集胞藻PCC6803膜脂循环关键基因酶学性质和生理功能

Characterization of a key gene in membrane lipid cycle in Synechocystis sp. PCC6803

摘要:

集胞藻PCC6803野生型和其脂酰ACP合酶敲除突变株的自由脂肪酸含量和组成表明膜脂的重构和降解是细胞内自由脂肪酸的来源之一。在这一过程中脂肪酶起到关键性作用。通过基因组数据库检索，发现集胞藻PCC6803基因组中只有一个脂肪酶编码基因sll1969，但是还没有其功能相关的生化证据。为了确定该基因的功能及其在脂肪酸代谢途径中的作用，加深对集胞藻PCC6803脂肪酸代谢途径的了解，文中将sll1969基因在大肠杆菌中过表达和体外纯化，得到重组蛋白Sll1969，并对其酶学性质进行初步分析。在30 ℃条件下，测得Sll1969以对硝基苯丁酸酯作为底物时的Km和kcat值分别为 (1.16±0.01) mmol/L和 (332.8±10.0)/min；该脂肪酶的最适反应温度为55 ℃。通过比较分析sll1969突变株中脂肪酸含量和组成变化，发现sll1969的表达量与细胞自由脂肪酸的产量呈正相关，但Sll1969不是细胞中唯一的脂肪酶。

Abstract:

Free fatty acid profiles of wild type and fatty acyl-ACP synthase deletion mutant strain of Synechocystis sp. PCC6803 indicated that one origin of these fatty acids is the process of lipid remodeling or lipid degradation. Lipase is the key enzyme involved in this process. The gene sll1969 is the sole gene encodes a putative lipase in Synechocystis sp. PCC6803. To identify the function of this gene and its role in fatty acid metabolism, we cloned the sll1969 from genomic DNA, overexpressed it in Escherichia coli BL21 (DE3) using pET expression system and purified this recombinant enzyme with Nickel-nitrilotriacetic acid affinity chromatography. The enzyme activity was assayed by spectrophotometric with p-nitro-phenylbutyrate as substrate. The Km and kcat of the enzyme is (1.16±0.01) mmol/L and (332.8±10.0)/min, respectively toward p-nitro-phenylbutyrate at 30 oC. The optimal temperature of the enzyme is 55 oC. To investigate the biological role of Sll1969 in fatty acid metabolism in cyanobacteria, we constructed sll1969 deletion and overexpression mutant strains in the background of fatty acyl-ACP synthase deletion mutant of Synechocystis sp. PCC6803. The analyses of the content of free fatty acids in different mutant strains showed that the contents of Sll1969 and free fatty acid are positively correlated. The free fatty acid profiles of the sll1969 mutant strains suggested this enzyme is not the sole enzyme for degrading lipid in Synechocystis sp. PCC6803.