PDF(Pubmed)
Abstract：
The evolution of the bacterial phosphotransferase system (PTS) linked to glycolysis is dependent on the availability of naturally occurring sugars. Although bacteria exhibit sugar specificities based on carbon catabolite repression, the acquisition and evolvability of the cellular sugar preference under conditions that are suboptimal for growth (e.g., environments rich in a rare sugar) are poorly understood. Here, we generated Escherichia coli mutants via a retro-aldol reaction to obtain progeny that can utilize the rare sugar d-tagatose. We detected a minimal set of adaptive mutations in the d-fructose-specific PTS to render E. coli capable of d-tagatose utilization. These E. coli mutant strains lost the tight regulation of both the d-fructose and N-acetyl-galactosamine PTS following deletions in the binding site of the catabolite repressor/activator protein (Cra) upstream from the fruBKA operon and in the agaR gene, encoding the N-acetylgalactosamine (GalNAc) repressor, respectively. Acquired d-tagatose catabolic pathways then underwent fine-tuned adaptation via an additional mutation in 1-phosphofructose kinase to adjust metabolic fluxes. We determined the evolutionary trajectory at the molecular level, providing insights into the mechanism by which enteric bacteria evolved a substrate preference for the rare sugar d-tagatose. Furthermore, the engineered E. coli mutant strain could serve as an in vivo high-throughput screening platform for engineering non-phosphosugar isomerases to produce rare sugars. IMPORTANCE Microorganisms generate energy through glycolysis, which might have preceded a rapid burst of evolution, including the evolution of cellular respiration in the primordial biosphere. However, little is known about the evolvability of cellular sugar preferences. Here, we generated Escherichia coli mutants via a retro-aldol reaction to obtain progeny that can utilize the rare sugar d-tagatose. Consequently, we identified mutational hot spots and determined the evolutionary trajectory at the molecular level. This provided insights into the mechanism by which enteric bacteria evolved substrate preferences for various sugars, accounting for the widespread occurrence of these taxa. Furthermore, the adaptive laboratory evolution-induced cellular chassis could serve as an in vivo high-throughput screening platform for engineering tailor-made non-phosphorylated sugar isomerases to produce low-calorigenic rare sugars showing antidiabetic, antihyperglycemic, and antitumor activities.
摘要：
与糖酵解相关的细菌磷酸转移酶系统（PTS）的进化取决于天然存在的糖的可用性。尽管细菌表现出基于碳分解代谢物抑制的糖特异性，在生长次优的条件下，细胞糖偏好的获得和进化(例如，富含稀有糖的环境)知之甚少。这里,我们通过反羟醛反应产生了大肠杆菌突变体，以获得可以利用稀有糖d-塔格糖的后代。我们在d-果糖特异性PTS中检测到最少一组适应性突变,以使大肠杆菌能够利用d-塔格糖。这些大肠杆菌突变菌株在fruBKA操纵子上游和agaR基因中的分解代谢物阻遏物/激活蛋白（Cra）的结合位点缺失后，失去了对d-果糖和N-乙酰半乳糖胺PTS的严格调节。编码N-乙酰半乳糖胺（GalNAc）阻遏物，分别。获得的d-塔格糖分解代谢途径然后通过1-磷酸果糖激酶中的额外突变进行微调适应以调节代谢通量。我们在分子水平上确定了进化轨迹,提供有关肠道细菌进化出稀有糖d-塔格糖的底物偏好的机制的见解。此外，这种经过改造的大肠杆菌突变菌株可以作为体内高通量筛选平台，用于改造非磷酸糖异构酶以生产稀有糖。重要性微生物通过糖酵解产生能量，这可能是在快速进化之前，包括原始生物圈中细胞呼吸的进化。然而,对细胞糖偏好的进化性知之甚少。这里,我们通过反羟醛反应产生了大肠杆菌突变体，以获得可以利用稀有糖d-塔格糖的后代。因此,我们确定了突变热点，并确定了分子水平的进化轨迹。这提供了对肠道细菌进化出各种糖的底物偏好的机制的见解，解释了这些类群的广泛发生。此外，适应性实验室进化诱导的细胞底盘可以作为体内高通量筛选平台，用于工程定制的非磷酸化糖异构酶，以生产显示抗糖尿病的低热稀有糖，抗高血糖,和抗肿瘤活性。
