黃素腺嘌呤二核苷酸 (FAD) 是許多生物氧化還原和自由基反應中的關鍵輔助因子。傳統上，核黃素的 FAD 生物合成遵循兩步驟酶途徑，需要 ATP，以黃素單核苷酸 (FMN) 為中間體。工業規模的 FAD 生產傳統上依賴微生物發酵，但這些方法通常需要繁瑣的淨化步驟。考慮到原子經濟性和產量效率的重要性，酵素體外方法提供了一種更可持續和有效的選擇。然而，由於依賴昂貴的 ATP 作為底物，酶促 FAD 合成的廣泛工業應用受到阻礙。為了解決這個限制，我開發了一種雙酶級聯繫統，利用來自廢水微藻的聚磷酸鹽作為磷酸鹽供體，從腺苷中再生 ATP。此再生機制透過雙功能核黃素激酶/FAD合成酶和焦磷酸酶的共同作用，使核黃素在2小時內完全轉化為FAD，最終濃度達到約1.2g/L（1.5mmol/L）。值得注意的是，唯一的副產品正磷酸鹽可以透過廢水微藻回收來再生聚磷酸鹽，進而可以作為磷酸鹽供體重新融入系統。這種閉環策略有利於實現環境永續的 FAD 合成過程，同時最大限度地減少廢棄物產生。;Flavin adenine dinucleotide (FAD) serves as a crucial cofactor in numerous biological redox and radical reactions. Conventionally, FAD biosynthesis from riboflavin follows a two-step enzymatic pathway requiring ATP, with flavin mononucleotide (FMN) as the intermediate. Industrial-scale FAD production has traditionally relied on microbial fermentation, but these methods often entail laborious purification steps. Given the importance of atomic economy and yield efficiency, an enzymatic in vitro approach presents a more sustainable and effective alternative. However, the widespread industrial application of enzymatic FAD synthesis is hindered by its dependence on costly ATP as a substrate. To address this limitation, I have developed a two-enzyme cascade system that regenerates ATP from adenosine, utilizing polyphosphate derived from wastewater microalgae as a phosphate donor. This regeneration mechanism enables the complete transformation of riboflavin into FAD within 2 h, achieving a final concentration of approximately 1.2 g/L (1.5 mmol/L) through the combined action of bifunctional riboflavin kinase/FAD synthetase and pyrophosphatase. Notably, the only byproduct, orthophosphate, can be recycled by wastewater microalgae to regenerate polyphosphate, which in turn can be reintegrated into the system as a phosphate donor. This closed-loop strategy facilitates an environmentally sustainable FAD synthesis process with minimal waste production.
