| Abstract: | Gamma-aminobutyric acid (GABA) 主要以抑制性神經傳遞物質的形式存在於人體中樞神經系統，具有多種不同的生理特性。除此之外，其他相關應用也相當廣泛，例如：飼料工業及保健食品，許多工業會使用 GABA 作為附加價值，而濃縮純化的GABA可做為保健食品。根據文獻，已知可以利用微生物發酵法進行生產 GABA ，主要以具有 Glutamate decarboxylase (GAD) 的乳酸菌為主要生產者，而短乳桿菌 (Levilactobacillus brevis) 為常見的具有高效 GABA 生產能力的物種。此實驗中，我們使用實驗室本土分離的短乳桿菌，命名為分離株 iso1 及 iso2 ，尋找菌株最佳生長條件、選殖並比較其GAD相關基因，並研究這些基因表現的調控。結果顯示： (1) 檢測兩分離株的生長培養基條件發現 iso1 可使用 pH 6.2 乳酸菌培養基進行培養；而 iso2 不論利用一般培養基、不同 pH 值與額外添加物、乳酸菌培養基測試後，生長狀態仍不佳。 (2) 利用基因庫中資料設計引子，接著以 PCR 選殖兩分離株的 GAD 基因 gadA, gadB, gadR, gadC, gts ，將兩分離株序列進行比較，兩者相似度如下： gadA 99.4% ； gadB 99.9% ； gadR 99.9%； gadC 99.9% 。將兩分離株胺基酸序列與其他乳酸菌進行親緣演化分析，結果表明兩分離株與菌株 BDGP6, TMW 1.2111, UCCLBS124 具有高度相似性。 (3) 分別選殖 gadA 及 gadB 啟動子區域，使其與無啟動子之 lacZ 基因報導系統結合。在大腸桿菌中檢測 GadR 調控因子對於兩基因啟動子表現的影響。 iso1 中的 PgadA 啟動子活性低於 iso2 ； iso1 中的 PgadB 啟動子活性在 GadR 和誘導物存在的情況下會顯著提升，而 iso2 中的 PgadB 活性沒有顯著變化。 (4) 將 gadA 基因送入大腸桿菌 DH5α及 Xanthomonas campestris pv. campestris (Xcc) 中進行表達。利用薄層層析法 (thin layer chromatography, TLC) 進行 GABA 生產能力之定性測試。遺憾的是，並沒有檢測到 GABA 的合成。
Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter in human central nervous system (CNS). GABA has various important physiological functions and can be used in feed industry as dietary supplement. According to the literature, microbial fermentation can be used to produce GABA. Lactic acid bacteria (LAB) with Glutamate decarboxylase (GAD) gene, such as Levilactobacillus brevis (L. brevis), are the main GABA producers. In this study, we used two L. brevis isolates, named as iso1 and iso2, to identify the suitable growth conditions, and clone and compare their GAD-related genes and their regulatory mechanisms. The results showed that (1) The culture conditions of the two isolates were tested. The iso1 strain could be cultured with pH 6.2 in a lactic acid bacteria medium; while iso2 was grew poorly even though many growth conditions have been tested. (2) Primer pairs were designed using the data in the gene bank for PCR amplification of the GAD genes (gadA, gadB, gadR and gadC) from the two isolates. Amplicons were cloned and their sequences were compared. The identity of GAD genes between the two isolates were as follows: gadA 99.4%; gadB 99.9%; gadR s 99.9%; gadC 99.9%. The phylogenetic tree showed that the amino acid sequences of GADs from iso1, iso2 shared the highest similarity with the strains BDGP6, TMW 1.2111 and UCCLBS124. (3) The gadA and gadB promoter regions were cloned and form a transcriptional fusion with a promoterless lacZ gene reporter system. To investigate the effect of GadR on the expression of two genes in Escherichia coli (E. coli). The activity of PgadA in iso1 was lower than iso2; the activity of PgadB in iso1 significantly increased in the presence of GadR, however, no significant change was observed in iso2. (4) The gadA gene was introduced into E. coli and Xanthomonas campestris pv. campestris (Xcc). Thin layer chromatography (TLC) were used to qualify their GABA-producing capacity. Unfortunately, no GABA synthesis were detected. |
