Enhanced accumulation of γ-aminobutyric acid by deletion of aminotransferase genes involved in γ-aminobutyric acid catabolism in engineered .

UNLABELLED

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 .

IMPORTANCE

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.