## 3 Results
### Production of Indole-3-Acetic Acid by the Keio Mutants  
Reaction disruptions with experimental evidence that deviated from the model's predictions were compared with reactions competing for their substrates and reactions predicted correctly by the model using Keio mutants. The reactions predicted by QPAML were directly associated with the genes annotated in the metabolic model. Two were selected when more than one gene was associated with a reaction, considering one could have lower activity. Although a gene can participate in more than one metabolic pathway, as is the case of the _tdcD_ gene annotated in producing propionate and acetate, we maintain the selected genes. It is possible that the silencing of a gene can have multiple effects by participating in more than one metabolic pathway, which is why the analysis we developed with QPAML focused on reactions. For instance, the _trpD_ gene encodes the bifunctional enzyme anthranilate synthase and anthranilate phosphoribosyltransferase in anthranilate production. The first function produces anthranilate, while the second consumes it, which is why a mutant with reduced anthranilate phosphoribosyltransferase activity was used for improving anthranilate production [64]. These types of complexities are outside the scope of our model, but it indicates that using reactions is a better approach than using genes. For  
Figure 7: Distribution of reaction steps based on their distance from the pFBA optimal path. The X-axis represents the shortest path length from reactions categorized for disruption (knockout and knockdown) to pFBA optima reactions. Blue bars depict reactions categorized for knockout, yellow bars represent target reactions for knockdown, and orange bars represent reactions with experimental evidence. this reason, the conversion of genetic modifications to the activity of metabolic reactions is performed as described in Section 2.7. The _tyrA_ and _pheA_ genes are responsible for L-tyrosine and L-phenylalanine synthesis and compete with the tryptophan pathway [7; 8]; _poxB_, _ackA_, and _tdcD_ are involved in two acetate production pathways that affect biomass production [11; 97]; _tnaA_ encodes the tryptophan-degradation enzyme tryptophananese [6; 7; 8; 11]; _mtr_ and _tnaB_ encode for tryptophan importers [6; 8; 9]; the _pykA_ and _pykF_ genes encode pyruvate kinase isoforms that compete for phosphoenolpyruvate, an essential precursor of chorismate [6; 8; 76]; and _gltB_ is a glutamate synthase that degrades glutamine an anthranilate amino donator [11]. The complexity of the media in which tryptophan is present hinders its quantification. Additionally, distinguishing free tryptophan from protein residues proves challenging. Conventional separation, such as high-performance liquid chromatography (HPLC), may result in poor throughput. Therefore, an alternative approach involves derivatizing tryptophan using enzymes to produce more specific metabolites for colorimetric quantification [39]. Tryptophan production was indirectly determined through conversion to IAA and quantified using the Salkowski reagent. The pIAAMHs plasmid expresses two enzymes of _Pseudomonas savastanoi_ that efficiently convert tryptophan into IAA. Figure 8 presents the IAA quantification data for relevant Keio mutants. Indole acetic acid (IAA) production in an _E. coli_ strain through the indole-3-acetamide pathway has been documented [98]. Moreover, the specificity of the Salkowski reagent for indoles derived from tryptophan, as demonstrated in previous studies [40], rationalizes the widespread occurrence of the Salkowski reaction across all tested strains. Upon transformation with pIAAMHs, the parental strain produced 3.12 ug/mL of IAA in the LB medium. Notably, when evaluating the Tukey test, the _pykA_, _tyrA_, and _poxB_ mutants did not display significant differences in IAA production compared to the parental strain. In contrast, the remaining mutants demonstrated a substantial and statistically significant increase in the production of IAA.