Department of Chemical and Biomolecular Engineering, Rice University, Houston, TX, USA.
Department of Bioengineering, Rice University, Houston, TX, USA.
Microbiology (Reading). 2010 Jun;156(Pt 6):1860-1872. doi: 10.1099/mic.0.036251-0. Epub 2010 Feb 18.
The fermentative metabolism of d-glucuronic acid (glucuronate) in Escherichia coli was investigated with emphasis on the dissimilation of pyruvate via pyruvate formate-lyase (PFL) and pyruvate dehydrogenase (PDH). In silico and in vivo metabolic flux analysis (MFA) revealed that PFL and PDH share the dissimilation of pyruvate in wild-type MG1655. Surprisingly, it was found that PDH supports fermentative growth on glucuronate in the absence of PFL. The PDH-deficient strain (Pdh-) exhibited a slower transition into the exponential phase and a decrease in specific rates of growth and glucuronate utilization. Moreover, a significant redistribution of metabolic fluxes was found in PDH- and PFL-deficient strains. Since no role had been proposed for PDH in the fermentative metabolism of E. coli, the metabolic differences between MG1655 and Pdh- were further investigated. An increase in the oxidative pentose phosphate pathway (ox-PPP) flux was observed in response to PDH deficiency. A comparison of the ox-PPP and PDH pathways led to the hypothesis that the role of PDH is the supply of reducing equivalents. The finding that a PDH deficiency lowers the NADH : NAD(+) ratio supported the proposed role of PDH. Moreover, the NADH : NAD(+) ratio in a strain deficient in both PDH and the ox-PPP (Pdh-Zwf-) was even lower than that observed for Pdh-. Strain Pdh-Zwf- also exhibited a slower transition into the exponential phase and a lower growth rate than Pdh-. Finally, a transhydrogenase-deficient strain grew more slowly than wild-type but did not show the slower transition into the exponential phase characteristic of Pdh- mutants. It is proposed that PDH fulfils two metabolic functions. First, by creating the appropriate internal redox state (i.e. appropriate NADH : NAD(+) ratio), PDH ensures the functioning of the glucuronate utilization pathway. Secondly, the NADH generated by PDH can be converted to NADPH by the action of transhydrogenases, thus serving as biosynthetic reducing power in the synthesis of building blocks and macromolecules.
我们研究了大肠杆菌中 D-葡萄糖醛酸（葡糖醛酸）的发酵代谢，重点研究了通过丙酮酸甲酸裂解酶（PFL）和丙酮酸脱氢酶（PDH）分解丙酮酸。基于计算机的代谢通量分析（MFA）和体内代谢通量分析揭示，野生型 MG1655 中，PFL 和 PDH 共同分解丙酮酸。令人惊讶的是，我们发现 PDH 在没有 PFL 的情况下也能支持葡糖醛酸的发酵生长。PDH 缺陷型菌株（Pdh-）进入指数生长期的速度较慢，比生长速率和葡糖醛酸利用速率都有所降低。此外，PDH 和 PFL 缺陷型菌株中的代谢通量也发生了显著重分布。由于 PDH 在大肠杆菌的发酵代谢中尚未被提出任何作用，我们进一步研究了 MG1655 和 Pdh-之间的代谢差异。PDH 缺陷导致氧化戊糖磷酸途径（ox-PPP）通量增加。PDH 缺陷与 ox-PPP 途径的比较导致了这样的假设，即 PDH 的作用是提供还原当量。PDH 缺陷降低 NADH：NAD(+) 比值的发现支持了 PDH 的拟议作用。此外，PDH 和 ox-PPP 均缺陷的菌株（Pdh-Zwf-）中的 NADH：NAD(+) 比值甚至低于 Pdh-。Pdh-Zwf- 菌株的生长速度也比 Pdh-更慢，进入指数生长期的速度也更慢。最后，转氢酶缺陷型菌株的生长速度比野生型慢，但与 Pdh- 突变体的特征性进入指数生长期的速度较慢不同。我们提出 PDH 有两个代谢功能。首先，通过创造适当的内部氧化还原状态（即适当的 NADH：NAD(+) 比值），PDH 确保了葡糖醛酸利用途径的正常运行。其次，PDH 产生的 NADH 可以通过转氢酶转化为 NADPH，从而为构建块和大分子的合成提供生物合成还原能力。
