Department of Bioengineering, Rice University, Houston, Texas, USA.
Metab Eng. 2012 Jul;14(4):380-7. doi: 10.1016/j.ymben.2012.03.007. Epub 2012 Mar 30.
Microbial biosynthesis of fatty acid like chemicals from renewable carbon sources has attracted significant attention in recent years. Free fatty acids can be used as precursors for the production of fuels or chemicals. Wild type E. coli strains produce fatty acids mainly for the biosynthesis of lipids and cell membranes and do not accumulate free fatty acids as intermediates in lipid biosynthesis. However, free fatty acids can be produced by breaking the fatty acid elongation through the overexpression of an acyl-ACP thioesterase. Since acetyl-CoA might be an important factor for fatty acid synthesis (acetate formation pathways are the main competitive pathways in consuming acetyl-CoA or pyruvate, a precursor of acetyl-CoA), and the long chain fatty acid CoA-ligase (FadD) plays a pivotal role in the transport and activation of exogenous fatty acids prior to their subsequent degradation, we examined the composition and the secretion of the free fatty acids in four different strains including the wild type MG1655, a mutant strain with inactivation of the fatty acid beta-oxidation pathway (fadD mutant (ML103)), and mutant strains with inactivation of the two major acetate production pathways (an ack-pta (acetate kinase/phosphotransacetylase), poxB (pyruvate oxidase) double mutant (ML112)) and a fadD, ack-pta, poxB triple mutant (ML115). The engineered E. coli cells expressing acyl-ACP thioesterase with glucose yield is higher than 40% of theoretical yield. Compared to MG1655(pXZ18) and ML103(pXZ18), acetate forming pathway deletion strains such as ML112(pXZ18) and ML115(pXZ18) produced similar quantity of total free fatty acids, which indicated that acetyl-CoA availability does not appear to be limiting factor for fatty acid production in these strains. However, these strains did show significant differences in the composition of free fatty acids. Different from MG1655(pXZ18) and ML103(pXZ18), acetate formation pathway deletion strains such as ML112(pXZ18) and ML115(pXZ18) produced similar level of C14, C16:1 and C16 free fatty acids, and the free fatty acid compositions of both strains did not change significantly with time. In addition, the strains bearing the fadD mutation showed significant differences in the quantities of free fatty acids found in the broth. Finally, we examined two potential screening methods for selecting and isolating high free fatty acids producing cells.
近年来，利用可再生碳源微生物生物合成脂肪酸样化合物引起了广泛关注。游离脂肪酸可用作生产燃料或化学品的前体。野生型大肠杆菌菌株主要用于脂质和细胞膜的生物合成而产生脂肪酸，并不将游离脂肪酸作为脂质生物合成的中间体积累。然而，通过过表达酰基辅酶 A 硫酯酶，可以打破脂肪酸的延伸，从而产生游离脂肪酸。由于乙酰辅酶 A 可能是脂肪酸合成的重要因素（乙酸盐形成途径是消耗乙酰辅酶 A 或丙酮酸（乙酰辅酶 A 的前体）的主要竞争途径），并且长链脂肪酸 CoA-连接酶（FadD）在随后的降解之前在外源脂肪酸的运输和激活中起着关键作用，我们检查了四种不同菌株（包括野生型 MG1655、脂肪酸β-氧化途径失活突变株（fadD 突变株（ML103））、两条主要乙酸盐产生途径失活突变株（ack-pta（乙酰激酶/磷酸转乙酰酶），poxB（丙酮酸氧化酶）双突变株（ML112））和 fadD、ack-pta、poxB 三突变株（ML115））中游离脂肪酸的组成和分泌。表达酰基辅酶 A 硫酯酶的工程大肠杆菌细胞的葡萄糖产率高于理论产率的 40%。与 MG1655(pXZ18)和 ML103(pXZ18)相比，如 ML112(pXZ18)和 ML115(pXZ18)等形成乙酸盐的途径缺失菌株产生了相似数量的总游离脂肪酸，这表明乙酰辅酶 A 的可用性似乎不是这些菌株中脂肪酸生产的限制因素。然而，这些菌株在游离脂肪酸的组成上确实存在显著差异。与 MG1655(pXZ18)和 ML103(pXZ18)不同，如 ML112(pXZ18)和 ML115(pXZ18)等形成乙酸盐的途径缺失菌株产生了相似水平的 C14、C16:1 和 C16 游离脂肪酸，并且这两种菌株的游离脂肪酸组成随时间变化不大。此外，携带 fadD 突变的菌株在发酵液中游离脂肪酸的数量上存在显著差异。最后，我们检查了两种用于选择和分离高产游离脂肪酸细胞的潜在筛选方法。
