五胺基酮戊酸 (5-ALA) 是一種內源性非蛋白質氨基酸，因為其多領域的廣泛應用性而受到關注。除了作為農業用藥與畜牧飼料添加劑以外，在癌症治療領域中 5-ALA 是一種前驅藥物用於光動力療法 (PDT) ，同時在近年的 COVID-19 傳染病中展現了其抗病毒活性藥劑的潛力。本研究利用來自莢膜紅桿菌的五胺基酮戊酸合成酶 (ALAS, EC 2.3.1.37) 作為生產 5-ALA 的主要基因，並以兩種途徑：增強三羧酸循環 (TCA cycle) 通量以及增加輔酶A (CoA) 供應，分別使用烏頭酸酶 (AcnA, EC 4.2.1.3) 與泛酸激酶 (CoaA, EC 2.7.1.33) 改善 5-ALA 之生物合成。最後，透過培養基優化、宿主篩選及生物反應器培養策略實現高水平的 5-ALA 生產菌株。
烏頭酸酶：以來自大腸桿菌 MG1655 與 Nissle 1917 之 AcnA (MGAcnA 與 EcNAcnA) 進行同源基因的比較，並且篩選得到較優異的 EcNAcnA。接著我們以雙質體系統調控不同表達水平的 EcNAcnA 以平衡細胞生長與 5-ALA 生物生產，結果顯示以低拷貝數 p15A ori驅動 EcNAcnA 為最佳的組合。為了減輕多質體合成帶來的能量負擔，設計了 all-in-one 質體並且以不同的培養基組分成功提升了 54% 生物量至 4.271 g/L。更探索在不同的基改菌株中能夠提升 5-ALA 生產量的優異宿主，在基因組上整合 ALAS 的菌株進一步將 5-ALA 產量提升至 8.3 g/L。同時，對菌株進行保種以確保後續培養的穩定性。最終，利用 1 L 批次饋料發酵結合培養基 pH 維持、連續式饋料及溶解氧氣水平的調整，於 27 小時達到了 10.4 g/L 的細胞量與 24.5 g/L 的高水平 5-ALA 產量，生產率為 0.907 g/L/h，產率為 74.8%。
泛酸激酶：首先利用四種不同表達水平的 CoaA 質體探索對 5-ALA 生產影響，並發現在添加泛酸的條件下以 pUC ori 搭配原生核糖體結合位點 (RBS) 得到 8.72 g/L 的 5-ALA 產量。隨後對泛酸添加的條件進行優化，得到最佳的添加濃度與時間。最後進行 1 L 批次饋料發酵，並嘗試共表達 EcNAcnA 與 CoaA 聯合發酵。
這項研究利用 EcNAcnA 與 CoaA 各自調控表達水平藉此提升菌株的細胞生長以及 5-ALA 生產量；在 EcNAcnA 的系統透過培養基組分、比較宿主特性，並優化生物反應器的培養策略，建立了能夠抵抗氧化壓力並生產高價值化學品的基改大腸桿菌；在 CoaA 的系統以最佳化泛酸添加的條件，得到具有放大培養潛力的菌株。

In recent years, 5-aminolevulinic acid (5-ALA) has attracted significant interest due to its roles as a photodynamic prodrug and an antiviral agent. In this study, we present a new approach utilizing aconitase A from E. coli Nissle 1917 (EcNAcnA), renowned for its exceptional activity and conjunction with ALA synthase from Rhodobacter capsulatus (RcALAS) to enhance 5-ALA production in an engineered chassis. Expression of EcNAcnA and RcALAS via dual plasmids led to a 60% increase in 5-ALA yield, reaching up to 6.645 g/L. Diverse 5-ALA production levels were observed with different combinations of promoters and replication origins for both genes. Subsequently, an all-in-one plasmid with a high copy number, designated as RcNN, was introduced into the genomic engineering RcI strain. This resulted in the production of 24.5 g/L 5-ALA with a productivity of 0.907 g/L/h in a bioreactor under pH control and glucose feeding over 27 h. To the best of our knowledge, this is the first study to enhance 5-ALA biosynthesis by applying a superior aconitase variant from E. coli Nissle 1917, which enhances isocitrate production in the TCA cycle and alleviates reactive oxygen species (ROS), thereby promoting 5-ALA accumulation effectively.

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