Module 1: Flux Analysis Protocols and MFA Application

Certificate three summary Module Two
Experiments and labeling
13C labeling experiment – Starts by growing the cell in labeled glucose, the first carbon atom of the glucose molecule is labeled. The solution via 13C MFA involves a recursive algorithm to solve for the fluxes (i.e. we get the flux profile using experimental data).
It involves adopting a mathematical algorithm or a set of equations where the flux profile can be used, for predicting the amino acid labeling. The result (simulated from the mathematical algorithm or flux profile) will then be compared with the experimental data.
13C fingerprinting – A procedure whereby the cell is allowed to grow with 1-C (one carbon) glucose, the yield analysis is then calculated by feeding the cells with labeled glucose; you then proceed to perform isotopomer analysis.
Nonstationary metabolic flux analysis (NMFA) – This is a procedure where the metabolite labeling is sampled and measured during the transient period before the system reaches an isotopic steady state. NMFA technique offers several advantages.
FBA optimization problem statement
How do we choose the biomass objective function (Z)?
Solution - We consider three mechanisms, which are:
1. Maximization of cellular growth
2. Specific metabolite engineering (maximization)
3. Energy consumption minimization
Biomass precursors
The biomass reaction accounts for all the fractional contributions from biosynthetic precursors and key cofactors to create 1 g (one gram) of biomass. The fractional contributions need to be determined experimentally for cells growing in the log phase.


Maintenance energy requirement
To stimulate growth, the energy required to maintain the cell growth must be accounted for. Two forms of energy are required.
1. Growth associated maintenance (GAM) energy represented in the model by
xATP + xH2O  xADP + xPi + H+

2. Nongrowth associated maintenance (NGAM) energy is represented in the model as:
ATP + H2O  ADP + Pi + H+
Applications of metabolic engineering
Some applications of MFA include:
1. Lipid biosynthesis of Yarrowia lipolytica
2. Fatty acids in E. coli using optforce
3. Limonene production from Synechocystis sp PCC 6803
4. Conversion of CO2 to isobutyraldehyde using cyanobacteria
5. Fatty alcohol production in S. cerevisiae