# Heterologous Production of Acrylic Acid: Current Challenges and Perspectives
## 2 Heterologous Production of 3-HP, a Frequent Intermediary from the AA Pathway
### Glycerol Route
In addition to the goal of limiting 3-HPA accumulation, [PERSON] et al. [50] tested cassettes containing synthetic regulation regions composed of promoters with different strengths and bicistronic ribosome-binding sites (RBSs). This allowed controlling the expression of the enzymes of interest (AldH expression was 8-fold higher than that of DhaB) and reduce the intermediary accumulation. In this study, 56.4 g/L of 3-HP was produced. To redirect the metabolic flux of the central metabolic pathway to 3-HP production, [PERSON] et al. [70] used a metabolic toggle switch [84]. This synthetic biology approach enabled the conditional repression of _gapA_ (encoding glyceraldehyde-3-phosphate (GAP) dehydrogenase) that led to an increase in 3-HP production. In this case, _gapA_ was deleted and a copy was overexpressed in a plasmid. The expression was controlled by the presence of IPTG. When IPTG was present, _lacI_ (lactose operon repressor) did not repress _lac_ promoter expression, which led to the expression of the 3-HP pathway genes and also of _tetR_ (tetracycline repressor) that repressed _gapA_ expression. In its turn, lack of expression of _gapA_ during the production phase prevented 1,3-biphosphoglycerate production from GAP, decreasing the metabolic flux to pyruvate. Since fed-batch operation mode was not used in this case, the productions (\(\approx\)6 g/L) were comparatively lower to previous studies (Table 1) but significantly higher than the controls, which proves that this synthetic biology approach can be beneficial to increase the 3-HP concentration. More recently, research has been also focusing on the use of xylose [38; 51; 83] as a carbon source to produce 3-HP due to its availability in agricultural residues wastes. [PERSON] et al. [83] relieved carbon catabolite repression by deletion of _ptsG_ (glucose-specific phosphoenolpyruvate:sugar transferring system enzyme), which represses _xyl_ operon genes, and overexpression of XylR (_xyl_ operon transcriptional regulator). They also deleted _glpK_ and _yqhD_ and overexpressed glycerol-3-phosphate dehydrogenase (GPD) and glycerol-3-phosphatase (GPP2) genes from _S. cerevisiae_ to direct the flux to glycerol and 3-HP biosynthesis. Finally, _dhuB_ and _dhaR_ from _L. brevis_ and _aldH_ from _Pseudomonas aeruginosa_ were used to produce 29.4 g/L of 3-HP from xylose and glucose. However, a high amount of glycerol was accumulated, which is not ideal as high concentrations of glycerol inhibit glycerol dehydratase enzyme. To circumvent this, the _gpsA_ gene, corresponding to glycerol-3-phosphate dehydrogenase from _E. coli_, was overexpressed instead of _GPD_ by [PERSON] et al. [51] in order to decrease the accumulation of glycerol. The overexpression of _galP_ (encoding _E. coli_ galactose transporter) also led to a decrease in glycerol accumulation during glucose and xylose co-fermentation, possibly due to an increase in biomass. All these modifications increased the 3-HP concentration to 37.6 g/L [51]. Later, the same research group performed more modifications to the pathway to reduce glycerol accumulation [38]. They opted for the expression of _GPD_ instead of _gpsA_ but also deleted _puuR_ (_puu_ operon repressor gene) and replaced the AldH endogenous promoter for a stronger one. Again, these modifications led to an improvement in the 3-HP concentration (53.7 g/L) from glucose and xylose. Finally, the use of crude glycerol for 3-HP production in _E. coli_ was recently reported [36]. This study allowed obtaining the highest 3-HP concentration reported in _E. coli_ (76.2 g/L from glycerol and 61 g/L from crude glycerol) with only a few genetic modifications. DhaB and GrdB were overexpressed together with several tested AldH enzymes from different organisms. \(\gamma\)-aminobutyraldehyde dehydrogenase from _K. pneumoniae_ (_ydcW_) was selected as it was found to be the best one to catalyze the pathway's second step. In addition, _lacl_ was deleted to constitutively express the pathway genes under _tac_ or _trc_ promoters without the need for supplementing IPTG.