# Heterologous Production of Acrylic Acid: Current Challenges and Perspectives
## 3 Heterologous Production of AA
### Without 3-HP as an Intermediary  
[PERSON] et al. [2] developed a pathway with several steps from the glycerol route to produce 3-HP but without producing 3-HP as an intermediary. This pathway required 4 enzymatic steps to convert glycerol to AA (Figure 7b). Glycerol was converted to 3-HPA by glycerol dehydatase, as previously described in Sections 2.1 and 3.1. Next, instead of being transformed to 3-HP, 3-HPA was directly converted to 3-HP-CoA by the enzyme propionaldehyde dehydrogenase. Afterwards, the next two steps were performed, as described in Figure 7a. Enoyl-CoA hydratase/3-HP-CoA hydratase converts 3-HP-CoA to AA-CoA, and a CoA transferase converts AA-CoA to AA. In this study, the authors used the previously described enzymes DhaB and GdrAB for the glycerol dehydatase and reactivation step. For the second step, they used a propionaldehyde dehydrogenase from _Salmonella typhimurium_ (PduP) [108]. The last step was performed by endogenous CoA transferases, and no gene was overexpressed. The focus of the study was the enoyl-CoA hydratase step. Several enzymes were tested: PcsII, the enoyl-CoA hydratase domain of the trifunctional enzyme _pcs_ from _C. aurantiacus_; PhaJ1 enoyl-CoA hydratase from _P. aeruginosa_; and hypothetical enoyl-CoA hydratase EcH from _P. aeruginosa_ and HcaD from _B. subtilis_. The pathway with PcsII was the one that allowed obtaining higher amounts of AA. Afterwards, the catalytic activity of this enzyme was improved by comparing its sequence with other enoyl-CoA hydrates and performing direct mutagenesis. A double mutation allowed increasing the concentrations up to 37.7 mg/L. The _prpR_ gene was also deleted to block propionate production, as in [41]. The authors also deleted _yhdH_ that encoded an NADPH-dependent AA-CoA reductase previously described to convert AA-CoA to propionyl-CoA [109]. However, instead of increasing, the concentrations decreased significantly, as well as the strain growth. This demonstrated that the _E. coli_ strain with this knockout is more sensitive to the presence of AA. Therefore, it was more beneficial to use a strain without the knockout that was also able to consume some of the intermediaries needed for AA production [2]. In addition, in 2016, [PERSON] et al. [27] patented the same pathway described by [PERSON] et al. [2]. They obtained similar concentrations (44 mg/L) but with a different combination of genes. After testing several genes for each step, they concluded that the pathway that performed better contained DhaB (DhaB1, DhaB2, and DhaB3) and GdrAB from _Ilyobacter polytropus_, PduP from _L. reuteri_, MELS_1449 (3-HP-CoA dehydratase) from _Megasphaera elsdenii_ and YciA from _E. coli_[27]. Acka, Pta and YqhD genes were deleted, as in other studies [31,32]. More recently, a pathway to produce AA was developed based on the \(\beta\)-alanine route but also without producing 3-HP [34]. In this biosynthetic pathway, \(\beta\)-alanine is converted to \(\beta\)-alanyl-CoA by a \(\beta\)-alanine CoA transferase (_act_), which is then converted to AA-CoA by \(\beta\)-alanyl-CoA:ammonia lyase (_acl2_), both enzymes from _Clostridium propionicum_. Finally, several enzymes were tested to convert AA-CoA to AA, but YciA was selected, as in the other pathways, due to its superior performance. This pathway is simpler as it only requires three heterologous steps, while the pathways that produce 3-HP as an intermediary require five steps and the pathway that converts glycerol to AA without producing 3-HP requires four steps. However, to obtain up to 237 mg/L of AA, the highest concentration reported so far, the authors overexpressed several genes and performed various knockouts. One of the strain modifications was the deletion of _yhdH_ (=_acul_) to prevent AA-CoA conversion to propionyl-CoA. As reported in [2], a decrease in the bacterial growth using the \(\Delta yhdH\) strain in the presence of AA was verified in comparison to the wild type.