Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, Tennessee, USA.
Appl Environ Microbiol. 2018 Oct 30;84(22). doi: 10.1128/AEM.01185-18. Print 2018 Nov 15.
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, and , which are involved in the degradation of the lignin-derived aromatic compound sinapic acid in the nonmodel bacterium 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. 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.
转座子诱变是微生物遗传学中用于鉴定未鉴定途径中基因的一种强大技术。最近，通过 DNA 条形码和高通量测序的结合，转座子诱变技术的通量得到了显著提高。在这里，我们表明，当应用于分解代谢途径时，条形码转座子文库可用于区分冗余途径、将复杂途径分解为替代模块、区分酶同源物，并在无偏基因组规模搜索中快速识别以前假设的酶。我们使用该技术鉴定了两个基因 和 ，它们参与了木质素衍生芳香化合物芥子酸在非模式细菌 中的降解。我们表明 DesC 是一种甲酯酶，作用于芥子酸代谢过程中形成的中间产物，为拟议的分解代谢途径提供了最后一种酶。这种方法在鉴定适合代谢工程异源表达的完整途径方面将特别有用。鉴定参与特定生化转化的基因是从核酸序列预测微生物功能和工程微生物赋予其新功能的关键步骤。我们已经表明，转座子诱变的新技术可以大大简化这个过程，并能够快速鉴定未鉴定途径中的基因。这些技术提供了充分阐明复杂生物网络所需的规模，例如那些用于降解木质素衍生芳香化合物混合物的生物网络。
