在人體腸胃道，益生菌扮演共生系統中重要的角色，菌叢數量將直接影響人體健康，因此，本研究旨在建立二種益生菌，即大腸桿菌Nissle (EcN) 與鼠李糖乳酸桿菌 (LGG)共生的平台及分析方法，亦進行挖掘EcN與MG1655模式大腸桿菌株於醣降解路徑、三羧酸循環、丙酮酸相關代謝與酸代謝等途徑的功能酶差異，瞭解酶動力學及對細胞生長的影響。
首先利用基因工程技術將綠色螢光蛋白 (sfGFP) 轉入EcN，可由螢光訊號作為菌體CFU的定量方法。其次，在共培養乳酸桿菌時，以qPCR的技術擬定一套鑑定乳酸桿菌的高通量定性分析。在EcN與LGG的共生培養中，發現乳酸可以大量被消耗，由於EcN的乳酸代謝酶 (YkgG) 與其他大腸桿菌相比，有6個胺基酸突變，將其進行基因重組外源表達後，測定EcN最大酶活速率 (Vmax) 為64.5 mM min-1、酶催化能力 (kcat/Km) 為2055 mM-1 min-1，較MG1655反應速率高出6倍。另外亦發現EcN與LGG的共生菌能使細胞的耐酸能力大幅提升，經由pH 2.5之磷酸鹽溶液酸化5及10分鐘後，菌落數分別提升了40 %及50 %存活率。
在挖掘功能性基因中，分別重組表達lacZ, acs, yahK, glpD, glpK,及sucABCD。 EcN的乳糖代謝酶LacZ (EC 3.2.1.23) 及醋酸降解酶Acs (EC 6.2.1.1) 酶活顯著優於MG1655。LacZ降解乳醣進入糖解路徑 (Glycolysis pathway)，而Acs則利用醋酸合成乙醯輔酶A進入酸縮酸循環中 (TCA cycle)。經由構建部分lacZ基因片段 (LacZ’) 測量其酶活特性，其酶活性為459.9 Unit (uM min-1 mg-1)。在酸代謝途徑中的acs基因，將醋酸有效的作為碳源，轉為在生長時重要的乙醯輔酶作為生長時所需能量。將EcN與MG1655經生長曲線擬合於Gompertz方程式，得到最大生長量 (YM) 分別為0.17及0.06 g/L和反應速率 (k) 0.17及0.22 1/h，EcN所含Acs酶的生長速率及能達到最高生長量高且生長速度快。
綜上所述，這些主要的降解與合成酶會影響EcN大腸桿菌的生長及抗酸能力，探討蛋白中含多突變點的氨基酸並將過量表達分析，未來將能更深入地瞭解EcN與其他大腸桿菌的差異。

In the human gastrointestinal tract, probiotics play a vital role in the symbiotic system and affect human health. Therefore, this study aims to establish probiotics symbiosis platform and analysis methods. Also, to explore the functional enzyme differences between EcN and MG1655 model E. coli strains in sugar degradation pathway (glycolysis pathway), tricarboxylic acid cycle (TCA cycle), pyruvate-related metabolism and acid metabolism. Firstly, the green fluorescent protein (sfGFP) transformed into EcN through genetic engineering techniques, enabling the use of the fluorescent signal as a quantitative method for measuring bacterial CFU. Secondly, a high-throughput qualitative analysis method via qPCR technology developed for the identification of Lactobacillus. In co-cultured EcN and LGG, the lactic acid can be consumed in significant amounts. Compared to other strains of Escherichia coli, EcN possesses six amino acid mutations in the lactic acid metabolizing enzyme (YkgG). After gene recombination and overexpression, the maximum enzyme activity rate (Vmax) of EcN was determined to be 64.5 mM min-1, and the enzyme catalytic ability (kcat/Km) was 2055 mM-1 min-1. Furthermore, the symbiotic relationship between EcN and LGG was found to significantly enhance the acid resistance of the cells. Following immersed in a pH 2.5 phosphate solution for 5 and 10 minutes, the colony count increased by 40% and 50%, respectively.
The enzymatic activities of two key enzymes, LacZ (EC 3.2.1.23) involved in lactose metabolism and Acs (EC 6.2.1.1) involved in acetate degradation, were significantly higher in EcN compared to MG1655. LacZ degrades lactose, which enters the glycolysis pathway, while Acs utilizes acetic acid to synthesize acetyl-CoA, which enters the tricarboxylic acid cycle (TCA cycle) for energy production. To measure the enzyme activities, a partial lacZ gene fragment (lacZ') was constructed, and its enzymatic activity was determined to be 459.9 Units (uM min-1 mg-1). The acs gene in the acid metabolism pathway effectively utilizes acetic acid as a carbon source, converting it to acetyl co-enzyme, which is crucial for growth energy. The growth patterns of EcN and MG1655 were fitted to the Gompertz equation. The maximum growth (YM) for EcN and MG1655 was measured as 0.17 g/L and 0.06 g/L, respectively. The reaction rate (k) was 0.17 1/h for EcN and 0.22 1/h for MG1655. Notably, the presence of the Acs enzyme in EcN allowed for higher growth rates and improved growth overall. In conclusion, these major degradation and synthesis enzymes have a significant impact on the growth and acid resistance of EcN. Exploring proteins with multiple mutation sites and analyzing their overexpression will provide a deeper understanding of EcN and its differences compared to other E. coli strains in the future.

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