Iacometti Camillo, Marx Katharina, Hönick Maria, Biletskaia Viktoria, Schulz-Mirbach Helena, Dronsella Beau, Satanowski Ari, Delmas Valérie A, Berger Anne, Dubois Ivan, Bouzon Madeleine, Döring Volker, Noor Elad, Bar-Even Arren, Lindner Steffen N
Max Planck Institute of Molecular Plant Physiology, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany.
Génomique Métabolique, Genoscope, Institut François Jacob, CEA, CNRS, Univ Evry, Université Paris-Saclay, 91057 Evry-Courcouronne, France.
Biodes Res. 2022 May 11;2022:9859643. doi: 10.34133/2022/9859643. eCollection 2022.
All living organisms share similar reactions within their central metabolism to provide precursors for all essential building blocks and reducing power. To identify whether alternative metabolic routes of glycolysis can operate in , we complementarily employed design, rational engineering, and adaptive laboratory evolution. First, we used a genome-scale model and identified two potential pathways within the metabolic network of this organism replacing canonical Embden-Meyerhof-Parnas (EMP) glycolysis to convert phosphosugars into organic acids. One of these glycolytic routes proceeds via methylglyoxal and the other via serine biosynthesis and degradation. Then, we implemented both pathways in strains harboring defective EMP glycolysis. Surprisingly, the pathway via methylglyoxal seemed to immediately operate in a triosephosphate isomerase deletion strain cultivated on glycerol. By contrast, in a phosphoglycerate kinase deletion strain, the overexpression of methylglyoxal synthase was necessary to restore growth of the strain. Furthermore, we engineered the "serine shunt" which converts 3-phosphoglycerate via serine biosynthesis and degradation to pyruvate, bypassing an enolase deletion. Finally, to explore which of these alternatives would emerge by natural selection, we performed an adaptive laboratory evolution study using an enolase deletion strain. Our experiments suggest that the evolved mutants use the serine shunt. Our study reveals the flexible repurposing of metabolic pathways to create new metabolite links and rewire central metabolism.
所有生物在其中心代谢过程中都有相似的反应，以提供所有必需组成部分的前体和还原力。为了确定糖酵解的替代代谢途径是否能在[具体生物]中发挥作用，我们互补地采用了设计、理性工程和适应性实验室进化方法。首先，我们使用了一个基因组规模的模型，并在该生物的代谢网络中确定了两条潜在途径，以取代经典的糖酵解途径（Embden-Meyerhof-Parnas，EMP），将磷酸糖转化为有机酸。其中一条糖酵解途径通过甲基乙二醛进行，另一条通过丝氨酸的生物合成和降解进行。然后，我们在EMP糖酵解存在缺陷的[具体生物]菌株中实施了这两条途径。令人惊讶的是，通过甲基乙二醛的途径似乎能在以甘油为培养基培养的磷酸丙糖异构酶缺失菌株中立即发挥作用。相比之下，在磷酸甘油酸激酶缺失菌株中，甲基乙二醛合酶的过表达对于恢复菌株生长是必要的。此外，我们构建了“丝氨酸分流”途径，该途径通过丝氨酸的生物合成和降解将3-磷酸甘油酸转化为丙酮酸，绕过了烯醇酶缺失。最后，为了探究这些替代途径中哪一条会通过自然选择出现，我们使用烯醇酶缺失菌株进行了适应性实验室进化研究。我们的实验表明，进化后的突变体使用丝氨酸分流途径。我们的研究揭示了代谢途径的灵活重新利用，以创建新的代谢物连接并重新构建中心代谢。
