鼠李糖脂減毒高產(chǎn)菌株的構(gòu)建和鹽霉素基因簇salBⅢ基因功能研究
發(fā)布時(shí)間：2018-10-30 15:58
【摘要】：鼠李糖脂是一種低毒性,易于生物降解的,具有良好選擇性和專一性的糖脂類陰離子生物表面活性劑,在醫(yī)藥、食品、化妝品、環(huán)境保護(hù)等眾多工業(yè)領(lǐng)域具有廣泛的應(yīng)用前景。銅綠假單胞菌(Pseudomas aeruginosa)是目前鼠李糖脂產(chǎn)量最高的菌株,但是其具有條件致病性,易感染免疫力低下的患者,臨床感染率可以達(dá)到30%以上,直接用于工業(yè)生產(chǎn)存在很嚴(yán)重的安全隱患。5-烯醇丙酮酞莽草酸-3-磷酸合成酶基因aroA是細(xì)菌和植物莽草酸途徑合成芳香族氨基酸的關(guān)鍵基因,而哺乳動(dòng)物不具備這種途徑。在前期研究中,本課題組利用在假單胞菌中建立的高效同源重組系統(tǒng),成功敲除了銅綠假單胞菌PAO1(Pseudomas aeruginosa PAO1)的aroA基因,從而構(gòu)建出了一株在添加有芳香族氨基酸的培養(yǎng)基中才能生長的減毒Pseudowas aeruginosa PAO1-△aroA。本課題在此基礎(chǔ)上構(gòu)建了一個(gè)鼠李糖脂表達(dá)質(zhì)粒,該質(zhì)粒包含鼠李糖脂合成相關(guān)基因鼠李糖脂轉(zhuǎn)移酶復(fù)合體RhlAB、自轉(zhuǎn)運(yùn)酯酶基因EstA、慶大霉素抗性基因、接合轉(zhuǎn)移起始位點(diǎn)oriT和一個(gè)廣譜性復(fù)制子pBBRI。將該質(zhì)粒電轉(zhuǎn)化進(jìn)入構(gòu)建好的減毒菌株P(guān)AO1-AaroA中,多拷貝質(zhì)�？梢栽黾邮罄钐侵铣上嚓P(guān)基因在該菌中的拷貝數(shù),且在發(fā)酵過程中不會(huì)丟失,通過鼠李糖脂甲苯乳化實(shí)驗(yàn)和測(cè)定鼠李糖脂發(fā)酵產(chǎn)量可確定該減毒菌株的鼠李糖脂產(chǎn)量可增加75%。最終,本課題成功構(gòu)建了該高產(chǎn)鼠李糖脂的減毒PAO1-△aroA。鹽霉素是由白色鏈霉菌DSM 41398產(chǎn)生的一種離子載體抗生素,它能夠妨礙細(xì)胞內(nèi)外Na+/Ca2+的傳遞,破壞膜內(nèi)外離子平衡,造成細(xì)胞死亡,對(duì)革蘭氏陽性菌(包括分枝桿菌和一些絲狀真菌)及各種球蟲有很強(qiáng)的抑制和殺滅作用。最近研究發(fā)現(xiàn),鹽霉素不僅能夠殺死乳腺癌干細(xì)胞,還能抑制前列腺癌、肝癌和肺癌等多種腫瘤細(xì)胞的生長,具有比普通抗癌藥物紫杉醇更好的藥效。前期研究將鹽霉素合成基因簇克隆至天藍(lán)色鏈霉菌A3(2)中進(jìn)行異源表達(dá)時(shí)發(fā)現(xiàn)并未表達(dá)出預(yù)期的鹽霉素(m/= 733.6[M-H20+H]+),而是產(chǎn)生了一個(gè)鹽霉素衍生物(m/z = 752.6[M+H]+)。本課題對(duì)鹽霉素生物合成途徑中的相關(guān)基因進(jìn)行測(cè)序發(fā)現(xiàn),salBⅢ基因發(fā)生突變,有六個(gè)核苷酸序列丟失,導(dǎo)致所編碼蛋白連續(xù)缺少一個(gè)蘇氨酸一個(gè)亮氨酸。之后對(duì)鹽霉素基因簇上該基因進(jìn)行遺傳操作,研究清楚salBⅢ基因的功能。通過體內(nèi)基因敲除和修復(fù)實(shí)驗(yàn)表明salBⅢ基因與鹽霉素的第一個(gè)吡喃環(huán)的成環(huán)反應(yīng)相關(guān)。
[Abstract]:Rhamnolipid is a kind of anionic biosurfactants with low toxicity, easy biodegradation, good selectivity and specificity. It has been widely used in many industrial fields such as medicine, food, cosmetics, environmental protection and so on. Pseudomonas aeruginosa (Pseudomas aeruginosa) is the strain with the highest yield of rhamnolipid, but it has a conditional pathogenicity and is susceptible to infection in patients with low immunity. The clinical infection rate can reach more than 30%. AroA gene is a key gene in the synthesis of aromatic amino acids by the pathway of shikimic acid in bacteria and plants. Mammals do not have this pathway. In previous studies, our team successfully knocked out the aroA gene of Pseudomonas aeruginosa (PAO1 (Pseudomas aeruginosa PAO1) by using the highly efficient homologous recombination system established in Pseudomonas aeruginosa (Pseudomonas aeruginosa). A attenuated Pseudowas aeruginosa PAO1- aroA. was constructed to grow in medium containing aromatic amino acids. In this study, a rhamnolipid expression plasmid was constructed, which contained the gene associated with rhamnolipid biosynthesis, RhlAB, self-transporter esterase gene EstA, and gentamicin resistance gene. The initiation site of conjugate transfer oriT and a broad-spectrum replicon pBBRI. The multicopy plasmid can increase the copy number of the genes associated with rhamnolipid biosynthesis in the strain PAO1-AaroA, and it will not be lost during fermentation. By emulsifying rhamnose glycolipid toluene and determining the fermentative yield of rhamnolipid, the rhamnolipid yield of the attenuated strain can be increased by 75%. Finally, the PAO1- aroA. with high yield of rhamnolipid was successfully constructed. Salinomycin is an ionic carrier antibiotic produced by Streptomyces albicans DSM 41398. It can block the transfer of Na / Ca2 in and out of cells, destroy the balance of ions inside and outside the membrane, and cause cell death. It can inhibit and kill Gram-positive bacteria (including Mycobacterium and some filamentous fungi) and various coccidia. Recent studies have found that salinomycin can not only kill breast cancer stem cells, but also inhibit the growth of many kinds of tumor cells, such as prostate cancer, liver cancer and lung cancer. In previous studies, when the salinomycin synthase gene cluster was cloned into Streptomyces aquilinum A3 (2) for heterologous expression, it was found that the expected salinomycin was not expressed (m / r = 733.6 [M-H20 H]). Instead, it produces a salinomycin derivative (m / z = 752.6 [M / M H]). In this study, we sequenced the related genes in the biosynthesis pathway of salinomycin and found that the salB 鈪，
本文編號(hào)：2300464
[Abstract]:Rhamnolipid is a kind of anionic biosurfactants with low toxicity, easy biodegradation, good selectivity and specificity. It has been widely used in many industrial fields such as medicine, food, cosmetics, environmental protection and so on. Pseudomonas aeruginosa (Pseudomas aeruginosa) is the strain with the highest yield of rhamnolipid, but it has a conditional pathogenicity and is susceptible to infection in patients with low immunity. The clinical infection rate can reach more than 30%. AroA gene is a key gene in the synthesis of aromatic amino acids by the pathway of shikimic acid in bacteria and plants. Mammals do not have this pathway. In previous studies, our team successfully knocked out the aroA gene of Pseudomonas aeruginosa (PAO1 (Pseudomas aeruginosa PAO1) by using the highly efficient homologous recombination system established in Pseudomonas aeruginosa (Pseudomonas aeruginosa). A attenuated Pseudowas aeruginosa PAO1- aroA. was constructed to grow in medium containing aromatic amino acids. In this study, a rhamnolipid expression plasmid was constructed, which contained the gene associated with rhamnolipid biosynthesis, RhlAB, self-transporter esterase gene EstA, and gentamicin resistance gene. The initiation site of conjugate transfer oriT and a broad-spectrum replicon pBBRI. The multicopy plasmid can increase the copy number of the genes associated with rhamnolipid biosynthesis in the strain PAO1-AaroA, and it will not be lost during fermentation. By emulsifying rhamnose glycolipid toluene and determining the fermentative yield of rhamnolipid, the rhamnolipid yield of the attenuated strain can be increased by 75%. Finally, the PAO1- aroA. with high yield of rhamnolipid was successfully constructed. Salinomycin is an ionic carrier antibiotic produced by Streptomyces albicans DSM 41398. It can block the transfer of Na / Ca2 in and out of cells, destroy the balance of ions inside and outside the membrane, and cause cell death. It can inhibit and kill Gram-positive bacteria (including Mycobacterium and some filamentous fungi) and various coccidia. Recent studies have found that salinomycin can not only kill breast cancer stem cells, but also inhibit the growth of many kinds of tumor cells, such as prostate cancer, liver cancer and lung cancer. In previous studies, when the salinomycin synthase gene cluster was cloned into Streptomyces aquilinum A3 (2) for heterologous expression, it was found that the expected salinomycin was not expressed (m / r = 733.6 [M-H20 H]). Instead, it produces a salinomycin derivative (m / z = 752.6 [M / M H]). In this study, we sequenced the related genes in the biosynthesis pathway of salinomycin and found that the salB 鈪，
本文編號(hào)：2300464
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