Ruffing Anne, Mao Zichao, Ruizhen Chen Rachel
School of Chemical and Biomolecular Engineering, Georgia Institute of Technology, 311 Ferst Drive, Atlanta, GA 30332-0100, USA.
Metab Eng. 2006 Sep;8(5):465-73. doi: 10.1016/j.ymben.2006.05.004. Epub 2006 Jun 7.
Curdlan-producing Agrobacterium sp. is unique in possessing a highly efficient UDP-glucose regeneration system. A broad-host-range expression strategy was successfully developed to exploit the unique metabolic capability for UDP-galactose regeneration during oligosaccharide synthesis. The engineered Agrobacterium cells functioned as a UDP-galactose regeneration system, allowing galactose-containing disaccharides to be synthesized from glucose or other simple sugars. Unexpectedly, a lag period of 24h preceded the active synthesis, which could be eliminated with rifampicin. An intracellular nucleotide profiling revealed that the UMP level was elevated by 3.8 fold in the presence of rifampicin, suggesting that rifampicin simulated a nitrogen-limitation condition that triggered the metabolic change. Product selectivity was improved nearly 40-fold by using high acceptor concentration and restricting glucose supply. N-acetyllactosamine concentration near 20 mM (7.5 g/l) was obtained, demonstrating the effectiveness of the engineered strain in UDP-galactose regeneration. This organism could be engineered to regenerate other UDP-sugar nucleotides using the same strategy as illustrated here.
产凝胶多糖的土壤杆菌具有高效的UDP-葡萄糖再生系统，这一点很独特。人们成功开发了一种广泛宿主范围的表达策略，以利用其在寡糖合成过程中UDP-半乳糖再生的独特代谢能力。工程改造的土壤杆菌细胞充当UDP-半乳糖再生系统，使得含半乳糖的二糖能够由葡萄糖或其他单糖合成。出乎意料的是，在活跃合成之前有24小时的延迟期，而利福平可以消除这一延迟期。细胞内核苷酸分析表明，在利福平存在的情况下，UMP水平提高了3.8倍，这表明利福平模拟了一种氮限制条件，从而引发了代谢变化。通过使用高受体浓度和限制葡萄糖供应，产物选择性提高了近40倍。获得了接近20 mM（7.5 g/l）的N-乙酰乳糖胺浓度，证明了工程菌株在UDP-半乳糖再生方面的有效性。可以使用此处所示的相同策略对这种生物体进行工程改造，以再生其他UDP-糖核苷酸。
