Analysis and Management of Fluxes in Bacillus Pathways for Pesticide and Protein Production

EPA Grant Number: R829589
Title: Analysis and Management of Fluxes in Bacillus Pathways for Pesticide and Protein Production
Investigators: Grossmann, Ignacio E. , Domach, Michael M.
Institution: Carnegie Mellon University
EPA Project Officer: Richards, April
Project Period: January 1, 2002 through December 31, 2004
Project Amount: $180,000
RFA: Technology for a Sustainable Environment (2001) RFA Text |  Recipients Lists
Research Category: Sustainability , Pollution Prevention/Sustainable Development


Acid by-product production by microorganisms lowers process yield and stability. To address the problem, we hypothesized that the coordination between energetic and synthetic reactions can be improved. Linear programming-based calculations suggested that attenuating pyruvate kinase activity would maintain cell function but less glucose would be directed to acids. B. subtilis cultures proved to produce nil acid when cultivated with a small amount of citrate. Data from 13C NMR studies were consistent with attenuated pyruvate kinase activity being linked to the desirable nil acid result thereby supporting the basic hypothesis. Recently, pyruvate kinase activity was directly eliminated from Escherchia coli and B. subtilis by genetic means. Both microbes exhibited nil acid production, which supports the hypothesis further. We now propose to investigate the application and extension of the metabolic engineering strategy in B. subtilis, and then parlay the results by metabolically engineering the agriculturally and commercially important microorganism, Bacillus thuringiensis (Bt). Some sub-species of Bt form g-endotoxins that are toxic to some insects. Over 200 Bt-derived pesticide products are registered in the US, and it is desirable to improve the production of these alternatives to synthetic organic pesticides.


Our plan has molecular biology and analytical components. In collaboration with another group, we aim to regulate the expression of pyruvate kinase in B. subtilis. Differential expression is intended to provide for faster growth than deletion mutants, but also minimize acid production. This experience will be transferred to Bt. Carnegie Mellon workers will further develop the metabolic network analysis algorithms and software to predict metabolic flux alternatives, design 13C NMR tracer experiments, and manage the performance of 13C NMR studies. The 13C NMR studies, will enumerate metabolic fluxes. This data, in turn, will document in detail the effects of strain manipulation as well as fully account for yield observations.

Expected Results:

We envision that we will develop high yield B. subtilis and Bt strains for recombinant protein and insecticide production, respectively. The data and constructs, in turn, will provide new insights on metabolic subsystem coordination. Additionally, advanced methods for integrating NMR-focused experimental design and data analysis will be developed. From a broader standpoint, the analytical tools we develop could aid the investigation of other environmental problems. For example, the pathways for polycyclic aromatic hydrocarbon metabolism are now known. However, how the pathways integrate into the intact cell from a whole physiological perspective remains to be determined. Our network analysis and 13C NMR experiment design/interpretation tools could be used by other workers to solve such a problem.

Publications and Presentations:

Publications have been submitted on this project: View all 17 publications for this project

Journal Articles:

Journal Articles have been submitted on this project: View all 4 journal articles for this project

Supplemental Keywords:

biology, modeling, measurement methods, engineering, agriculture., RFA, Scientific Discipline, Toxics, Sustainable Industry/Business, Chemical Engineering, cleaner production/pollution prevention, Environmental Chemistry, Sustainable Environment, Chemistry, pesticides, Technology for Sustainable Environment, Biology, Engineering, Environmental Engineering, Agricultural Engineering, pesticide production, cleaner production, environmentally friendly technology, fluxes in bacillus pathwasy, sustainable development, clean technology, pesticide products, proteins, modeling, B. subtilis, innovative technology, protein production, agriculture, Bacillus thuringiensis (Bt), pollution prevention, environmentally conscious design

Progress and Final Reports:

  • 2002 Progress Report
  • 2003 Progress Report
  • Final Report