Department of Biological and Environmental Engineering, College of Agriculture and Life Sciences, Cornell University, Ithaca, New York, USA.
Department of Biological and Environmental Engineering, College of Agriculture and Life Sciences, Cornell University, Ithaca, New York, USA
Appl Environ Microbiol. 2018 Dec 13;85(1). doi: 10.1128/AEM.02084-18. Print 2019 Jan 1.
The genetic characterization of Pf-5 was recently completed. However, the inferred metabolic network structure has not yet been evaluated experimentally. Here, we employed C-tracers and quantitative flux analysis to investigate the intracellular network for carbohydrate metabolism. In lieu of the direct phosphorylation of glucose by glucose kinase, glucose catabolism was characterized primarily by the oxidation of glucose to gluconate and 2-ketogluconate before the phosphorylation of these metabolites to feed the Entner-Doudoroff (ED) pathway. In the absence of phosphofructokinase activity, a cyclic flux from the ED pathway to the upper Embden-Meyerhof-Parnas (EMP) pathway was responsible for routing glucose-derived carbons to the non-oxidative pentose phosphate (PP) pathway. Consistent with the lack of annotated genes in Pf-5 for the transport or initial catabolism of pentoses and galactose, only glucose was assimilated into intracellular metabolites in the presence of xylose, arabinose, or galactose. However, when glucose was fed simultaneously with fructose or mannose, co-uptake of these hexoses was evident, but glucose was preferred over fructose (3 to 1) and over mannose (4 to 1). Despite gene annotation of mannose catabolism to fructose-6-phosphate, metabolite labeling patterns revealed that mannose was assimilated into fructose-1,6-bisphosphate, similarly to fructose catabolism. Remarkably, carbons from mannose and fructose were also found to cycle backward through the upper EMP pathway toward the ED pathway. Therefore, the operational metabolic network for processing carbohydrates in Pf-5 prioritizes flux through the ED pathway to channel carbons to EMP, PP, and downstream pathways. Species of the genus thrive in various nutritional environments and have strong biocatalytic potential due to their diverse metabolic capabilities. Carbohydrate substrates are ubiquitous both in environmental matrices and in feedstocks for engineered bioconversion. Here, we investigated the metabolic network for carbohydrate metabolism in Pf-5. Metabolic flux quantitation revealed the relative involvement of different catabolic routes in channeling carbohydrate carbons through a cyclic metabolic network. We also uncovered that mannose catabolism was similar to fructose catabolism, despite the annotation of a different pathway in the genome. Elucidation of the constitutive metabolic network in is important for understanding its innate carbohydrate processing, thus laying the foundation for targeting metabolic engineering of this untapped species.
Pf-5 的遗传特征最近已经完成。然而,推断的代谢网络结构尚未通过实验进行评估。在这里,我们采用 C 示踪剂和定量通量分析来研究碳水化合物代谢的细胞内网络。葡萄糖激酶不能直接磷酸化葡萄糖,葡萄糖分解代谢主要通过将葡萄糖氧化为葡萄糖酸和 2-酮葡萄糖酸,然后将这些代谢物磷酸化以供给 Entner-Doudoroff (ED)途径。在不存在磷酸果糖激酶活性的情况下,ED 途径到上式的循环通量 Embden-Meyerhof-Parnas (EMP)途径负责将葡萄糖衍生的碳路由到非氧化戊糖磷酸 (PP)途径。与 Pf-5 中缺乏用于戊糖和半乳糖运输或初始分解代谢的注释基因一致,只有在存在木糖、阿拉伯糖或半乳糖的情况下,才能将葡萄糖同化到细胞内代谢物中。然而,当同时给葡萄糖喂食果糖或甘露糖时,这些己糖的共摄取是明显的,但葡萄糖优先于果糖(3 比 1)和甘露糖(4 比 1)。尽管有 mannose 代谢为 fructose-6-phosphate 的基因注释,但代谢物标记模式表明 mannose 被同化到 fructose-1,6-bisphosphate 中,类似于 fructose 代谢。值得注意的是,来自 mannose 和 fructose 的碳也被发现通过上式的 EMP 途径向后循环到 ED 途径。因此, Pf-5 处理碳水化合物的操作代谢网络优先通过 ED 途径将碳输送到 EMP、PP 和下游途径。属的物种在各种营养环境中茁壮成长,并且由于其多样化的代谢能力而具有强大的生物催化潜力。碳水化合物底物在环境基质和工程生物转化的原料中都很普遍。在这里,我们研究了 Pf-5 中碳水化合物代谢的代谢网络。代谢通量定量揭示了不同分解代谢途径在通过循环代谢网络引导碳水化合物碳方面的相对参与。我们还发现,尽管在基因组中注释了不同的途径,但 mannose 代谢类似于 fructose 代谢。 Pf-5 组成型代谢网络的阐明对于理解其固有的碳水化合物处理很重要,从而为该未开发物种的代谢工程靶向奠定了基础。
Antonie Van Leeuwenhoek. 1985
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