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海洋木霉对磺胺类抗生素的转化作用研究
Alternative TitleBiotransformation of sulfonamides by marine-derived Trichoderma strains
于汶莉
Subtype硕士
Thesis Advisor季乃云
2023-05-26
Training institution中国科学院烟台海岸带研究所
Degree Grantor中国科学院大学
Place of Conferral中国科学院烟台海岸带研究所
Degree Name生物与医药硕士
Degree Discipline生物与医药
Keyword海洋木霉 棘孢木霉 磺胺类抗生素 生物转化 转录组分析
Abstract环境中的抗生素污染已成为亟待解决的难题之一。在长期药物使用、储存和废物处理过程中,抗生素残留对自然环境和生物体造成了不同程度的污染,其中磺胺类抗生素(SAs)因环境检出率高而受到广泛关注。生物转化(包括降解)的方法是解决抗生素污染问题的研究热点,利用微生物转化/降解抗生素具有极大优势。现已报道的磺胺类抗生素转化菌株多以细菌为主,对高浓度磺胺类抗生素的耐受能力较差,关于转化产物的结构、生物转化机制,以及转化产物生物活性等方面研究较少。海洋来源的木霉属真菌(Trichoderma spp.)具有较强的环境适应性,次生代谢途径丰富多样、结构独特,并富含丰富的酶系统,可作为研究磺胺类抗生素转化的重要微生物来源。此外,木霉具有生物转化能力,是对有机化合物进行结构修饰的重要工具,被广泛应用于合成新型杂环化合物和具有生物活性的化合物。 为了探究海洋木霉对SAs的生物转化作用,本研究利用海洋真菌菌株库中的海洋木霉,以RBBR活性蓝平板筛选、磺胺类抗生素去除效果筛选及愈创木酚平板筛选方法对160株海洋木霉进行化学筛选,获得具有转化潜力的菌株7株。在上述菌种筛选的基础之下,对该7株菌以磺胺甲恶唑(SMX)、磺胺嘧啶(SDZ)、磺胺二甲基嘧啶(SMZ)为底物进行培养,提取转化粗物进行生物筛选,得到能够降低磺胺类抗生素生态毒性的优势菌株A-YMD-9-1,形态学和分子生物学鉴定结果表明该菌株为棘孢木霉(Trichoderma asperellum)。 本研究对棘孢木霉A-YMD-9-1生物转化SMX过程中的生物学性质进行了初步探索,包括对SMX的耐受性、菌丝吸附作用及培养过程中pH值的变化,并对转化最佳培养条件进行研究。结果表明,该菌株对SMX具有高度耐受性,在浓度为400 mg/L的SMX-PDA平板上仍可生长。菌丝吸附实验证明该菌株对SMX的去除能力与菌丝吸附作用相关甚微。培养过程中发酵液pH值的结果表明,新转化产物的生成可能与酸催化过程无关。通过对底物浓度、培养基组成和培养时间的研究获得了棘孢木霉A-YMD-9-1对SMX的最佳转化条件,即SMX浓度为200 mg/L、培养基为Ⅱ(含适量碳/氮源)、培养时间为21天。在该培养条件下,棘孢木霉A-YMD-9-1对SMX具有一定去除能力并且转化产物含量相对较高。 以最佳转化培养条件进行大规模发酵培养,并运用各种现代色谱技术分离得到转化产物,通过现代波谱技术对转化产物进行结构解析及鉴定。在避光条件下得到了1个新的SMX连萜转化产物A-1;在自然光条件下分离得到5个化合物,包括与A-1平面结构相同的新连萜转化产物B-4,1个常见N4-乙酰化转化产物B-3,1个没药烷类倍半萜B-2,1个SMX立体异构体B-5及未转化完全的底物 SMX (B-1)。对获得的化合物生物活性进行研究,结果表明化合物A-1对费氏弧菌(Vibrio fischeri)、杜氏盐藻(Dunaliella salina)及三角褐指藻(Phaeodactylum tricornutum)的生长抑制作用与SMX相比略低,而N4-乙酰化产物B-3相较于SMX而言,对两种海洋赤潮藻(东海原甲藻Prorocentrum donghaiense、赤潮异弯藻Heterosigma akashiwo)表现出较好的抑制作用,且所有化合物对丰年虾(Artemia salina)均显示低毒性。 针对棘孢木霉A-YMD-9-1对SMX的转化过程,进行了转录组学分析,探究了该菌株对磺胺类抗生素的响应关系或生物转化机制。结果表明,棘孢木霉A-YMD-9-1受到SMX的胁迫时,通过上调与跨膜转运蛋白有关基因,以应对环境的变化;在此过程中,参与编码谷胱甘肽转移酶(GS)、甲基转移酶、醛酮还原酶等解毒蛋白基因同样显著上调,以减少对细胞的损害。此外,与氧化还原、氨基转移等过程相关的氧化还原酶、单加氧酶、转移酶、乙醇脱氢酶等基因表达水平显著上调,可能与SMX降解及转化产物的生成密切相关。 综上所述,本文以海洋木霉为菌源开展对磺胺类抗生素生物转化研究,通过对菌株的筛选、转化培养条件的优化、转化产物尤其是复杂未知转化产物的结构分离鉴定及生物活性评价、转化过程可能涉及的转化机制开展相应研究,并首次报道了磺胺类抗生素的连萜转化产物,有助于充分认识磺胺类抗生素的环境归趋,为磺胺类抗生素的深度生态风险评估提供了新思路。
Other AbstractOne of the most pressing problems that needs to be resolved is antibiotic pollution of the environment. During the long-term drug usage, storage, and waste disposal processes, varying degrees of environmental and biological pollution were caused by residual antibiotics. Among them, Sulfonamides (SAs) have drawn widespread attention due to their high environmental detection rates.The biotransformation (including degradation) approaches are considered as a hot research issue to resolve antibiotic contamination, and employment of microorganisms to transform or degrade antibiotics has many benefits. Most of the reported sulfonamide transforming strains are bacteria that have a low tolerance for high sulfonamide concentrations. Less research has been done on the biological activities of biotransformation products, as well as their structures and biotransformation pathways. Marine-derived Trichoderma spp. have strong environmental adaptability and the structure of its secondary metabolites is novel, diversity and unique. They also have abundant enzyme systems. As a result, Trichoderma species can be an important microbial source for studying the transformation of sulfonamides. Additionally, due to the biotransformation ability and importance of Trichoderma species in the structural modification of organic compounds, they are frequently utilized in the synthesis of new heterocyclic compounds and that with biological activity. To investigate the biotransformation of SAs by marine-derived Trichoderma species, about 160 strains of marine-derived Trichoderma species were chemically screened by RBBR plate screening, sulfonamide antibiotic removal screening, and guaiacol plate screening. Through the aforementioned chemical screening, seven strains with transformation potential were discovered. The seven strains were cultivated with sulfamethoxazole (SMX), sulfadiazine (SDZ), and sulfamethazine (SMZ) as substrates based on the strain screening described above. The crude extracts of the biotransformation were extracted for biological screening, and a dominant strain A-YMD-9-1 was found that might reduce the ecotoxicity of sulfonamides. The results of morphological and molecular biological identification showed that the dominant strain is Trichoderma asperellum. The biological characteristics of Trichoderma asperellum A-YMD-9-1 during biotransformation of SMX were investigated in this work, including tolerance to SMX, mycelial adsorption, the change of pH during culture, and the optimal culture conditions for transformation. It was found that the strain was highly tolerant to SMX and was still able to grow on SMX-PDA plates at a concentration of 400 mg/L. The mycelial adsorption experiments showed that the strain's capacity to remove SMX was unrelated to mycelial adsorption. The results of the change of pH indicated that the production of the new transformation compound be irrelevant connected to the acid catalytic process. The optimum culture conditions for Trichoderma asperellum A-YMD-9-1 in SMX biotransformation were obtained from the work of SMX concentration, medium composition, and incubation period. The most optimal conditions were SMX concentration of 200 mg/L, medium II (with appropriate carbon/nitrogen supply), and 21-day incubation period. Under these conditions, Trichoderma asperellum A-YMD-9-1 could remove SMX and the content of transformed products is relatively high. The biotransformation compounds were isolated by various modern chromatographic techniques, moreover, the structures were resolved and identified by modern spectroscopic techniques. One new complex biotransformation compound A-1, which had not been discovered in previous studies, was obtained under the conditions of protection from light. Besides, five compounds were isolated under the conditions of natural light, including a new complex biotransformation compound B-4 with the same planar structure as A-1, a common N4-acety transformation compound B-3, a bisabolane sesquiterpene B-2, an SMX stereoisomer B-5, and incompletely transformed SMX (B-1). The compounds' biological activity research showed that compared with SMX, compound A-1 slightly less effectively inhibited the growth of Vibrio fischeri, Dunaliella salina, and Phaeodactylum tricornutum, N4-acetyl-SMX (B-3) showed superior growth inhibition on Prorocentrum donghaiense and Heterosigma akashiwo. Furthermore, all compounds showed low toxicity to Artemia salina. Transcriptome analysis was performed to investigate the relationship or biotransformation mechanism of Trichoderma asperellum A-YMD-9-1 response to SMX. The results demonstrated that genes related to transmembrane transport proteins were up-regulated when the strain was stressed by SMX in response to environmental changes. To lessen cell damage, the genes involved in detoxification proteins such glutathione transferase, methyltransferase, and aldo-keto reductase were also dramatically up-regulated. In addition, the genes for oxidoreductase, monooxygenase, transferase, and ethanol dehydrogenase were significantly up-regulated, which may be closely related to the degradation of SMX and the production of biotransformation compounds. In summary, this paper studied biotransformation of sulfonamides by marine Trichoderma species. The research includes the selection of SAs transformed strains, the optimization of culture conditions for biotransformation, the isolation and structural identification of transformation products (especially complex unknown transformation products), biological activity research of compounds, and transcriptome analysis. This study presents the first report on the transformation compounds of sulfonamides, which contributes to a comprehensive understanding of the environmental trend of sulfonamides and provides new insights for the in-depth ecological risk assessment of these compounds.
Pages117
Language中文
Document Type学位论文
Identifierhttp://ir.yic.ac.cn/handle/133337/32050
Collection中国科学院烟台海岸带研究所知识产出_学位论文
Recommended Citation
GB/T 7714
于汶莉. 海洋木霉对磺胺类抗生素的转化作用研究[D]. 中国科学院烟台海岸带研究所. 中国科学院大学,2023.
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