Kim Youngnyun, Ingram L O, Shanmugam K T
Department of Microbiology and Cell Science, Box 110700, University of Florida, Gainesville, FL 32611, USA.
J Bacteriol. 2008 Jun;190(11):3851-8. doi: 10.1128/JB.00104-08. Epub 2008 Mar 28.
Under anaerobic growth conditions, an active pyruvate dehydrogenase (PDH) is expected to create a redox imbalance in wild-type Escherichia coli due to increased production of NADH (>2 NADH molecules/glucose molecule) that could lead to growth inhibition. However, the additional NADH produced by PDH can be used for conversion of acetyl coenzyme A into reduced fermentation products, like alcohols, during metabolic engineering of the bacterium. E. coli mutants that produced ethanol as the main fermentation product were recently isolated as derivatives of an ldhA pflB double mutant. In all six mutants tested, the mutation was in the lpd gene encoding dihydrolipoamide dehydrogenase (LPD), a component of PDH. Three of the LPD mutants carried an H322Y mutation (lpd102), while the other mutants carried an E354K mutation (lpd101). Genetic and physiological analysis revealed that the mutation in either allele supported anaerobic growth and homoethanol fermentation in an ldhA pflB double mutant. Enzyme kinetic studies revealed that the LPD(E354K) enzyme was significantly less sensitive to NADH inhibition than the native LPD. This reduced NADH sensitivity of the mutated LPD was translated into lower sensitivity of the appropriate PDH complex to NADH inhibition. The mutated forms of the PDH had a 10-fold-higher K(i) for NADH than the native PDH. The lower sensitivity of PDH to NADH inhibition apparently increased PDH activity in anaerobic E. coli cultures and created the new ethanologenic fermentation pathway in this bacterium. Analogous mutations in the LPD of other bacteria may also significantly influence the growth and physiology of the organisms in a similar fashion.
在厌氧生长条件下，由于烟酰胺腺嘌呤二核苷酸（NADH）产量增加（>2个NADH分子/葡萄糖分子），活性丙酮酸脱氢酶（PDH）预计会在野生型大肠杆菌中造成氧化还原失衡，这可能导致生长抑制。然而，在该细菌的代谢工程中，PDH产生的额外NADH可用于将乙酰辅酶A转化为还原型发酵产物，如醇类。最近，作为ldhA pflB双突变体的衍生物，分离出了以乙醇为主要发酵产物的大肠杆菌突变体。在所有测试的六个突变体中，突变均发生在编码二氢硫辛酰胺脱氢酶（LPD）的lpd基因中，LPD是PDH的一个组分。三个LPD突变体携带H322Y突变（lpd102），而其他突变体携带E354K突变（lpd101）。遗传和生理学分析表明，任一基因座的突变都支持ldhA pflB双突变体的厌氧生长和同型乙醇发酵。酶动力学研究表明，LPD（E354K）酶对NADH抑制的敏感性明显低于天然LPD。突变型LPD对NADH敏感性的降低转化为相应的PDH复合物对NADH抑制的较低敏感性。PDH的突变形式对NADH的抑制常数（K(i)）比天然PDH高10倍。PDH对NADH抑制较低的敏感性显然增加了厌氧大肠杆菌培养物中的PDH活性，并在该细菌中产生了新的乙醇生成发酵途径。其他细菌的LPD中的类似突变也可能以类似方式显著影响生物体的生长和生理学。
