結合計算模型與生化分析以探索非核糖體肽與 Microcin J25 的生合成機制

Abstract

抗生素抗藥性已成為全球醫療系統的重大威脅，當前急需發掘新型抗菌藥物。在微生物所產生的代謝物中，尤其是非核糖體肽（NRPs）以及核糖體合成及轉譯後修飾肽（RiPPs）具有很大的潛力。本論文聚焦於此兩類抗菌物進行研究：首先，針對兩個新發現的非核糖體肽合成酶（NRPS）模組 A5742 與 Mr_4320 進行鑑定與分析；其次，深入探討合成套索型多肽 microcin J25（MccJ25）之酵素結構與功能。 我們利用 antiSMASH 5.1.0 進行基因體挖掘，在 Mycobacterium fortuitum 中找到了 A5742，以及在 Mycobacterium rhodesiae 中發現了 Mr_4320，這兩個基因皆可能參與雜環多肽的製造。分析結果顯示，A5742 對絲胺酸（Ser）與蘇胺酸（Thr）表現出高度專一性，證實其在雜環多肽合成中的角色。相較之下，Mr_4320 對任何一種所謂蛋白胺基酸都無活性，顯示其可能需要其他胺基酸作為受質。我們之後進一步在大腸桿菌BAP1菌株中表現 Mr_4320，證實了其 T2 domain可被磷酸泛硫辛酯化（phosphopantetheinylation），這對 NRPS 系統中的受質結合至關重要。本研究並提出策略來鑑定 Mr_4320 的天然受質，例如從原始宿主菌裂解液中抓取受質、以及運用較小區塊的Mr_4320來促進表現等方法。 除此之外，本研究也探討了具備高溫與酸鹼穩定性的套索型多肽 MccJ25，並透過 AlphaFold 3 的結構預測，模擬了整個 MccJ25 生合成複合體（McjA/McjB/McjC/Mg2+/ATP）之間的相互作用。研究中鑑定了關鍵的「蛋白—蛋白」以及「蛋白—配體」結合位置，並特別著重於前導多肽中倒數第二個蘇胺酸（T-2）在辨識上的重要性。定點突變（site-directed mutagenesis）實驗針對此位置及其周邊胺基酸進行改造，成功透過恢復或抑制 MccJ25 產量的實驗結果，驗證了理論模擬的準確度。 最後，我們嘗試在 McjA/McjB/McjC 複合體中引入經過精心設計的突變，以減少蛋白聚集並利於後續結晶研究。雖然目前仍難以獲得理想的晶體，但隨著此處的種種嘗試及結果，我們已成功於理解 MccJ25 生合成的分子機制上邁進了一步，也為未來套索型多肽的結構改良提供了指引、開啟了機會。 透過結合理論模擬與生化驗證，本論文在 NRPS 受質特異性、酵素催化機制以及多肽改造等面向皆有新的發現，並逐步開啟新型抗生素的研發，以持續在抗藥性問題上做出實質的進展。

Antibiotic resistance is a growing global health crisis that necessitates the discovery of novel antimicrobial agents. Among one of the most promising leads are specialized microbial metabolites, particularly nonribosomal peptides (NRPs) and ribosomally synthesized and post-translationally modified peptides (RiPPs). This thesis focuses on two aspects of antimicrobial metabolites research: the characterization of two newly identified nonribosomal peptide synthetase (NRPS) modules, A5742 and Mr_4320, and the structural and functional investigation of the biosynthetic enzymes involved in the production of the lasso peptide microcin J25 (MccJ25). Using a genome mining approach with antiSMASH 5.1.0, we identified A5742 (from Mycobacterium fortuitum) and Mr_4320 (from Mycobacterium rhodesiae) as potential genes that involved in heterocyclic peptides production. Functional assays revealed that A5742 strongly prefers serine and threonine as substrates, underscoring its role in heterocycle formation. By contrast, Mr_4320 did not exhibit activity toward any of the proteinogenic amino acids, suggesting the incorporation of non-proteinogenic substrates. Subsequent expression trials in an engineered Escherichia coli (E. coli) strain BAP1 confirmed phosphopantetheinylation of the T2 domain, which is vital for substrate attachment in NRPS processes. Strategies to identify Mr_4320’s natural substrates, such as capturing them from native bacterial lysates and employing partial domain constructs, are highlighted. Additionally, this work explores the biosynthesis of MccJ25, a lasso peptide known for its thermal and pH stability and unique inhibitory mechanisms against Gram-negative pathogens. Through AlphaFold 3–based modeling of the MccJ25 biosynthetic complex (McjA/McjB/McjC/Mg2+/ATP), we identified key protein–protein and protein–ligand interactions, including the importance of the penultimate threonine (T-2) (T minus-two) in the leader peptide. Site-directed mutagenesis experiments targeting this residue and its surrounding environment helped validate the computational model by recovering or abolishing MccJ25 production. Finally, we attempted to stabilize the McjA/McjB/McjC complex for crystallographic studies by introducing strategically chosen mutations to minimize protein aggregation. Although challenges remain in obtaining well-diffracting crystals, our findings mark significant progress toward understanding the molecular basis of MccJ25 maturation and guiding future rational engineering of lasso peptides. By integrating computational predictions with biochemical validation, this thesis provides new insights into NRPS substrate specificity, enzymatic catalysis, and peptide engineering strategies, moving closer to next-generation antibiotics to combat antibiotic resistance.