| 其他摘要 | One 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. |
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