Department of Biochemistry and Molecular Biology, Graduate School of Science and Engineering, Saitama University, Saitama, 338-8570 Japan.
Department of Environmental Science and Technology, Graduate School of Science and Engineering, Saitama University, Saitama, 338-8570 Japan.
Plant Cell Physiol. 2020 Sep 1;61(9):1537-1547. doi: 10.1093/pcp/pcaa069.
Although cyanobacteria do not possess wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT), the bacterial enzyme for triacylglycerol (TAG) production, there have been several studies reporting the accumulation of TAG-like compounds in cyanobacteria. In this study, we aimed to evaluate TAG productivity of the ΔrecJ::atfA strain of Synechocystis sp. PCC 6803 generated by inserting atfA encoding WS/DGAT from Acinetobacter baylyi ADP1 into recJ (sll1354), together with the wild type (WT) and the gene-disrupted strain of slr2103 having homology with eukaryotic DGAT2 gene family (Δ2103). Thin-layer chromatography (TLC) of neutral lipids or isolation of the neutral lipid-enriched fraction followed by gas chromatography or liquid chromatography-tandem mass spectrometry was employed for analyses. The ΔrecJ::atfA strain accumulated 0.508 nmol ml-1OD730-1 of TAG after a week of incubation at 100 μmol photons m-2 s-1. The saturated fatty acids C16:0 and C18:0 accounted for about 50% and 20% of the TAG fatty acids, respectively, suggesting that de novo-synthesized fatty acids were preferentially incorporated into TAG molecules. When the neutral lipid profile of the lipid extracts was examined by TLC, a spot located in a slightly lower position compared with the TAG standard was detected in WT but not in the Δ2103 strain. TAG accumulation levels of both strains was only 0.01-0.03 nmol ml-1OD730-1, but the fatty acid composition was substantially different from that of the background. These results suggest that trace amounts of TAG can be produced in Synechocystis cells by enzymes other than Slr2103, and major constituents of the TAG-like spot are unknown lipid species produced by Slr2103.
尽管蓝藻不具有蜡酯合酶/酰基辅酶 A：二酰基甘油酰基转移酶 (WS/DGAT)，这是细菌三酰基甘油 (TAG) 生产的酶，但已有几项研究报告称在蓝藻中积累了类似 TAG 的化合物。在这项研究中，我们旨在评估插入 Acinetobacter baylyi ADP1 编码 WS/DGAT 的 atfA 基因的 Synechocystis sp. PCC 6803 ΔrecJ::atfA 菌株的 TAG 生产力，该基因插入到 recJ (sll1354) 中，同时还评估了具有与真核 DGAT2 基因家族同源的 slr2103 基因缺失株（Δ2103）的野生型（WT）和基因缺失株。采用薄层层析 (TLC) 分析中性脂质或分离富含中性脂质的级分，然后进行气相色谱或液相色谱-串联质谱分析。在 100 μmol 光子 m-2 s-1 的光照下培养一周后，ΔrecJ::atfA 菌株积累了 0.508 nmol ml-1OD730-1 的 TAG。TAG 脂肪酸中饱和脂肪酸 C16:0 和 C18:0 分别约占 50%和 20%，表明从头合成的脂肪酸优先掺入 TAG 分子中。当用 TLC 检查脂质提取物的中性脂质图谱时，在 WT 中检测到一个与 TAG 标准相比略低位置的斑点，但在 Δ2103 菌株中未检测到。两种菌株的 TAG 积累水平均仅为 0.01-0.03 nmol ml-1OD730-1，但脂肪酸组成与背景有很大不同。这些结果表明，Synechocystis 细胞中可以通过 Slr2103 以外的酶产生痕量的 TAG，并且 TAG 样斑点的主要成分是 Slr2103 产生的未知脂质种类。
