# Heterologous Production of Acrylic Acid: Current Challenges and Perspectives
## 2 Heterologous Production of 3-HP, a Frequent Intermediary from the AA Pathway
### Glycerol Route
Glucose was used as a substrate in the growth phase, and glycerol was supplemented in the production stage as it was concluded that high concentrations of glycerol inhibit cell growth and its consumption rate. This allowed reducing the accumulation of fermentation by-products (lactate and acetate). The benefits of adding glucose as a substrate were also verified by [PERSON] et al. [74]. AldH (KGSADH) activity was enhanced in the presence of glucose. Moreover, glucose also increased ATP availability for cell growth and NAD\({}^{+}\) for the conversion of 3-HPA to 3-HP. Later, the strain designed by [PERSON] et al. [73] was genetically modified by [PERSON] et al. [82]. The genes _glpK_ and _yqhD_, encoding glycerol kinase and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-dependent aldehyde reductase, respectively, were deleted to improve the flux to 3-HP. GlpK deviates glycerol to glycerol-3-phosphate, and YqhD converts the intermediary 3-HPA to 1,3-propanediol (Figure 4). These deletions allowed increasing the concentration from 14.3 g/L to 57.3 g/L, demonstrating the need to eliminate competing pathways to increase 3-HP production. \begin{table}
\begin{tabular}{c c c c c c c c} \hline \hline
**Host** & **Substate** & **Pathway Genes** & **Other Genes** & **Crosses** & **Classification Modification** & **Operational Mode;** & **3-HP** & **Concentration (g/g) 4** & **Ref. ** \\  & **(Concentration)** & **Overpresseses in Plasmid** & **Overpresses in Plasmid** & **Crosses modification** & **Correlation Time** & **Concentration (g/g) 4**  
Since 3-HP and 3-HPA can be toxic to cells in high amounts, especially 3-HPA, research has also focused on improving _E. coli_ (and other hosts) tolerance to these compounds or to find more resistant strains [52]. [PERSON] et al. [52] verified that 3-HP production and enzyme expression and activities significantly differ among several _E. coli_ strains. They selected the _E. coli_ W strain for 3-HP production as it presents a high maximum growth rate and final cell density in the presence of 3-HP. Using the previous described pathway (DhaB, GDRAB and KGSADH), they were able to produce 41.5 g/L of 3-HP. A similar concentration was obtained by [69] (42.1 g/L) using DhaB, GDRAB and AldH. In this study, four genes were also deleted: _ackA_ (acetate kinase), _pta_ (phosphate acetyltransferase) and _yqhD_ in order to reduce the accumulation of 3-HP production by-products (acetate, 1,3-propanediol) and _glpR_ (glycerol catabolic pathway repressor) to increase the flux towards glycerol. _AckA_ and _pta_ are responsible for the conversion of acetyl-CoA to acetate (Figure 4). GlpF (glycerol facilitation) was also overexpressed to facilitate glycerol transport in the cell. Later, [PERSON] et al. [35] screened several aldehyde dehydrogenase enzymes in order to identify one that allows reducing 3-HPA accumulation. While most of the enzymes tested did not present increased catalytic activity, GabD4 from _Cupiravidus reactor_ demonstrated high catalytic activity towards the pathway intermediary. Moreover, the activity was improved by site-directed mutagenesis. In addition to expressing DhaB, GDRAB and mutant GabD4, they also deleted _ackA_, _pta_ and _yqhD_ genes to direct the pathway towards 3-HP production. These modifications allowed increasing the concentrations to 71.9 g/L. [PERSON] et al. [53] also used rational untranslated region (UTR) engineering to control the accumulation of toxic 3-HPA by balancing the expression of the pathway enzymes. UTR Designer ([[https://sbi.postech.ac.kr/utr_designer/](https://sbi.postech.ac.kr/utr_designer/)]([https://sbi.postech.ac.kr/utr_designer/](https://sbi.postech.ac.kr/utr_designer/))) was used to design UTR regions with targeted expression levels of _dhaB1_, _dhaB2_ and _kssadh_. The optimal balance, correspondent to high expression of KGSADH and lower expression of DhaB1, enhanced 3-HP concentration (40.51 g/L) compared to the control and allowed obtaining a yield of 0.97 g 3-HP/g glycerol.