A Systems Biology Approach to Quantify the Metabolism of Escherichia Coli for Generating 3-Hydroxypropionic Acid From Glycerol

A Systems Biology Approach to quantify the metabolism of Escherichia coli for Generating 3-Hydroxypropionic Acid from Glycerol

Kilho Lee, Zuyi (Jacky) Huang

Abstract

3-hydroxypropionic acid (3-HP; C₃H₆O₃ – MW 90.08) has the potential for a broad range of industrial applications like deriving acrylic acid, 1,3-propanediol, methyl acrylate, propiolactone, malonic acid, acrylamide, and hydroxyamides (Paster et al., 2003; USITC, 2008). However, 3-HP production via chemical synthesis is not commercially feasible due to numerous technological and environmental concerns (Wolff, 1991). Instead, genetically modified Escherichia coli strains have been engineered that produce 3-HP from glycerol (Rathnasingh et al., 2009). Due to a significant increase in the amount of crude glycerol generated as a by-product during the biodiesel production process, the market value of crude glycerol is low and its value is expected to further diminish in correspondence to its increased supply. Therefore, a comprehensive understanding of the recombinant E. coli strains to further enhance the production level and provide a cost effective strategy for producing 3-HP. Rathnasingh et al., 2009 has reported that two recombinant E. coli strains, SH-BGA and SH-BGK, has capability of successfully converting glycerol into 3-HP. Both strains of E. coli were genetically modified to express glycerol dehydratase (DhaB) and glycerol dehydratase reactivase (GDR). The protein expression of DhaB and GDR enables the conversion from glycerol to 3-hydroxypropionaldehyde, or 3-HPA. SH-BGA contains aldehyde dehydrogenase (ALDH), while SH-BGK includes a-ketoglutaric semialdehyde dehydrogenase (KGSADH). Both ALDH and KGSADH converts 3-HPA into 3-HP. The experimental approach demonstrated biological 3-HP production from glycerol feedstock. However, the production level of 3-HP is not high enough to be commercially or industrially feasible. Since the metabolism of E. coli depends on hundreds to thousands of highly-interacted metabolic reactions, a mathematical modeling approach is required to identify the reactions that can be used to further engineer E. coli for a higher yield of 3-HP.  

In this work, we developed the first mathematical model to quantify 3-HP produced by engineered E. coli strain from glycerol. The model can be further used to suggest experimental strategies, such as gene-knockout, for increasing 3-HP production level. The proposed approach is based on a previously published E. coli kinetic model that includes glycerol metabolic network (Cintolesi et al., 2011). This kinetic model consists of the glycerol fermentation pathway with ethanol as the primary product. The model has been extended to incorporate the metabolic reactions that produce 3-HP from glycerol. Specifically, three reactions have been added into the model presented by Cintolesi et al., 2011: 1) glycerol dehydratase dependent reaction that converts glycerol to 3-HPA, 2) aldehyde dehydrogenase dependent reaction that converts 3-HPA to 3-HP, and 3) a reaction converting 3-HPA to 1,3-propanediol. 1,3-propanediol, or 1,3-PDO, is a by-product that is produced in a significant amount due to the accumulation of NADH from 3-HPA (Rathnasingh et al., 2009). Differential equations for the newly added metabolites were developed based on Michaelis-Menten kinetics and added to the model.

Appropriate experimental data were provided by Dr. Park's group at Pusan University for their initially developed E. coli strains. The data were used to estimate the newly added parameters via a nonlinear least square approach. The outputs of the estimated model match the experimental data for the glycerol consumption and the production of several metabolites, such as 3-HP and 1,3-PDO. Based upon the developed model, sensitivity analysis was conducted to identify the metabolic reactions that have influence the 3-HP production. Furthermore, we analyzed the flux change of each enzymatic reaction in the glycerol fermentation pathway for E. coli to produce 3-HP. It was found that the fluxes through the pathway from glycerol to pyruvate were decreased in the engineered E. coli that can produce 3-HP.

This work presents a computational approach to quantify the metabolism of a genetically modified E. coli strain that produces non-native metabolite, 3-HP. In-depth experimental analysis of a recombinant E. coli is often difficult to carry out due to technical limitations as well as the viability of the microorganism. The proposed modeling approach can overcome these limitations and provide valuable information on how to enhance the 3-HP production level that can extend to the experimental strategies.

References

Cintolesi, A., Clomburg, J. M., Rigou, V., Zygourakis, K. and Gonzalez, R. 2012, Quantitative analysis of the fermentative metabolism of glycerol in Escherichia coli. Biotechnol. Bioeng., 109: 187–198. 

Paster M, Pellegrino JL, Carole TM. 2003. Industrial bioproducts: Today and tomorrow, US DOE report. http://www.brdisolutions.com/pdfs/ BioProductsOpportunitiesReportFinal.pdf.

Rathnasingh, C., Raj, S. M., Jo, J.-E. and Park, S. 2009, Development and evaluation of efficient recombinant Escherichia coli strains for the production of 3-hydroxypropionic acid from glycerol. Biotechnol. Bioeng., 104: 729–739.

USITC. 2008. Industrial biotechnology: Development and adoption by the U.S. Chemical and biofuel industries, Investigation No.332–481.Publ.4020. July 2008. http://hotdocs.usitc.gov/docs/pubs/332/pub4020.pdf.

Wolff GT. 1991. Kirk-Othmer encyclopedia of chemical technology. In: Kroschwitz J I, Howe-Grant M, editors. Air pollution. Vol. 1, 4th edn. New York: Wiley. p 711–749.