# Heterologous Production of Acrylic Acid: Current Challenges and Perspectives
## 3 Heterologous Production of AA
### Without 3-HP as an Intermediary
However, in this study, the deleted strain was selected to perform the production experiments as the inhibition effects reported were much lower than in [2], since after a significant lag phase, the deleted strain reached lower growth than the wild type but still significant. The _iclR_ and _funAC/funB_ genes were also deleted to increase the flux through a glyoxylate shunt and to increase fumarate availability, which, consequently, increase aspartate production, the precursor of \(\beta\)-alanine (Figure 7c). Then, as previously reported in pathways from Section 2.3, the AspA gene from _E. coli_ and PanD from _C. glutamicum_ were overexpressed to convert fumarate to \(\beta\)-alanine. AspA and ACS chromosomal promoters were also replacedby stronger constitutive promoters. ACS overexpression led to an increase in acetyl-CoA and, consequently, to an increased flux in the TCA cycle and then in \(\beta\)-alanine precursors. In addition, acetyl-CoA is also needed to convert \(\beta\)-alanine to \(\beta\)-alanyl-CoA. The production process using this pathway was also patented [30]. Recently, a new pathway from glycerol that produces acrolein as an intermediary was described (Figure 7d) [105]. This pathway is based on the pathway from _Gluconobacter oxydans_ that is able to produce 3-HP and AA using acrolein as an intermediate. In this study, the enzymes involved in acrolein production from 3-HPA were identified: two isoenzymes named 3-hydroxyacyl-(ACP) dehydatase (3 HAD) and 3-hydroxydecanoyl-(ACP) dehydatase (3 HDD). Next, the authors transferred parts of the identified pathway to _E. coli_ to produce AA using resting cells. For the first step, glycerol dehydatase from _K. pneumoniae_ was used (_dhaB_) to convert glycerol to 3-HPA. The expression of glycerol dehydatase reactivase was not mentioned. Then, 3-HPA was converted to acrolein by 3 HAD and/or 3 HDD from _G. oxydans_. No enzyme was expressed to convert acrolein to AA, so it is assumed that an endogenous aldehyde dehydrogenase (e.g., ALDH) performed this step. The expression of _3 had_ alone with _dhaB_ allowed obtaining a higher AA concentration (144 mg/L in shake-flask culture) than when _3 had_ and _3 hdd_ were co-expressed. Resting cells were used in this study in order to decrease the inhibition of _E. coli_ growth by AA. This is in accordance with the study by [PERSON] et al. [110] who concluded that the AA effect is specific on growth and not in the central carbon metabolism and that, consequently, a high concentration of AA can be tolerated under resting conditions. In addition, in [105], acrolein accumulation was not observed, which is positive due to its reported toxicity. This 3-step pathway allowed obtaining the highest concentration so far in batch fermentation. Since apparently, there was no expression of a glycerol dehydatase reactivase and also no expression of a specific enzyme to convert acrolein to AA, these results are promising. It is expected that more investment in this simple pathway can lead to much higher concentrations in the future.