【作者】 赵春华；

【导师】 周秀芬；

【作者基本信息】 上海交通大学 ， 微生物学， 2008， 博士

【摘要】 垩唑霉素(oxazolomycin,OZM)及相关化合物是由链霉菌产生的抗生素。其中OZM是由链霉菌sp.KBFP-2025和白色链霉菌JA3453产生的,其结构特征为:螺旋链接的β-内酯/γ-内酰胺,垩唑环,(E,E)二烯和一个(Z,Z,E)三烯链。OZM具有抗肿瘤、抗病毒和一定的抗细菌的生物活性。通过简并引物PCR的方法从白色链霉菌JA3453基因组中扩增得到编码甲氧丙二酰ACP的HADH和ADH基因。在白色链霉菌JA3453染色体上用基因置换手段缺失相应的12kb BglII片段后得到不产OZM的突变株,证明克隆的基因与OZM合成相关。对该区域的测序揭示了负责甲氧丙二酰ACP生物合成位点的6个基因。对基因ozmC和ozmF失活以及互补实验证实其与OZM生物合成相关。进一步的染色体步移得到五个重叠粘粒,它们覆盖的135kb的DNA区域包含整个ozm基因簇。全基因簇测序是在国家人类基因组南方中心完成的,总共得到了127,755bp连续的碱基,其总的GC含量为73.8%,具有明显的链霉菌DNA特征。对测序区域的生物信息学分析显示了55个开放阅读框,每个开放阅读框的功能都通过推测的基因产物和数据库里已知功能的蛋白质相比较得到确定并注释。通过基因置换实验初步确定了垩唑霉素合成基因簇的边界。ozm生物合成基因簇跨越了大约79.5kb DNA区域并涉及了20个开放阅读框,分别命名为ozmA、ozmB至ozmU。其中,ozmJ、ozmK、ozmM、ozmN、ozmQ为聚酮合酶,而ozmH是聚酮聚肽杂合酶,ozmL和ozmO是非核糖体肽合成酶,ozmA和ozmS可能是抗性基因。基因ozmB-G组成的操纵元是甲氧丙二酰ACP生物合成位点。ozmP和ozmT是未知功能的基因,而ozmR和ozmU可能是调节基因。有意思的是,ozm生物合成中的聚酮合酶ozmJKNQ和杂合聚酮聚肽合酶ozmH总共10个模块都缺少内部的酰基转移酶。相应的,在OZM生物合成基因簇中发现了一个反式独立的酰基转移酶ozmM,其基因产物编码一个1057个氨基酸的蛋白质。Blast搜索显示OzmM可以分为三个区域。OzmM的N端(氨基酸1-322)(命名为OzmM-AT1)和中间部分(氨基酸333-631)(命名为OzmM-AT2)都包含了酰基转移酶活性位点,而OzmM的C端(氨基酸残基613-1057)可能编码氧化还原区域,与反式AT LnmG的C端有较高的同源性。通过对基因ozmM基因置换,互补和定点诱变实验揭示OzmM-AT2为垩唑霉素合成所必需的而OzmM-AT1并不是垩唑霉素合成所需要的。因此,我们推测而OzmM-AT2能够将两种不同的延伸单位丙二酰辅酶A和甲氧丙二酰ACP加载到所有ACP上。另外,在OZM生物合成基因簇中发现了一些有别于I型聚酮合酶装配线模型的不寻常的特征,如模块中的额外区域,缺失的区域和区域移位。这些研究发现对于我们深入理解“缺少AT”的聚酮合酶并通过组合化学手段改造新的“非天然的”天然产物会提供很大的帮助。更多还原

【Abstract】 The oxazolomycins (OZMs) are a growing family of antibiotics produced by several Streptomyces species. OZM, produced by Streptomyces sp. KBFP-2025 and Streptomyces albus JA3453, is structurally characterized by a unique spiro-linkedβ-lactone/γ-lactam moiety, a 5-substituted oxazole ring, an (E, E)-diene, and a (Z, Z, E)-triene chain. OZM exhibits potent antitumor, antiviral, but limited antibacterial activities. Using degenerated primers to clone genes HADH and ADH encoding methoxymalonyl-acyl carrier protein (ACP) biosynthesis as probes, a 135-kb DNA region from S. albus JA3453 was cloned and localized to cover the entire OZM biosynthetic gene cluster. The involvement of the cloned gene cluster in OZM biosynthesis was confirmed by deletion of a 12-kb DNA fragment from the specific region of the chromosome to generate OZM non-producing mutants. Sequence analysis of this fragment revealed six genes likely responsible for methoxymalonyl-ACP biosynthesis. The involvement of the latter in OZM biosynthesis was proven directly by mutational and complementary analysis of selected genes, ozmF encoding a methyltransferase and ozmC encoding a ketoacyl synthase, within this locus.Chromosomal walking from the methoxymalonyl-ACP biosynthesis locus followed by extensive restriction endonuclease analysis resulted in the identification of the overlapping cosmids covering the 135-kb contiguous DNA region. A 127,755-bp DNA involved in oxazolomycin (OZM) biosynthesis was sequenced from Streptomyces albus JA3453 in South Center of National Human Genome, with an overall GC content of 73.8% characteristic of Streptomyces DNA. Bioinformatic analysis of the sequenced region revealed 55 open reading frames (ORFs), whose putative functions were individually assigned by comparing the proposed gene products with proteins of known functions in the databases as annotated. The boundaries of ozm gene cluster were preliminarily determined by gene inactivation. The ozm cluster covers about 79.5-kb genes encoding 20 genes: nonribosomal peptide synthetase (NRPS; OzmL and OzmO), polyketide synthetase (PKS; OzmJ, OzmK, OzmM, OzmN and OzmQ), hybrid NRPS-PKS (OzmH), resistance (OzmA and OzmS), regulatory (OzmR and OzmU) and tailoring enzymes (OzmBCDEFG).A model for OZM biosynthesis is proposed. Surprising of all, the only AT activity identified within the ozm gene cluster is a discrete trans AT (acyl carrier protein) encoded by ozmM whose deduced product is a protein of 1057 amino acids. Blast research suggested that OzmM contains three distinctive domains. N-terminal of OzmM (amino acid residues 1-332), designated as OzmM-AT1 and central of this protein (amino acid residues 333-631), designated as OzmM-AT2, both have similarity to most discovered modular or discrete acyltransferase. C-terminal of OzmM (amino acid residues 613-1057) encode a putative oxidoreductase domain show high homologous to trans AT LnmG C-terminus. Mutational analysis, complementary experiments and site-directed mutagenesis suggested that AT2 are essential for OZM biosynthesis but AT1 seems to be redundant. We propose that AT2 was recruited ten times required for transferring of malonyl-CoA and methoxymalonyl-ACP extender units onto all ACPs. Also, unusual features in polyketide synthase were characterized in OZM gene cluster, e.g. extra domain, domain missing and domain misposition. Above studies about OZM PKS and NRPS genetic organization will provide us new insights into“AT-less”PKS engineering to generate new“unnatural”natural compound by combinatorial chemistry.更多还原