Department of Chemical Engineering, Pennsylvania State University, University Park, PA 16802, USA.
Biotechnol Prog. 2011 Mar-Apr;27(2):333-41. doi: 10.1002/btpr.559. Epub 2011 Feb 22.
Escherichia coli engineered to uptake xylose while metabolizing glucose was previously shown to produce high levels of xylitol from a mixture of glucose and xylose when expressing NADPH-dependent xylose reductase from Candida boidinii (CbXR) (Cirino et al., Biotechnol Bioeng. 2006;95:1167-1176). We then described the effects of deletions of key metabolic pathways (e.g., Embden-Meyerhof-Parnas and pentose phosphate pathway) and reactions (e.g., transhydrogenase and NADH dehydrogenase) on resting-cell xylitol yield (Y RPG: moles of xylitol produced per mole of glucose consumed) (Chin et al., Biotechnol Bioeng. 2009;102:209-220). These prior results demonstrated the importance of direct NADPH supply by NADP+-utilizing enzymes in central metabolism for driving heterologous NADPH-dependent reactions. This study describes strain modifications that improve coupling between glucose catabolism (oxidation) and xylose reduction using two fundamentally different strategies. We first examined the effects of deleting the phosphofructokinase (pfk) gene(s) on growth-uncoupled xylitol production and found that deleting both pfkA and sthA (encoding the E. coli-soluble transhydrogenase) improved the xylitol Y RPG from 3.4 ± 0.6 to 5.4 ± 0.4. The second strategy focused on coupling aerobic growth on glucose to xylitol production by deleting pgi (encoding phosphoglucose isomerase) and sthA. Impaired growth due to imbalanced NADPH metabolism (Sauer et al., J Biol Chem. 2004;279:6613-6619) was alleviated upon expressing CbXR, resulting in xylitol production similar to that of the growth-uncoupled precursor strains but with much less acetate secretion and more efficient utilization of glucose. Intracellular nicotinamide cofactor levels were also quantified, and the magnitude of the change in the NADPH/NADP+ ratio measured from cells consuming glucose in the absence vs. presence of xylose showed a strong correlation to the resulting Y RPG.
先前已经证明，当大肠杆菌摄取木糖并代谢葡萄糖时，通过表达来自 Candida boidinii（CbXR）的 NADPH 依赖性木糖还原酶，可以从葡萄糖和木糖的混合物中产生高水平的木糖醇（Cirino 等人，Biotechnol Bioeng. 2006;95:1167-1176）。然后，我们描述了删除关键代谢途径（例如，Embden-Meyerhof-Parnas 和戊糖磷酸途径）和反应（例如，转氢酶和 NADH 脱氢酶）对静止细胞木糖醇产率（Y RPG：每消耗 1 摩尔葡萄糖产生的摩尔木糖醇）的影响（Chin 等人，Biotechnol Bioeng. 2009;102:209-220）。这些先前的结果表明，在中心代谢中，利用 NADP+-的酶直接提供 NADPH 对于驱动异源 NADPH 依赖性反应非常重要。本研究描述了两种从根本上不同的策略来改善葡萄糖分解代谢（氧化）和木糖还原之间的偶联的菌株修饰。我们首先研究了删除磷酸果糖激酶（pfk）基因对生长非偶联木糖醇生产的影响，发现删除 pfkA 和 sthA（编码大肠杆菌可溶性转氢酶）可将木糖醇 Y RPG 从 3.4±0.6 提高到 5.4±0.4。第二种策略侧重于通过删除 pgi（编码磷酸葡萄糖异构酶）和 sthA 将有氧生长与木糖生产偶联。由于 NADPH 代谢失衡（Sauer 等人，J Biol Chem. 2004;279:6613-6619）导致生长受损，在表达 CbXR 后得到缓解，导致木糖产率与生长非偶联前体菌株相似，但乙酸盐分泌减少，葡萄糖利用率更高。还定量了细胞内烟酰胺辅酶水平，并且从消耗葡萄糖的细胞中测量的在不存在和存在木糖的情况下 NADPH/NADP+ 比的变化幅度与所得 Y RPG 强烈相关。
