3-Hydroxyacids are a group of valuable fine chemicals with numerous applications, and 3-hydroxybutyrate (3-HB) represents the most common species with acetyl-CoA as a precursor. Due to the lack of propionyl-CoA in most, if not all, microorganisms, bio-based production of 3-hydroxyvalerate (3-HV), a longer-chain 3-hydroxyacid member with both acetyl-CoA and propionyl-CoA as two precursors, is often hindered by high costs associated with the supplementation of related carbon sources, such as propionate or valerate. Here, we report the derivation of engineered Escherichia coli strains for the production of 3-HV from unrelated cheap carbon sources, in particular glucose and glycerol. Activation of the sleeping beauty mutase (Sbm) pathway in E. coli enabled the intracellular formation of non-native propionyl-CoA. A selection of enzymes involved in 3-HV biosynthetic pathway from various microorganisms were explored for investigating their effects on 3-HV biosynthesis in E. coli. Glycerol outperformed glucose as the carbon source, and glycerol dissimilation for 3-HV biosynthesis was primarily mediated through the aerobic GlpK-GlpD route. To further enhance 3-HV production, we developed metabolic engineering strategies to redirect more dissimilated carbon flux from the tricarboxylic acid (TCA) cycle to the Sbm pathway, resulting in an enlarged intracellular pool of propionyl-CoA. Both the presence of succinate/succinyl-CoA and their interconversion step in the TCA cycle were identified to critically limit the carbon flux redirection into the Sbm pathway and, therefore, 3-HV biosynthesis. A selection of E. coli host TCA genes encoding enzymes near the succinate node were targeted for manipulation to evaluate the contribution of the three TCA routes (i.e. oxidative TCA cycle, reductive TCA branch, and glyoxylate shunt) to the redirected carbon flux into the Sbm pathway. Finally, the carbon flux redirection into the Sbm pathway was enhanced by simultaneously deregulating glyoxylate shunt and blocking the oxidative TCA cycle, significantly improving 3-HV biosynthesis. With the implementation of these biotechnological and bioprocessing strategies, our engineered E. coli strains can effectively produce 3-HV up to 3.71 g l⁻¹ with a yield of 24.1% based on the consumed glycerol in shake-flask cultures.

3-羟基酸是一组应用广泛的有价值的精细化学品，3-羟基丁酸酯（3-HB）代表了最常见的以乙酰辅酶A为前体的物质。由于在大多数（如果不是全部）微生物中缺乏丙酰辅酶 A，基于生物的 3-羟基戊酸 (3-HV) 生产是一种较长链的 3-羟基酸成员，同时具有乙酰辅酶 A 和丙酰辅酶 A 作为两个前体，通常受到与补充相关碳源（如丙酸盐或戊酸盐）相关的高成本的阻碍。在这里，我们报告了从不相关的廉价碳源（特别是葡萄糖和甘油）生产 3-HV的工程大肠杆菌菌株的衍生。大肠杆菌中睡美人变位酶 (Sbm) 通路的激活使非天然丙酰辅酶A的细胞内形成成为可能。探索了一系列参与来自各种微生物的 3-HV 生物合成途径的酶，以研究它们对大肠杆菌中3-HV 生物合成的影响. 甘油作为碳源的表现优于葡萄糖，并且用于 3-HV 生物合成的甘油异化主要通过有氧 GlpK-GlpD 途径介导。为了进一步提高 3-HV 产量，我们开发了代谢工程策略，将更多的异化碳通量从三羧酸 (TCA) 循环重定向到 Sbm 途径，导致细胞内丙酰辅酶 A 池扩大。琥珀酸/琥珀酰-CoA 的存在及其在 TCA 循环中的相互转化步骤被确定为严格限制碳通量重定向到 Sbm 途径，因此，3-HV 生物合成。精选大肠杆菌将编码琥珀酸节点附近酶的宿主 TCA 基因作为操作目标，以评估三种 TCA 途径（即氧化性 TCA 循环、还原性 TCA 分支和乙醛酸分流）对重新定向的碳通量进入 Sbm 途径的贡献。最后，通过同时解除乙醛酸分流和阻断氧化 TCA 循环，增强了碳通量重定向到 Sbm 途径，显着改善了 3-HV 生物合成。随着这些生物技术和生物加工策略的实施，我们的工程大肠杆菌菌株可以有效地产生高达 3.71 g l ^{-1 的}3-HV，基于摇瓶培养物中消耗的甘油，产量为 24.1%。