural products of a given fungus.

背景:非还原性聚酮合成酶(NR-PKSs)在Asco-和担子菌科产生的天然产物多样性中占主要份额。目前进化多样化为11支进一步强调了这些多结构域酶的相关性。根据目前的知识,NR-PKSs通过n端起始单元:酰基转移酶(SAT)结构域启动聚酮组装,催化乙酰基起始物从乙酰辅酶a硫酯转移到酰基载体蛋白(ACP)上。结果:通过对NR-PKSs的综合系统发育分析,建立了第12枝,并从该枝中鉴定了3个代表性的CrPKS1-3酶。这些担子菌合成酶缺乏SAT结构域,但在体内是功能齐全的七肽和八肽合成酶。对担子菌nr - pks的另一个分支(分支VIII)的三个成员进行了无sat结构域版本的制备,并在体内和体外进行了分析。它们保留了充分的活性,从而证实了SAT结构域对于许多担子菌nr - pks是必不可少的。相比之下,子囊菌八肽合成酶atrochrysone羧酸合成酶(ACAS)也作为无sat结构域的酶被生产出来,但被证明是完全无活性的。然而,一项文献调查显示,子囊菌的一些NR-PKSs在SAT结构域的催化基序中携带突变。在这些情况下,结构域的作用和正式醋酸酯单元的起源仍然是开放的。结论:在asco-和担子菌NR-PKSs中,SAT结构域的作用是不同的。对于后者,它不是NR-PKS结构域最小集的一部分,也不是功能所必需的。这一知识可能有助于设计更紧凑的NR-PKSs,以获得更有效的资源。从基因组的角度来看,编码sat -domain - less nr - pks的看似不完整或损坏的基因不应被自动视为非功能假基因,而应在基因组分析中考虑,以破译给定真菌的潜在天然产物库。
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引用次数: 0
Antimicrobial activities of metabolites isolated from endophytic Aspergillus flavus of Sarcophyton ehrenbergi supported by in-silico study and NMR spectroscopy. 石藻内生黄曲霉代谢物的抑菌活性及核磁共振研究。
Q1 Agricultural and Biological Sciences Pub Date : 2023-08-02 DOI: 10.1186/s40694-023-00161-2
Abdel Nasser B Singab, Yasmin A Elkhawas, Eman Al-Sayed, Ahmed M Elissawy, Iten M Fawzy, Nada M Mostafa

Background: Endophytic Aspergillus species produce countless valuable bioactive secondary metabolites. In the current study, Aspergillus flavus an endophyte from the soft coral Sarcophyton ehrenbergi was chemically explored and the extracted phytoconstituents were subsequently evaluated for antimicrobial activity. This is accomplished by employing nuclear magnetic resonance (NMR) spectroscopy and computational techniques. Additionally, An in vitro anticancer analysis of A. flavus total extract against breast cancer cells (MCF-7) was investigated.

Result: Six compounds were separated from the crude alcohol extract of the endophytic Aspergillus flavus out of which anhydro-mevalonolactone was reported for the first time. The anti-fungal and anti-Helicobacter pylori properties of two distinct compounds (Scopularides A and B) were assessed. Additionally, computational research was done to identify the binding mechanisms for all compounds. Both the compounds were found to be active against H. pylori with minimum inhibitory concentration (MIC) values ranging from 7.81 to 15.63 µg/ mL as compared with clarithromycin 1.95 µg/ mL. Scopularides A was potent against both Candida albicans and Aspergillus niger with MIC values ranging from 3.9 to 31.25 µg/ mL, while scopularides B only inhibits Candida albicans with MIC value of 15.63 µg/ mL and weak inhibitory activity against A. niger (MIC = 125 µg/ mL). Furthermore, cytotoxic activity showed a significant effect (IC50: 30.46 mg/mL) against MCF-7 cells.

Conclusion: Our findings report that cytotoxic activity and molecular docking support the antimicrobial activity of Aspergillus flavus, which could be a promising alternative source as a potential antimicrobial agent.

背景:内生曲霉产生无数有价值的生物活性次生代谢物。本研究对软珊瑚中黄曲霉的一种内生菌进行了化学探索,并对其提取的植物成分进行了抑菌活性评价。这是通过使用核磁共振(NMR)光谱学和计算技术来完成的。此外,还研究了黄芪总提取物对乳腺癌细胞(MCF-7)的体外抗癌作用。结果:从内生真菌黄曲霉粗醇提取物中分离得到6个化合物,其中无水美伐内酯为首次报道。对两种不同化合物(Scopularides A和B)的抗真菌和抗幽门螺杆菌性能进行了评估。此外,还进行了计算研究,以确定所有化合物的结合机制。两种化合物对幽门螺杆菌均有抑制作用,最小抑制浓度(MIC)值为7.81 ~ 15.63µg/ mL,克拉霉素为1.95µg/ mL。scopscopides A对白色念珠菌和黑曲霉均有抑制作用,MIC值为3.9 ~ 31.25µg/ mL, scopscopides B仅对白色念珠菌有抑制作用,MIC值为15.63µg/ mL,对黑曲霉的抑制作用较弱(MIC = 125µg/ mL)。此外,对MCF-7细胞具有显著的细胞毒活性(IC50: 30.46 mg/mL)。结论:黄曲霉的细胞毒活性和分子对接支持其抗菌活性,是一种很有前景的潜在抗菌药物。
{"title":"Antimicrobial activities of metabolites isolated from endophytic Aspergillus flavus of Sarcophyton ehrenbergi supported by in-silico study and NMR spectroscopy.","authors":"Abdel Nasser B Singab,&nbsp;Yasmin A Elkhawas,&nbsp;Eman Al-Sayed,&nbsp;Ahmed M Elissawy,&nbsp;Iten M Fawzy,&nbsp;Nada M Mostafa","doi":"10.1186/s40694-023-00161-2","DOIUrl":"https://doi.org/10.1186/s40694-023-00161-2","url":null,"abstract":"<p><strong>Background: </strong>Endophytic Aspergillus species produce countless valuable bioactive secondary metabolites. In the current study, Aspergillus flavus an endophyte from the soft coral Sarcophyton ehrenbergi was chemically explored and the extracted phytoconstituents were subsequently evaluated for antimicrobial activity. This is accomplished by employing nuclear magnetic resonance (NMR) spectroscopy and computational techniques. Additionally, An in vitro anticancer analysis of A. flavus total extract against breast cancer cells (MCF-7) was investigated.</p><p><strong>Result: </strong>Six compounds were separated from the crude alcohol extract of the endophytic Aspergillus flavus out of which anhydro-mevalonolactone was reported for the first time. The anti-fungal and anti-Helicobacter pylori properties of two distinct compounds (Scopularides A and B) were assessed. Additionally, computational research was done to identify the binding mechanisms for all compounds. Both the compounds were found to be active against H. pylori with minimum inhibitory concentration (MIC) values ranging from 7.81 to 15.63 µg/ mL as compared with clarithromycin 1.95 µg/ mL. Scopularides A was potent against both Candida albicans and Aspergillus niger with MIC values ranging from 3.9 to 31.25 µg/ mL, while scopularides B only inhibits Candida albicans with MIC value of 15.63 µg/ mL and weak inhibitory activity against A. niger (MIC = 125 µg/ mL). Furthermore, cytotoxic activity showed a significant effect (IC<sub>50</sub>: 30.46 mg/mL) against MCF-7 cells.</p><p><strong>Conclusion: </strong>Our findings report that cytotoxic activity and molecular docking support the antimicrobial activity of Aspergillus flavus, which could be a promising alternative source as a potential antimicrobial agent.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"10 1","pages":"16"},"PeriodicalIF":0.0,"publicationDate":"2023-08-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10394880/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9929822","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 1
Rapid and robust squashed spore/colony PCR of industrially important fungi. 工业上重要真菌的快速和稳健的压扁孢子/菌落PCR。
Q1 Agricultural and Biological Sciences Pub Date : 2023-07-08 DOI: 10.1186/s40694-023-00163-0
Guoliang Yuan, Jeffrey J Czajka, Ziyu Dai, Dehong Hu, Kyle R Pomraning, Beth A Hofstad, Joonhoon Kim, Ana L Robles, Shuang Deng, Jon K Magnuson

Background: Fungi have been utilized for centuries in medical, agricultural, and industrial applications. Development of systems biology techniques has enabled the design and metabolic engineering of these fungi to produce novel fuels, chemicals, and enzymes from renewable feedstocks. Many genetic tools have been developed for manipulating the genome and creating mutants rapidly. However, screening and confirmation of transformants remain an inefficient step within the design, build, test, and learn cycle in many industrial fungi because extracting fungal genomic DNA is laborious, time-consuming, and involves toxic chemicals.

Results: In this study we developed a rapid and robust technique called "Squash-PCR" to break open the spores and release fungal genomic DNA as a template for PCR. The efficacy of Squash-PCR was investigated in eleven different filamentous fungal strains. Clean PCR products with high yields were achieved in all tested fungi. Spore age and type of DNA polymerase did not affect the efficiency of Squash-PCR. However, spore concentration was found to be the crucial factor for Squash-PCR in Aspergillus niger, with the dilution of starting material often resulting in higher PCR product yield. We then further evaluated the applicability of the squashing procedure for nine different yeast strains. We found that Squash-PCR can be used to improve the quality and yield of colony PCR in comparison to direct colony PCR in the tested yeast strains.

Conclusion: The developed technique will enhance the efficiency of screening transformants and accelerate genetic engineering in filamentous fungi and yeast.

背景:真菌在医学、农业和工业上的应用已经有几个世纪了。系统生物学技术的发展使这些真菌的设计和代谢工程能够从可再生原料中生产新的燃料、化学品和酶。为了操纵基因组和快速创造突变体,已经开发了许多遗传工具。然而,在许多工业真菌的设计、构建、测试和学习周期中,筛选和确认转化体仍然是一个效率低下的步骤,因为提取真菌基因组DNA既费力又耗时,而且涉及有毒化学物质。结果:在这项研究中,我们开发了一种快速而强大的技术,称为“南瓜PCR”,可以打开孢子并释放真菌基因组DNA作为PCR的模板。研究了Squash-PCR在11株不同丝状真菌中的应用效果。所有被试真菌均获得了纯度高的PCR产物。孢子年龄和DNA聚合酶类型对南瓜pcr的效率没有影响。然而,在黑曲霉中,孢子浓度被发现是南瓜PCR的关键因素,起始材料的稀释通常会导致更高的PCR产物产量。然后,我们进一步评估了压扁程序对九种不同酵母菌株的适用性。我们发现,与直接集落PCR相比,squsquash -PCR可以提高所测试酵母菌的集落PCR的质量和产量。结论:该技术将提高丝状真菌和酵母菌转化子的筛选效率,加快基因工程的发展。
{"title":"Rapid and robust squashed spore/colony PCR of industrially important fungi.","authors":"Guoliang Yuan,&nbsp;Jeffrey J Czajka,&nbsp;Ziyu Dai,&nbsp;Dehong Hu,&nbsp;Kyle R Pomraning,&nbsp;Beth A Hofstad,&nbsp;Joonhoon Kim,&nbsp;Ana L Robles,&nbsp;Shuang Deng,&nbsp;Jon K Magnuson","doi":"10.1186/s40694-023-00163-0","DOIUrl":"https://doi.org/10.1186/s40694-023-00163-0","url":null,"abstract":"<p><strong>Background: </strong>Fungi have been utilized for centuries in medical, agricultural, and industrial applications. Development of systems biology techniques has enabled the design and metabolic engineering of these fungi to produce novel fuels, chemicals, and enzymes from renewable feedstocks. Many genetic tools have been developed for manipulating the genome and creating mutants rapidly. However, screening and confirmation of transformants remain an inefficient step within the design, build, test, and learn cycle in many industrial fungi because extracting fungal genomic DNA is laborious, time-consuming, and involves toxic chemicals.</p><p><strong>Results: </strong>In this study we developed a rapid and robust technique called \"Squash-PCR\" to break open the spores and release fungal genomic DNA as a template for PCR. The efficacy of Squash-PCR was investigated in eleven different filamentous fungal strains. Clean PCR products with high yields were achieved in all tested fungi. Spore age and type of DNA polymerase did not affect the efficiency of Squash-PCR. However, spore concentration was found to be the crucial factor for Squash-PCR in Aspergillus niger, with the dilution of starting material often resulting in higher PCR product yield. We then further evaluated the applicability of the squashing procedure for nine different yeast strains. We found that Squash-PCR can be used to improve the quality and yield of colony PCR in comparison to direct colony PCR in the tested yeast strains.</p><p><strong>Conclusion: </strong>The developed technique will enhance the efficiency of screening transformants and accelerate genetic engineering in filamentous fungi and yeast.</p>","PeriodicalId":52292,"journal":{"name":"Fungal Biology and Biotechnology","volume":"10 1","pages":"15"},"PeriodicalIF":0.0,"publicationDate":"2023-07-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10329332/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10166712","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
引用次数: 0
Bifurcate evolution of quinone synthetases in basidiomycetes. 基枝菌中醌合成酶的分叉进化。
Q1 Agricultural and Biological Sciences Pub Date : 2023-07-03 DOI: 10.1186/s40694-023-00162-1
Paula Sophie Seibold, Stefanie Lawrinowitz, Ihar Raztsou, Markus Gressler, Hans-Dieter Arndt, Pierre Stallforth, Dirk Hoffmeister

Background: The terphenylquinones represent an ecologically remarkable class of basidiomycete natural products as they serve as central precursors of pigments and compounds that impact on microbial consortia by modulating bacterial biofilms and motility. This study addressed the phylogenetic origin of the quinone synthetases that assemble the key terphenylquinones polyporic acid and atromentin.

Results: The activity of the Hapalopilus rutilans synthetases HapA1, HapA2 and of Psilocybe cubensis PpaA1 were reconstituted in Aspergilli. Liquid chromatography and mass spectrometry of the culture extracts identified all three enzymes as polyporic acid synthetases. PpaA1 is unique in that it features a C-terminal, yet catalytically inactive dioxygenase domain. Combined with bioinformatics to reconstruct the phylogeny, our results demonstrate that basidiomycete polyporic acid and atromentin synthetases evolved independently, although they share an identical catalytic mechanism and release structurally very closely related products. A targeted amino acid replacement in the substrate binding pocket of the adenylation domains resulted in bifunctional synthetases producing both polyporic acid and atromentin.

Conclusions: Our results imply that quinone synthetases evolved twice independently in basidiomycetes, depending on the aromatic α-keto acid substrate. Furthermore, key amino acid residues for substrate specificity were identified and changed which led to a relaxed substrate profile. Therefore, our work lays the foundation for future targeted enzyme engineering.

背景:三联苯醌类化合物是基枝菌中具有生态学意义的一类天然产物,它们是色素和化合物的核心前体,通过调节细菌的生物膜和运动性对微生物联合体产生影响。本研究探讨了醌合成酶的系统发育起源,该酶组装了关键的三苯基醌类化合物多孔菌酸和阿托门汀:结果:在曲霉菌中重组了 Hapalopilus rutilans 合成酶 HapA1、HapA2 和 Psilocybe cubensis PpaA1 的活性。培养物提取物的液相色谱法和质谱法鉴定出这三种酶都是多孔菌酸合成酶。PpaA1 的独特之处在于它有一个 C-末端但无催化活性的二氧化酶结构域。结合生物信息学重建系统发育,我们的研究结果表明,基枝菌多孔菌酸合成酶和阿托菌素合成酶是独立进化的,尽管它们具有相同的催化机制,并释放出结构上非常接近的产物。在腺苷酸化结构域的底物结合袋中进行有针对性的氨基酸置换,可产生同时产生多孔菌酸和阿托菌素的双功能合成酶:我们的研究结果表明,醌合成酶在基枝菌中根据芳香族α-酮酸底物的不同独立进化了两次。此外,我们还发现了底物特异性的关键氨基酸残基,这些氨基酸残基的改变导致了底物特征的放宽。因此,我们的工作为未来有针对性的酶工程奠定了基础。
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引用次数: 0
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Fungal Biology and Biotechnology
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