2002 Progress Report: Analysis and Management of Fluxes in Bacillus Pathways for Pesticide and Protein ProductionEPA 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 Period Covered by this Report: January 1, 2002 through December 31, 2003
Project Amount: $180,000
RFA: Technology for a Sustainable Environment (2001) RFA Text | Recipients Lists
Research Category: Sustainability , Pollution Prevention/Sustainable Development
The overall objective of this research project is to modify the metabolic network of Bacillus species to attain near normal growth without acid byproduct production. Bacillus species are a target because they generally are regarded as safe and can serve as a means for producing natural pesticides. Achieving this objective will improve the prospects for Bacillis-based bioprocesses because waste products are reduced. Specifically, we are putting pyruvate kinase under inducible control in Bacillus species. Additionally, cloning the E. coli enzyme that converts phosphoenolpyruvate to oxaloacetate (PPC) into Bacillus is underway. A second supportive effort involves augmenting a general metabolic engineering design (computer) platform that will enable the characterization of the metabolically engineered cells and provide a general tool for other workers. There are three objectives for the platform: (1) identifying flux alternatives based on a network picture entered by the user; (2) automating the design of contrast 13C nuclear magnetic resonance (NMR) experiments such that alternative flux distributions can be better resolved; and (3) improving the capability of going from spectra-to-fluxes, which would enable trouble shooting the biological "designs."
Cloning/Biology. Using green fluorescent protein (GFP) as a tracer, we have found vectors that appear to enable the integration of a desired gene into the Bacillus subtilis genome. We aim for integration into a gene that B. subtilis can "live without" under standard laboratory cultivation conditions (e.g., amylase). Thus far, we have confirmed integration by the following positive results after attempting transformation: (1) the correct alteration of antibiotic resistance was obtained; (2) the chromosomal gene into which the new gene was inserted was not expressed; and (3) some cells acquired GFP expression capability.
A series of chemostat experiments were completed with Bacillus thuringiensis (Bt). These experiments showed that the acid reduction strategy that works for acid reduction in B. subtilis (add a small amount of citrate) also works for Bt.
Software. A version with a graphical user interface has been completed. This version predicts all flux scenarios possible for mutation and/or objective. The scenarios are, in turn, used to drive the design of 13 NMR experiments such that the best labeled precursor is chosen so that one can maximally discriminate between flux distribution alternatives. Based on this work, we were invited to contribute to a special edition of the journal, Computers in Chemical Engineering.
Side Discoveries. We have found that under some circumstances, Bacillus can form helical or other types of filaments that are comprised on 5-100 cells. There are sketchy reports of this in the literature. We wonder if this phenomena may affect transformation and whether it can be harnessed for cell-culture broth separations. Thus, we will further explore this avenue.
Future activities include the following:
Cloning. We expect to have two Bacillus constructs by the end of the summer: (1) inducible pyruvate kinase; and (2) inducible polyenylphosphatidylcholine (PPC). These constructs will be characterized in terms of physiology and fluxes via cultivation experiments and 13C NMR.
Communications. We are submitting the Bt results for publication. A contribution to a special edition of Computers and Chemical Engineering will be completed in early fall of 2003. Abstracts have been submitted to the American Institute of Chemical Engineers Meeting to be held in November 2003.
Software. We recently adopted a new algorithm for using NMR spectra information for flux determination. The challenge with this problem is some "optimal fit" algorithms find a "local" (i.e., pretty good) versus the "actual" (i.e., most optimal) solution. The new algorithm bypasses this problem. However, depending on the analytes measured, different combinations of fluxes and reaction reversibilities may produce the same spectra. That is, there may be more than one "correct" answer. Our algorithm is very good at finding the "correct" net fluxes for the test problem constructed and it also yields many "correct" values of reaction reversibility (exchange coefficients). We will refine the computation further to tackle the problem of determining exchange coefficients, and expect that further insights on experimental design will be obtained.
Journal Articles on this Report : 1 Displayed | Download in RIS Format
|Other project views:||All 17 publications||4 publications in selected types||All 4 journal articles|
||Zhu T, Phalakornkule C, Ghosh S, Grossmann IE, Koepsel RR, Ataai MM, Domach MM. A metabolic network analysis & NMR experiment design tool with user interface-driven model construction for depth-first search analysis. Metabolic Engineering 2003;5(2):74-85.||