Fungal degradation of phenylacetate focusing on CRISPR/Cas9-assisted characterization of two oxidative enzyme genes of Akanthomyces muscarius AM1091

IF 6.1 1区 生物学 Q1 MICROBIOLOGY
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Abstract

The degradation of phenylacetate (PA) was investigated as a model to explore aromatic compound breakdown in the fungal system. Fungal strains capable of utilizing PA as their sole carbon source were isolated using a minimal solid medium supplemented with 0.5 % PA. Subsequent cultivation in minimum liquid medium revealed that selected fungal strains, including Trametes versicolor TV0876 and TV3295, Paecilomyces hepiali PH4477, and Akanthomyces muscarius AM1091, efficiently removed PA within 24 h. HPLC analysis of culture supernatants from various fungal strains revealed a time-dependent accumulation of 2-hydroxyphenylacetate (2-HPA) and 4-hydroxyphenylacetate (4-HPA), two key major metabolic products primarily found in ascomycetes and basidiomycetes, respectively. This suggests that the first hydroxylation of PA is catalyzed by two distinct hydroxylases, one for each fungal group. Furthermore, fungal species that make 4-HPA also produce phenylethanol (PE), indicating a distinct catabolic mechanism to remove PA by direct reduction of PA to PE. A. muscarius AM1091, identified as the most efficient PA degrader in this study, was studied further to determine the biochemical pathway of PA degradation. RNA-Seq and RT-PCR analyses of AM1091 revealed two oxidative enzyme genes, CYP1 and DIO4, upregulated in the presence of PA. Targeted disruption utilizing preassembled Cas9-gRNA ribonucleoprotein complexes and homologous DNAs harboring the URA3 gene as an auxotrophic marker resulted in the cyp1 and dio4 mutant strains. The cyp1 mutant was incapable of converting PA to 2-HPA, indicating its involvement in the C2 hydroxylation, whereas the dio4 mutant was unable to degrade 2,5-dihydroxyphenylacetate (2,5-DHPA), resulting in the accumulation of 2,5-DHPA. Our findings indicate that A. muscarius AM1091 degrades PA through the activities of CYP1 and DIO4 for the C2 hydroxylation and subsequent ring-opening reactions, respectively.
以 CRISPR/Cas9 辅助鉴定 Akanthomyces muscarius AM1091 的两种氧化酶基因为重点的苯乙酸真菌降解。
研究人员以苯乙酸盐(PA)的降解为模型,探索了真菌系统中芳香化合物的分解过程。使用添加了 0.5 % PA 的最小固体培养基,分离出了能够利用 PA 作为唯一碳源的真菌菌株。随后在最小液体培养基中的培养结果表明,所选真菌菌株(包括多色曲霉 TV0876 和 TV3295、Paecilomyces hepiali PH4477 和 Akanthomyces muscarius AM1091)可在 24 小时内有效去除 PA。对不同真菌菌株培养上清液的 HPLC 分析表明,2-羟基苯乙酸酯(2-HPA)和 4-羟基苯乙酸酯(4-HPA)的积累随时间变化,这两种主要代谢产物分别存在于子囊菌和基枝菌中。这表明 PA 的第一次羟基化是由两种不同的羟化酶催化的,每个真菌类群都有一种。此外,制造 4-HPA 的真菌种类也会产生苯乙醇(PE),这表明存在一种不同的分解代谢机制,通过直接将 PA 还原成 PE 来清除 PA。A. muscarius AM1091 在本研究中被确定为最有效的 PA 降解菌,我们对其进行了进一步研究,以确定 PA 降解的生化途径。AM1091 的 RNA-Seq 和 RT-PCR 分析显示,在 PA 存在的情况下,两个氧化酶基因 CYP1 和 DIO4 上调。利用预组装的 Cas9-gRNA 核糖核蛋白复合物和携带 URA3 基因作为辅助营养标记的同源 DNA 进行靶向破坏,产生了 cyp1 和 dio4 突变株。cyp1 突变体不能将 PA 转化为 2-HPA,表明它参与了 C2 羟基化,而 dio4 突变体不能降解 2,5- 二羟基苯乙酸(2,5-DHPA),导致 2,5-DHPA 的积累。我们的研究结果表明,A. muscarius AM1091 通过 CYP1 和 DIO4 的活性分别进行 C2 羟基化和随后的开环反应来降解 PA。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Microbiological research
Microbiological research 生物-微生物学
CiteScore
10.90
自引率
6.00%
发文量
249
审稿时长
29 days
期刊介绍: Microbiological Research is devoted to publishing reports on prokaryotic and eukaryotic microorganisms such as yeasts, fungi, bacteria, archaea, and protozoa. Research on interactions between pathogenic microorganisms and their environment or hosts are also covered.
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