Grantee Research Project Results

2006 Progress Report: Collaborative Research: Metabolic Engineering of E. coli Sugar-Utilization Regulatory Systems for the Consumption of Plant Biomass Sugars

EPA Grant Number: R831441
Title: Collaborative Research: Metabolic Engineering of E. coli Sugar-Utilization Regulatory Systems for the Consumption of Plant Biomass Sugars
Investigators: San, Ka-Yiu
Institution: Rice University
EPA Project Officer: Richards, April
Project Period: December 22, 2003 through December 21, 2007
Project Period Covered by this Report: December 22, 2005 through December 21, 2006
Project Amount: $200,000
RFA: Interagency Announcement of Opportunities in Metabolic Engineering (2001) RFA Text |  Recipients Lists
Research Category: Technology for a Sustainable Environment , Human Health , Sustainable and Healthy Communities

Objective:

The overall objective of this collaborative project is to metabolically engineer the E. coli sugar-utilization regulatory systems (SURS) to utilize sugar mixtures obtained from plant biomass. We will introduce specific genetic modifications to the SURS and analyze the consequences of these changes at the cellular level. The specific goals are to: (1) construct E. coli strains with modified SURS and evaluate their capability to efficiently utilize sugar mixtures (glucose, xylose and arabinose); (2) evaluate changes in the regulation of gene expression at the genomic scale resulting from engineering E. coli SURS; (3) identify functional metabolic pathways and quantify their fluxes in SURS mutants and wild type strains by using a novel flux analysis technique based on bondomer analysis of 2D NMR bond-labeling experiments; (4) integrate these results using a novel genetic network-based MFA model that combines gene expression and metabolic flux data; and (5) propose further genetic/environmental modification for improving the capacity of SURS mutants for fermenting sugar mixtures.

The specific aims of this project are: (1) to develop an integrated quantitative model that captures existing mechanistic knowledge about SURS from literature and exiting data; and (2) to refine the model with in-house experiments from Iowa State University.

Progress Summary:

Lignocellulosic biomass such as agricultural and wood residues, crops, and byproduct streams provides a low cost and uniquely sustainable resource for producing many fuels and chemicals. The microbial transformation of its constituent sugars (5-and 6-carbon sugars) into commodity chemicals is one of the most important steps in this process. In fact, this has been identified as a high-priority research area in the “Technology Vision 2020” for the U.S. Chemical industry published by the Department of Energy. The process requires an organism, such as E. coli, that is capable of fermenting sugar mixtures containing 5- and 6-carbon sugars.

When E. coli is grown on sugar mixtures, the delay and frequently incomplete consumption of other sugars due to the presence of glucose, results in low yields and productivities of the desired product. E. coli possesses sophisticated regulatory systems (referred to as sugar-utilization regulatory systems, SURS) that control the utilization of different sugars. SURS regulate the preferential utilization of specific sugars from a sugar mixture, the induction of several genes due to the presence of glucose and the direction of carbon flow.

We have developed a framework for the development of a mathematical model based on first principle. The current model is capable of capturing the inner working of sugar-utilization regulatory systems in response to the availability of various carbon sources. We are currently embarking in two major efforts:

1. In the first project, we have constructed a mechanistic based model to describe the xylose utilization system under the control of a global transcription factor Crp and its own regulator xylR. As a result, the gene expression and enzymatic levels of those genes related to xylose uptake and utilization will be regulated by Crp and XylR. This model will be fine-tuned by using in-house experimental data.
2. Concurrently, we are extending the model to include a mechanistic based glucose utilization model which involves several regulatory molecules, such as cAMP and Crp, as well as the global regulator Mlc. With this extension, one can examine the effect of the Ptsg system and Mlc on xylose uptake and utilization.

An important feature of this modeling approach is that the output from the glucose utilization model will provide pertinent information about the transcription factor Crp which will in turns regulates the xylose utilization system. The current model is capable of describing the dynamics of simultaneous presence of both carbon sources once the two sub-models are integrated together. The current model structure was built on exist knowledge about the regulatory mechanisms of the glucose and xylose systems.

The current SURS model consists of 24 ordinary differential equations (ODEs), about 100 algebraic equations, and 110 parameters. The differential equations are used to capture the dynamics of the state variables of the system, such as the concentrations of various carbon sources. The main part of the model is the algebraic equations used to describe the regulatory nature of the system. These algebraic equations mainly involve chemical equilibrium between the transcription factors and their binding partners, and the total mass balance of various transcription factors. The inclusion of the Mlc sub-system resulted in a net increase of one ODE and 42 algebraic equations from our previous model. Most of the parameter values used in the model are based on data reported in the literature.

Conclusions

We have developed a mechanistic based model to describe the xylose utilization system under the control of a global transcription factor Crp and its own regulator xylR. The model is capable of describing the dynamics of xylose utilization. The current model integrates two sub-models that expand the modeling capability to describe the effect of the Ptsg system and Mlc on xylose uptake and utilization.

Journal Articles:

No journal articles submitted with this report: View all 9 publications for this project

Supplemental Keywords:

RFA, Scientific Discipline, Sustainable Industry/Business, Sustainable Environment, Technology for Sustainable Environment, Ecology and Ecosystems, gene expression patterns, plant biomass sugars, E coli Sugar-Utilization Regulatory Systems, genetic engineering

Progress and Final Reports:

Original Abstract

2004 Progress Report

2005 Progress Report

Final Report