Toyoda Koichi, Sugaya Riki, Domon Akihiro, Suda Masako, Hiraga Kazumi, Inui Masayuki
Research Institute of Innovative Technology for the Earth (RITE), 9-2 Kizugawadai, Kizugawa 619-0292, Japan.
Division of Biological Science, Graduate School of Science and Technology, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma 630-0192, Japan.
Microorganisms. 2022 May 10;10(5):1002. doi: 10.3390/microorganisms10051002.
Understanding the metabolic pathways of amino acids and their regulation is important for the rational metabolic engineering of amino acid production. The catabolic pathways of L-asparagine and L-aspartate are composed of transporters for amino acid uptake and asparaginase and aspartase, which are involved in the sequential deamination to fumarate. However, knowledge of the catabolic genes for asparagine in bacteria of the Actinobacteria class has been limited. In this study, we identified and characterized the operon required for L-Asn catabolism in R. The operon consisted of genes encoding a transcriptional regulator (AnsR), asparaginase (AnsA2), aspartase (AspA2), and permease (AnsP). The enzymes and permease encoded in the operon were shown to be essential for L-Asn utilization, but another asparaginase, AnsA1, and aspartase, AspA1, were not essential. Expression analysis revealed that the operon was induced in response to extracellular L-Asn and was transcribed as a leaderless mRNA. The DNA-binding assay demonstrated that AnsR acted as a transcriptional repressor of the operon by binding to the inverted repeat at its 5'-end region. The AnsR binding was inhibited by L-Asn. This study provides insights into the functions and regulatory mechanisms of similar operon-like clusters in related bacteria.
了解氨基酸的代谢途径及其调控对于氨基酸生产的合理代谢工程至关重要。L-天冬酰胺和L-天冬氨酸的分解代谢途径由氨基酸摄取转运蛋白以及天冬酰胺酶和天冬氨酸酶组成，它们参与向富马酸的顺序脱氨作用。然而，放线菌纲细菌中天冬酰胺分解代谢基因的相关知识一直有限。在本研究中，我们鉴定并表征了R中L-天冬酰胺分解代谢所需的操纵子。该操纵子由编码转录调节因子（AnsR）、天冬酰胺酶（AnsA2）、天冬氨酸酶（AspA2）和通透酶（AnsP）的基因组成。结果表明，操纵子中编码的酶和通透酶对于L-天冬酰胺的利用至关重要，但另一种天冬酰胺酶AnsA1和天冬氨酸酶AspA1并非必需。表达分析表明，该操纵子在细胞外L-天冬酰胺的诱导下表达，并转录为无帽mRNA。DNA结合试验表明，AnsR通过结合其5'-末端区域的反向重复序列，作为该操纵子的转录阻遏物发挥作用。AnsR的结合受到L-天冬酰胺的抑制。本研究为相关细菌中类似操纵子样簇的功能和调控机制提供了见解。
