Development of microbiome modification using lactic acid bacterial phages and the artificial synthesis technology of phages using lactic acid bacteria as hosts

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Title:	Development of microbiome modification using lactic acid bacterial phages and the artificial synthesis technology of phages using lactic acid bacteria as hosts
Authors:	吉村友希;Yoshimura, Tomoki
Contributors:	化學工程與材料工程學系
Keywords:	腸道微生物組;乳酸杆菌;噬菌體;微生物組調控;噬菌体人工合成;Gut microbiome;Lactic acid bacteria;Bacteriophage specificity;Microbiome modulation;Phage synthesis
Date:	2025-04-28
Issue Date:	2025-10-17 11:09:32 (UTC+8)
Publisher:	國立中央大學
Abstract:	人體腸道中共棲有超過 1,000 種微生物，總數約為 100 兆個細胞。腸道菌相失衡已知與多種疾病有關，因此理解個別細菌的功能以及其在腸道中的交互作用具有重要意義。在腸道菌群中，乳酸菌（LAB）被公認為有益的共生菌。雖然將乳酸菌納入腸道菌群所帶來的健康益處已被廣泛證實，但若腸道中原生乳酸菌數量減少，對整體菌相的影響以及與其他細菌之間的交互作用仍不清楚。因此，理解乳酸菌在腸道菌相中的角色至關重要。噬菌體（phage）因其高度的宿主特異性，被視為潛在的腸道菌相調控工具。噬菌體可自廢水、土壤等環境來源中分離而得。在本研究室中，我們成功分離出兩種可感染Lactiplantibacillus plantarum NCIMB 8826 的噬菌體，分別為 ΦLpTT1 和 ΦLpTT2。這些噬菌體已被證實能在由四種不同細菌（大腸桿菌Escherichia coli、普特假單胞菌 Pseudomonas putida、枯草桿菌 Bacillus subtilis 及 L.plantarum）構成的合成菌相中，專一性地抑制 L. plantarum 的生長。然而，若要有效利用這些噬菌體來進行菌相調控，亦需評估其對腸道中其他乳酸菌的特異性。此外，由於環境樣本中是否存在目標噬菌體具有不確定性，且噬菌體的分離須依賴可培養的宿主細菌，因此分離噬菌體的過程耗時且具挑戰性。因此，發展以噬菌體DNA為基礎的合成方法，以取代傳統從環境中分離噬菌體的方式，顯得尤為關鍵。在本研究中，我使用斑塊分析法（plaque assay）評估了ΦLpTT1 與 ΦLpTT2 對其宿主 L.plantarum 以外的多種乳酸菌及五種不同菌株的感染特異性。隨後，我測試了這兩株噬菌體在由八種乳酸菌組成的合成菌相中對 L. plantarum 生長的抑制能力。此外，為實現乳酸菌噬菌體的合成，我將所需的同源重組酶導入 L. plantarum。包含RecE/T 同源酶基因的質體 pLH01 在誘導肽（MAGNSSNFIHKIKQIFTHR）添加後能表現該酶，並成功導入 L. plantarum。接著，為確認 RecE/T 同源酶可識別的最佳同源序列長度，我將pKO-2000 質體分割成具 50、100、200 與 500 bp 同源序列的兩段，並分別導入 L. plantarum，以確認是否發生同源重組。根據這些實驗結果，最適合 RecE/T 同源酶識別的同源序列長度得以確定。;Abstract The human gut harbors over 1,000 species of microorganisms, totaling approximately 100 trillion cells. Imbalances in this microbiome are known to contribute to a wide range of diseases, highlighting the importance of understanding the roles of individual bacteria and their interactions within the gut. Among the gut microbiota, lactic acid bacteria (LAB) are recognized as beneficial commensals. While the health benefits of incorporating LAB into the microbiome are well established, the impact of a reduction in native LAB populations on the microbiome and their interactions with other bacteria remains unclear. Therefore, understanding the role of LAB in the gut microbiome is essential. Bacteriophages (phages), which exhibit high host specificity, have garnered attention as potential tools for microbiome manipulation. Phages can be isolated from environmental sources such as wastewater and soil. In our laboratory, we have successfully isolated two phages, ΦLpTT1 and ΦLpTT2, that infect Lactiplantibacillus plantarum NCIMB 8826. These phages have been shown to specifically inhibit L. plantarum in a synthetic microbiome composed of four different bacteria (Escherichia coli, Pseudomonas putida, Bacillus subtilis, and L. plantarum). However, to effectively utilize these phages for microbiome manipulation, it is also necessary to evaluate their specificity toward other LAB species present in the gut. Moreover, the isolation of phages is time consuming and challenging due to the uncertainty of their presence in environmental samples and the need for cultivable host bacteria. Therefore, it is critical to develop methods to synthesize phages rather than isolate them from environmental sources, using phage DNA. In this study, I evaluated the specificity of ΦLpTT1 and ΦLpTT2 against several other LAB species and five different bacterial strains, in addition to their host L. plantarum, using plaque assays. Subsequently, I tested the ability of these phages to inhibit the growth of L. plantarum in an eight LAB species synthetic microbiome. Additionally, the homologous recombination enzyme required for synthetic LAB phage synthesis was introduced into L. plantarum. The plasmid pLH01, which contains the RecE/T homologue gene and is expressed upon the addition of the inducer peptide (MAGNSSNFIHKIKQIFTHR), was introduced into L. plantarum. Subsequently, to determine the optimal length of the homologous sequence recognized by the RecE/T homologue, the pKO-2000 plasmid was divided into two fragments with homologous sequences of 50, 100, 200, and 500 bp. Each fragment was introduced into L. plantarum, and homologous recombination was confirmed. Based on these experiments, the optimal homologous sequence length for RecE/T homologue recognition was determined.
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