Production of extracellular fatty acid using engineered Escherichia coli
Production of extracellular fatty acid using engineered Escherichia coli
复制标题
使用工程大肠杆菌生产细胞外脂肪酸
DOI：
10.1186/1475-2859-11-41
复制
发表时间：
2012-04-03
影响因子：
6.4
通讯作者：
Xian, Mo
中科院分区：
文献类型：
--
作者：
Liu, Hui;Yu, Chao;Xian, Mo
背景
作为经济生物柴油生产的替代方案，从可再生资源中微生物生产细胞外脂肪酸最近受到更多关注，因为从微生物细胞中分离脂肪酸通常涉及一系列能源密集型步骤。人们已经做出了许多尝试，通过靶向脂肪酸生物合成途径中的基因来构建脂肪酸生产菌株，但很少有研究关注培养过程和传质动力学。
结果
在这项研究中，菌株改良和培养工艺策略均被应用于增加工程大肠杆菌的细胞外脂肪酸产量。我们的结果表明，在大肠杆菌 BL21 (DE3) 中过度表达 ‘TesA 和删除 fadL 可以改善细胞外脂肪酸的产生，而删除 fadD 并不能增强细胞外脂肪酸的产生，其机制尚未确定。此外，通过调节温度、添加氨苄青霉素和采用在线提取，培养过程控制对细胞外脂肪酸的滴度、细胞生长和生产率做出了巨大贡献。在最佳条件下，大肠杆菌菌株（pACY-‘tesA-ΔfadL）可产生4.8 g L−1胞外脂肪酸，其比生产力为0.02 g h−1 g−1干细胞团，葡萄糖产率为4.4%，培养15 h后细胞相关脂肪酸与胞外脂肪酸的比率保持在0.5以下。脂肪酸包括C12:1、C12:0、C14:1、C14:0、C16:1、C16:0、C18:1、C18:0。其组成主要是C14和C16饱和和不饱和脂肪酸。使用菌株 pACY-‘tesA，在相同的培养条件下出现了类似的结果，并且滴度也比以前报道的要高得多，这表明所谓的优越菌株不一定在有效生产所需产物方面表现最好。 pACY-‘tesA菌株也可以作为下一步基因操作的原始菌株。
结论
细胞外脂肪酸生产代谢工程的总体策略应该是培养性能和菌株改良之间的循环优化。在我们优化栽培工艺的基础上，还需进一步进行菌种改良，以获得有效且经济的生产工艺。
Background
As an alternative for economic biodiesel production, the microbial production of extracellular fatty acid from renewable resources is receiving more concerns recently, since the separation of fatty acid from microorganism cells is normally involved in a series of energy-intensive steps. Many attempts have been made to construct fatty acid producing strains by targeting genes in the fatty acid biosynthetic pathway, while few studies focused on the cultivation process and the mass transfer kinetics.
Results
In this study, both strain improvements and cultivation process strategies were applied to increase extracellular fatty acid production by engineered Escherichia coli. Our results showed overexpressing ‘TesA and the deletion of fadL in E. coli BL21 (DE3) improved extracellular fatty acid production, while deletion of fadD didn’t strengthen the extracellular fatty acid production for an undetermined mechanism. Moreover, the cultivation process controls contributed greatly to extracellular fatty acid production with respect to titer, cell growth and productivity by adjusting the temperature, adding ampicillin and employing on-line extraction. Under optimal conditions, the E. coli strain (pACY-‘tesA-ΔfadL) produced 4.8 g L−1 extracellular fatty acid, with the specific productivity of 0.02 g h−1 g−1dry cell mass, and the yield of 4.4% on glucose, while the ratios of cell-associated fatty acid versus extracellular fatty acid were kept below 0.5 after 15 h of cultivation. The fatty acids included C12:1, C12:0, C14:1, C14:0, C16:1, C16:0, C18:1, C18:0. The composition was dominated by C14 and C16 saturated and unsaturated fatty acids. Using the strain pACY-‘tesA, similar results appeared under the same culture conditions and the titer was also much higher than that ever reported previously, which suggested that the supposedly superior strain did not necessarily perform best for the efficient production of desired product. The strain pACY-‘tesA could also be chosen as the original strain for the next genetic manipulations.
Conclusions
The general strategy of metabolic engineering for the extracellular fatty acid production should be the cyclic optimization between cultivation performance and strain improvements. On the basis of our cultivation process optimization, strain improvements should be further carried out for the effective and cost-effective production process.