# Heterologous Production of Acrylic Acid: Current Challenges and Perspectives
## 2 Heterologous Production of 3-HP, a Frequent Intermediary from the AA Pathway
### Glycerol Route
Therefore, 3-HPA accumulation should be as limited as possible. Figure 4: 3-Hydroxypionic acid (3-HP) heterologous pathway in _Escherichia coli_ using the glycerol route. Enzymes in pink are from _E. coli_: AckA: acetate kinase; AdhE: bifunctional aldehyde/alcohol dehydrogenase; AldH: aldehyde dehydrogenase; GapA: glyceraldehyde-3-phosphate dehydrogenase; GlpF: glycerol facilitator; GlpK: glycerol kinase; GlpR: glycerol catabolic pathway repressor; GpsA: glycerol-3-phosphate dehydrogenase; LdhA: lactate dehydrogenase; PoxB: pyruvate oxidase; Pta: phosphate acetyltransferase; YqhD: NADPH-dependent aldehyde reductase. Enzymes in blue correspond to heterologous enzymes commonly overexpressed: DhaB: B\({}_{12}\)-dependent glycerol dehydratase; GPD: glycerol-3-phosphate dehydrogenase; GPP: glycerol-3-phosphate; GrdAB/DhaR: glycerol dehydratase reactivase; KGSAGH/GabD4/YcdW: aldehyde dehydrogenase. Other abbreviations: Co-B\({}_{12}\): coenzyme B\({}_{12}\); “Co-B\({}_{12}\): damaged Co-B\({}_{12}\); DHAP: dihydroxyacetone-phosphate; 1,3-BPG: 1,3-bisphosphoglycerate; G3P: glycerol-3-phosphate; GAP: glyceraldehyde-3-phosphate; NAD\({}^{+}\): nicotinamide adenine dinucleotide adenine dinucleotide; NADH: reduced form of NAD\({}^{+}\); NADP\({}^{+}\): nicotinamide adenine dinucleotide phosphate; NADPH: reduced form of NADP\({}^{+}\). 3-HP heterologous production from glycerol was first reported in _E. coli_ SH254 [72]. _K. pneumoniae_ is known to naturally accumulate 3-HPA [80] that can be mostly converted to 1,3-propanediol and also to 3-HP but in low amounts [81]. Therefore, the DhaB operon (_dhaB1_, _dhaB2_, _dhaB3_, _dhaB4_) from _K. pneumoniae_ DSM 2026 encoding glycerol dehydatase was used to perform the first step of the heterologous pathway. The second step was catalyzed by AldH (also known as PuuC) from _E. coli_ K-12 MG1655 encoding an aldehyde dehydrogenase, more specifically a \(\gamma\)-glutamyl-\(\gamma\)-aminobutyraldehyde dehydrogenase. This was the first time AldH was described to have specificity towards 3-HPA. In this study, 0.58 g/L of 3-HP was obtained from 100 mM glycerol. The concentration obtained was highly dependent on the concentration of vitamin B\({}_{12}\) supplemented. Later [67], the concentrations using the same biosynthetic pathway were increased to 4.4 g/L through the optimization of isopropyl \(\beta\)-D-1 thiogalactopyranoside (IPTG) and substrate concentrations, pH and liquid-to-flask ratio. Afterwards, in a fed-batch process for 72 h, the concentrations increased to 31 g/L. This study reported an instability of DhaB enzyme in the presence of glycerol and an imbalanced activity between DhaB and AldH due a low expression of AldH. Therefore, the same research group optimized the constructed strain to eliminate the imbalance between the two enzymes [68] (Table 1). To stabilize DhaB activity, glycerol dehydatase reactivase (DhaR) from _K. pneumoniae_ DSM 2026 (GDRAB) was used. GDRAB is encoded by _gdrA_ (formally referred as _dduB4_) and _gdrB_ genes and can reactivate glycerolinactivated DhaB through the replacement of damaged coenzyme B\({}_{12}\) with a new one. To balance the enzymes, DhaB was expressed in a low-copy plasmid with GDRAB, while AldH was cloned in a high-copy plasmid. This pathway [68] allowed to improve the production by 2.5-fold compared to [72] under shake-flask conditions. In addition to AldH from _E. coli_, they also tested a different aldehyde dehydrogenase-\(\alpha\)-ketoglutaric semialdehyde dehydrogenase, from _Azospirillum brasilense_ (KGSADH), that was demonstrated to have higher catalytic efficiency. This enzyme allowed improving the 3-HP production by 2.5-fold in shake flask, and in a bioreactor, up to 38.5 g/L of 3-HP was produced [68]. In a different study, [PERSON] et al. [73] used DhaB and DhaR from _Lactobacillus brevis_ and AldH from _E. coli_ and produced 14.3 g/L of 3-HP in a 2-step fed-batch process.