Constraint-Based Flux Distribution Estimation
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Solution Overview
Problem
Current methods for predicting intracellular flux distributions in organisms rely heavily on costly carbon isotope-based metabolic flux analysis (MFA) data, which is limited, and alternative methods like FBA struggle with accurate parental strain flux estimation, leading to errors in predicting fluxes in derived strains.
Innovation Solution
A constraint-based modeling approach that uses easily obtained experimental data from reference strains to calculate extracellular and intracellular flux distributions in parental strains, improving the accuracy of flux predictions in newly derived strains without requiring MFA data.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If carbon isotope-based metabolic flux analysis (MFA) is used to quantify central metabolic fluxes, then measurement precision is improved, but cost increases and data availability is limited
Solution Approach 1:
The patent uses FBA to create a computational copy of the metabolic flux distribution that approximates the expensive MFA measurements. By building a genome-scale metabolic model and applying flux balance analysis with extracellular flux constraints, the system generates predicted intracellular flux distributions that serve as substitutes for costly MFA data, enabling flux estimation without direct isotopic labeling experiments
Solution Approach 2:
The patent replaces expensive, limited MFA data with inexpensive, easily obtained extracellular flux measurements combined with computational FBA. The extracellular flux data (substrate uptake and product secretion rates) are cheap to obtain through standard bioreactor experiments, and when combined with the metabolic model, provide sufficient information to predict intracellular fluxes without requiring costly MFA experiments
2Productivity
If flux balance analysis (FBA) is used to predict flux distributions, then computational efficiency is improved and data requirements are reduced, but prediction accuracy deteriorates
Solution Approach 1:
The patent incorporates experimentally measured extracellular fluxes as feedback constraints into the FBA model. By using actual measured substrate uptake rates and product secretion rates to constrain the computational model, the system adjusts the predicted flux distributions to better match experimental reality, improving accuracy while maintaining computational efficiency
Solution Approach 2:
The patent performs preliminary experimental measurements of extracellular fluxes before running the FBA computation. These pre-measured extracellular flux values are then used to constrain and guide the FBA calculation, ensuring that the computational prediction starts from experimentally validated boundary conditions rather than theoretical assumptions alone
3Measurement precision
If alternative FBA methods (MOMA, ROOM, RELATCH) are used to predict fluxes in derived strains, then prediction accuracy for mutants is improved, but reliability deteriorates when parental strain fluxes are incorrect
Solution Approach 1:
The patent performs preliminary optimization to determine accurate parental strain flux distributions before using them as the basis for predicting mutant fluxes. By first constraining the FBA model with experimental extracellular flux data from the parental strain and optimizing the objective function to match these measurements, the system ensures that the parental flux distribution is experimentally validated before serving as the reference for deriving mutant predictions
Solution Approach 2:
The patent makes the parental strain flux distribution self-correcting by using the same FBA framework with extracellular flux constraints to simultaneously determine both the parental and mutant fluxes. The system uses the measured extracellular fluxes from the parental strain to automatically optimize and determine its intracellular flux distribution without requiring external MFA data, making the entire process self-consistent and reducing propagation of errors
Data Source
AI summary
Provided are systems and methods for accurately determining flux distribution in organisms or cells without use of metabolic flux analysis data. The methods include estimating flux distributions in multiple reference strains (variants of a parental strain) using experimentally determined extracellular flux data from the reference strains, and determining a flux distribution for the parental strain from the estimated flux distributions for the reference strains and from experimentally measured extracellular fluxes for the parental strain. The systems are configured for carrying out the methods.