# Heterologous Production of Acrylic Acid: Current Challenges and Perspectives
## 2 Heterologous Production of 3-HP, a Frequent Intermediary from the AA Pathway
### \(\beta\)-Alanine Route  
The \(\beta\)-alanine route is the third route explored to produce 3-HP. First, \(\beta\)-alanine is converted to malonic semialdehyde by \(\beta\)-alanine aminotransferase. In this step, pyruvate is consumed and \(L\)-alanine is produced. Then, as in the malonyl-CoA route, malonic semialdehyde is transformed to 3-HP by MCR or YdfG (Figure 6). Figure 6: 3-Hydroxypropionic acid (3-HP) heterologous pathway in _Escherichia coli_ using the \(\beta\)-alanine route. Enzymes in pink are from _E. coli_: AspA: aspartate; FumAC: fumarase A and C; FumB: fumarase B; PPC: phosphoenolpyruvate carboxylase; YdfG: 3-hydroxy acid dehydrogenase. Enzymes in blue correspond to heterologous enzymes commonly overexpressed (including enzymes expressed when _Saccharomyces cerevisiae_ is the host): AAT2: aspartate aminotransferase; BAPAT/PA0132: \(\beta\)-alanine aminotransferase/\(\beta\)-alanine pyruvate transaminase; MCR: malonyl-CoA reductase; PanD: aspartate-1-decarboxylase; PYC1/PYC2: pyruvate carboxylases. Other abbreviations: NADP\({}^{\pi+}\): nicotinamide adenine dinucleotide phosphate; NADPH: reduced form of NADP\({}^{+}\); TCA: tricarboxylic acid. This route has been explored mostly in _S. cerevisiae_[40, 98, 99] but also in _E. coli_[46] (Table 3). However, although this route was academically less explored than the other two routes, in comparison, more patents have been filed by Cargill and Novozymes using _E. coli_, _S. cerevisiae_ or _Isattchenkia orientalis_ as the host (e.g., [100, 101, 102, 103]). _S. cerevisiae_ was the first organism to heterologously produce 3-HP from \(\beta\)-alanine [98]. Aminotransferase from _Bacillus cereus_ (BAPAT) and YdfG (from _E. coli_) were used to convert \(\beta\)-alanine to 3-HP. Additionally, _S. cerevisiae_ aspartate aminotransferase (AAT2), pyruvate carboxylases (PYC1/PYC2) and _Tribeleum castanium_ aspartate-1-decarboxylase (PanD) were overexpressed with the goal of increasing \(\beta\)-alanine availability [40, 98, 99] (Figure 6). Using these genetic modifications, up to 25 g/L of 3-HP was obtained from glucose [40] and 7.37 g/L of 3-HP from xylose [99]. For _S. cerevisiae_ to be able to efficiently metabolize xylose, several genes from _Pichia stipitis_ were overexpressed (Table 3). 3-HP highest production via \(\beta\)-alanine was obtained in _E. coli_ (31.1 g/L) [46]. \(\beta\)-alanine was converted to malonic semialdehyde by \(\beta\)-alanine pyruvate transaminase from _P. aeruginosa_, and malonic semialdehyde was converted to 3-HP by YdfG from _E. coli_. PanD from _C. glutamicum_ and _E. coli_ aspartase (AspA) and phosphoenolpyruvate carboxylase (PPC) were also overexpressed to increase the flux to \(\beta\)-alanine (Figure 6). The fumaras genes (_fumAC_, _fumB_), which convert fumarate to malate, were deleted to increase fumarate availability and, consequently, aspartate and \(\beta\)-alanine. The isocitrate lyase repressor (_iclR_) gene was deleted to improve the flux through the glyoxylate shunt. The flux towards fumarate and aspartate was also increased by replacing succinate:quinone oxidoreductasethase (SdhC) and AspA endogenous promoters by stronger ones. _I. orientalis_ was also demonstrated to be a suitable host to produce 3-HP using the \(\beta\)-alanine route as it produced 22.8 g/L in fed-batch operation mode [101]. In this patent, PanD from the insect _Aedes aegypti_ was expressed. The other genetic modifications were not specified in detail but included the expression of aspartate aminotransferase, \(\beta\)-alanine aminotransferase and dehydrogenases, among others, and the deletion of genes that deviated the pathway to other undesirable compounds. The 3-HP concentrations obtained using this route (up to 31.1 g/L in _E. coli_) are, in general, lower than the ones obtained by the other two routes (up to 76.2 g/L in the glycerol route and 49.04 g/L in the malonyl-CoA route in _E. coli_). Nevertheless, this route is much less explored.