Graduate School of Fisheries and Environmental Sciences, Nagasaki University, Nagasaki, Japan.
Institute of Integrated Science and Technology, Nagasaki University, Nagasaki, Japan.
Appl Environ Microbiol. 2024 Sep 18;90(9):e0073424. doi: 10.1128/aem.00734-24. Epub 2024 Aug 12.
OUT30018 is a moderately halophilic bacterium that synthesizes and accumulates ectoine as an osmolyte by activities of the enzymes encoded by the high salinity-inducible operon. Previously, we engineered a γ-aminobutyric acid (GABA)-producing GOP-Gad (Δ) from an ectoine-deficient mutant of this strain due to its ability to use high-salinity biomass waste as substrate. Here, to further increase GABA accumulation, we deleted which encodes GABA aminotransferase (GABA-AT) that catalyzes the first step of the GABA catabolic pathway, from the GOP-Gad genome. The resulting strain ZN3 (Δ Δ) accumulated 291 µmol/g cell dry weight (CDW) of GABA in the cells, which is a 1.5-fold increase from GOP-Gad's 190 µmol/g CDW. This result has confirmed the role of GABA-AT in the GABA catabolic pathway. However, redundancy in endogenous GABA-AT activity was detected in a growth test, where a -deletion mutant of OUT30018 was cultured in a medium containing GABA as the sole carbon and nitrogen sources. Because L-2,4-diaminobutyric acid aminotransferase (DABA-AT), encoded by an gene of the operon, shares sequence similarity with GABA-AT, a complementation analysis of the and the genes was performed in the ZN3 genetic background to test the involvement of DABA-AT in the redundancy of GABA-AT activity. Our results indicate that the expression of DABA-AT can restore GABA-AT activity in ZN3 and establish DABA-AT's aminotransferase activity toward GABA .
In this study, we were able to increase the yield of GABA by 1.5 times in the GABA-producing ZN3 strain by deleting the gene, which encodes GABA-AT, the initial enzyme of the GABA catabolic pathway. We also report the first evidence for GABA aminotransferase activity of an -encoded DABA-AT, confirming a longstanding speculation based on the reported GABA-AT activity of DABA-AT. According to our findings, the DABA-AT enzyme can catalyze the initial step of GABA catabolism, in addition to its known function in ectoine biosynthesis. This creates a cycle that promotes adequate substrate flow between the two pathways, particularly during the early stages of high-salinity stress response when the expression of the gene is upregulated.
OUT30018 是一种中度嗜盐菌，通过高盐诱导 操纵子编码的酶的活性合成并积累作为渗透物的章鱼胺。先前，我们由于其能够利用高盐生物质废物作为底物，从该菌株的章鱼胺缺陷突变体中工程改造出产生 γ-氨基丁酸 (GABA) 的 GOP-Gad (Δ)。在这里，为了进一步增加 GABA 的积累，我们从 GOP-Gad 基因组中删除了编码 GABA 转氨酶 (GABA-AT) 的 ，该酶催化 GABA 分解代谢途径的第一步。由此产生的菌株 ZN3 (ΔΔ) 在细胞中积累了 291µmol/g 细胞干重 (CDW) 的 GABA，比 GOP-Gad 的 190µmol/g CDW 增加了 1.5 倍。这一结果证实了 GABA-AT 在 GABA 分解代谢途径中的作用。然而，在含有 GABA 作为唯一碳源和氮源的培养基中培养 OUT30018 的 -缺失突变体的生长试验中检测到内源性 GABA-AT 活性的冗余。由于 操纵子的 基因编码的 L-2,4-二氨基丁酸转氨酶 (DABA-AT) 与 GABA-AT 具有序列相似性，因此在 ZN3 遗传背景下对 和 基因进行了互补分析，以测试 DABA-AT 是否参与 GABA-AT 活性的冗余。我们的结果表明，DABA-AT 的表达可以恢复 ZN3 中的 GABA-AT 活性，并确定 DABA-AT 对 GABA 的转氨酶活性。
在这项研究中，我们通过删除编码 GABA 分解代谢途径初始酶 GABA-AT 的 基因，使 GABA 产生的 ZN3 菌株的 GABA 产量增加了 1.5 倍。我们还首次报道了 编码的 DABA-AT 具有 GABA 转氨酶活性的证据，证实了基于报道的 DABA-AT 对 GABA-AT 活性的长期推测。根据我们的发现，DABA-AT 酶除了在章鱼胺生物合成中的已知功能外，还可以催化 GABA 分解代谢的第一步。这创建了一个循环，促进了两条途径之间的足够底物流动，特别是在高盐胁迫反应早期 基因上调表达时。
