## 6 Metabolic Flux Analysis
### Metabolic Pathways Analysis--A Quantitative Approach  
Using MFA, the intracellular metabolic fluxes of every reaction can be estimated. This is achieved with the help of elementary biochemical equations and their stoichiometric \begin{table}
\begin{tabular}{p{56.9 pt} p{113.8 pt} p{113.8 pt} p{113.8 pt}} \hline \hline
**Molecule** & **Host** & **Genetical Importance** & **Ref.** \\ \hline Isoprene & _E. coli_ & Identify isoprene synthases from _Ipomoa batatas, Populus alba,_ and _Pueraria montana_ that could be used in _E. coli._ & [119] \\ \hline Isoprene & _E. coli_ & OleTJE from _Jeqtallicoccus_ sp.; OhyAEM from _Elizabethkingia meningoseptica_ & [120] \\ \hline Isoprene & _Syntechocystis_ & Plasmid-based system containing kudzu ispS expressed in low _PCC6803_ & [121] \\ \hline Isoprene & _Syntechocystis_ & fni gene from _Streptococcus pneumoniae_ to increase the cellular DMAPP:IPP ratio. & [107] \\ \hline Farnesene & _S. cerevisiae_ & Formerly engineered for artemisinin acid production further modified to produce farresene by allowing biosynthesis of cytosolic acetyl-CoA with reduced ATP requirement. & [122] \\ \hline Farnesene & _Y. lipolytica_ & Overexpression of HMG1, idi, ERG20, and codon-optimized farresene synthase & [123] \\ \hline Isoprene & _B. subtilis_ & ispS gene from _Pueraria_ spp. & [124] \\ \hline Isoprene & _Syntechocystis_ & Fusion of the IspS gene to the \(\beta\)-subunit of phycocyanin. & [110] \\ \hline Limonene & _Syntechocystis_ sp. _PCC 6803_ & Overexpression of ribose 5-phosphate isomerase and ribulose 5-phosphate 3-epiomerase gene in pentose phosphate pathway; limonene synthase from _Mentha spicata_ and geranyl diphosphate synthase from _Abies grandis_. & [125] \\ \hline Farnesene & _E. coli_ & Over-expression of IPP isomerase and FPP synthase. & [117] \\ \hline Farnesene & _S. cerevisiae_ & nphT7 from _Streptomyces_ sp. to catalyze irreversible condensation of acetyl CoA and Malonyl CoA to produce acetoacetyl CoA hence directing the flux to the MVA pathway. & [126] \\ \hline Pinene & _E. coli_ & Mutated _Abies grandis_ GPPS through error-prone PCR and DNA shuffling; insertion of a tunable intergenic region GPPS and PS genes. & [12] \\ \hline Limonene & _E. coli_ & Heterologous expression of mutated _Enterococcus faecalis_ MvaS, _Methanosarcina mazei_ MK, _Solanum lycopersicum_ SINPPS (neryl pyrophosphate synthase), and _M. spicata_ MsLS. & [127] \\ \hline Limonene & _S. cerevisiae_ & Orthogonal limonene biosynthetic pathway containing exogenous _S. lycopersicum_ SINDPS1 (catalyzes IPP and DMAPP to cis-GPP) and _Citrus limon_ limonene synthase. & [128] \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Metabolic engineering for biofuels from isoprenoids through various microorganisms (source: [65]). models. MFA also helps in understanding the regulatory mechanisms like the knockout or integration of genes in cells. For instance, in the MFA studies conducted on acetone/acid synthesizing and the wild type of _Clostridium acetobutylicum_ strains by [PERSON] et al., they revealed the quantities and the crucial roles played by pathways that form acid in their metabolisms [137]. Furthermore, MFA not only helps in understanding the regulatory mechanisms of a pathway and evidences regarding the crucial reactions, but it also suggests a possible way to enhance the yield with the help of approaches like metabolic engineering. MFA studies were more useful in understanding the roles of pivotal reactions involved in the metabolic pathways of _C. acetobutylicum_ to identify targets that can produce butanol from sugars like xylose, glucose, etc., obtained from hydrolys lignocellulosic biomass [138]. There are different kinds of MFA methods like (i) 13C MFA, (ii) flux balance analysis (FBA), and (iii) classical/standard stoichiometric MFA (Figure 6). In the case of regular parallel pathways, to estimate the branch fluxes (v2 and v1), regular MFA can be applied (Figure 6).