**2. Glycine betaine and metabolism engineering**

Glycinebetaine is a quaternary ammonium compound that appears commonly in a large diversity of plants, animals and microorganisms, the first betaine discovered was trimethylglycine (**Figure 1**) named also N, N,N-trimethylglycine [8, 12]. The glycine betaine as a osmolytes is a crucial non-toxic molecule that is accumulated in various plant species under environmental stresses [15].

### **2.1 Glycine betaine biosynthesis**

GB synthesis begins with an essential molecule named choline, synthesized through three sequential adenosyl-methionine dependent methylations of phospho-ethanolamine catalyzed by the cytosolic enzyme phosphoethanolamine methyltransferase (phosphoethanolamine N-methyltransferase) [30]. In plant, the biosynthesis of GB is two steps of oxidation initiated with choline and then betaine aldehyde (**Figure 2**). In plant such as *Arabidopsis* the biosynthesis of choline can be resume by this following line: L-serine → ethanolamine → *O*-phosphoethanolamine → N-methylethanolamine phosphate → N-dimethylethanolamine phosphate → phosphocholine → choline [31–33]. Pursuing the transformation of N-methylethanolamine phosphate by phosphoethanolamine methyltransferase (PeMt) the byproduct differs according to the plant species, for instance in that stage the spinach produce choline like in *Arabidopsis* choline biosynthesis pathway, meanwhile in tobacco PeMt catalyzed a reaction that synthesize phosphatidylcholine in the first place then metabolized to choline [8, 15]. The first stage of GB biosynthesis is modulated by CMO which is an Fd-dependent monooxygenase with a Rieske-type iron–sulfur (2Fe-2S) cluster-binding motif. The second stage of GB

**Figure 1.** *N,N,N-trimethylglycine.*

**Figure 2.** *Diagram of GB biosynthesis in brief.*

biosynthesis is catalyzed by BADH, an enzyme belong to the superfamily of aldehyde dehydrogenases which is an NAD+ or NADP+ dependent [17, 34].
