Construction of Plug-and-Play Modules Designed using an Integrated Metabolic and Regulatory Model of B. subtilis for Production of High Value Bioproducts Including Isoprene
EMSL Project ID
47477
Abstract
Rising concerns about fossil fuel shortage and climate change have evoked increasing interest in renewable bioproducts including biofuels. While microbial metabolism can be reengineered for the production of bioproducts, this work remains a challenge due to the complexity of microbial metabolism, the hard-wired tendency of these systems to resist alteration, and lengthy debugging process associated with optimizing flux through a newly engineered pathway. We are proposing to develop and demonstrate a novel model-driven approach to the design and integration of modules for the decoupled production of isoprene by Bacillus subtilis DSM10.We will construct a genome-scale transcription regulatory network model for B. subtilis DSM10, which we will integrate with an existing highly-curated metabolic model of B. subtilis, the iBsu1103V2. Constraints that capture the effect of transcriptional regulation will be integrated into our metabolic model using both probabilistic constraints (PROM) and Boolean constraints (rFBA). Our integrated model will be assembled and validated based on existing regulatory network and gene expression data, as well as using the unique combination of advanced high-throughput technology developed at EMSL on new data sets in transcriptomics, proteomics, and metabolomics. This works falls under the EMSL call topic Biological Interactions & Dynamics, directly pertaining to “systems biology data collection and analysis that serve as a basis for predictive modeling of metabolic pathways and regulatory networks”.
We will apply our integrated model to design and analyze new experiments which we are proposing to perform using the EMSL facilities. Such experiments include: (i) the knockout of both regulatory and metabolic genes identified by the model as having an impact on native isoprene production, with proteomic, metabolomic, and transcriptomic analysis of our knockout mutants to validate model predictions of microbial response to knockouts; and (ii) the construction, insertion, and omic characterization of an isoprene production module designed by our model. For the first experiment type, our integrated model will be applied to predict enzyme and transcription factor knockouts that should increase or disrupt the native isoprene production. Knockout strains will be implemented with omics experiments performed to experimentally characterize the impact that these mutations have, which will be compared with model predictions to support validation and refining of the model. In the second set of experiments, our refined and validated model will then be applied to design an insertion module for the production of isoprene in a manner that is decoupled from the native metabolism and regulation. This module will be inserted at Washington State University, and the resulting strain will be characterized for further model validation and correction. Finally, the model will be applied to support the improvement of our isoprene module design to further improve isoprene production.
This work will develop and demonstrate approaches to utilize a metabolic model to support the metabolic engineering and module design, showing how to incorporate transcriptional regulation into an expanded model in the process. We anticipate the approaches explored here for the use of an integrated model for knockout predictions, omics data interpretation, module design, and module optimization will be widely applicable to many other metabolic engineering efforts. We will also produce a highly curated, validated, and refined integrated model of B. subtilis DSM10 that will be a valuable resource to the metabolic engineering community.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2012-10-01
End Date
2014-09-30
Status
Closed
Released Data Link
Team
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