天蓝色链霉菌中长链3-酮脂酰ACP合成酶的功能

Function of the long chain 3-ketoacyl-ACP synthase in Streptomyces coelicolor

摘要:

【背景】链霉菌属于放线菌科，在土壤环境中广泛分布。链霉菌具有复杂的形态分化和多样性的次生代谢网络，能产生大量具有生物活性的次级代谢产物，被广泛深入研究。【目的】天蓝色链霉菌是链霉菌的模式菌株，其脂肪酸合成代谢与次级代谢联系紧密，但目前脂肪酸合成代谢途径还不清楚，其长链3-酮脂酰ACP合成酶还未见报道。【方法】利用大肠杆菌FabF序列进行同源比对，发现天蓝色链霉菌A3(2)的基因组中，SCO2390 (ScoFabF1)、SCO1266 (ScoFabF2)、SCO0548 (ScoFabF3)和SCO5886 (ScoRedR)具有较高的相似性，并具有保守的Cys-His-His催化活性中心，可能具有长链3-酮脂酰ACP合成酶活性。采用PCR扩增方法分别获得以上基因，连入表达载体pBAD24M后分别互补大肠杆菌fabB(ts)突变株和fabB(ts)fabF双突变株，并检测转化子的生长情况。以上基因与pET-28b连接后，在大肠杆菌BL21(DE3)中表达，并利用Ni-NTA纯化获得蛋白，体外测定其催化活性。将以上基因分别互补大肠杆菌fabF突变株后，GC-MS测定互补株的脂肪酸组成。【结果】4个同源基因中，只有ScofabF1能恢复fabB(ts)fabF双突变株42 °C时在添加油酸条件下的生长，其他3个基因均不能恢复生长。而这4个基因都不能恢复fabB(ts)突变株42 °C时生长。体外活性测定ScoFabF1具有长链3-酮脂酰ACP合成酶活性，其他3个蛋白都不具有该活性。仅ScofabF1能显著提高大肠杆菌fabF突变株的顺-11-十八碳烯酸(C18:1)比例，其他3个基因都不具有该功能。【结论】天蓝色链霉菌中ScofabF1编码长链3-酮脂酰ACP合成酶II，在脂肪酸利用过程中发挥重要作用。天蓝色链霉菌中没有发现编码长链3-酮脂酰ACP合成酶I的基因，其可能通过其他途径合成少量的不饱和脂肪酸。以上研究结果为进一步研究天蓝色链霉菌中脂肪酸合成机制奠定了基础。

Abstract:

[Background] Streptomyces strains are widely distributed in soil environment. With a complex morphological differentiation and a large diversity of secondary metabolic networks, Streptomyces can produce many bioactive secondary metabolites. [Objective] Fatty acid biosynthesis and secondary metabolism are closely related in the model strain Streptomyces coelicolor, but the fatty acid synthetic mechanism is still unclear, and its long-chain 3-ketoacyl ACP synthase has not been reported. [Methods] Through sequence alignment with Escherichia coli FabF (EcFabF), SCO2390 (ScoFabF1), SCO1266 (ScoFabF2), SCO0548 (ScoFabF3) and SCO5886 (ScoRedR) were found in the genome of Streptomyces coelicolor A3(2), which showed high similarity with EcFabF, and contained the conserved Cys-His-His sites, indicating that they may have the 3-ketoacyl-ACP synthase activity. The four genes were amplified by PCR, and ligated into the expression vector pBAD24M, and transferred into E. coli fabB(ts) and E. coli fabB(ts)fabF mutants. The growth of transformants was analyzed. The four genes were also ligated into pET-28b, and expressed in E. coli BL21(DE3). The four EcFabF homologues with hexahistidine-tag were purified by Ni-NTA, and the activities were analyzed in vitro. The fatty acid profiles of E. coli fabF mutants completed with the four genes were also analyzed by GC-MS. [Results] Only ScofabF1 conferred the E. coli fabB(ts)fabF mutant to grow with oleatic acid supplemented at 42 °C, and all failed to complete E. coli fabB(ts) at 42 °C. In vitro enzymatic analysis also demonstrated that only ScoFabF1 has 3-ketoacyl-ACP synthase activity, while the other three proteins showed no similar activity. E. coli fabF mutant harboring ScofabF1 increased the amount of unsaturated fatty acid C18:1 significantly. [Conclusion] All of above suggested that Streptomyces coelicolor ScofabF1 encodes 3-ketoacyl-ACP synthase II, and plays an important role in fatty acid synthesis. However, no gene encoding 3-ketoacyl-ACP synthase I was found in the genome, indicating that Streptomyces coelicolor may have other mechanism to synthesize small amount of unsaturated fatty acids. Achievement in this study will contribute to further research about the mechanism of fatty acid synthesis in Streptomyces coelicolor.