# Biological Production of 3-Hydroxpropionic Acid: An Update on the Current Status
## 2 Production of 3 HP from Glycerol
### Production Using Genes That Are Part of the Dha Operon  
The naturally occurring _dha_ operon encodes the necessary enzymes for the conversion of glycerol to 1,3-propanediol. A detailed description of the genes in the _dha_ operon and their action is given in our previous review article [3]. Briefly, the operon mainly consists of genes encoding (1) a glycerol dehydatase, which catalyzes the conversion of glycerol to 3-hydroxypropionaldehyde; (2) a reactivase of the glycerol dehydatase; and (3) a 1,3-propanediol oxidoreductase, which catalyzes the conversion of 3-hydroxypropionaldehyde to 1,3-propanediol, with some structural differences in different microorganisms. Conversion of glycerol to 3 HP using the _dha_ operon is performed in two stages, whereby glycerol is initially converted to 3-hydroxypropionaldehyde through the action of the enzyme glycerol dehydatase, followed by conversion of the 3-hydroxypropionaldehyde to 3 HP by the action of an aldehyde dehydrogenase (ald) enzyme (Figure 1). A strategy that is commonly employed is to use a microorganism that already possess the _dha_ operon, and genetically modify it to produce 3 HP. In that case, the gene encoding the 1,3-propanediol oxidoreductaseus should be deleted or underexpressed if the co-production of 1,3-propanediol is not wanted, but special care should be taken to maintain a balance between NADH and NAD\({}^{+}\). Regeneration of NAD\({}^{+}\) is important, as it is required for the action of the ald enzyme. NAD\({}^{+}\) can be regenerated in the electron transport chain, which requires increased aeration, but the presence of oxygen inhibits the synthesis of coenzyme B\({}_{12}\), which is also involved in the process [12]. Moreover, as will be discussed later, oxygen also inactivates the glycerol dehydatase. Regeneration of NAD\({}^{+}\) can also take place during the conversion of 3-hydroxypropionaldehyde to 1,3-propanediol by the action of the 1,3-propanediol oxidoreductaseusae, which consumes NADH [13]. If the 1,3-propanediol oxidoreductaseusae gene is knocked out, another pathway for NAD\({}^{+}\) should be found in order to maintain the balance between NADH and NAD\({}^{+}\), such as lactate or acetate formation [12]. On the other hand, co-production of 3 HP and 1,3-propanediol can also be desirable, as 1,3-propanediol also has significant applications and these two molecules can be separated easily, due to the presence of different functional groups on the molecules [14].  
Another consideration when the _dha_ operon is used is that the first step of the reaction requires coenzyme B\({}_{12}\), which is produced naturally de novo by some bacterial strains, such as _Klebsiella pneumoniae_[15; 16], whereas other bacteria, such as _Escherichia coli_, cannot produce it de novo [16]. When the host microorganism is not capable of producing this coenzyme, external addition of it should be included in the cultivation--which will affect the cost of the process. An effort is therefore being made to create new bacterial strains that are capable of producing coenzyme B\({}_{12}\). Another consideration when using the _dha_ operon is the inactivation of the glycerol dehydatase during the catalysis of glycerol conversion [17; 18], so proper expression of the glycerol dehydatase reactivase is important to maintain the activity of glycerol dehydatase and, in turn, 3 HP production. Glycerol dehydatase can also be inactivated by oxygen [19], which on the other hand, is necessary for efficient regeneration of the NAD\({}^{+}\)[20] that is required for the action of the ald enzymes.