1-Decanol has great value in the pharmaceutical and fragrance industries and plays an important role in the chemical industry. In this study, we engineered Escherichia coli to selectively synthesize 1-decanol by using enzymes of the core reverse β-oxidation (rBOX) pathway and termination module with overlapping chain-length specificity. Through screening for acyl-CoA reductase termination enzymes and proper regulation of rBOX pathway expression, a 1-decanol titer of 1.4 g/L was achieved. Further improvements were realized by engineering pyruvate dissimilation to ensure the generation of NADH through pyruvate dehydrogenase (PDH) and reducing byproduct synthesis via a tailored YigI thioesterase knockout, increasing 1-decanol titer to 1.9 g/L. The engineered strain produced about 4.4 g/L 1-decanol with a yield of 0.21 g/g in 36 h in a bi-phasic fermentation that used a dodecane overlay to increase 1-decanol transport and reduce its toxicity. Adjustment of pathway expression (varying inducer concentration) and cell growth (oxygen availability) enabled 1-decanol production at 6.1 g/L (0.26 g/g yield) and 10.05 g/L (0.2 g/g yield) using rich medium in shake flasks and bioreactor, respectively. Remarkably, the use of minimal medium resulted in 1-decanol production with 100% specificity at 2.8 g/L (0.14 g/g yield) and a per cell mass yield higher than rich medium. These 1-decanol titers, yields and purity are at least 10-fold higher than others reported to date and the engineered strain shows great potential for industrial production. Taken together, our findings suggest that using rBOX pathway and termination enzymes of proper chain-length specificity in combination with optimal chassis engineering should be an effective approach for the selective production of alcohols.

1-癸醇在医药和香料工业中具有巨大的价值，在化学工业中发挥着重要作用。在本研究中，我们对大肠杆菌进行了改造，利用核心反向β-氧化（rBOX）途径的酶和具有重叠链长特异性的终止模块来选择性合成1-癸醇。通过筛选酰基辅酶A还原酶终止酶和适当调节rBOX通路表达，获得了1.4 g/L的1-癸醇滴度。通过工程化丙酮酸异化以确保通过丙酮酸脱氢酶 (PDH) 生成 NADH，并通过定制的 YigI 硫酯酶敲除减少副产物合成，将 1-癸醇滴度提高至 1.9 g/L，从而实现了进一步的改进。该工程菌株在双相发酵中在 36 小时内产生了约 4.4 g/L 1-癸醇，产量为 0.21 g/g，并使用十二烷覆盖来增加 1-癸醇转运并降低其毒性。调整途径表达（不同的诱导剂浓度）和细胞生长（氧气利用率）使得在摇动中使用丰富培养基能够以 6.1 g/L（0.26 g/g 产量）和 10.05 g/L（0.2 g/g 产量）生产 1-癸醇分别为烧瓶和生物反应器。值得注意的是，使用基本培养基可产生 1-癸醇，特异性为 2.8 g/L（0.14 g/g 产率），并且每个细胞质量产率高于丰富培养基。这些 1-癸醇的滴度、产量和纯度比迄今为止报道的其他菌株至少高 10 倍，并且该工程菌株显示出巨大的工业生产潜力。综上所述，我们的研究结果表明，使用 rBOX 途径和具有适当链长特异性的终止酶与最佳底盘工程相结合应该是选择性生产醇的有效方法。