School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, Atlanta, GA 30332-0100, USA.
Appl Biochem Biotechnol. 2011 Jul;164(6):851-66. doi: 10.1007/s12010-011-9179-1. Epub 2011 Feb 8.
Agrobacterium sp. ATCC 31749 was previously shown to be an advantageous host for oligosaccharide production. Unexpectedly, the addition of citrate to the oligosaccharide synthesis reaction resulted in up to a sixfold improvement in the production N-aceytl-lactosamine, a disaccharide. The possible mechanisms for this citrate-induced stimulation of oligosaccharide production were investigated, including the consumption of citrate as a carbon and energy source, enhanced metal ion solubility from citrate chelation, and the ability of citrate to act as a buffer. The main mechanisms for the effect of citrate on oligosaccharide production were determined to be carbon and energy provision from citrate consumption and pH maintenance. ATCC 31749 was shown to co-metabolize citrate along with sucrose, a preferred carbon source, indicating the lack of a catabolite repression system in this Agrobacterium. Metabolic flux analysis suggested an increase in flux through TCA cycle for the citrate-containing reaction, which may provide additional energy supply to support enhanced oligosaccharide production. The citrate stimulation of oligosaccharide synthesis was shown to be unique to the Agrobacterium strain, as a similarly engineered Escherichia coli strain did not show significant improvement in oligosaccharide production with citrate addition. This work provides insight into the metabolism of Agrobacterium sp. ATCC 31749 and highlights important factors in whole-cell oligosaccharide synthesis.
根瘤农杆菌 sp. ATCC 31749 先前被证明是生产寡糖的有利宿主。出乎意料的是，在寡糖合成反应中添加柠檬酸可使 N-乙酰乳糖胺（一种二糖）的产量提高多达六倍。研究了这种柠檬酸诱导的寡糖产量刺激的可能机制，包括柠檬酸作为碳和能源的消耗、柠檬酸螯合增强金属离子的溶解度，以及柠檬酸作为缓冲剂的能力。确定柠檬酸对寡糖生产的影响的主要机制是柠檬酸消耗提供碳和能量以及 pH 值维持。根瘤农杆菌能够与蔗糖（首选碳源）一起共代谢柠檬酸，表明该菌中缺乏分解代谢物阻遏系统。代谢通量分析表明，含柠檬酸的反应中通过 TCA 循环的通量增加，这可能为增强的寡糖生产提供额外的能量供应。含有柠檬酸的反应可刺激寡糖的合成，这是根瘤农杆菌菌株所特有的，因为类似工程化的大肠杆菌菌株在添加柠檬酸时并没有显示出寡糖产量的显著提高。这项工作深入了解了根瘤农杆菌 sp. ATCC 31749 的代谢，并强调了全细胞寡糖合成中的重要因素。
