# Heterologous Production of Acrylic Acid: Current Challenges and Perspectives
## 4 Key Points to Optimize AA Heterologous Production
### Unravelling More Efficient Enzymes and Pathways  
Currently, there is a need to search for even more efficient enzymes. Two different approaches are important and should be used: the search for new enzymes in other organisms never considered and the modification of known enzymes to reach a desirable activity or specificity or even to eliminate some types of present regulation. As established, more is not always better, and when toxic compounds are present, such as 3-HPA, it is important to design pathways with limited accumulation of intermediaries [88]. In these cases, enzymes with different activities or the use of different promoters with different strengths is ideal to balance the pathways, as previously demonstrated [121, 34, 122, 123]. New synthetic biology tools, such as CRISPR/a (CRISPR interference/activation), can also be extremely important to balance the pathway and increase or decrease the expression of a specific enzyme [25]. The use of methods such as EvolvR, a CRISPR-Cas9-based targeted mutagenesis method [124], to create gene libraries can also be useful to modify enzymes activities. This modification is created by engineered error-prone DNA polymerases that target defined locations via CRISPR-guided nickases (nCas9) and generate mutations. Similar methods (e.g., iCREATE) were previously used to modify the _E. coli_ genome and increase 3-HP concentrations [42]. Finally, the use of scaffold proteins can also be beneficial for these cases as they help to decrease intermediary accumulation due to co-localization and also decrease the conversion of intermediaries to other undesirable by-products, such as propionyl-CoA or 1,3-propanediol in the case of the AA pathway [26; 84]. Therefore, this type of strategy could be used to improve AA concentrations.  
In addition to the pathways previously described in Sections 2 and 3, there are other pathways less explored to produce 3-HP and AA that can be a source of more efficient enzymes even if the pathways are not the most efficient. For example, AA-CoA, in addition to being an intermediate in the 3-HP cycle in some organisms, is also an intermediate in the fermentation of lactate by _C. propionicum_, _Propionibacterium freudeneichihi_, _Clostidium homopropionicum_, _Clostidium neopropionicum_ or _Veillonella parula_, among others [125; 126]. In this pathway, lactate is converted to lactoyl-CoA, which is then converted to AA-CoA. Afterwards, AA-CoA can be converted to propionyl-CoA or AA [127]. This pathway has not been explored for AA production, as the production is low and transient in the endogenous organism and only occurs in certain circumstances. Still, the enzyme that performs the last step could be evaluated as the pathway can be highly regulated in the endogenous organism. Another pathway that has been less explored for 3-HP production is the one that uses propionate as a precursor, producing as intermediates propionyl-CoA, AA-CoA, 3-HP-CoA and, finally, 3-HP [63; 128]. Part of this pathway is the reverse of the pathway used to produce AA from 3-HP (Figure 7). Nevertheless, this pathway could be adapted to produce AA from propionate, having as intermediates propionyl-CoA and AA-CoA. Therefore, the enzymes previously used could be evaluated in new pathways.