Quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli.

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 20 g/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.

Cintolesi A ，Clomburg JM ，Rigou V ，Zygourakis K ，Gonzalez R ... -  《-》

Metabolic engineering of Escherichia coli for the production of 1,2-propanediol from glycerol.

Due to its availability, low-price, and high degree of reduction, glycerol has become an attractive carbon source for the production of fuels and reduced chemicals. Using the platform we have established from the identification of key pathways mediating fermentative metabolism of glycerol, this work reports the engineering of Escherichia coli for the conversion of glycerol into 1,2-propanediol (1,2-PDO). A functional 1,2-PDO pathway was engineered through a combination of overexpression of genes involved in its synthesis from the key intermediate dihydroxyacetone phosphate (DHAP) and the manipulation of the fermentative glycerol utilization pathway. The former included the overexpression of methylglyoxal synthase (mgsA), glycerol dehydrogenase (gldA), and aldehyde oxidoreductase (yqhD). Manipulation of the glycerol utilization pathway through the replacement of the native E. coli PEP-dependent dihydroxyacetone kinase (DHAK) with an ATP-dependent DHAK from C. freundii increased the availability of DHAP allowing for higher 1,2-PDO production. Analysis of the major fermentative pathways identified ethanol as a required co-product while increases in 1,2-PDO titer and yield were achieved through the disruption of the pathways for acetate and lactate production. Combination of these key metabolic manipulations resulted in an engineered E. coli strain capable of producing 5.6 g/L 1,2-PDO, at a yield of 21.3% (w/w). This strain also performed well when crude glycerol, a by-product of biodiesel production, was used as the substrate. The titer and yield achieved in this study were favorable to those obtained with the use of E. coli for the production of 1,2-PDO from common sugars.

Clomburg JM ，Gonzalez R 《-》

Understanding and harnessing the microaerobic metabolism of glycerol in Escherichia coli.

Given its availability, low prices, and high degree of reduction, glycerol has become an ideal feedstock for the production of reduced compounds. The anaerobic fermentation of glycerol by Escherichia coli could be an excellent platform for this purpose but it requires expensive nutrients such as tryptone and yeast extract. In this work, microaerobic conditions were used as a means of eliminating the need for rich nutrients. Availability of low amounts of oxygen enabled redox balance while preserving the ability to synthesize reduced products. A fermentation balance analysis showed approximately 95% recovery of carbon and reducing equivalents. The pathways involved in glycerol dissimilation were identified using different genetic and biochemical approaches. Respiratory (GlpK-GlpD/GlpABC) and fermentative (GldA-DhaKLM) routes mediated the conversion of glycerol to glycolytic intermediates. Although pyruvate formate-lyase (PFL) and pyruvate dehydrogenase contributed to the synthesis of acetyl-CoA from pyruvate, most of the carbon flux proceeded through PFL. The pathways mediating the synthesis of acetate and ethanol were required for the efficient utilization of glycerol. The microaerobic metabolism of glycerol was harnessed by engineering strains for the co-production of ethanol and hydrogen (EH05 [pZSKLMgldA]), and ethanol and formate (EF06 [pZSKLMgldA]). High ethanol yields were achieved by genetic manipulations that reduced the synthesis of by-products succinate, acetate, and lactate. Co-production of hydrogen required the use of acidic pH while formate co-production was facilitated by inactivation of the enzyme formate-hydrogen lyase. High rates of product synthesis were realized by overexpressing glycerol dehydrogenase (GldA) and dihydroxyacetone kinase (DhaKLM). Engineered strains efficiently produced ethanol and hydrogen and ethanol and formate from glycerol in a minimal medium without rich supplements.

Durnin G ，Clomburg J ，Yeates Z ，Alvarez PJ ，Zygourakis K ，Campbell P ，Gonzalez R ... -  《-》

Production of ethanol from thin stillage by metabolically engineered Escherichia coli.

Gonzalez R ，Campbell P ，Wong M 《-》

Engineering Escherichia coli for the efficient conversion of glycerol to ethanol and co-products.

Shams Yazdani S ，Gonzalez R 《-》