# Heterologous Production of Acrylic Acid: Current Challenges and Perspectives
## 4 Key Points to Optimize AA Heterologous Production
### Testing a B\({}_{12}\)-Independent Enzyme in the Glycerol Route  
B\({}_{12}\)-dependent glycerol dehydatase, which performs the first step in the glycerol route with or without 3-HP as an intermediary, represents a limiting step of the AApathway. Although the concentrations obtained with this enzyme are high, up to 76.2 g/L in _E. coli_, the use of this pathway at an industrial scale is unsustainable due to the need of supplementing B12. In addition, the engineering of the B12 pathway in _E. coli_ is not ideal due to the challenge of expressing more than 20 genes [111, 112]. Therefore, new enzymes ought to be discovered and tested as the glycerol pathway is the one with the most potential to produce AA (and 3-HP), and on top of that, it can use as a substrate an industry by-product (crude glycerol from the biodiesel industry).  
In the past years, a B12-independent glycerol dehydatase was described for the heterologous production of 1,3-propanediol [113, 114, 115]. 1,3-Propanediol is an important building block and can be produced from 3-HPA, as previously mentioned. The step described for 1,3-propanediol production is exactly the same from the 3-HP pathway where glycerol is converted to 3-HPA. This type of B12-independent enzyme was first described in _Clostridium butylricum_[116, 117] that is used for 1,3-propanediol biosynthesis. Since then, several bioinformatic studies have identified other putative enzymes, all from organisms that also contain B12-dependent enzymes [118]. B12-independent enzymes from _C. butylricum_, as B12-dependent enzymes, also contain several different units in an operon: _dhaB1_, _dhaB2_ and _dhaT_. The first two encode B12-independent glycerol dehydatase and its activator factor (an S-adenosyl-L-methionine (SAM)-dependent enzyme), and _dhaT_ encodes an enzyme specific for 1,3 propanediol biosynthesis [114].  
It is unclear why this enzyme has not yet been used for 3-HP/AA biosynthesis. However, a patent was filed regarding glycerol conversion to 3-HP or 1,3-propanediol, where the possibility of using a B12-independent enzyme is mentioned [119]. Since this enzyme, as B12-dependent enzyme, is sensitive to oxygen (and glycerol) [117], the dissolved oxygen concentration should be in the range of 30% or less, preferably 10% or less, at the time of initiation of its expression [119]. The adjustment of the oxygen concentration can be performed by controlling the aeration and agitation rates in a bioreactor. In addition, sequential inoculation of strains carrying separate modules of the pathway may be a possibility if the oxygen demands for the different steps of the pathway are not compatible. Moreover, the expression of a B12-independent enzyme could be triggered by the absence of oxygen using the inducible _nar_ promoter [119, 120].  
Nevertheless, the results obtained with B12-independent enzymes for 1,3-propanediol biosynthesis are encouraging (up to 104.4 g/L was produced [115]), whereby these enzymes should be used in the future in the development of an economical B12-free bioprocess for AA and even 3-HP biosynthesis using renewable substrates. In addition, the engineering of this enzyme using synthetic biology techniques, as described in the next section (Section 4.2), would be of upmost importance to decrease its sensitivity to oxygen and glycerol and in turn simplify the bioprocess using this pathway.