杀念菌素生物合成中糖基转移酶的研究
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摘要
杀念菌素是由链霉菌FR-008(Streptomyces sp. FR-008)所产生的具有广谱抗真菌活性的多烯类抗生素。该类化合物主要来自细菌的次级代谢产物，并且实验证实这类化合物是通过与真菌细胞膜内的甾醇分子相互作用形成通透性孔道来发挥抗真菌活性的。由于多烯类抗生素的广谱抗菌活性以及耐药性产生几率小使得它们在临床上的应用已经超过半个世纪，成为治疗许多危重深部真菌感染的重要药物。多烯类抗生素结构中普遍存在一个特殊的氨基海藻糖，它不仅在该类抗生素发挥抗真菌活性中起到了重要作用，同时也影响了该类化合物的水溶性等理化性质。鉴于氨基海藻糖的重要性，长期以来科学家们对负责其加载的糖基转移酶表现出了浓厚的研究兴趣，但多烯类糖基转移酶体外表达的困难使得对它的底物特异性以及影响酶催化活性的重要氨基酸残基等方面的研究难以顺利展开。本研究利用链霉菌FR-008生长周期短，遗传操作简单，产素水平高等优点，建立了一个体内研究多烯类糖基转移酶的生物化学检测方法，并利用该方法揭示了多烯类糖基转移酶的底物特异性以及影响其催化活性的重要氨基酸残基，为利用组合生物合成手段产生具有更高药理活性的多烯类衍生物提供了理论依据。
首先，敲除杀念菌素生物合成基因簇中可能负责编码糖基转移酶的基因fscMI获得了不再携带糖基的杀念菌素糖苷配基，并且在用红霉素启动子控制下的fscMI基因进行回补实验之后恢复了产生杀念菌素的能力，证明FscMI参与催化了氨基海藻糖的加载，是一个多烯类糖基转移酶。同时，多烯类抗生素制霉菌素、两性霉素以及匹马霉素生物合成基因簇中的同源蛋白(NysDI、AmphDI、PimK)也都能够识别杀念菌素糖苷配基，并且在不同程度上恢复fscMI缺失突变株LX1中杀念菌素的产量，证明多烯类糖基转移酶对糖苷配基的底物特异性并不是非常严格。
为了揭示糖基转移酶在糖苷配基也即聚酮骨架上的识别基团，本研究构建了DH11的点突变菌株LX8。DH11位于杀念菌素生物合成基因簇的聚酮合酶FscD上，负责杀念菌素结构中C22-C23位上双键的形成，该键处于多烯类抗生素的结构保守区并且是距离氨基海藻糖最近的一个双键。突变株LX8不再产生杀念菌素，经LC-MS、Q-TOF以及特征紫外吸收峰的检测确定该突变株产生了两个具有相同分子量([M+H]+=1,127.3)以及预测结构式的杀念菌素六烯衍生物，[M+H]+1,127.3证明C23位已经被羟基取代，并且氨基海藻糖仍然能够加载，推测这两个新的化合物是由糖基加载在原始的C21位或新形成的C23位羟基上所引起的。该结果也进一步说明多烯类糖基转移酶对糖苷配基底物具有一定的选择宽泛性。
随后，通过对糖基转移酶FscMI的同源建模找到可能在糖基化过程中发挥重要作用的氨基酸残基，并用定点突变的方法分别将这些氨基端残基进行了突变，得到一系列糖基转移酶FscMI的突变体。体内对这些FscMI突变体识别天然底物的能力进行了定量检测，发现Ser346、Ser361、His362和Cys387显著影响FscMI催化能力。这是首例对多烯类糖基转移酶中重要氨基酸残基的研究，为今后通过定向突变的方式改变多烯类糖基转移酶的底物特异性，扩大其糖苷配基和糖基的识别范围从而得到具有更高药用价值的多烯类抗生素衍生物提供了理论依据。
另外，本研究还对杀念菌素生物合成基因簇中存在的ABC转运蛋白编码基因fscTI和fscTII的生物学功能进行了研究。通过遗传操作手段，构建了一个用于敲除转运基因fscTI和fscTII的同源重组双交换质粒，并通过接合转移获得了转运蛋白缺失突变株LX10，对该突变株的发酵检测显示它不再产生多烯类抗生素；此外，通过将转运蛋白FscTI和FscTII在杀念菌素产生菌中进行过量表达得到了基因工程菌株LX11，LX11中杀念菌素的产量得到了提高。本研究证实了FscTI和FscTII是ABC转运蛋白，FscTI和FscTII的过量表达增加了基因工程菌株中杀念菌素的产量，为利用转运蛋白过量表达的方法提高两性霉素等其他多烯类抗生素的产量提供了例证。
Candicidin is a heptaene macrolide produced by Streptomyces sp.FR-008. This kind of compounds mainly comes from the secondarymetabolites of bacterials and exhibits antifungal activity by formingtransmembrane pores in the fungus membrane. Polyenes have been usedin human therapy for treatment of severe fungal infections for over50years due to their broad spectrum of activity and the low frequency ofappearance of resistrant fungal pathogens. Mycosamine exists in mostpolyenes, it contributes a lot to the antifungal activity and solubility ofpolyenes. Condersiding of this, scientists exhibit great research interestsin glycosyltransferases (GTs), however, the substrate specificity andactivity affecting amino acid residues have not been elucidated due to thedifficulty of expression polyene GTs. In this study, we made use of therapid growth, simple genetic manipulation and high production of S. spFR-008and generated an in vivo biochemical detection method tocharacterize polyene GTs, illustrated that polyene GTs have loosesubstrate specificity toward aglycones and found the activity affectingamino acid residues of polyene GTs, thus providing an opportunity to generate libraries of polyene derivatives with improved pharmaceuticalactivity.
Disruption of GT gene fscMI in the biosynthesis of candicidin led tothe accumulation of deglycosylated candicidin aglycone andcomplementation experiment confirmed FscMI is a polyene GT. Besides,expression of homologous polyene GTs (NysDI、AmphDI、PimK) infscMI mutant could all restore the production of candicidin. These resultsillustrated that polyene GTs have some tolerance toward their substrates.
In order to identify which moiety of polyene structures is essentialfor GTs recognition, we constructed a DH11point mutation mutant LX8.DH11is located in FscD and is accredited for the formation of the doublebond between C22and C23, which lies in the conserved region ofpolyenes and adjacent to mycosamine. The fermentation culture extractsof LX8were analyzed by LC-MS、Q-TOF and characteritic UV detection,two new hexaene derivatives with the same mass of1,127.3([M+H]+)and formular appeared. The [M+H]+of1,127.3suggests that the sugarmoiety is still present in the compounds and the double bond betweenC22and C23has changed to a hydroxyl group at C23, thus the hydroxylgroups at C21and C23may provide two possible attachment site formycosamine, leading to the two new peaks in the fermentation cultureextracts of LX8. These results further confirm that polyene GTs havesome tolerance toward their substrates.
To gain insight into the amino acid residues that contribute to theglycosylation process of FscMI, we used homology modeling and finallygot a set of site-directed mutated FscMI. Quantitative HPLC analysis ofthe site-directed mutants illustrated that Ser346, Ser361, His362andCys387contribute a lot to the catalytic activity of FscMI. This is the firsttime to report the critical amino acid residues in polyene GTs. In thefuture, we could expect to change the critical amino acid residues toappropriate ones and make use of the loose substrate specificity featuresof polyene GTs, generating novel polyene derivatives with improvedbioactivities by manipulating with an ample pool of sugar donors andacceptors with different structural originality.
Also, we analyzed the ABC transporter genes fscTI and fscTII thatexist in the biosynthetic gene cluster of candicidin. pJTU4137wasconstructed for disruption of fscTI and fscTII and it was transferred intoStreptomyces sp. FR-008derived strain ZYJ-6by conjugation. Theresulting mutant LX10was unable to produce polyenes. In addition, thetransporter genes were overexpressed in strain ZYJ-6and the productionof candicidin in the resultant engineered strain LX11increased to1.5-foldwith that of the control. We confirmed that FscTI and FscTII are putativeABC(ATP-binding cassette)transporters and overexpression of transportergenes increased candicidin production provides a positive example forimproving other polyene production.
首先，敲除杀念菌素生物合成基因簇中可能负责编码糖基转移酶的基因fscMI获得了不再携带糖基的杀念菌素糖苷配基，并且在用红霉素启动子控制下的fscMI基因进行回补实验之后恢复了产生杀念菌素的能力，证明FscMI参与催化了氨基海藻糖的加载，是一个多烯类糖基转移酶。同时，多烯类抗生素制霉菌素、两性霉素以及匹马霉素生物合成基因簇中的同源蛋白(NysDI、AmphDI、PimK)也都能够识别杀念菌素糖苷配基，并且在不同程度上恢复fscMI缺失突变株LX1中杀念菌素的产量，证明多烯类糖基转移酶对糖苷配基的底物特异性并不是非常严格。
为了揭示糖基转移酶在糖苷配基也即聚酮骨架上的识别基团，本研究构建了DH11的点突变菌株LX8。DH11位于杀念菌素生物合成基因簇的聚酮合酶FscD上，负责杀念菌素结构中C22-C23位上双键的形成，该键处于多烯类抗生素的结构保守区并且是距离氨基海藻糖最近的一个双键。突变株LX8不再产生杀念菌素，经LC-MS、Q-TOF以及特征紫外吸收峰的检测确定该突变株产生了两个具有相同分子量([M+H]+=1,127.3)以及预测结构式的杀念菌素六烯衍生物，[M+H]+1,127.3证明C23位已经被羟基取代，并且氨基海藻糖仍然能够加载，推测这两个新的化合物是由糖基加载在原始的C21位或新形成的C23位羟基上所引起的。该结果也进一步说明多烯类糖基转移酶对糖苷配基底物具有一定的选择宽泛性。
随后，通过对糖基转移酶FscMI的同源建模找到可能在糖基化过程中发挥重要作用的氨基酸残基，并用定点突变的方法分别将这些氨基端残基进行了突变，得到一系列糖基转移酶FscMI的突变体。体内对这些FscMI突变体识别天然底物的能力进行了定量检测，发现Ser346、Ser361、His362和Cys387显著影响FscMI催化能力。这是首例对多烯类糖基转移酶中重要氨基酸残基的研究，为今后通过定向突变的方式改变多烯类糖基转移酶的底物特异性，扩大其糖苷配基和糖基的识别范围从而得到具有更高药用价值的多烯类抗生素衍生物提供了理论依据。
另外，本研究还对杀念菌素生物合成基因簇中存在的ABC转运蛋白编码基因fscTI和fscTII的生物学功能进行了研究。通过遗传操作手段，构建了一个用于敲除转运基因fscTI和fscTII的同源重组双交换质粒，并通过接合转移获得了转运蛋白缺失突变株LX10，对该突变株的发酵检测显示它不再产生多烯类抗生素；此外，通过将转运蛋白FscTI和FscTII在杀念菌素产生菌中进行过量表达得到了基因工程菌株LX11，LX11中杀念菌素的产量得到了提高。本研究证实了FscTI和FscTII是ABC转运蛋白，FscTI和FscTII的过量表达增加了基因工程菌株中杀念菌素的产量，为利用转运蛋白过量表达的方法提高两性霉素等其他多烯类抗生素的产量提供了例证。
Candicidin is a heptaene macrolide produced by Streptomyces sp.FR-008. This kind of compounds mainly comes from the secondarymetabolites of bacterials and exhibits antifungal activity by formingtransmembrane pores in the fungus membrane. Polyenes have been usedin human therapy for treatment of severe fungal infections for over50years due to their broad spectrum of activity and the low frequency ofappearance of resistrant fungal pathogens. Mycosamine exists in mostpolyenes, it contributes a lot to the antifungal activity and solubility ofpolyenes. Condersiding of this, scientists exhibit great research interestsin glycosyltransferases (GTs), however, the substrate specificity andactivity affecting amino acid residues have not been elucidated due to thedifficulty of expression polyene GTs. In this study, we made use of therapid growth, simple genetic manipulation and high production of S. spFR-008and generated an in vivo biochemical detection method tocharacterize polyene GTs, illustrated that polyene GTs have loosesubstrate specificity toward aglycones and found the activity affectingamino acid residues of polyene GTs, thus providing an opportunity to generate libraries of polyene derivatives with improved pharmaceuticalactivity.
Disruption of GT gene fscMI in the biosynthesis of candicidin led tothe accumulation of deglycosylated candicidin aglycone andcomplementation experiment confirmed FscMI is a polyene GT. Besides,expression of homologous polyene GTs (NysDI、AmphDI、PimK) infscMI mutant could all restore the production of candicidin. These resultsillustrated that polyene GTs have some tolerance toward their substrates.
In order to identify which moiety of polyene structures is essentialfor GTs recognition, we constructed a DH11point mutation mutant LX8.DH11is located in FscD and is accredited for the formation of the doublebond between C22and C23, which lies in the conserved region ofpolyenes and adjacent to mycosamine. The fermentation culture extractsof LX8were analyzed by LC-MS、Q-TOF and characteritic UV detection,two new hexaene derivatives with the same mass of1,127.3([M+H]+)and formular appeared. The [M+H]+of1,127.3suggests that the sugarmoiety is still present in the compounds and the double bond betweenC22and C23has changed to a hydroxyl group at C23, thus the hydroxylgroups at C21and C23may provide two possible attachment site formycosamine, leading to the two new peaks in the fermentation cultureextracts of LX8. These results further confirm that polyene GTs havesome tolerance toward their substrates.
To gain insight into the amino acid residues that contribute to theglycosylation process of FscMI, we used homology modeling and finallygot a set of site-directed mutated FscMI. Quantitative HPLC analysis ofthe site-directed mutants illustrated that Ser346, Ser361, His362andCys387contribute a lot to the catalytic activity of FscMI. This is the firsttime to report the critical amino acid residues in polyene GTs. In thefuture, we could expect to change the critical amino acid residues toappropriate ones and make use of the loose substrate specificity featuresof polyene GTs, generating novel polyene derivatives with improvedbioactivities by manipulating with an ample pool of sugar donors andacceptors with different structural originality.
Also, we analyzed the ABC transporter genes fscTI and fscTII thatexist in the biosynthetic gene cluster of candicidin. pJTU4137wasconstructed for disruption of fscTI and fscTII and it was transferred intoStreptomyces sp. FR-008derived strain ZYJ-6by conjugation. Theresulting mutant LX10was unable to produce polyenes. In addition, thetransporter genes were overexpressed in strain ZYJ-6and the productionof candicidin in the resultant engineered strain LX11increased to1.5-foldwith that of the control. We confirmed that FscTI and FscTII are putativeABC(ATP-binding cassette)transporters and overexpression of transportergenes increased candicidin production provides a positive example forimproving other polyene production.
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