Kato Akihiro, Takatani Nobuyuki, Ikeda Kazutaka, Maeda Shin-Ichi, Omata Tatsuo
Graduate School of Bioagricultural Sciences, Nagoya University, Nagoya, 464-8601 Japan.
Japan Science and Technology Agency, CREST, Tokyo, Japan.
Biotechnol Biofuels. 2017 May 31;10:141. doi: 10.1186/s13068-017-0831-z. eCollection 2017.
Cyanobacterial mutants engineered for production of free fatty acids (FFAs) secrete the products to the medium and hence are thought to be useful for biofuel production. The dAS1T mutant constructed from PCC 7942 has indeed a large capacity of FFA production, which is comparable to that of triacylglycerol production in green algae, but the yield of secreted FFAs is low because the cells accumulate most of the FFAs intracellularly and eventually die of their toxicity. To increase the FFA productivity, enhancement of FFA secretion is required.
Growth of dAS1T cells but not WT cells was inhibited in a liquid medium supplemented with 0.13 g L of palmitic acid. This suggested that when FFA accumulates in the medium, it would inhibit the release of FFA from the cell, leading to FFA accumulation in the cell to a toxic level. To remove FFAs from the medium during cultivation, an aqueous-organic two-phase culture system was developed. When the dAS1T culture was overlaid with isopropyl myristate (IM), the final cell density, cellular chlorophyll content, and the photosynthetic yield of PSII were greatly improved. The total amount of extracellular FFA was more than three times larger than that in the control culture grown without IM, with most of the secreted FFAs being recovered in the IM layer. The cellular FFA content was decreased by more than 85% by the presence of the IM layer. Thus, the two-phase culture system effectively facilitated FFA secretion out of the cell. An average FFA excretion rate of 1.5 mg L h was attained in the 432 h of cultivation, with a total amount of excreted FFA being 0.64 g L of culture. These figures were more than three times higher than those reported previously for the cyanobacteria-based FFA production systems.
Removal of FFA from the culture medium is important for improving the productivity of the FFA production system using cyanobacteria. Further increase in productivity would require an increase in both the rates of FFA production in the cell and active FFA export across the plasma membrane.
经基因工程改造用于生产游离脂肪酸（FFA）的蓝藻突变体将产物分泌到培养基中，因此被认为对生物燃料生产有用。由集胞藻PCC 7942构建的dAS1T突变体确实具有很大的FFA生产能力，这与绿藻中三酰甘油的生产能力相当，但分泌的FFA产量较低，因为细胞在细胞内积累了大部分FFA，并最终因毒性而死亡。为了提高FFA生产力，需要增强FFA分泌。
在添加0.13 g/L棕榈酸的液体培养基中，dAS1T细胞的生长受到抑制，而野生型（WT）细胞不受影响。这表明当FFA在培养基中积累时，它会抑制FFA从细胞中的释放，导致FFA在细胞内积累到有毒水平。为了在培养过程中从培养基中去除FFA，开发了一种水-有机两相培养系统。当dAS1T培养物覆盖肉豆蔻酸异丙酯（IM）时，最终细胞密度、细胞叶绿素含量和PSII的光合产量都有很大提高。细胞外FFA的总量比没有IM的对照培养物中多三倍以上，大部分分泌的FFA在IM层中被回收。IM层的存在使细胞内FFA含量降低了85%以上。因此，两相培养系统有效地促进了FFA向细胞外的分泌。在432小时的培养中，平均FFA排泄率达到1.5 mg/L·h，分泌的FFA总量为0.64 g/L培养物。这些数字比之前报道的基于蓝藻的FFA生产系统高出三倍多。
从培养基中去除FFA对于提高使用蓝藻的FFA生产系统的生产力很重要。进一步提高生产力需要提高细胞内FFA的生产速率和FFA跨质膜的主动输出。
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