Project Research Results
Grantee Research Project Results
Final 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 Amount: $180,000
RFA: Technology for a Sustainable Environment (2001)
Research Category: 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, but without acid by-product production. Bacillus species are a target because they are regarded generally as safe, and they can serve a means for producing natural pesticides. Achieving this goal will improve the prospects for Bacillis-based bioprocesses because waste products are reduced. Specifically, we are putting pyruvate kinase (PYK) under inducible control in Bacillus species to diminish the flow of glycolytic metabolites to acetate and lactate. Additionally, cloning the Escherichia coli enzyme that converts phosphoenolpyruvate to oxaloacetate (phosphoenolpyruvate carboxylase [PPC]) into PYK-deficient Bacillus has been investigated. This second metabolic engineering strategy, which involves deleting pyruvate carboxykinase from PPC+/PYK- Bacillus, also is envisioned to lead to low acetate production, but an exogeneous inducer (e.g., IPTG is used to control PYK induction) will not be required. Obviating the need for an inducer to control metabolic fluxes is desirable because recombinant protein expression can be controlled instead with common inducers such as IPTG. A supportive effort involves augmenting a general metabolic engineering design (computer) platform that will enable the characterization of fluxes in metabolically engineered cells, as well as providing a general tool for other workers. There are three general aims for the platform: (1) identifying flux alternatives based on a network “model picture” that is entered easily into the computer; (2) automating the design of 13C nuclear magnetic resonance (NMR) experiments such that alternative flux distributions can be better resolved; and (3) improving the accuracy of going from spectra to fluxes, which would enable the validation of how fluxes were altered by metabolic engineering.Summary/Accomplishments (Outputs/Outcomes):
Concerning the experimental component, we have achieved the following:
(1) Low acid-producing Bacillus thuringiensis chemostat cultivations have been demonstrated successfully through medium design.
(2) Inducible PYK has been successfully introduced into B. subtilis, and more than 70 percent of the wild-type growth rate can be retained while acetate production is reduced significantly when a low level of PYK is induced.
(3) PPC from E. coli has been demonstrated to be active in Bacillus, which paves the way for developing low acetate-producing Bacillus strains that do not require an exogenous inducer for the desirable high growth rate/low byproduct phenotype to be attained.
(4) Compared to wild-type, recombinant protein expression is approximately 50 percent higher or more in the B. subtilis that we constructed to have inducible PYK. The same is true for E. coli that is deficient in PYK.
Concerning the computational component, we have achieved the following:
(1) All common NMR analytes (i.e., abundant cytosolic molecules or protein-derived amino acids) can be considered in a computer-driven design of 13C NMR experiments. Our initial work was limited to glutamate.
(2) We developed an analyte screening methodology that ranks analytes according to how exactly they will reveal fluxes. That is, some analyte sets can produce similar NMR spectra for different underlying fluxes, which could yield misleading results. Thus, we have developed a uniqueness test by enumerating how many flux solutions an analyte set can produce upon reversion when a tolerance (i.e., acceptable error) is prescribed for the flux values. Analytes that yield only one solution or similar solutions (e.g., with a 5% tolerance) are those accepted into the experimental set.
(3) The above screening theory and computation has been integrated into a comprehensive analyte and glucose labeling selection algorithm. Overall, optimal experimental design is based on selecting a maximal combination of the following factors: analyte abundance (i.e., isolation ease), spectra intensity (i.e., high signal-to-noise ratio), uniqueness between fluxes and NMR signature (i.e., no false alternative flux solutions), and contrast power (i.e., can distinguish between different flux solutions that satisfy mass and energy balances subject to agreeing also with experimental flux measurements).Conclusions:
The main conclusions from this project are:
(1) Through medium design or using molecular biology (ITPG control) to control PYK activity at a low level, B. thuringiensis and B. subtilis can be made to grow rapidly and not produce carboxylic acid waste products. Moreover, recombinant protein expression is increased, indicating that more efficient use of resources occurs along with lessened by-products.
(2) A major step towards making Bacillus have the metabolic network and low by-product attributes of PYK-deficient E. coli has been made: E. coli PPC can be effectively expressed in Bacillus. The next step, deleting pyruvate carboxykinase from PPC-endowed Bacillus is expected to yield a low acid-producing Bacillus platform that requires no exogeneous inducer to control the activities of central pathway enzymes such as PYK.
(3) A flux solution identification and 13C NMR experimental design tool has been developed that allows one to choose the best combination of labeled glucose and intracellular analytes for the purpose of accurately and robustly identifying the central pathway fluxes in wild-type and engineered cells.
Journal Articles on this Report : 2 Displayed | Download in RIS Format
|Other project views:||All 15 publications||2 publications in selected types||All 2 journal articles|
||Ghosh S, Zhu T, Grossmann IE, Ataai MM, Domach MM. Closing the loop between feasible flux scenario identification for construct evaluation and resolution of realized fluxes via NMR. Computers & Chemical Engineering 2005;29(3):459-466||
||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||
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