Medium-chain length methyl ketones are potential blending fuels due to their cetane numbers and low melting temperatures. Biomanufacturing offers the potential to produce these molecules from renewable resources such as lignocellulosic biomass. In this work, we designed and tested metabolic pathways in Escherichia coli to specifically produce 2-heptanone, 2-nonanone and 2-undecanone. We achieved substantial production of each ketone by introducing chain-length specific acyl-ACP thioesterases, blocking the β-oxidation cycle at an advantageous reaction, and introducing active β-ketoacyl-CoA thioesterases. Using a bioprospecting approach, we identified fifteen homologs of E. coli β-ketoacyl-CoA thioesterase (FadM) and evaluated the in vivo activity of each against various chain length substrates. The FadM variant from Providencia sneebia produced the most 2-heptanone, 2-nonanone, and 2-undecanone, suggesting it has the highest activity on the corresponding β-ketoacyl-CoA substrates. We tested enzyme variants, including acyl-CoA oxidases, thiolases, and bi-functional 3-hydroxyacyl-CoA dehydratases to maximize conversion of fatty acids to β-keto acyl-CoAs for 2-heptanone, 2-nonanone, and 2-undecanone production. In order to address the issue of product loss during fermentation, we applied a 20% (v/v) dodecane layer in the bioreactor and built an external water cooling condenser connecting to the bioreactor heat-transferring condenser coupling to the condenser. Using these modifications, we were able to generate up to 4.4 g/L total medium-chain length methyl ketones.

由于其十六烷值和低熔化温度，中链甲基酮是潜在的混合燃料。生物制造提供了从可再生资源（如木质纤维素生物质）生产这些分子的潜力。在这项工作中，我们设计并测试了大肠杆菌中的代谢途径，以专门生产 2-庚酮、2-壬酮和 2-十一酮。我们通过引入链长特异性酰基-ACP 硫酯酶、在有利反应中阻断 β-氧化循环以及引入活性 β-酮酰基-CoA 硫酯酶，实现了每种酮的大量生产。使用生物勘探方法，我们确定了大肠杆菌β-酮脂酰辅酶 A 硫酯酶 (FadM) 的15 种同源物，并在体内评估了每种对各种链长底物的活性。来自Providencia sneebia的FadM变种产生最多的 2-庚酮、2-壬酮和 2-十一酮，表明它对相应的 β-酮酰基-CoA 底物具有最高的活性。我们测试了酶变体，包括酰基辅酶 A 氧化酶、硫解酶和双功能 3-羟酰基辅酶 A 脱水酶，以最大限度地将脂肪酸转化为 β-酮酰基辅酶 A，从而生产 2-庚酮、2-壬酮和 2-十一酮. 为了解决发酵过程中产品损失的问题，我们在生物反应器中应用了 20% (v/v) 十二烷层，并构建了一个外部水冷冷凝器，连接到生物反应器传热冷凝器耦合到冷凝器。使用这些修改，我们能够生成高达 4.4 g/L 的总中链长度甲基酮。