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Biosynthesis of Alkylcitric Acids in Aspergillus niger Involves Both Co-localized and Unlinked Genes.
Frontiers in Microbiology ( IF 4.0 ) Pub Date : 2020-05-28 , DOI: 10.3389/fmicb.2020.01378
Sylvester Palys 1 , Thi Thanh My Pham 1 , Adrian Tsang 1
Affiliation  

Filamentous fungi are an abundant source of bioactive secondary metabolites (SMs). In many cases, the biosynthetic processes of SMs are not well understood. This work focuses on a group of SMs, the alkylcitric acids, each of which contains a saturated alkyl “tail,” and a citrate-derived “head.” We initially identified their biosynthetic gene cluster and the transcriptional regulator (akcR) involved in the biosynthesis of alkylcitrates in the filamentous fungus Aspergillus niger by examining the functional annotation of SM gene clusters predicted from genomic data. We overexpressed the transcription regulator gene akcR and obtained from one liter of culture filtrate 8.5 grams of extract, which are represented by seven alkylcitric acids as determined by NMR. Hexylaconitic acid A comprised 94.1% of the total production, and four of the seven identified alkylcitrates have not been reported previously. Analysis of orthologous alkylcitrate gene clusters in the Aspergilli revealed that in A. oryzae and A. flavus an in-cluster gene displays sequence similarity to cis-aconitate decarboxylase, the orthologue of which in A. niger, NRRL3_00504, is located on a different chromosome. Overexpression of the A. niger NRRL3_00504 and akcR genes together shifted the profile of alkylcitrates production from primarily hexylaconitic acids to mainly hexylitaconic acids, suggesting that NRRL3_00504 encodes an enzyme with hexyl aconitate decarboxylase activity. We also detected two additional, previously unreported, alkylcitric acids in the double overexpression strain. This study shows that phylogenomic analysis together with experimental manipulations can be used to reconstruct a more complete biosynthetic pathway in generating a broader spectrum of alkylcitric compounds. The approach adopted here has the potential of elucidating the complexity of other SM biosynthetic pathways in fungi.



中文翻译:


黑曲霉中烷基柠檬酸的生物合成涉及共定位基因和非连锁基因。



丝状真菌是生物活性次生代谢物(SM)的丰富来源。在许多情况下,SM 的生物合成过程尚不清楚。这项工作重点关注一组 SM,即烷基柠檬酸,每种 SM 都包含一个饱和烷基“尾部”和一个柠檬酸盐衍生的“头部”。我们通过检查从基因组数据预测的 SM 基因簇的功能注释,初步鉴定了丝状真菌黑曲霉中参与烷基柠檬酸生物合成的生物合成基因簇和转录调节因子 (akcR)。我们过表达转录调节基因akcR,并从1升培养物滤液中获得8.5克提取物,经NMR测定,其代表为7个烷基柠檬酸。己基乌头酸 A 占总产量的 94.1%,并且已鉴定的 7 种柠檬酸烷基酯中有 4 种以前未曾报道过。对曲霉中直系同源烷基柠檬酸基因簇的分析表明,在米曲霉和黄曲霉中,簇内基因与顺乌头酸脱羧酶表现出序列相似性,其在黑曲霉中的直系同源物 NRRL3_00504 位于不同的染色体上。黑曲霉 NRRL3_00504 和 akcR 基因的过度表达共同将烷基柠檬酸的产生从主要己基乌头酸转变为主要己基衣康酸,表明 NRRL3_00504 编码具有乌头酸己酯脱羧酶活性的酶。我们还在双过表达菌株中检测到了另外两种以前未报道的烷基柠檬酸。这项研究表明,系统发育分析与实验操作相结合可用于重建更完整的生物合成途径,以产生更广泛的烷基柠檬酸化合物。 这里采用的方法有可能阐明真菌中其他 SM 生物合成途径的复杂性。

更新日期:2020-06-30
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