阿维菌素和安丝菌素生物合成相关基因的功能研究
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摘要
阿维菌素是由阿维链霉菌（Streptomyces avermtilis）产生的16元大环内酯类抗生素。它具有高效、广谱的抗各种线虫和人类寄生虫的活性，被广泛地用于农业和畜牧业的病虫害防治。
avtAB（sav_933、sav_934）是紧邻阿维菌素生物合成基因簇的两个基因，它们分别编码ABC转运蛋白的ATP结合亚基（AvtA）和跨膜亚基（AvtB）。根据生物信息学的分析，AvtAB极有可能与阿维菌素的外排相关。本研究构建了avtAB的缺失突变株，发现avtAB的缺失并不能影响阿维菌素的产量。但以野生型菌株NRRL8165和工业菌株3-115作为出发菌株构建avtAB高表达菌株，固体发酵时阿维菌素的产量约有一倍左右的提高。 avtAB工业高拷贝菌株（3-115/pJTU3470）在工业液体培养基中发酵时，阿维菌素B1a的产量约有50%的提高，可从3.3g/L提高到4.8g/L。利用YMG液体培养基定量分析胞内胞外阿维菌素产量的变化，结果发现avtAB拷贝数的增加使得胞内与胞外阿维菌素B1a的比例从6:1下降到4.5:1。利用Real-time RT-PCR的方法定量分析ABC转运蛋白基因avtAB、结构基因aveA3和调节基因aveR在avtAB高拷贝菌株中的转录水平，发现avtAB的转录量提高了30-500倍，而aveA3和aveR的转录量却没有明显变化。这说明avtAB高表达提高阿维菌素的产量是通过及时将其泵出胞外，以减少胞内的反馈抑制，而不是在转录水平激活结构基因和调节基因的表达。此外我们还发现，AvtAB对于阿维菌素具有底物专一性，avtAB的高表达并没有影响阿维链霉菌中另一种抗生素寡霉素的外排；对链霉菌ABC药物转运蛋白进行进化分析，发现AvtAB与天蓝色链霉菌产生的钙依赖抗生素（CDA）的外排蛋白同源性极高，但在天蓝色链霉菌中高表达avtAB并没有明显提高CDA的产量。
aveE基因位于阿维菌素生物合成基因簇的内部，它编码P450单加氧酶，推测其负责催化阿维菌素后修饰的第一步反应——C6-C8a位的呋喃环闭合。为了阐明AveE的功能，深入研究AveE的作用机制，我们首先构建了aveE基因中断突变株，从中分离到了AveE体外催化底物。我们还构建了aveE回补菌株和meiE异源回补菌株，发现aveE和meiE只能恢复阿维菌素A组分，并不能恢复阿维菌素B组分的产生，暗示AveE可能还具有脱去阿维菌素C5位甲基的功能。在获得AveE可溶蛋白后，我们进行了体外反应尝试，并检测到了反应的发生，从体外的角度初步证明了AveE具有催化呋喃环形成的功能。
聚酮化合物中常会出现双键位置发生转移的现象，即双键的位置从两个延伸单位相邻两个碳原子之间（α-β位）转移到了前一个延伸单位的两个碳原子之间（β-γ位）。安丝菌素是从珍贵束丝放线菌橙色变种（Actinosynnema pretiosum ssp. auranticum）ATCC31565中分离到的安莎类抗生素。安丝菌素生物合成的第2和第3个模块中的脱水酶结构域（AsmDH2、AsmDH3）所对应催化的双键都发生了α-β位向β-γ位转换的现象。本研究通过定点突变失活了AsmDH2和AsmDH3，但定点突变株不能积累安丝菌素的完整延伸产物，说明下游酮脂酰-ACP-合成酶（KS）具有严格的底物严谨性，不能催化非天然的三酮或四酮中间产物。另外，我们还构建了将红霉素硫酯酶（EryTE）基因融合加载在安丝菌素模块2之后的突变株，结果显示EryTE并不能高效释放三酮中间产物，安丝菌素中间产物的体内捕获具有一定的难度。体外表达安丝菌素模块2，将约200kDa的可溶蛋白与安丝菌素天然二酮中间产物的类似物甲基氢肉桂酸-SNAC进行反应，得到了甲基氢肉桂酸等反应产物，证明该蛋白具有一定的催化活性。
Avermectins are16-membered macrocyclic polyketides with potentantiparasitic activities, produced by Streptomyces avermitilis. Avermectinand its derivatives have been widely used in livestock industry andagriculture.
Upstream of the avermectin biosynthetic gene cluster, there is theavtAB operon (sav_933, sav_934) encoding the ABC transporterATP-binding subunit (AvtA) and transmembrane subunit (AvtB).According to bioinformatic analysis, AvtAB is most likely related withavermectin export. Inactivation of avtAB had no effect, but increasing thecopies of avtAB, using a high-copy-number plasmid in S. avermitilis,enhanced avermectin production about two-fold both in the wild-type andin a high-yielding producer strain on agar plates. In liquid industrialfermentation medium, the overall productivity of avermectin B1a in theengineered high-yielding producer was improved for about50%, from3.3to4.8g/L. In liquid YMG medium, the ratio of intracellular toextracellular accumulation of avermectin B1a was dropped from6:1to4.5:1in response to the high copies of avtAB. Additionally, theoverexpression of avtAB increased the concentration of avtAB mRNA30-500folds, but did not cause any increased expression of theavermectin biosynthetic genes detected by real-time RT-PCR analysis.We propose that the AvtAB transporter exports avermectin, and thus reduces the feedback inhibition on avermectin production in the cell.Moreover, the AvtAB transporter seemed to be specific for avermectinsbecause the production of oligomycin A, other macrolide produced by S.avermitilis, was not affected. According to phylogenetic analysis of ABCexporters located in antibiotic biosynthetic gene clusters, AvtAB is highlyhomologous to the multidrug efflux pump of calcium-dependentantibiotic (CDA) produced by S. coelicolor. However, CDA productionwas not increased by overexpression of avtAB in S. coelicolor.
The gene aveE, encoding an enzyme with the consensus sequence ofP450monooxygenase, was identified in the avermectin biosynthetic genecluster. It is suggested that furan ring closure at C6-C8a is catalyzed byAveE. To clarify the function of AveE, we inactivated aveE and separated6,8a-seco-6,8a-deoxy-5-oxoavermectin B1a aglycon, the predictedsubstrate of AveE, from the mutant. Complementation of the mutant withaveE and meiE from the meilingmycin biosynthetic gene cluster onlyrestored the production of avermectin A components, suggesting aputative demethylation function of AveE on the C5O-methyl group. Invitro characterization of AveE showed detectable activity for theconversion of6,8a-seco-6,8a-deoxy-5-oxoavermectin B1a aglycon into5-oxoavermectin B1a aglycon, verifying its function for the furan ringclosure.
Double bonds placed at atypical positions, mainly shifted from α-β toβ-γ-positions, are found in the structures of various polyketides.Ansamitocins, produced by Actinosynnema pretiosum ssp. auranticumATCC31565, are maytansinoids of microbial origin. The dehydratase(DH) domains of Asm-module2and module3would be involved in theformation of the shifted double bonds of ansamitocin P-3. Site-directedmutations of active site residues of AsmDH2and AsmDH3resulted in mutants that lack the ability to produce full-length ansamitocinderivatives, suggesting that downstream KS3and KS4, acting asgatekeepers, accept only the natural intermediates for further processing.Additionally, fusion of the erythromycin thioesterase (EryTE) domain atthe end of Asm-module2could not release the assembled triketide chainfrom the PKS. Engineered Asm-module2was expressed in E. coli andpurified. Incubation of Asm-module2with unnatural substrate2-methyl-3-phenylpropanoic-SNAC resulted in several productsincluding α-methylhydrocinnamic acid, suggesting that Asm-module2had catalytic activity.
avtAB（sav_933、sav_934）是紧邻阿维菌素生物合成基因簇的两个基因，它们分别编码ABC转运蛋白的ATP结合亚基（AvtA）和跨膜亚基（AvtB）。根据生物信息学的分析，AvtAB极有可能与阿维菌素的外排相关。本研究构建了avtAB的缺失突变株，发现avtAB的缺失并不能影响阿维菌素的产量。但以野生型菌株NRRL8165和工业菌株3-115作为出发菌株构建avtAB高表达菌株，固体发酵时阿维菌素的产量约有一倍左右的提高。 avtAB工业高拷贝菌株（3-115/pJTU3470）在工业液体培养基中发酵时，阿维菌素B1a的产量约有50%的提高，可从3.3g/L提高到4.8g/L。利用YMG液体培养基定量分析胞内胞外阿维菌素产量的变化，结果发现avtAB拷贝数的增加使得胞内与胞外阿维菌素B1a的比例从6:1下降到4.5:1。利用Real-time RT-PCR的方法定量分析ABC转运蛋白基因avtAB、结构基因aveA3和调节基因aveR在avtAB高拷贝菌株中的转录水平，发现avtAB的转录量提高了30-500倍，而aveA3和aveR的转录量却没有明显变化。这说明avtAB高表达提高阿维菌素的产量是通过及时将其泵出胞外，以减少胞内的反馈抑制，而不是在转录水平激活结构基因和调节基因的表达。此外我们还发现，AvtAB对于阿维菌素具有底物专一性，avtAB的高表达并没有影响阿维链霉菌中另一种抗生素寡霉素的外排；对链霉菌ABC药物转运蛋白进行进化分析，发现AvtAB与天蓝色链霉菌产生的钙依赖抗生素（CDA）的外排蛋白同源性极高，但在天蓝色链霉菌中高表达avtAB并没有明显提高CDA的产量。
aveE基因位于阿维菌素生物合成基因簇的内部，它编码P450单加氧酶，推测其负责催化阿维菌素后修饰的第一步反应——C6-C8a位的呋喃环闭合。为了阐明AveE的功能，深入研究AveE的作用机制，我们首先构建了aveE基因中断突变株，从中分离到了AveE体外催化底物。我们还构建了aveE回补菌株和meiE异源回补菌株，发现aveE和meiE只能恢复阿维菌素A组分，并不能恢复阿维菌素B组分的产生，暗示AveE可能还具有脱去阿维菌素C5位甲基的功能。在获得AveE可溶蛋白后，我们进行了体外反应尝试，并检测到了反应的发生，从体外的角度初步证明了AveE具有催化呋喃环形成的功能。
聚酮化合物中常会出现双键位置发生转移的现象，即双键的位置从两个延伸单位相邻两个碳原子之间（α-β位）转移到了前一个延伸单位的两个碳原子之间（β-γ位）。安丝菌素是从珍贵束丝放线菌橙色变种（Actinosynnema pretiosum ssp. auranticum）ATCC31565中分离到的安莎类抗生素。安丝菌素生物合成的第2和第3个模块中的脱水酶结构域（AsmDH2、AsmDH3）所对应催化的双键都发生了α-β位向β-γ位转换的现象。本研究通过定点突变失活了AsmDH2和AsmDH3，但定点突变株不能积累安丝菌素的完整延伸产物，说明下游酮脂酰-ACP-合成酶（KS）具有严格的底物严谨性，不能催化非天然的三酮或四酮中间产物。另外，我们还构建了将红霉素硫酯酶（EryTE）基因融合加载在安丝菌素模块2之后的突变株，结果显示EryTE并不能高效释放三酮中间产物，安丝菌素中间产物的体内捕获具有一定的难度。体外表达安丝菌素模块2，将约200kDa的可溶蛋白与安丝菌素天然二酮中间产物的类似物甲基氢肉桂酸-SNAC进行反应，得到了甲基氢肉桂酸等反应产物，证明该蛋白具有一定的催化活性。
Avermectins are16-membered macrocyclic polyketides with potentantiparasitic activities, produced by Streptomyces avermitilis. Avermectinand its derivatives have been widely used in livestock industry andagriculture.
Upstream of the avermectin biosynthetic gene cluster, there is theavtAB operon (sav_933, sav_934) encoding the ABC transporterATP-binding subunit (AvtA) and transmembrane subunit (AvtB).According to bioinformatic analysis, AvtAB is most likely related withavermectin export. Inactivation of avtAB had no effect, but increasing thecopies of avtAB, using a high-copy-number plasmid in S. avermitilis,enhanced avermectin production about two-fold both in the wild-type andin a high-yielding producer strain on agar plates. In liquid industrialfermentation medium, the overall productivity of avermectin B1a in theengineered high-yielding producer was improved for about50%, from3.3to4.8g/L. In liquid YMG medium, the ratio of intracellular toextracellular accumulation of avermectin B1a was dropped from6:1to4.5:1in response to the high copies of avtAB. Additionally, theoverexpression of avtAB increased the concentration of avtAB mRNA30-500folds, but did not cause any increased expression of theavermectin biosynthetic genes detected by real-time RT-PCR analysis.We propose that the AvtAB transporter exports avermectin, and thus reduces the feedback inhibition on avermectin production in the cell.Moreover, the AvtAB transporter seemed to be specific for avermectinsbecause the production of oligomycin A, other macrolide produced by S.avermitilis, was not affected. According to phylogenetic analysis of ABCexporters located in antibiotic biosynthetic gene clusters, AvtAB is highlyhomologous to the multidrug efflux pump of calcium-dependentantibiotic (CDA) produced by S. coelicolor. However, CDA productionwas not increased by overexpression of avtAB in S. coelicolor.
The gene aveE, encoding an enzyme with the consensus sequence ofP450monooxygenase, was identified in the avermectin biosynthetic genecluster. It is suggested that furan ring closure at C6-C8a is catalyzed byAveE. To clarify the function of AveE, we inactivated aveE and separated6,8a-seco-6,8a-deoxy-5-oxoavermectin B1a aglycon, the predictedsubstrate of AveE, from the mutant. Complementation of the mutant withaveE and meiE from the meilingmycin biosynthetic gene cluster onlyrestored the production of avermectin A components, suggesting aputative demethylation function of AveE on the C5O-methyl group. Invitro characterization of AveE showed detectable activity for theconversion of6,8a-seco-6,8a-deoxy-5-oxoavermectin B1a aglycon into5-oxoavermectin B1a aglycon, verifying its function for the furan ringclosure.
Double bonds placed at atypical positions, mainly shifted from α-β toβ-γ-positions, are found in the structures of various polyketides.Ansamitocins, produced by Actinosynnema pretiosum ssp. auranticumATCC31565, are maytansinoids of microbial origin. The dehydratase(DH) domains of Asm-module2and module3would be involved in theformation of the shifted double bonds of ansamitocin P-3. Site-directedmutations of active site residues of AsmDH2and AsmDH3resulted in mutants that lack the ability to produce full-length ansamitocinderivatives, suggesting that downstream KS3and KS4, acting asgatekeepers, accept only the natural intermediates for further processing.Additionally, fusion of the erythromycin thioesterase (EryTE) domain atthe end of Asm-module2could not release the assembled triketide chainfrom the PKS. Engineered Asm-module2was expressed in E. coli andpurified. Incubation of Asm-module2with unnatural substrate2-methyl-3-phenylpropanoic-SNAC resulted in several productsincluding α-methylhydrocinnamic acid, suggesting that Asm-module2had catalytic activity.
引文
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