Enzymatic and physiological characterization of fatty acid activation in Synechocystis sp PCC6803

Free fatty acids are typically activated by thioesterification processes and catalyzed by the fatty acyl-CoA synthetase or fatty acyl-ACP synthetase. However, the routes for fatty acid activation in cyanobacteria are not well understood. In this investigation, the slr1609 gene, which encodes the fatty acid activation enzyme, was cloned from Synechocystis sp. PCC6803. This gene was identified by heterologous expression and in vitro enzymatic activity analyses. Different from previous reports stating that free fatty acids are only activated through the fatty acyl-ACP synthetases encoded by these genes in cyanobacteria, this gene was also proven to possess a fatty acyl-CoA synthetase activity, by in vitro enzymatic activity analyses and in vivo complementation experiments. The protein Slr1609 is located in both the cell membrane and the cytosol of Synechocystis sp. PCC6803. The differences in the transcriptional profiles between the wild type and the slr1609 deletion mutant strain were evaluated using microarray analyses. These analyses indicated that 299 differentially expressed genes are involved in fatty acid metabolism, photosynthesis, carbon fixation, stress tolerance and other metabolic processes. Our experiments demonstrate the observed compositional changes in the unsaturated fatty acids from the membrane lipids of the slr1609 deletion mutant when shifted from 30 to 24 degrees C.

游离脂肪酸通常通过硫酯化过程被激活，由脂肪酰基辅酶A合成酶或脂肪酰基 - 酰基载体蛋白合成酶催化。然而，蓝藻中脂肪酸激活的途径尚未被充分了解。在这项研究中，从集胞藻属PCC6803中克隆了编码脂肪酸激活酶的slr1609基因。该基因通过异源表达和体外酶活性分析得以鉴定。不同于先前报道称蓝藻中游离脂肪酸仅通过这些基因编码的脂肪酰基 - 酰基载体蛋白合成酶激活，通过体外酶活性分析和体内互补实验，该基因也被证明具有脂肪酰基辅酶A合成酶活性。蛋白质Slr1609位于集胞藻属PCC6803的细胞膜和细胞质中。利用微阵列分析评估了野生型和slr1609缺失突变株之间转录图谱的差异。这些分析表明，299个差异表达基因涉及脂肪酸代谢、光合作用、碳固定、胁迫耐受性和其他代谢过程。我们的实验证明了当从30℃转变到24℃时，slr1609缺失突变体膜脂中不饱和脂肪酸组成发生了变化。