# Heterologous Production of Acrylic Acid: Current Challenges and Perspectives
## 4 Key Points to Optimize AA Heterologous Production
### Improving Chassis Tolerance to AA  
It is well described in the literature that 3-HPA, 3-HP and AA are toxic to cells in high concentrations, with AA being more toxic than 3-HP [32; 105]. It is known that AA presence in high concentrations increases the lag phase and decreases the bacterial growth rate and final cell concentration [34; 129], demonstrating potential antimicrobial properties. Several factors contribute to AA toxicity. One factor is general to short-chain acids. These acids are known to decrease the rate of DNA, RNA, protein and lipid synthesis. They affect genes and proteins involved in the transcription-translation machinery, regulation and response to stress [130]. In addition, the AA double-bond structure can conjugate with glutathione, causing its depletion [1]. This may lead to toxicity since glutathione protects bacteria from low pH, chlorine, and also osmotic and oxidative stresses [131]. The importance of glutathione availability was demonstrated for 3-HP production in _S. cerevisiae_[132]. In addition, the AA \(\alpha\),\(\beta\)-conjugated unsaturated carbonyl group was demonstrated to be toxic to bacterial metabolism [133]. Due to the reported AA toxicity, high productions can be challenging in _E. coli_ or other organisms. To improve product concentrations, several metabolic engineering strategies can be used. As previously mentioned, 3-HP tolerance was increased through the modification of the _E. coli_ genome using iCREATE [42]. Genes associated with low pH, transporters, organic acid tolerance, transcription factors and regulators were targeted in [42] and other studies (e.g., [43; 134]). Strategies to increase the unsaturated fatty acid content in the membrane have also been successful in increasing _E. coli_ tolerance to 3-HP [135]. In addition, it was demonstrated that _E. coli_ is more sensitive to AA under anaerobic conditions [110] and this sensitivity is related to the inhibition of pyruvate formate lyase (PfIB) that is required for _E. coli_ anaerobic growth. Therefore, this enzyme gene could be an interesting target to increase AA tolerance. In the future, other target genes need to be identified and transcriptome and proteome studies can be useful to identify the genes affected by AA presence. After identification of the target genes, several synthetic biology tools may be used to create mutations in the selected genes, including the abovementioned EvolvR [124]. Random mutagenesis can also be used to unravel new targets. After mutagenesis, which can be performed using different methods (e.g., UV irradiation, ethidium bromide or N-methyl-N\({}^{\prime}\)-nitro-N-nitrosoguanidine exposure), clones can be screened for changes in the tolerance to AA. Strains with altered tolerance profiles can then be sequenced to identify the location of the mutation using next-generation sequencing. Additionally, AA biosynthesis can be optimized at the operational level. Different operation modes may decrease possible metabolite inhibitions and, therefore, increase the productivity. For example, in situ product removal strategies may be evaluated to remove AA from the culture broth using solid-liquid extraction methods and, consequently, avoid the AA toxic effect [136; 137]. Adsorption with solid resins (e.g., Amberlite) was recently successfully used to remove AA from an aqueous medium [138]. These resins can be used directly in a fermenter since they are non-toxic to microorganisms. This approach may allow simultaneous AA production and purification. In addition, liquid-liquid extraction methods using natural oils or ionic liquids is also reported to remove AA [139; 140]. Moreover, if the glycerol route is selected, the dissolved oxygen concentration should be carefully optimized due to the enzyme sensitivity to oxygen (glycerol dehydatase) and to the NAD\({}^{+}\) regeneration need.