戊二酸是一种重要的C5直链二羧酸，广泛用于化学工业中的聚酯和聚酰胺，如尼龙-4,5和尼龙-5,5。戊二酸目前由衍生自石油的原料生产，特别是通过氧化由硝酸催化的环己酮和环己醇的混合物生产。然而，化学合成导致高污染和显著的温室气体排放。 因此，直接从底物生物产生戊二酸是非常重要的。虽然有报道使用谷氨酸棒杆菌生产戊二酸，但由于赖氨酸供应有限和发酵时间长，它有严重的局限性。为了解决这个问题，本研究构建了一种新的合成途径。
在本研究中报道了利用在Thermobifida fusca中鉴定的五步反向己二酸降解途径（RADP）在大肠杆菌中生产戊二酸。通过表达RADP的酶，菌株Bgl146摇瓶检测到戊二酸。发酵优化后，Bgl146的戊二酸摇瓶产量增加到4.7±0.2mM。通过CRISPR / Cas9，进一步消除了竞争碳通量的主要代谢物的途径（ΔarcA，ΔldhA，ΔatoB和ΔpflB）。 而且，最终菌株Bgl4146通过补料分批发酵得到36.5±0.3mM戊二酸。这些结果是大肠杆菌中报道的最高戊二酸产量。
本文以“EngineeringEscherichia colifor Glutarate Production as the C5Platform Backbone”为题发表于“Applied and Environmental Microbiology”期刊，国工赵梅为第一作者，邓禹教授为通讯作者。这一研究受到了江苏省自然科学基金（No.BK20150136），国家自然科学基金项目（No.31500070），江苏省生物质能源与酶技术重点实验室开放基金（No.BEETKA1801）和江苏省杰出教授项目支持。
A novel synthetic pathway for glutarate production in E. coli. The reverse adipate degradation pathway (RADP) includes five steps inThermobifida fusca:β-ketothiolase (Tfu_0875), 3 hydroxyacyl-CoA dehydrogenase (Tfu_2399), 3-hydroxyadipyl-CoA dehydrogenase (Tfu_0067), 5-carboxy-2-pentenoyl-CoA reductase (Tfu_1647), and adipyl-CoA synthetase (Tfu_2576-7). The arcA, ldhA, atoB, sucD, and pflB genes were deleted using the CRISPR/Cas9 system. A rough arrow represents genes or enzymes subject to overexpression, “X” represents those deleted, and the “┴”symbol represents suppression. Solid arrows indicate single steps, three arrows indicate multiple steps, and dashed arrows indicate that a step does not exist inE. coli. ldhA, L-lactate dehydrogenase; atoB, acetyl-CoA C-acetyltransferase; arcA, aerobic respiration control protein; pflB, formate C-acetyltransferase 1; TCA, tricarboxylic acid cycle; EMP, the glycolysis pathway; OAA, oxaloacetate; PEP, phosphoenolpyruvate; PYR, pyruvate; SuccCoA, succinyl-CoA; ACP, acyl carrier protein.
Fed-batch fermentation with strain Bgl4146. Substrate consumptions, metabolites, and cell growth during fed-batch fermentation are indicated. The highest titer was achieved in 5-liter bioreactor with a 0.5-vvm aeration rate and a 400-rpm agitation rate in SOB medium. Error bars represent the SD from three independent assays.
