【作者】 宋春旭；

【导师】 孙明；

【作者基本信息】 华中农业大学 ， 生物化学与分子生物学， 2008， 硕士

【摘要】 Zwittermicin A,简称ZwA,中文名双效菌素,是由部分蜡状芽胞杆菌（Bacilluscereus）和苏云金芽胞杆菌（Bacillus thuringiensis）产生的一种新型广谱的氨基多元醇类抗生素,化学式C₁₃H₂₈N₆O₈。已知ZwA对卵菌纲、海藻、原生生物等比较低等的真核生物具有很强抑制作用,对大部分革兰氏阴性菌和一些特定的革兰氏阳性菌有中等的抑制作用,同时还可以抑制或杀灭一系列植物病原真菌。此外,ZwA还可以与Bt毒素协同作用从而增加Bt的杀虫活性。对ZwA合成所需要的前体物的预测已经有报道,推测ZwA合成需要5种前体物:L-丝氨酸（L-serine）,丙二酰辅酶A（malonyl CoA）,氨基丙二酰-ACP（Amino-malonyl-ACP）,羟基丙二酰-ACP（Hydroxy-malonyl-ACP）,2,3-二氨基丙酸（2,3-diamino-propionate）。本课题组在国内率先开展其生物合成基因簇的克隆及生物合成途径的研究,但ZwA的生物合成途径还没有一个完善的学说,只是基于16 kb DNA序列的假说。此16 kb的序列只包含假想前体物氨基丙二酰-ACP和羟基丙二酰-ACP合成的相关基因,没有对2,3-二氨基丙酸这种前体物合成基因进行预测和验证。所以本研究旨在完善对ZwA前体物的认识,从而推动对ZwA合成基因簇以及生物合成途径的研究,具有重要的理论研究意义。本研究是以获得的苏云金芽胞杆菌菌株YBT-1520 ZwA生物合成的67 kb的基因簇为基础,对此基因簇进行了生物信息学的分析,发现ZwA5A,ZwA5B和orf3三个基因与2,3-二氨基丙酸的合成相关。同源性比较显示ZwA5A和ZwA5B分别是半胱氨酸合成酶和鸟氨酸环化脱氨酶的同源物。通过同源交换插入,获得ZwA5A基因缺失突变体ZwA5A^-。该突变体不再产生ZwA,用化学合成的D/L-2,3-二氨基丙酸添加到突变体ZwA5A^-的发酵生长过程中,突变体ZwA5A^-可以恢复产ZwA。这说明ZwA5A基因对ZwA的产生是必需的;在ZwA的产生过程中,化合物D/L-2,3-二氨基丙酸可以替代基因ZwA5A的功能。通过pGEX-6P-1载体,在大肠杆菌中超量表达了ZwA合成基因簇ORF3中的腺苷酰化功能域ZwAA。依赖于ATP的焦磷酸交换实验表明,腺苷酰化功能域ZwAA2可以识别并活化2,3-二氨基丙酸。上述结果证实,2,3-二氨基丙酸是ZwA生物合成过程中的直接前体物之一。更多还原

【Abstract】 Zwittermicin A（ZwA） is a novel,broad-spectrum linear aminopolyol antibiotic produced by some Bacillus cereus and Bacillus thuringiensis,whose chemical formula is C₁₃H₂₈N₆O₈.Zwittermicin A is highly active against the Oomycetes and their relatives, the algal,protists,and has moderate activity against diverse Gram-negative bacteria and certain Gram-positive bacteria as well as against a wide range of plant pathogenic fungi. Meanwhile,ZwA could acilitates the insecticidal activity of the protein toxin produced by B.thuringiensis.Jo Handelsman group pedicted that ZwA could be synthesised by five proposed precursors:L-serine,malonyl CoA,Amino-malonyl-ACP, Hydroxy-malonyl-ACP and 2,3-diamino-propionate.Its biosynthesis pathway is different to both polyketides synthases（PKS） and NRPS（nonribosomal peptide synthetases） way, but a hybrid pathway.Since the whole biosynthesis gene cluster is not complete and the biosynthesis pathway is unknown,we carried on this work.The reported 16 kb sequence includes the relative genes of Amino-malonyl-ACP and Hydroxy-malonyl-ACP,but there is no evidence on the relative biosynthesis gene of 2,3-diamino-propionate.This work is to verify 2,3-diamino-propionate is the direct precursor of Zwittermicin A and completes the whloe gene biosynthesis pathway.This research is based on the 67 Kb biosynthesis gene cluster of Bacillus thuringiensis strain YBT-1520 in our lab.After bioinformatics analysis,we found three genes ZwA5A,ZwA5B and orf3 are relative with the precursor 2,3-diaminopropionate. ZwA5A and ZwA5B are homologs of cysteine synthetase and ornithine cyclodeaminase respectively.The ZwA5A-mutant could not produce ZwA when ZwA5A,the 2, 3-diaminopropionate biosynthesis related gene,was interrupted by homologous exchange, while the ZwA5A-mutant resumed to produce ZwA when 2,3-diaminopropionate was fed.It indicates that ZwA5A gene is necessary for the biosynthesis of ZwA and 2, 3-diaminopropionate can replace ZwA5A in the biosynthesis of ZwA.The adenylation domain ZWAA in the ZwA biosynthesis cluster ORF3 was over expressed in E.coli by pGEX-6P-1.ATP-dependent PPi exchange experiment result showed that ZwAA2 can active 2,3-diaminopropionate.Taken together,we have demonstrated that 2, 3-diaminopropionate is one of the precursors during the biosynthesis of ZwA.更多还原