# Heterologous Production of Acrylic Acid: Current Challenges and Perspectives
## 2 Heterologous Production of 3-HP, a Frequent Intermediary from the AA Pathway
### Malonyl-CoA Route  
The malonyl-CoA route to produce 3-HP has also been thoroughly studied in _E. coli_[41; 42; 43; 86; 87]. Compared to the glycerol route, this route has the advantage of not needing B\({}_{12}\) vitamin as a cofactor. However, it is well known that malonyl-CoA availability is limited in microorganisms due to tight regulation of fatty acid biosynthesis [88; 89; 90]. This low availability limits 3-HP production, whereby most of the studies focus on increasing malonyl-CoA concentration, for example, by overexpressing the enzyme acetyl-CoA carboxylase (ACC) [41; 42; 43; 86; 87]. In addition, biotin and NaHCO\({}_{3}\) are also supplemented to increase malonyl-CoA availability. However, biotin, as vitamin B\({}_{12}\), is expensive and its use is industrially not sustainable. From malonyl-CoA, this route needs only one enzyme, malonyl-CoA reductase (MCR), and requires two NADPH molecules to produce 3-HP (Figure 5). MCR enzyme can be found in several organisms, including _C. aurantiacus_, that assimilate CO2 through the 3-HP cycle. This enzyme contains two distinct regions, identified as MCR-N and MCR-C according to the N- and C- terminals, respectively [91]. MCR-C catalyzes the conversion of malonyl-CoA to the intermediate malonic semialdehyde, which is further converted to 3-HP by MCR-N. The first step is rate-limiting [91]. So far, 3-HP heterologous production using the malonyl-CoA route was reported at least in _E. coli_, _S. cerevisiae_, _Pichia pastoris_, _Schizosaccharomyces pombe_ and _Synechococcus elongatus_[42; 86; 92; 93; 94; 95]. [PERSON] et al. [86] were the first to report 3-HP production in _E. coli_ using this route (Table 2). In addition to overexpressing MCR from _C. aurantiacus_, they overexpressed ACC (_accADBC_) and BirA (biotinilase) genes to increase malonyl-CoA availability and pyridine nucleotide transhydrogenase subunits \(a\) and \(B\) (PntAB from _E. coli_) gene to convert reduced nicotinamide adenine dinucleotide (NADH) to NADPH (all genes from _E. coli_ K-12). Using this strategy, they were able to produce 0.193 g/L from  
Figure 5: 3-Hydrooxypropionic acid (3-HP) heterologous pathway in _Escherichia coli_ using the malonyl-CoA route. Enzymes in pink are from _E. coli_: AccADBC: acetyl-CoA carboxylase; AckA: acetate kinase; ACS: acetyl-CoA synthase; AdhE: bifunctional aldehyde/alcohol dehydrogenase; BirA: biotinylated. Fabl: enoyl-acyl carrier protein; LdhA: lactate dehydrogenase; MgsA: methylglyoxal synthase; PIB: pyruvate formate-lyase; PntAB: pyridine nucleotide transhydrogenase subunits \(a\) and \(B\); PoxB: pyruvate oxidase; Pta: phosphate acetyltransferase; YdfG: 3-hydroxy acid dehydrogenase. Enzymes in blue correspond to heterologous enzymes commonly overexpressed: ACC/DstR1: acetyl-CoA carboxylase; ACS: acetyl-CoA synthase; Other abbreviations: ADP: adenosine di-phosphate; ATP: adenosine triphosphate; BCCP: biotin carboxyl carrier protein; NAD\({}^{+}\): nicotinamide adenine dinucleotide; NADH: reduced form of NAD\({}^{+}\); NADP\({}^{+}\): nicotinamide adenine dinucleotide phosphate; NADPH: reduced form of NADP\({}^{+}\). glucose. In a later study [87], 3-HP was produced in _E. coli_ through the overexpression of codon-optimized MCR from _C. aurantiacus_ and overexpression of ACC from _C. glutamicum_. This ACC is simpler as it contains only two subunits, AccBC and DtR1, and is reported to be a better option to increase malonyl-CoA bioavailability in _E. coli_ possibly because it is not feedback-inhibited as the one of _E. coli_. Using fed-batch operation mode, 10.08 g/L of 3-HP was obtained from glucose. [PERSON] et al. [91], who studied MCR enzyme from _C. aurantiacus_, concluded that MCR activity increases when the two enzyme fragments MCR-C and MCR-N are separated and that MCR-N expression is significantly higher than that of MCR-N. This separation led to an increase of 1.5 times in 3-HP production (0.15 g/L in shake-flask fermentation) in relation to the control carrying the full-length MCR gene. ACC from _E.