Sandia National Laboratories, Department of Bioenergy and Defense Technologies, MS 1413, P,O, Box 5800, 87185-1413, Albuquerque, NM, USA.
Biotechnol Biofuels. 2013 Aug 6;6(1):113. doi: 10.1186/1754-6834-6-113.
High-energy-density biofuels are typically derived from the fatty acid pathway, thus establishing free fatty acids (FFAs) as important fuel precursors. FFA production using photosynthetic microorganisms like cyanobacteria allows for direct conversion of carbon dioxide into fuel precursors. Recent studies investigating cyanobacterial FFA production have demonstrated the potential of this process, yet FFA production was also shown to have negative physiological effects on the cyanobacterial host, ultimately limiting high yields of FFAs.
Cyanobacterial FFA production was shown to generate reactive oxygen species (ROS) and lead to increased cell membrane permeability. To identify genetic targets that may mitigate these toxic effects, RNA-seq analysis was used to investigate the host response of Synechococcus elongatus PCC 7942. Stress response, nitrogen metabolism, photosynthesis, and protein folding genes were up-regulated during FFA production while genes involved in carbon and hydrogen metabolisms were down-regulated. Select genes were targeted for mutagenesis to confirm their role in mitigating FFA toxicity. Gene knockout of two porins and the overexpression of ROS-degrading proteins and hypothetical proteins reduced the toxic effects of FFA production, allowing for improved growth, physiology, and FFA yields. Comparative transcriptomics, analyzing gene expression changes associated with FFA production and other stress conditions, identified additional key genes involved in cyanobacterial stress response.
A total of 15 gene targets were identified to reduce the toxic effects of FFA production. While single-gene targeted mutagenesis led to minor increases in FFA production, the combination of these targeted mutations may yield additional improvement, advancing the development of high-energy-density fuels derived from cyanobacteria.
高能量密度生物燃料通常来源于脂肪酸途径，因此游离脂肪酸（FFA）成为了重要的燃料前体。利用蓝藻等光合微生物生产 FFAs，可以实现二氧化碳向燃料前体的直接转化。最近研究表明，该过程具有生产 FFAs 的潜力，但 FFAs 的生产也对蓝藻宿主产生了负面影响，最终限制了 FFAs 的高产量。
蓝藻 FFAs 的生产会产生活性氧（ROS）并导致细胞膜通透性增加。为了确定可能减轻这些毒性作用的遗传靶标，我们使用 RNA-seq 分析来研究 Synechococcus elongatus PCC 7942 的宿主反应。在 FFAs 生产过程中，应激反应、氮代谢、光合作用和蛋白质折叠基因上调，而参与碳和氢代谢的基因下调。选择了一些基因进行突变，以确认它们在减轻 FFAs 毒性方面的作用。两种孔蛋白的基因敲除以及 ROS 降解蛋白和假设蛋白的过表达，减轻了 FFAs 生产的毒性作用，从而提高了生长、生理和 FFAs 产量。比较转录组学分析，分析了与 FFAs 生产和其他应激条件相关的基因表达变化，确定了参与蓝藻应激反应的其他关键基因。
共鉴定出 15 个基因靶标，以减轻 FFAs 生产的毒性作用。虽然单基因靶向突变导致 FFAs 产量略有增加，但这些靶向突变的组合可能会带来额外的改善，从而推进源自蓝藻的高能量密度燃料的开发。
