真菌沉默基因簇激活策略研究进展

真菌次生代谢产物一直是天然药物的重要源泉,然而目前人们从真菌中发现新天然产物的几率越来越低。近年来,随着基因组测序技术的飞速发展,越来越多的真菌基因组被解密。生物信息分析显示,真菌基因组中编码天然产物的基因簇数目远远大于已从中发现的天然产物数量,提示真菌中尚存在大量的沉默基因簇有待深入发掘。因此,如何高效地激活真菌沉默基因簇已成为后基因组时代深度利用真菌资源的关键。系统总结了近年来真菌沉默基因簇激活策略的研究进展,以期为真菌资源的高效利用提供参考。     

激活细菌沉默基因簇的研究进展

目的综述了近年来细菌沉默基因簇激活策略的研究进展。方法通过查阅文献对激活细菌沉默基因簇的研究进展进行了总结。结果微生物基因组序列的持续揭示表明,细菌在合成各种天然产物方面蕴藏着巨大的、尚未开发的潜能。一般来说,这些天然产物一直是药物先导化合物的重要来源。在标准的实验室条件下,大多数生物合成基因簇处于低表达或沉默状态。为此,我们回顾了近年来已开发的三种激活沉默生物合成基因簇的方法:使用CRISPR/Cas9插入活性启动子,高通量筛选激活剂用以鉴定小分子诱导剂,以及报告基因指导下的突变体筛选用以构建高产菌株。结论结合以前实施的策略,这些方法有望在未来几年内获得由这些沉默基因簇编码的天然产物。     

OSMAC策略探索海洋真菌次生代谢产物的研究进展

海洋生态环境(如高盐、高压、低温和寡营养等)以及海洋生物物种间复杂广泛关系赋予了海洋真菌独特的新陈代谢途径及适应机制,因此相比于陆地来源的相同种属真菌,海洋来源真菌能够产生结构独特、丰富多样、生物活性显著的化合物。迄今为止,已经有上万种化合物从海洋真菌中分离出来,但受限于传统培养技术,新结构和活性化合物的发现几率和速度都有所下降。“一株多化合物”(one stain-many compounds, OSMAC)策略是传统培养方法的发展,为寻找新型天然产物提供了新的方法,提高挖掘海洋真菌合成次级代谢产物的能力,逐渐被应用于海洋天然产物的发现。本文对OSMAC策略在海洋真菌次生代谢产物发现及活性研究中的应用进行总结,旨在为提高海洋真菌次生代谢产物的新颖性和多样性,加快先导化合物和生物资源的研究提供参考。     

激活沉默基因以产生新抗生素的研究进展

基因工程技术的不断成熟使得人们寻找新抗生素和新生理活性物质的途径更加多样化,更具针对性。本文将具体介绍一种利用重金属选择压力激活金属耐受放线菌的沉默基因以获得新抗生素的方法,以及人们分析基因组测序发现很多隐藏的生物合成基因簇得到大量新的生物活性物质的方法,为今后科研工作做参考。     

2株深海来源真菌次级代谢产物的研究D

目的 对2株深海来源真菌Graphostroma sp. MCCC 3A00421和Aspergillus sp. MCCC 3A400414进行化学成分和生物活性研究。方法 采用溶剂萃取、柱色谱层析及半制备HPLC等方法对2株深海真菌的发酵产物进行化学分离,通过理化性质及波谱学方法并参阅文献进行化合物结构鉴定,并初步评价其细胞毒活性。结果 从菌株3A00421的发酵产物中分离得到了4个化合物,经鉴定其结构分别为3,5-dimethyl-8-methoxy-3,4-dihydroisocoumarin（1）、6-O-methylreticulol（2）、nectriapyrone（3）、demethyl nectriapyrone A（4）;从菌株3A00414中分离得到了5个化合物,经鉴定其结构分别为(22E,24R) -麦角甾-5,7,22-三烯-3β-醇（5）、WIN 64821（6）、diorcinol（7）、9-十八碳烯酸（8）、9,12-十八碳二烯酸（9）。其中化合物6具有中等强度的细胞毒活性,其IC50为7.14～15.78 μmol/L。结论 深海真菌是重要的活性物质来源和生物资源,本研究首次从深海来源Graphostroma属真菌的代谢产物中发现了2个异香豆素和2个苯并吡喃酮类化合物。     

链霉菌沉默生物合成基因簇调控的研究进展

本文以天蓝色链霉菌(Streptomyces     

链霉菌沉默生物合成基因簇调控的研究进展

本文以天蓝色链霉菌(Streptomyces coelicolor)中黄色色素coelimycin生物合成调控及开发策略的研究进展为代表,介绍了链霉菌中沉默生物合成基因簇调控激活的新进展,为链霉菌天然产物基因簇的挖掘和新次级代谢产物的发现提供研究思路。     

海洋真菌分离及菌株Aspergillus versicolor ZLQ-43的次级代谢产物的研究D

目的 对来源于渤海地区样品进行海洋真菌分离,并深入研究目标菌株杂色曲霉Aspergillus versicolor ZLQ-43的抗肿瘤活性成分。方法 采用稀释涂布平板法进行海洋真菌的分离;应用溶剂萃取,TLC分析,柱色谱层析及制备HPLC等方法对菌株ZLQ-43的发酵产物进行研究;通过理化性质及波谱学方法并参阅文献进行化学结构鉴定;采用CPE MTT法评价其抗H1N1流感病毒活性,采用SRB法评价其抗肿瘤的活性。结果 从渤海地区样品中最终筛选分离到海洋真菌90株,从菌株ZLQ-43发酵提取物中分离得到聚酮类化合物6个,经结构鉴定分别为sterigmatocystin (1), dihydrosterigmatocystin (2), vericolorin B (3), paeciloquinone C (4), versiconol (5)和2,4-dihydroxy-6-((R)-4-hydroxy-2-oxopentyl)-3-methylbenzaldehyde (6)。化合物4、6具有中等强度的抗H1N1病毒活性,抑制率分别为67.6%和51.5%;化合物5在1 mg?mL-1浓度下对P388细胞增殖抑制率67.79%。 结论 菌株ZLQ-43能够产生结构多样的活性次级代谢产物,发现化合物1、3、5具有P388细胞增殖抑制活性,并首次报道化合物4、6的抗H1N1病毒活性。     

表观遗传修饰应用于真菌次级代谢调控研究的最新进展

真菌次级代谢产物的生物合成与其基因簇所在染色体的表观遗传状态密切相关,通过表观遗传修饰能够调控真菌的次级代谢过程.分子表观遗传修饰方法主要通过敲除或过表达表观遗传相关酶类的编码基因,而化学表观遗传修饰方法则是外源加入化学表观遗传修饰酶抑制剂.二者都能促进基因的转录,进而激活沉默的生物合成基因簇,提高真菌次级代谢产物的化...     

激活微生物沉默基因簇合成新型天然产物的研究

随着微生物基因组测序项目开展,越来越多的沉默基因簇被发现。采用激活微生物沉默基因簇的方法,可以充分发掘微生物次级代谢的潜力并可以获得大量新结构天然产物,为新药发现提供更多天然化合物来源。本文简要介绍激活沉默基因簇合成微生物次级代谢产物的方法。     

Expression of biosynthetic gene clusters in heterologous hosts for natural product production and combinatorial biosynthesis

Expression of biosynthetic gene clusters in heterologous hosts for natural product production and combinatorial biosynthesis is playing an increasingly important role in natural product-based drug discovery and development programmes. This review highlights the requirements and challenges associated with this conceptually simple strategy of using surrogate hosts for the production of natural products in good yields and for the generation of novel analogues by combinatorial biosynthesis methods, taking advantage of the recombinant DNA technologies and tools available in the model hosts. Specific topics addressed include: i) the mobilisation of biosynthetic gene clusters using different vector systems; ii) the selection of suitable model heterologous hosts; iii) the requirement of post-translational protein modifications and precursor supply within the model hosts; iv) the influence of promoters and pathway regulators; and v) the choice of suitable fermentation conditions. Lastly, the use of heterologous expression in combinatorial biosynthesis is addressed. Future directions for model heterologous host engineering and the optimisation of natural product biosynthetic gene cluster expression in heterologous hosts are also discussed.     

Delivery approaches for CRISPR/Cas9 therapeutics in vivo: advances and challenges

Introduction: Therapeutic gene editing is becoming a viable biomedical tool with the emergence of the CRISPR/Cas9 system. CRISPR-based technologies have promise as a therapeutic platform for many human genetic diseases previously considered untreatable, providing a flexible approach to high-fidelity gene editing. For many diseases, such as sickle-cell disease and beta thalassemia, curative therapy may already be on the horizon, with CRISPR-based clinical trials slated for the next few years. Translation of CRISPR-based therapy to in vivo application however, is no small feat, and major hurdles remain for efficacious use of the CRISPR/Cas9 system in clinical contexts.

Areas covered: In this topical review, we highlight recent advances to in vivo delivery of the CRISPR/Cas9 system using various packaging formats, including viral, mRNA, plasmid, and protein-based approaches. We also discuss some of the barriers which have yet to be overcome for successful translation of this technology.

Expert opinion: This review focuses on the challenges to efficacy for various delivery formats, with specific emphasis on overcoming these challenges through the development of carrier vehicles for transient approaches to CRISPR/Cas9 delivery in vivo.     

Anti-angiogenic gene therapy for hepatocellular carcinoma mediated by microbubble-enhanced ultrasound exposure: An in vivo experimental study

Objective: The aim of this study was to assess the effect of anti-angiogenic gene therapy for hepatocelluar carcinoma (HCC) treated by microbubble-enhanced ultrasound exposure.

Methods: Forty C57BL/6J female mice were inoculated s.c. with Hepa1-6 tumor cell line. Herpes simplex virus thymidine kinase under the control of kinase domain-containing receptor (KDR, angiogenic growth factor's corresponding receptor) promoter was used. Plasmid DNA with or without microbubble contrast agent of SonoVue? was i.v. injected. Ultrasound (1 MHz, 2 W/cm², 5 min) was delivered to hepatic carcinomas in mice. The KDR-tk gene transfer was followed by ganciclovir (GCV) injection for 10 days and then the diameters of tumors were measured every 4 days till 28 days. The survivals of tumor-bearing mice were observed. PCR analysis and immunohistochemistry measurements revealed expression of the transfected gene. Transferase-mediated dUTP nick end labeling staining was used to detect apoptotic cells.

Results: Compared with the group treated by ultrasound alone, KDR-tk gene treatment treated by ultrasound combined with SonoVue restrained tumor growth and increased survival time of tumor-bearing mice; microvessel density in group mediated by ultrasound and SonoVue was significantly lower than that in group ultrasound alone (12.3 ± 1.4 vs. 27.4 ± 3.2, P < 0.05). An apoptosis index increased in the group treated by ultrasound and SonoVue compared with the group treated by ultrasound alone (25 ± 3.6 vs. 36 ± 3.8, P < 0.05), whereas there was no significant difference between group mediated by SonoVue alone and group phosphate-buffered saline alone (17 ± 1.8 vs. 14 ± 1.2, P>0.05).

Conclusions: Gene therapy mediated by ultrasound exposure enhanced by a microbubble contrast agent may become a new treatment option for persistent HCC.