# Heterologous Production of Acrylic Acid: Current Challenges and Perspectives
## 3 Heterologous Production of AA
### Using 3-HP as an Intermediary  
3-HP is converted to AA using three enzymes (Figure 7a): a CoA transferase to convert 3-HP to 3-hydroxypropionyl-CoA (3-HP-CoA), a 3-HP-CoA hydratase to convert 3-HP to AA-CoA and a CoA hydrolase to convert AA-CoA to AA. The first two steps of this pathway are based on the 3-HP cycle found in several organisms, such as C. _auranticus_, _Acidiamus brieferlyi_, _Acidiamus ambivlens_, _M. sedula_, _Megasphera elsdenii_, _Sulfolobus_ sp. strain VE6 and _Sulfolobus metallicus_[105; 106; 107]. Some of these organisms have already been explored as a source of enzymes for this pathway, but there are still several enzymes than can be evaluated. The first study reporting AA heterologous production was by [PERSON] et al. [32] in 2015. AA was produced in _E. coli_ from glucose using the glycerol route, while producing 3-HP as an intermediary. Glycerol was converted to 3-HP by the expression of _dhaB_, _gdrAB_ and _gabD4_, which were integrated into the chassis genome by homologous recombination (Table 4). Next, several genes from different organisms were screened for the three steps from 3-HP to AA. The enzymes that performed best were overexpressed in a plasmid: YciF (CoA transferase) from _C. necator_ was used to convert 3-HP to 3-HP-CoA, Aflv_0566 (CoA dehydatase) from _Anoxyhacillus fluithermus_ to convert 3-HP-CoA to AA-CoA and YciA (CoA thioesterase) from _E. coli_ to convert AA-CoA to AA. To increase the flux towards glycerol, the _gpsA_ gene from _E. coli_ and _gpp2_ from _S. cerevisiae_ were overexpressed in a plasmid. Additionally, _pta_, _ackA_ and _yahD_ were deleted to reduce the production of the by-products acetate and 1,3-propanediol and, consequently, increase the 3-HP concentration. Using this strain, it was possible to produce 0.12 g/L of AA from glucose. In addition, 2.2 g/L of 3-HP was produced, which is quite low compared to the concentrations previously described using this route (Section 2.1). This might be related to the integration of the pathway genes required to produce 3-HP from glycerol into the _E. coli_ genome that decreases the number of copies to one. This AA production method was patented by [PERSON] et al. [29]. Later, [PERSON] et al. [33] used the malonyl-CoA route via 3-HP to produce AA. They constructed an AA biosensor based on fluorescence to evaluate the several strains they constructed using high-throughput methods. The biosensor was based on _acuR_, a transcription regulator from _Rlodobacter shareoids_ that responds to AA presence. To convert 3-HP to AA-CoA, they used a truncated version of propionyl-CoA synthase (_pcs_) from _C. aurantiacus_. The original version of this enzyme allows converting 3-HP to 3-HP-CoA, 3-HP-CoA to AA-CoA and then AA-CoA to propionyl-CoA. Therefore, the functional domain responsible for the last step was removed. To convert AA-CoA to AA, they co-expressed AA-CoA hydrolase (_ach_) from _Acinetobacter baylyi_. 3-HP was produced from malonyl-CoA by _mcr_. To improve malonyl-CoA accumulation, cerulenin was supplemented in 96-well plates. Cerulenin inhibits fatty acid biosynthesis, which in turn leads to an increase in malonyl-CoA availability. However, this approach is not sustainable at a higher scale as cerulenin is expensive. Nevertheless, they demonstrated that malonyl-CoA availability limits AA production as much higher concentrations were obtained when cerulenin was supplemented. Up to 4.32 mg/L of AA was produced. This reinforces that this routecan only be successfully used in a strain engineered to increase malonyl-CoA availability. Afterwards, [PERSON] and [PERSON] [31] also produced AA from glucose via malonyl-CoA and 3-HP mostly using enzymes from _M. sedula_: To convert malonyl-CoA to 3-HP, an MCR and an malonate semialdehyde reductase (MSR) were used; then, a 3-hydroxypropionyl-CoA synthetase (3 HPCS) was used to convert 3-HP to 3-HP-CoA and a 3-hydroxypropionyl-CoA dehydratase (3 HPCD) to synthesize 3-HP-CoA from AA-CoA. Succinyl-CoA synthase (SucC) from _E. coli_ was used to remove the CoA moiety from AA-CoA.