# Heterologous Production of Acrylic Acid: Current Challenges and Perspectives
## 2 Heterologous Production of 3-HP, a Frequent Intermediary from the AA Pathway
### Glycerol Route  
The glycerol route (CoA-independent) is probably the route most studied as 3-HP can be produced directly from glycerol in two steps (Figure 3) [36; 52; 67; 68; 69; 70]. Additionally, 3-HP can also be produced from glucose using glycerol as an intermediary [32; 34; 35; 38; 39; 44; 45; 47; 51; 71; 72; 73; 74]. In this case, the carbon flux is often redirected to glycerol. From glycerol, the biosynthetic pathway starts with the B\({}_{12}\)-dependent enzyme glycerol dehydatase that in the presence of coenzyme B\({}_{12}\) transforms glycerol into 3-hydroxypropionaldehyde (3-HPA). The next step of this pathway is catalyzed by an aldehyde dehydrogenase that converts 3-HPA to 3-HP, with NAD\({}^{+}\) (nicotinamide adenine dinucleotide) as electron acceptor. In this route, 1,3-propanediol is produced as a by-product [72] (Figure 4). In addition to these two enzymes, the enzyme to reactivate glycerol dehydatase, known as glycerol dehydatase reactivase, a chaperone, must be expressed to repair coenzyme B\({}_{12}\)[58]. The glycerol route has some identified advantages and disadvantages. First, this pathway is simple as it only requires two enzymatic steps to convert glycerol to 3-HP, and consequently, the concentrations and productivities are, in general, higher than the ones obtained in malonyl-CoA and \(\beta\)-alanine routes [53]. The direct use of glycerol as a substrate can also be seen as an advantage as this opens the door to the use of crude glycerol. Crude glycerol is a cheap and highly available by-product from the biodiesel industry that is growing at a CAGR of 5.87% (USD 46.79 billion at the end of 2021, USD 51.48 billion by 2026) [75]. Currently, close to 40 billion tons of biodiesel per year are produced [76], leading to more than 4 billion tons of crude glycerol. This glycerol is impure (70-80%) [77] and is expensive to purify for use in the food, pharmaceutical, or cosmetics industry. Consequently, it is crucial to explore crude glycerol transformation to value-added chemicals in order to sustain price and growth in the demand for glycerol. The use of crude glycerol to produce 3-HP [45] or AA may allow not only a reduction in these building blocks' cost but also a reduction in environmental and economic problems associated with waste storing and treatment and will contribute to the viability of biodiesel, reducing its production cost and increasing the use of renewable and sustainable energy. Figure 3: 3-Hydrooxyppropionic acid (3-HP) biosynthetic pathway using three different intermediaries naturally produced in microorganisms from glucose: glycerol, \(\beta\)-alanine and malonyl-CoA. Co-B\({}_{12}\) represents coenzyme B\({}_{12}\), and *Co-B\({}_{12}\) represents damaged Co-B\({}_{12}\). NAD\({}^{+}\): nicotinamide adenine dinucleotide; NADH: reduced form of NAD\({}^{+}\); NADP\({}^{+}\): nicotinamide adenine dinucleotide phosphate; NADPH: reduced form of NADP\({}^{+}\). Regarding the glycerol route disadvantages, as the name indicates, B\({}_{12}\)-dependent glycerol dehydratase requires vitamin B\({}_{12}\) as a cofactor. However, not all microbes are able to produce it. For example, B\({}_{12}\) supplementation is required when _E. coli_ is used as a host. Since vitamin B\({}_{12}\) is expensive, its supplementation would be undesirable at an industrial scale, which makes _E. coli_ a less suitable host to carry this pathway compared to _K. pneumoniae_ and _P. denitrificans_ that naturally produce this vitamin [47; 78]. There are also other problems related to the first step of the heterologous pathway. Glycerol dehydratase enzyme is sensitive to glycerol accumulation and also to oxygen [79]. Therefore, 3-HP production should be preferably performed in microaerophilic or aerobic conditions. However, low oxygen concentration impacts NAD\({}^{+}\) regeneration that is needed for the second reaction. Another identified problem is the accumulation of the intermediary produced, 3-HPA, that is toxic to the cells if accumulated in high amounts [52; 53].