(626f) 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.