開發以穿梭載體系統應用於枯草桿菌中的脂肽生產工程

Abstract

表面活性素（surfactin）與豐原素（fengycin）是由枯草桿菌所產生的脂肽類生物界面活性劑，分別具有抗菌、抗病毒與抗絲狀真菌的活性，應用潛力橫跨生醫、農業與環境工程等多元領域。然而，由於天然生產菌株的產量普遍偏低，限制了其工業化應用的可行性。本研究首先針對枯草桿菌野生型菌株Bacillus subtilis NTU-14進行產能測試與發酵條件優化，測試兩種不同培養基對脂肽產量之影響，結果顯示YGF培養基具有最佳表現（培養48小時後，surfactin與fengycin產量分別為288.39 mg/L與40.41 mg/L）。隨後，本研究建構一個雙宿主穿梭載體系統，使其能同時適用於大腸桿菌與枯草桿菌，用於異源表現導入功能性基因sfp與degQ，分別應用於重建枯草桿菌模式菌株Bacillus subtilis 168的脂肽生合成能力，以及強化NTU-14的脂肽生合成調控效能。本研究確認質體可穩定轉形進入枯草桿菌並成功放大培養，並於後續整合驗證階段，初步比較改造菌株與對照株於不同培養條件下之脂肽產量。儘管異源表現尚未成功展現明確增效，推測可能與誘導條件、表現系統設計或蛋白折疊效率有關，本研究已建立一個具可行性之基因工程與菌株開發平台。未來若結合調控元件優化與培養參數微調，將可建構出具備實用價值的脂肽工業化生產系統。

Surfactin and fengycin are lipopeptide-type biosurfactants produced by Bacillus subtilis, exhibiting antibacterial, antiviral, and antifungal activities against filamentous fungi. These compounds hold significant application potential in diverse fields including biomedicine, agriculture, and environmental engineering. However, the generally low production levels of natural producer strains have limited their feasibility for industrial-scale applications. In this study, we first evaluated the production capacity of the wild-type Bacillus subtilis NTU-14 and optimized its fermentation conditions by testing two different culture medium. The results showed that NTU-14 achieved the highest lipopeptide production when cultivated in YGF medium (288.39 mg/L surfactin and 40.41 mg/L fengycin after 48 hours). Subsequently, a dual-host shuttle vector system compatible with both Escherichia coli and Bacillus subtilis was constructed to enable the heterologous expression of functional genes sfp and degQ. These genes were respectively applied to restore the lipopeptide biosynthetic capacity of the model strain B. subtilis 168 and to enhance the biosynthetic regulation in NTU-14. The plasmids were confirmed to be stably transformed into B. subtilis and successfully maintained during scale-up cultivation. In the final stage, the gene engineering and fermentation strategies were integrated to preliminarily assess the production performance of engineered strains under different cultivation conditions. Although the heterologous expression did not result in significant enhancement of lipopeptide yields, possibly due to induction conditions, expression system limitations, or protein folding inefficiency, this study has established a feasible platform for genetic engineering and strain development. With further improvements in regulatory element design and fermentation parameter refinement, this work may facilitate the development of a practically applicable and scalable lipopeptide production system for industrial use.