【摘要】：Macrolactins是一類(lèi)新型大環(huán)內(nèi)酯類(lèi)化合物,具有抗菌、免疫抑制、抗腫瘤等多種生物學(xué)活性,在醫(yī)藥領(lǐng)域具有極大的應(yīng)用前景。目前野生菌發(fā)酵產(chǎn)macrolactins的產(chǎn)量較低,限制了對(duì)此類(lèi)化合物的研究與應(yīng)用,因此改造相關(guān)生物合成基因簇以提高macrolactins的產(chǎn)量具有十分重要的意義。本文以實(shí)驗(yàn)室前期篩選獲得的一株能產(chǎn)macrolactins的野生菌ZJU-2011為研究對(duì)象,通過(guò)測(cè)序確定其為一株新型的解淀粉芽孢桿菌,將其PKS基因簇中高保守的AT結(jié)構(gòu)域及可能的啟動(dòng)子區(qū)域與參考基因簇進(jìn)行比對(duì),確定了該基因簇的存在并發(fā)現(xiàn)其啟動(dòng)子元件位于第一模塊前1Kb左右的位置。以綠色熒光蛋白為報(bào)告基因構(gòu)建啟動(dòng)子檢測(cè)載體,與P43啟動(dòng)子比較確定該基因簇原始啟動(dòng)子為弱啟動(dòng)子,進(jìn)一步研究發(fā)現(xiàn)原始啟動(dòng)子是一個(gè)受葡萄糖調(diào)節(jié)的啟動(dòng)子,最適作用溫度為37℃。以P43啟動(dòng)子為基礎(chǔ)通過(guò)飽和突變構(gòu)建啟動(dòng)子突變文庫(kù)。優(yōu)化確定聚合酶環(huán)形延伸克隆(CPEC)最適的退火溫度為61.6℃,最佳的PCR循環(huán)數(shù)為25個(gè)。以LacZ作為報(bào)告基因通過(guò)CPEC克隆構(gòu)建啟動(dòng)子突變文庫(kù),以β-半乳糖苷酶的酶活表征啟動(dòng)子的強(qiáng)度,篩選得到11個(gè)正向突變株,其中標(biāo)號(hào)為“7”的突變株β-半乳糖苷酶酶活為P43啟動(dòng)子控制下的167.7%。對(duì)采用同源重組法在ZJU-2011中進(jìn)行基因敲除的可行性進(jìn)行研究。確定解淀粉芽孢桿菌ZJU-2011的最適轉(zhuǎn)化條件為甘氨酸添加量為1%,菌體OD600為0.9左右,質(zhì)粒進(jìn)行去甲基化處理,電場(chǎng)強(qiáng)度為21KV/cm,電阻為200Ω。構(gòu)建以mazF毒蛋白為反篩標(biāo)記的整合載體pIEFMLN,通過(guò)同源重組成功篩選到了單交換陽(yáng)性轉(zhuǎn)化子,但經(jīng)實(shí)驗(yàn)驗(yàn)證雙交換重組的概率低于1/1500,因此初步判斷同源重組法不適用于解淀粉芽孢桿菌ZJU-2011的基因敲除。嘗試構(gòu)建解淀粉芽孢桿菌中的CRISPR-Cas基因編輯系統(tǒng)。成功構(gòu)建CRISPR-Cas9相關(guān)的敲除載體pHYpCasMLN,經(jīng)驗(yàn)證該質(zhì)粒已成功轉(zhuǎn)入ZJU-2011 中。
[Abstract]:Macrolactins is a new class of macrolides with many biological activities, such as antibacterial, immunosuppressive, antitumor and so on. At present, the yield of fermentative macrolactins from wild bacteria is relatively low, which limits the research and application of these compounds. Therefore, it is of great significance to modify the related biosynthetic gene clusters to increase the yield of macrolactins. In this paper, a new strain of Bacillus amylolyticus, ZJU-2011, which can produce macrolactins, was selected as a new strain of Bacillus amylolyticus by sequencing. The highly conserved AT domain and possible promoter region in the PKS gene cluster were compared with the reference gene cluster. The existence of the gene cluster was determined and the position of the promoter element located about 1Kb in front of the first module was found. Green fluorescent protein (GFP) was used as a reporter gene to construct promoter detection vector. Compared with P43 promoter, the primordial promoter of the gene cluster was identified as weak promoter, and the primordial promoter was found to be a glucose regulated promoter. The optimum action temperature is 37 鈩，

本文編號(hào)：2153816