qdoI基因對枯草菌BCRC80517進行黃酮醇化合物磷酸化之影響

Abstract

本研究室先前已知利用源自於Bacillus subtilis BCRC80517的類黃酮磷酸酯合成酶可將quercetin、kaempferol及fisetin轉化生成相對應的磷酸酯衍生物。然而，利用微生物本身直接對上述三種化合物進行生物轉化時，卻僅有fisetin可得到與in vitro酵素轉化時相同的磷酸酯衍生物。由化學結構及轉化產物分析轉化基質的結果，推測原因為quercetin、kaempferol在結構上共同具有5號碳之羥基，相較於類黃酮磷酸酯合成酶，與B. subtilis中的quercetin 2,3-dioxygenase親和性較高，而quercetin 2,3-dioxygenase會在化合物的2號及3號碳進行氧化反應，導致其開環，無法轉化生成磷酸酯衍生物，fisetin無5號碳之羥基，則能以磷酸化途徑進行代謝。 本研究利用磷酸酯合成酶轉化fisetin，產物純化後再經由NMR及LC-MS/MS鑑定，確認其為磷酸酯衍生物，磷酸酯鍵分別位於3’及4’號碳上。由於fisetin在B. subtilis BCRC80517中能以磷酸化途徑進行轉化，推測將quercetin 2,3-dioxygenase活性抑制，則5號碳上具羥基之黃酮醇類同樣能以此途徑進行代謝。本研究第二部分建立同源重組及CRISPR-Cas9系統，以基因剔除方式去除quercetin 2,3-dioxygenase。B. subtilis 168為模式菌株，以其作為基因編輯之對象。首先，確認B. subtilis 168具有類黃酮磷酸酯合成酶及quercetin 2,3-dioxygenase後，再建構同源重組及CRISPR-Cas9系統之載體，並配合電穿孔進行突變，最後分別建立篩選模式挑選突變菌株。同源重組以抗生素進行篩選，CRISPR-Cas9系統則以quercetin作為指標，未經過基因編輯之菌株於含quercetin的固態培養基上會產生透明圈，且突變後主要的代謝途徑被剔除，推測會導致菌株生長速度較慢，因此挑選生長慢且未產生透明圈之菌株，再進行定序。

Flavonols are a group of secondary metabolites in plants and possess versatile physiological activities for human health. However, most of them are water insoluble, resulting in the limitation of oral administration applications. Our previous studies revealed Bacillus subtilis BCRC80517 was capable of converting several types of flavonoids into highly water-soluble phosphate conjugates. Nevertheless, flavonols with C-5-hydroxyl group in structure, such as quercetin and kaempferol, could not be transformed into the corresponding phosphate conjugates by the bacterium, but ring-opened in the C-ring of structure by bacterial constitutive quercetin 2,3-dioxygenase (qdoI) instead. Interestingly, when B. subtilis BCRC80517 cultivated with flavonols without C-5-hydroxyl group in structure, such as fisetin, the main transformation products would be the phosphate conjugates rather than the ring-opened products. In this work, we isolated the bioconversion products and identified two novel derivatives, fisetin 4’-O-phosphate and fisetin 3’-O-phosphate, by LC-MS/MS and 1H, 13C and 31P NMR spectral data. For flavonols with C-5-hydroxyl group, knocking out the qdoI gene might be a crucial approach to produce phosphorylated conjugates. B. subtilis 168-type strain was used as the host for eliminating the qdoI gene through homologous recombination and CRISPR-Cas9. For homologous recombination, neomycin resistance was used to positively select transformed colonies. For CRISPR-Cas9 system, the clear zone formed by quercetin degradation was used as negative selection criteria to screen the mutant strains.