Department of Chemical and Biological Engineering, Iowa State University, Ames, IA 50011, USA; NSF Engineering Research Center for Biorenewable Chemicals, Iowa State University, Ames, IA 50011, USA.
Department of Chemical and Biomolecular Engineering, University of Maryland, College Park, MD 20742, USA.
Metab Eng. 2017 Nov;44:1-12. doi: 10.1016/j.ymben.2017.08.006. Epub 2017 Sep 1.
Economically competitive microbial production of biorenewable fuels and chemicals is often impeded by toxicity of the product to the microbe. Membrane damage is often identified as a major mechanism of this toxicity. Prior efforts to strengthen the microbial membrane by changing the phospholipid distribution have largely focused on the fatty acid tails. Herein, a novel strategy of phospholipid head engineering is demonstrated in Escherichia coli. Specifically, increasing the expression of phosphatidylserine synthase (+pssA) was found to significantly increase both the tolerance and production of octanoic acid, a representative membrane-damaging solvent. Tolerance of other industrially-relevant inhibitors, such as furfural, acetate, toluene, ethanol and low pH was also increased. In addition to the increase in the relative abundance of the phosphoethanolamine (PE) head group in the +pssA strain, there were also changes in the fatty acid tail composition, resulting in an increase in average length, percent unsaturation and decreased abundance of cyclic rings. This +pssA strain had significant changes in: membrane integrity, surface potential, electrochemical potential and hydrophobicity; sensitivity to intracellular acidification; and distribution of the phospholipid tails, including an increase in average length and percent unsaturation and decreased abundance of cyclic rings. Molecular dynamics simulations demonstrated that the +PE membrane had increased resistance to penetration of ethanol into the hydrophobic core and also the membrane thickness. Further hybrid models in which only the head group distribution or fatty acid tail distribution was altered showed that the increase in PE content is responsible for the increase in bilayer thickness, but the increased hydrophobic core thickness is due to altered distribution of both the head groups and fatty acid tails. This work demonstrates the importance of consideration of the membrane head groups, as well as a modeling approach, in membrane engineering efforts.
经济上有竞争力的微生物生产生物可再生燃料和化学品通常受到产物对微生物的毒性的阻碍。膜损伤通常被认为是这种毒性的主要机制。先前通过改变磷脂分布来增强微生物膜的努力主要集中在脂肪酸尾部。本文展示了一种在大肠杆菌中进行磷脂头工程的新策略。具体来说，发现增加磷脂酰丝氨酸合酶（+pssA）的表达可显著提高辛酸的耐受性和产量，辛酸是一种代表性的破坏膜溶剂。其他工业相关抑制剂（如糠醛、乙酸、甲苯、乙醇和低 pH）的耐受性也得到了提高。除了+pssA 菌株中磷酸乙醇胺（PE）头基的相对丰度增加外，脂肪酸尾部组成也发生了变化，导致平均长度、不饱和百分比增加，环状物丰度降低。与对照菌株相比，+pssA 菌株在：膜完整性、表面电势、电化学势和疏水性；对细胞内酸化的敏感性；以及磷脂尾部的分布方面都有显著变化，包括平均长度和不饱和百分比增加，环状物丰度降低。分子动力学模拟表明，+PE 膜对乙醇渗透进入疏水区的阻力增加，膜厚度也增加。进一步的混合模型表明，只有改变头基团分布或脂肪酸尾部分布，增加 PE 含量可导致双层厚度增加，但增加的疏水区厚度是由于头基团和脂肪酸尾部分布的改变所致。这项工作证明了在膜工程努力中考虑膜头基团以及建模方法的重要性。
