Grantee Research Project Results
2005 Progress Report: Collaborative Research: Metabolic Engineering of E. coli Sugar-Utilization Regulatory Systems for the Consumption of Plant Biomass Sugars
EPA Grant Number: R831441Title: 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, 2004 through December 21, 2005
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 Escherichia 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 objectives of this research project 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 two-dimensional nuclear magnetic resonance bond-labeling experiments; (4) integrate these results using a novel genetic network-based metabolic flux analysis 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) develop an integrated quantitative model that captures existing mechanistic knowledge about SURS from literature and exiting data; and (2) 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 U.S. 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 delayed and frequently incomplete consumption of other sugars caused by the presence of glucose results in low yields and productivities of the desired product. E. coli possesses sophisticated regulatory systems (referred to as 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 caused by the presence of glucose, and the direction of carbon flow.
We have developed a framework for the development of an algorithm that is capable of capturing the inner working of SURS in response to the availability of various carbon sources. We are currently embarking on 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 that 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 turn regulate the xylose utilization system. The current model is capable of describing the dynamics of simultaneous presence of both carbon sources once the two submodels are integrated together. The current model structure was built on existing knowledge about the regulatory mechanisms of the glucose and xylose systems. The parameter values used in the model are based on data reported in the literature. These models will be refined with in-house data from Drs. Gonzalez’s and Shanks’ groups.
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 current model is capable of describing the dynamics of xylose utilization. Extension of the current model by integrating the two sub-models will expand the model capability to describe the effect of the Ptsg system and Mlc on xylose uptake and utilization.
Future Activities:
The investigators did not report any future activities.
Journal Articles:
No journal articles submitted with this report: View all 9 publications for this projectSupplemental Keywords:
engineering, metabolic engineering, genetic engineering, genetic regulatory circuit, bacteria, sugar, glucose, xylose, sugar mixture, sugar utilization, NMR, metabolic flux analysis, modeling, gene array, genetic regulation, pathway engineering, renewable resources, E. coli, sugar-utilization regulatory systems, gene expression patterns, metabolic engineer,, 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 engineeringProgress and Final Reports:
Original AbstractThe perspectives, information and conclusions conveyed in research project abstracts, progress reports, final reports, journal abstracts and journal publications convey the viewpoints of the principal investigator and may not represent the views and policies of ORD and EPA. Conclusions drawn by the principal investigators have not been reviewed by the Agency.