Phocaeicolavulgulgatus属于肠道微生物组,它发酵分解食物衍生的生物聚合物,因此,有助于肠道代谢组。此外,由于其产品范围,P.vulgatus是可持续生产基本有机化学品的潜在非标准平台生物。补充最近的生理-蛋白质组学报告破译菌株的通用发酵网络[1],本研究的重点是全球生长阶段依赖性反应。在过程控制的生物反应器中用葡萄糖厌氧培养。进行近距离采样以测量生长参数(OD,CDW,ATP含量,底物/产品概况)以确定生长化学计量。较粗的采样(½ODmax,ODmax,和ODstat)用于全球生长阶段依赖性反应的分子分析,应用蛋白质组学(可溶性和膜部分,nanoLC-ESI-MS/MS)和靶向/非靶向代谢组分析。确定的生长性能的特征1.74h倍增时间,0.06gCDW/mmolGlc生物量产量,0.36(琥珀酸)和0.61(乙酸)mmolP/mmolGlc作为主要的发酵产物产量,和0.43mmolATP/mmolC作为理论计算的ATP产率。发酵途径显示出生长阶段依赖性动力学:构成糖酵解上部的蛋白质及其伴随的代谢物的水平在½ODmax达到峰值,而糖酵解下部和发酵途径,特别是主要的乙酸盐和琥珀酸盐的发酵途径在ODmax和ODstat最高。虽然已鉴定的单体生物合成蛋白质显示出相当非特异性的特征,大多数细胞内氨基酸在ODmax达到峰值。相比之下,与应激反应和群体感应相关的蛋白质和代谢物在ODmax和ODstat显示出丰度增加。外代谢组的组成从1/2ODmax的2,317个分子式扩展到ODmax的4,258到ODstat的4,501,与特定于生长期的子集平行增加。本研究提供了对生物反应器中培养过程中P.vulgatus的独特生长阶段依赖性行为的见解。这可以作为进一步开发P.vulgatus作为用于生物技术应用的非常规平台生物的有价值的知识库。此外,这些发现为P.vulgatus在肠道微生物环境中潜在的生长阶段依赖性印记提供了新的思路,例如,通过群体感应指示相互作用,并通过解开发酵产物和次生代谢产物形成的复杂的外代谢背景。
BACKGROUND: Phocaeicola vulgatus (basonym Bacteroides vulgatus) belongs to the intestinal microbiome of healthy humans and animals, where it participates in the fermentative breakdown of biopolymers ingested with food. In doing so, P. vulgatus contributes to the shaping of the gut metabolome, which benefits the host health. Moreover, considering the fermentation product range (short chain fatty acids), P. vulgatus suggests itself as a potential nonstandard platform organism for a sustainable production of basic organic chemicals. Complementing a recent physiologic-proteomic report deciphering the strain\'s versatile fermentation network, the present study focusses on the global growth phase-dependent response of P. vulgatus.
METHODS: P. vulgatus was anaerobically cultivated with glucose as sole source of carbon and energy in process-controlled bioreactors operated in parallel. Close sampling was conducted to measure growth parameters (OD, CDW, ATP-content, substrate/product profiles) as basis for determining growth stoichiometry in detail. A coarser sampling (½ODmax, ODmax, and ODstat) served the molecular analysis of the global growth phase-dependent response, studied by means of differential proteomics (soluble and membrane fractions, nanoLC-ESI-MS/MS) as well as targeted metabolite (GC-MS and LC-MS/MS) and untargeted exometabolome (FT-ICR-MS) analyses.
RESULTS: The determined growth performance of P. vulgatus features 1.74 h doubling time, 0.06 gCDW/mmolGlc biomass yield, 0.36 (succinate) and 0.61 (acetate) mmolP/mmolGlc as predominant fermentation product yields, and 0.43 mmolATP/mmolC as theoretically calculated ATP yield. The fermentation pathway displayed distinct growth phase-dependent dynamics: the levels of proteins and their accompanying metabolites constituting the upper part of glycolysis peaked at ½ODmax, whereas those of the lower part of glycolysis and of the fermentation routes in particular toward the predominant products acetate and succinate were highest at ODmax and ODstat. While identified proteins of monomer biosynthesis displayed rather unspecific profiles, most of the intracellular amino acids peaked at ODmax. By contrast, proteins and metabolites related to stress response and quorum sensing showed increased abundances at ODmax and ODstat. Finally, the composition of the exometabolome expanded from 2,317 molecular formulas at ½ODmax via 4,258 at ODmax to 4,501 at ODstat, with growth phase-specific subsets increasing in parallel.
CONCLUSIONS: The present study provides insights into the distinct growth phase-dependent behavior of P. vulgatus during cultivation in bioreactors on the physiological and molecular levels. This could serve as a valuable knowledge-base for further developing P. vulgatus as a nonconventional platform organism for biotechnological applications. In addition, the findings shed new light on the potential growth phase-dependent imprints of P. vulgatus on the gut microbiome environment, e.g., by indicating interactions via quorum sensing and by unraveling the complex exometabolic background against which fermentation products and secondary metabolites are formed.