Klebsiella pneumoniae can naturally synthesize 3‐hydroxypropionic acid (3‐HP), 1,3‐propanediol (1,3‐PD), and 2,3‐butanediol (2,3‐BD) from glycerol. However, biosynthesis of these industrially important chemicals is constrained by troublesome byproducts. To clarify the influences of byproducts on 3‐HP production, in this study, a total of eight byproduct‐producing enzyme genes including pmd, poxB, frdB, fumC, dhaT, ilvH, adhP, and pflB were individually deleted from the K. pneumoniae genome. The resultant eight mutants presented different levels of metabolites. In 24‐h shake‐flask cultivation, the adhP‐ and pflB‐deletion mutants produced 0.41 and 0.44 g/L 3‐HP, respectively. Notably, the adhP and pflB double deletion mutant K. pneumoniaeΔadhPΔpflB produced 1.58 g/L 3‐HP in 24‐h shake‐flask cultivation. When K. pneumoniaeΔadhPΔpflB was harnessed as a host strain to overexpress PuuC, a native aldehyde dehydrogenase (ALDH) catalyzing 3‐hydroxypropionaldehyde (3‐HPA) to 3‐HP, the resulting recombinant strain K. pneumoniaeΔadhPΔpflB(pTAC‐puuC) (pTAC‐puuC is PuuC expression vector) generated 66.91 g/L 3‐HP with a cumulative yield of 70.84% on glycerol in 60‐h bioreactor cultivation. Additionally, this strain showed 2.3‐, 5.1‐, and 0.67‐fold decrease in the concentrations of 1,3‐PD, 2,3‐BD, and acetic acid compared with the reference strain K. pneumoniae(pTAC‐puuC). These results indicated that the byproducts exerted differential impacts on the production of 3‐HP, 1,3‐PD, and 2,3‐BD. Although combinatorial elimination of byproduct pathways could reprogram glycerol flux, the enzyme 1,3‐propanediol oxidoreductase (DhaT) that catalyzes 3‐HPA to 1,3‐PD and the enzymes ALDHs, especially, PuuC are most pivotal for 3‐HP production. This study provides a deep understanding of how byproducts affect the production of 3‐HP, 1,3‐PD, and 2,3‐BD in K. pneumoniae.

中文翻译：

---

副产物途径对肺炎克雷伯菌3-羟基丙酸产生影响的生理学研究

肺炎克雷伯菌可以从甘油自然合成 3-羟基丙酸 (3-HP)、1,3-丙二醇 (1,3-PD) 和 2,3-丁二醇 (2,3-BD)。然而，这些工业上重要的化学品的生物合成受到麻烦的副产品的限制。为了阐明副产物对 3-HP 产生的影响，本研究从肺炎克雷伯菌中分别删除了 pmd、poxB、frdB、fumC、dhaT、ilvH、adhP 和 pflB 共 8 个产生副产物的酶基因基因组。由此产生的八个突变体呈现出不同水平的代谢物。在 24 小时摇瓶培养中，adhP 和 pflB 缺失突变体分别产生 0.41 和 0.44 g/L 3-HP。值得注意的是，adhP 和 pflB 双缺失突变体肺炎克雷伯菌ΔadhPΔpflB 在 24 小时摇瓶培养中产生 1.58 g/L 3-HP。当 K。肺炎克雷伯菌ΔadhPΔpflB 被用作宿主菌株过表达 PuuC，一种天然醛脱氢酶 (ALDH)，催化 3-羟基丙醛 (3-HPA) 生成 3-HP，由此产生的重组菌株肺炎克雷伯菌ΔadhPΔpflB(pTAC-puuC) (pTAC-puuC表达载体）在 60 小时生物反应器培养中使用甘油产生 66.91 g/L 3-HP，累积产量为 70.84%。此外，与参考菌株肺炎克雷伯菌 (pTAC-puuC) 相比，该菌株的 1,3-PD、2,3-BD 和乙酸浓度降低了 2.3、5.1 和 0.67 倍。这些结果表明副产物对 3-HP、1,3-PD 和 2,3-BD 的生产产生不同的影响。尽管副产物途径的组合消除可以重新编程甘油流量，但催化 3-HPA 为 1 的酶 1,3-丙二醇氧化还原酶 (DhaT)，3-PD 和酶 ALDH，尤其是 PuuC，对于 3-HP 的生产最为关键。本研究深入了解副产物如何影响肺炎克雷伯菌中 3-HP、1,3-PD 和 2,3-BD 的产生。