Quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli
Quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli
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DOI：
10.1002/bit.23309
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发表时间：
2012-01-01
影响因子：
3.8
通讯作者：
Gonzalez, Ramon
中科院分区：
文献类型：
--
作者：
Cintolesi, Angela;Clomburg, James M.;Gonzalez, Ramon
可得性、低价格和高还原度使甘油成为生产燃料和还原化学品的极具吸引力和可开发的碳源。本文报道了通过动力学建模和代谢控制分析（MCA）对大肠杆菌中甘油发酵代谢的定量分析，以更好地了解甘油发酵，并确定基因操作的关键靶点，从而提高产品的合成。采用由甘油、乙醇、生物质和11种细胞内代谢物的质量平衡组成的动力学模型，以及每个物种代谢的相应动力学表达式，模拟了间歇培养中甘油发酵的动力学。然后利用该模型计算代谢控制系数，阐明甘油利用和乙醇合成途径的控制结构。计算的通量控制系数表明，甘油发酵过程中的糖酵解通量几乎完全由甘油脱氢酶（由gldA编码）和二羟丙酮激酶（dhaKLM编码）控制。与MCA结果一致，gldA和dhaKLM的过表达导致甘油利用率和乙醇合成通量显著增加。此外，参与介导甘油利用及其转化为乙醇的途径的其他酶的过表达对甘油利用和乙醇合成没有显著影响，进一步验证了MCA的预测。这些发现随后被应用于增加乙醇产量的手段：甘油脱氢酶和DHAK的过度表达使粗甘油（生物柴油生产的副产品）生产20g/L乙醇，这表明在厌氧条件下将废甘油转化为乙醇具有工业规模的潜力。Biotechnol。Bioeng。2012;109: 187198。(c) 2011 Wiley期刊公司
Availability, low price, and high degree of reduction have made glycerol a highly attractive and exploited carbon source for the production of fuels and reduced chemicals. Here we report the quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli through the use of kinetic modeling and metabolic control analysis (MCA) to gain a better understanding of glycerol fermentation and identify key targets for genetic manipulation that could enhance product synthesis. The kinetics of glycerol fermentation in a batch culture was simulated using a dynamic model consisting of mass balances for glycerol, ethanol, biomass, and 11 intracellular metabolites, along with the corresponding kinetic expressions for the metabolism of each species. The model was then used to calculate metabolic control coefficients and elucidate the control structure of the pathways involved in glycerol utilization and ethanol synthesis. The calculated flux control coefficients indicate that the glycolytic flux during glycerol fermentation is almost exclusively controlled by the enzymes glycerol dehydrogenase (encoded by gldA) and dihydroxyacetone kinase (DHAK) (encoded by dhaKLM). In agreement with the MCA findings, overexpression of gldA and dhaKLM led to significant increase in glycerol utilization and ethanol synthesis fluxes. Moreover, overexpression of other enzymes involved in the pathways that mediate glycerol utilization and its conversion to ethanol had no significant impact on glycerol utilization and ethanol synthesis, further validating the MCA predictions. These findings were then applied as a means of increasing the production of ethanol: overexpression of glycerol dehyrdogenase and DHAK enabled the production of 20g/L ethanol from crude glycerol, a by-product of biodiesel production, indicating the potential for industrial scale conversion of waste glycerol to ethanol under anaerobic conditions. Biotechnol. Bioeng. 2012;109: 187198. (c) 2011 Wiley Periodicals, Inc.