# Biological Production of 3-Hydroxpropionic Acid: An Update on the Current Status
## 2 Production of 3 HP from Glycerol
### Engineering of E. coli Cells for the Conversion of Glycerol to 3 HP  
_E. coli_ is commonly used as a host microorganism for genetic modifications, and there is a wide range of commercial genetic tools available, making it easy to handle with efficient control of gene transfer. In addition, _E. coli_ has a large number of aids in its genome--genes that could be studied and overexpressed for the conversion of glycerol to 3 HP. However, the disadvantage of _E. coli_ is that it is not able to produce coenzyme B\({}_{12}\), leading to the necessity of external addition of this expensive compound, thus affecting process economics and industrial applications. As when _K. pneumoniae_ is used as the host strain, when using _E. coli_, one common strategy is to use the genes of the _dha_ operon. As has been discussed, glycerol dehydatase undergoes rapid inactivation, which can result in shutdown of 3 HP production. [PERSON] et al. [PERSON] et al. (2016) demonstrated that when the glycerol dehydatase reactivase gene was cloned together with the glycerol dehydatase gene, the 3 HP concentration increased fivefold, due to the prevention of glycerol dehydatase inactivation. In an attempt to increase the 3 HP production yield, the authors also expressed the gene encoding the NAD\({}^{+}\)-regenerating enzyme, glycerol-3-phosphate dehydrogenase (Table 2). However, the concentration of 3 HP was reduced, with increased production of malic acid, due to the lack of NAD\({}^{+}\). Enhancement of 3 HP production by regulation of glycerol metabolism and minimizing of by-product formation was attempted by [PERSON] et al. [PERSON] et al. (2016). During their work, they tried to eliminate the formation of major by-products, such as acetate and 1,3-propanediol, and to increase the metabolic flow of glycerol towards 3 HP by upregulating the glycerol kinase (_glpK_) and the glycerol facilitator (_glpF_), and by deleting the regulatory factor that repressed the use of glycerol (_glpR_). After these modifications, the generation of by-products was minimized, and the uptake of glycerol was improved, resulting in a production of 3 HP as high as 42.1 g/L. The average yield was 0.268 g/g. When aiming to produce 3 HP through the 3-hydroxypropionaldehyde intermediate, proper balancing of the steps should be attempted, to avoid accumulation of the 3-hydroxypropionaldehyde. 3-Hydrooxypropionaldehyde is very toxic for microorganisms, even at concentrations of 15-30 mM, and its accumulation can result in inhibition of 3 HP production, with concentrations as low as 10 mM or even lower having a significant negative effect on growth and enzyme activity [PERSON] et al. (2016); [PERSON] et al. (2016). One solution to the problem could be the selection of an efficient aldehyde dehydrogenase that would rapidly act on 3-hydroxypropionaldehyde and convert it to 3 HP. Based on this strategy, [PERSON] et al. [PERSON] et al. (2016) tried 17 candidate aldehyde dehydrogenases for their activity against 3-hydroxypropionaldehyde, with the _gabD4_ from _Cupriavidus necator_ turning out to be the most effective one. In an attempt to further improve the aldehyde dehydrogenase selected, the authors performed site-directed and saturation mutagenesis, based on homologous modeling. The mutant enzyme obtained had 1.4-fold higher activity compared to the wild type one, and a high 3 HP production of 71.9 g/L (with a productivity of 1.8 g/L-h) was achieved in fed-batch bioreactor culture, which, to the best of our knowledge, is the highest reported concentration reported with _E. coli_ as a host growing on glycerol. Another strategy to avoid 3-hydroxypropionaldehyde accumulation is the use of promoters with different strength in controlling the expression of the enzymes [PERSON] et al. (2016), although the number of genetic elements can limit the control over gene expression [PERSON] et al. (2016). The use of the in silico design tool \"UTR Designer\" has also been proposed; it can provide precise predictions of the translation initiation efficiency [PERSON] et al. (2016).