【作者】 章亦荣；

【导师】 邓子新；

【作者基本信息】 上海交通大学 ， 微生物学， 2009， 博士

【摘要】 井冈霉素是从吸水链霉菌井冈变种5008(Streptomyces hygroscopicus var. jinggangensis 5008)中分离得到的一种弱碱性C7N氨基环醇类抗生素。它是一种海藻糖酶抑制剂,在中国及东南亚国家被广泛用在控制水稻叶鞘枯萎病和黄瓜幼苗枯萎病上。井冈霉素生物合成基因簇已被成功克隆并测序,这些序列信息有助于我们研究它的生物合成机理。我们在井冈霉素生物合成基因簇中确定了valC基因,它编码的酶与阿卡泊糖生物合成途径中的2-表-5-表-有效醇酮激酶(AcbM)同源。valC的失活使突变株丧失了合成井冈霉素的能力。用携带全长valC的质粒的互补实验使突变株ZYR-1中井冈霉素的产生得到恢复。这证明了valC在井冈霉素生物合成中的关键作用。ValC蛋白的体外实验揭示了ValC是一种新型的C7-环醇激酶,负责磷酸化有效烯酮和有效酮,但不能磷酸化2-表-5-表-有效醇酮、5-表-有效醇酮及葡萄糖。结果揭示了这个酶的新活性也证明了阿卡波糖和井冈霉素化学结构中共同的有效烯胺结构是通过不同的途径合成的。我们还在井冈霉素生物合成基因簇中确定了一个编码VOC超家族蛋白的基因valD。valD的失活显著降低了井冈霉素的产量,这种产量的降低可以在互补全长valD之后恢复,而互补N端或C端的ValD蛋白只能使产量部分恢复。异源表达的ValD负责催化2-表-5-表-有效醇酮异构生成5-表-有效醇酮。通过生物信息学分析发现ValD的N端区域有4个可能的活性位点H44、E107、H130和E183;C端区域也有4个可能的活性位点H229、E291、H315和E366。对ValD蛋白中8个可能的位点进行了单突变或组合突变结果显示,N端H44/E107和C端H315/E366比内部的4个氨基酸残基H130、E183、H229和E291更重要。我们还对基因簇中的valB、valK和valO三个基因进行基因置换。基因valB编码一个可能的核苷转移酶。我们推测ValB在井冈霉素生物合成中可能负责1-表-有效烯醇-1-磷酸的腺苷化。valB基因失活突变株丧失了产生井冈霉素的能力,而这种产量的丧失可以在互补全长基因valB后得到恢复。基因valK编码的蛋白与糖异构化酶/脱水酶具有显著的同源性。它可能负责5-表-有效醇酮C-5和C-6位的脱水反应。valK基因失活突变株丧失了合成井冈霉素的能力。这种产量丧失可在互补全长valK之后可到恢复。以上结果证明valB和valK是井冈霉素生物合成的必需基因。valO编码一个可能的磷酸酶/磷酸变位酶。我们推测它可能负责将磷酸基从C-7位转移到C-1位的羟基上。然而,valO基因失活突变株仍能产生井冈霉素,但积累了大量有效氧胺。互补全长valO之后,突变株中有效氧胺减少而井冈霉素增多。以上结果暗示了valO可能与有效氧胺和井冈霉素之间的转化有关。在一些抗生素产生菌中,对氨基苯甲酸(PABA)和它的前体4-氨基-4-脱氧分支酸(ADC)不仅参与初级代谢中叶酸的合成,也参与次级代谢产物抗生素的生物合成。在杀念菌素产生菌FR-008中对融合的ADC合成酶基因pabAB的缺失使得杀念菌素产量几乎完全丧失,这种产量的丧失可以通过互补克隆的pabAB或喂养外源PABA得到恢复。一个位于PKS区域上游的可能的IV型PLP依赖性酶的编码基因pabC-1的失活导致杀念菌素的产量约下降到野生型的20%。用pabC-1对大肠杆菌pabC突变株的成功互补证明了pabC-1与PABA的生物合成相关。此外,我们从同一菌株中克隆了另一个可能的IV型PLP依赖性酶的编码基因pabC-2。pabC-2的失活导致杀念菌素产量约降低到野生型水平的57%。PabC-2对大肠杆菌pabC突变株的成功互补证明了它参与PABA的生物合成。而pabC-1/pabC-2双突变株几乎完全丧失了合成杀念菌素的能力,说明pabC-2也参与了杀念菌素的生物合成。令我们吃惊的是,双突变株在基本培养基上生长明显延迟,这说明pabC-1和pabC-2都参与了初级代谢中PABA的合成。最后,我们通过检测丙酮酸的释放证明了PabC-1和PabC-1都是有功能的ADC裂解酶。到目前为止,pabC-1和pabC-2是放线菌中首先确定的两例ADC裂解酶基因。两个ADC裂解酶基因既参与细胞生长又参与杀念菌素的生物合成,是细菌中是初级代谢与次级代谢之间相互作用的一个很好的例子。更多还原

【Abstract】 Validamycins, weakly basic C7N-aminocyclitol-containing antibiotics, were isolated from Streptomyces hygroscopicus var. jinggangensis 5008. It is a trehalase inhibitor widely used as a prime control agent against sheath blight disease of rice plants and dumping-off of cucumber seedlings in China and many other eastern Asian countries. The sequence information of the cloned biosynthetic gene cluster helps us to investigate the biosynthesis of this antibiotic.The gene valC, which encodes an enzyme homologous to the 2-epi-5-epi-valiolone kinase (AcbM) of the acarbose biosynthetic pathway, was identified in the validamycin biosynthetic gene cluster. Inactivation of valC resulted in mutants that lack the ability to produce validamycin A. Complementation with full-length valC restored the production of validamycin, thus suggesting a critical function of valC in validamycin biosynthesis. In vitro characterization of ValC revealed a new type of C7-cyclitol kinase, which phosphorylates valienone and validone-but not 2-epi-5-epi-valiolone, 5-epi-valiolone, or glucose-to afford their 7-phosphate derivatives. The results provide new insights into the activity of this enzyme and also confirm the existence of two different pathways leading to the same end-product: the valienamine moiety common to acarbose and validamycin.The gene valD, which encodes a large Vicinal Oxygen Chelate(VOC)superfamily protein, has been identified in the validamycin biosynthetic gene cluster. Inactivation of valD significantly reduced validamycin production, which was fully restored with the full-length valD and partially restored with either N-terminal or C-terminal half by complementation. Heterologously expressed ValD catalyzed the epimerization of 2-epi-5-epi-valiolone to 5-epi-valiolone. Individual and combined site-directed mutations of eight putative active site residues revealed that the N-terminal H44/E107 and the C-terminal H315/E366 are more critical for the activity than the internal H130, E183, H229 and E291.Three biosynthetic genes valB, valK and valO were inactivated respectively. The gene valB encodes a putative nucleotidyl transferase, which may catalyze the adenylation of 1-epi-valienol-1-phosphate. Inactivation of valB abolished validamycin biosynthesis, which was restored by complementation of full-length valB. The gene valK encodes a putative dehydratase, which may catalyze the dehydration of C-5 and C-6 of 5-epi-valiolone to generate valienone. Inactivation of valK abolished validamycin production, which was restored by complementation with cloned valK. These results indicate that valB and valK are essential for validamycin biosynthesis. The gene valO encodes a putative phosphatase/phosphohexo- mutase, which may catalyze the transfer of phosphate group from the C-7 to the C-1 hydroxyl group. However, inactivation of valO did not abolish validamycin production but resulted in significant accumulation of validoxylamine, which could be partially complemented by cloned valO. This result suggests that valO may be involved in conversion of validoxylamine to validamycin.In some antibiotic producers, p-aminobenzoic acid (PABA) or its immediate precursor, 4-amino-4-deoxychorismate (ADC), is involved in primary metabolism and antibiotic biosynthesis. A fused pabAB gene was identified in FR-008/candicidin biosynthetic gene cluster from Streptomyces sp. FR-008. Its deletion abolished FR-008/candicidin biosynthesis, which was restored either by complementation with cloned pabAB or feeding with exogenous PABA. A gene pabC-1 putatively encoding fold-type IV of PLP-dependent enzyme was found in the upstream PKS region, whose inactivation significantly reduced the productivity of antibiotic FR-008 to ca. 20% of the wild-type level. Its specific role for PABA formation was further demonstrated through the successful complementation of an E. coli pabC mutant. Moreover, a free standing gene pabC-2, probably encoding another fold-type IV of PLP-dependent enzyme, was cloned from the same strain. Inactivation of pabC-2 rendered antibiotic FR-008 yield to about 57% of the wild-type level in the mutant, and the complementation of the E. coli pabC mutant established its involvement in PABA biosynthesis. Furthermore, a pabC-1/pabC-2 double mutant only kept about 4% of the wild-type productivity, clearly indicating that pabC-2 also contributed to antibiotic FR-008 biosynthesis. Surprisingly, apparent retarded growth of the double mutant was observed on minimal medium, which suggested that both pabC-1 and pabC-2 were involved in PABA biosynthesis for primary metabolism. Finally, both PabC-1 and PabC-2 were proved to be functional ADC lyases through in vitro enzymatic lysis with the release of pyruvate. To date, pabC-1 and pabC-2 represent the first two functional ADC lyase genes indentified in actinomycetes. The involvement of these two ADC lyase genes in both cell growth and antibiotic FR-008 biosynthesis sets an example for the interplay between primary and secondary metabolisms in bacteria.更多还原