Rapid, Parallel Identification of Catabolism Pathways of Lignin-Derived Aromatic Compounds in Novosphingobium aromaticivorans
Rapid, Parallel Identification of Catabolism Pathways of Lignin-Derived Aromatic Compounds in Novosphingobium aromaticivorans
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DOI：
10.1128/aem.01185-18
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发表时间：
2018-11-01
影响因子：
4.4
通讯作者：
Michener, Joshua K.
中科院分区：
文献类型：
--
作者：
Cecil, Jacob H.;Garcia, David C.;Michener, Joshua K.
转座子诱变是微生物遗传学中用于鉴定未知途径中的基因的强有力的技术。最近，转座子诱变技术的通量已经通过DNA条形码化和高通量测序的组合而显著增加。在这里，我们表明，当应用于分解代谢途径，条形码转座子库可以用来区分冗余的途径，复杂的途径分解成取代基模块，酶同系物之间的歧视，并迅速识别以前假设的酶在一个公正的基因组规模的搜索。我们使用这种技术来确定两个基因，desC和desD，这是参与降解的木质素衍生的芳香族化合物芥子酸的非模型细菌Novosphingobium aromaticivorans。我们表明，DesC是一种甲基酯酶作用于芥子酸催化过程中形成的中间体，提供了一个拟议的分解代谢途径中的最后一个酶。这种方法将是特别有用的，在识别适合异源表达在代谢engineering.IMPORTANCE的完整途径参与特定的生化转化的基因的识别是一个关键步骤，在预测微生物的功能，从核酸序列和工程微生物赋予它们新的功能。我们已经证明，转座子突变的新技术可以显着简化这一过程，并能够快速识别未表征途径中的基因。这些技术提供了必要的规模，以充分阐明复杂的生物网络，如那些用于降解混合物的木质素衍生的芳香族化合物。
Transposon mutagenesis is a powerful technique in microbial genetics for the identification of genes in uncharacterized pathways. Recently, the throughput of transposon mutagenesis techniques has been dramatically increased through the combination of DNA barcoding and high-throughput sequencing. Here, we show that when applied to catabolic pathways, barcoded transposon libraries can be used to distinguish redundant pathways, decompose complex pathways into substituent modules, discriminate between enzyme homologs, and rapidly identify previously hypothetical enzymes in an unbiased genome-scale search. We used this technique to identify two genes, desC and desD, which are involved in the degradation of the lignin-derived aromatic compound sinapic acid in the nonmodel bacterium Novosphingobium aromaticivorans. We show that DesC is a methyl esterase acting on an intermediate formed during sinapic acid catabolism, providing the last enzyme in a proposed catabolic pathway. This approach will be particularly useful in the identification of complete pathways suitable for heterologous expression in metabolic engineering.IMPORTANCE The identification of the genes involved in specific biochemical transformations is a key step in predicting microbial function from nucleic acid sequences and in engineering microbes to endow them with new functions. We have shown that new techniques for transposon mutagenesis can dramatically simplify this process and enable the rapid identification of genes in uncharacterized pathways. These techniques provide the necessary scale to fully elucidate complex biological networks such as those used to degrade mixtures of lignin-derived aromatic compounds.