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Trehalose lipids are glycolipids containing trehalose hydrophilic moiety. In fact, structurally, trehalose lipids consist of a hydrophilic moiety (trehalose) formed by two glucose units linked through the α,α-1, 1-glycosidic linkage and a hydrophobic moiety represented by chains of fatty acids, such as succinic, octanoic, decanoic, and mycolic acids (Paulino et al. 2016). Trehalose is a non-reducing disaccharide, it is the carbohydrate group of the cell wall glycolipids in Mycobacteria and Corynebacteria (Franzetti et al. 2010). Trehalose displays thermostability, resistance to acid hydrolysis and non-reactivity to the Maillard reaction.

Trehalose lipids were discovered in 1933 (Anderson and Newman 1933) and purified in 1956 (Soberón-Chávez 2011). They are among the best known biosurfactants, as rhamnolipids and sophorolipids, in both composition and activity (Soberón-Chávez 2011). In comparison to other microbial glycolipids, trehalose lipids have shown contrasting results and achievements in both cases of inhibition and enhancement of biodegradation rates (Kügler et al. 2014; Silva et al. 2014). They can reduce the surface tension of aqueous solutions and the interfacial tension between aqueous and oil phases to levels observed with synthetic surfactants, and have low critical micelle concentrations.

A wide variety of trehalose lipids can be found in several forms (Figure 1.4), namely: trehalose monomycolates, dimycolates, trimycolates, non-ionic acylated trehalose derivatives, anionic trehalose tetraesters, and succinoyl trehalolipids (Franzetti et al. 2010; Kügler et al. 2014; Paulino et al. 2016).

Figure 1.4 The main chemical structure of trehalose lipids (adaped from Franzetti et al. 2010).

Therefore the different types of trehalose lipids can be classified into two general subclasses: The first group is the 6,6-trehalose diesters such as fatty acid trehalose diesters (TDEs, 1), trehalose dicorynomycolates (TDCMs, 2) and trehalose dimycolates (TDMs, 3). The most quantified trehaloselipid is trehalose 6,6´-dimycolate which is an a-branched chain mycolic acid esterified to the C6 position of each glucose (Franzetti et al. 2010). Although TDEs are the simplest glycolipids found in the trehalose 6,6´-diester series, they can be differentiated by lipid length and branching of the fatty acids, which can be divided into anionic or non-anionic. It is known that anionic surface-active TDEs have a higher surface activity than non-ionic (Silva et al. 2014).

The second group is the 2,3-trehalose diesters such as diacyl trehalose sulfates (STL) and sulfolipid-1 (SLL) (Kügler et al. 2014). One of the studies showed that succinoyl trehalose lipids (SCTLs) are promising biosurfactants since they can be efficiently produced from n-alkanes by Rhodococcus bacteria and recovered by precipitation. Among SCTL producers, Rhodococcus sp. SD-74 provided the best yield (40 g L^-1) from n-hexadecane under high osmotic conditions. These SCTLs also have also been demonstrated to show growth inhibition against the influenza virus and cell-differentiation induction towards human leukemia cells (Isoda et al. 1997; Sudo et al. 2000).

Mycolic acids are 2-alkyl-3-hydroxy fatty acids of high molecular mass, present exclusively in the cell envelope of bacteria of the mycolata taxa, including Rhodococcus species (Paulino et al. 2016).

The synthesis of mycolic residues, such as trehalose lipids, is believed to be a Claisen condensation. The key reaction for the formation of trehalose-6-phosphate is catalyzed by trehalose-6-phosphate synthetase (TPS). TPS links the two D-glycopyranosyl units at C1 and C1ʹ (Franzetti et al. 2010). Uridine diphosphate-glucose and glucose-6 phosphate participate in that reaction, forming trehalose phosphate (Franzetti et al. 2010):

In alkanotrophic Rhodococci, TPS is induced by n-alkanes. Further reactions for the formation of trehalose lipids have been clearly elucidated for trehalose dimycolates (TDMs) in M. tuberculosis. The mycobacterial glycolipid has been proposed to play a key role in the immunopathogenesis of tuberculosis (Hoq et al. 1997; Jain and Roy 2009; Paulino et al. 2016). The production of threalose lipids established during the final stages of the cell wall synthesis of M.tuberculosis. In this step newly synthesized mycolic acids are transported and attached to the peptidoglycan-arabinogalactan complex of the cell wall, followed by the formation of trehalose dimycolates. This formation occurs in four different reactions (Franzetti et al. 2010, p. 25; Takayama et al. 2005).

The first reaction of the synthesis, is the transfer of the mycolyl group to D-mannopyranosyl-1-phosphoheptaprenol by an as-yet unidentified cytoplasmic mycolyltransferase, forming 6-O-mycolyl-β-D-mannopyranosyl-1-phosphoheptaprenol (Franzetti et al. 2010; Takayama et al. 2005).

The mycolyl group is then transferred to trehalose 6-phosphate by a membrane-associated mycolyltransferase II to form trehalose mono mycolate (TMM)-phosphate. After a dephosphorylation, the resulting product is TMM (Franzetti et al. 2010) (reaction 2) (Figure 1.5).

Figure 1.5 Synthesis of trehalose dimycolates, in four different reactions, the first two reactions are performed inside the cell, the third reaction forms the transport out of the cell and the fourth is outside the cell. Pks13: polyketide synthase 13; Man-P-heptaprenol: D-mannopyranosyl-1-phosphoheptaprenol; Myc-PL: 6-O-mycolyl-β-D-mannopyranosyl-1-phosphoheptaprenol; Treh 6-P: trehalose 6-phosphate; TMM: trehalose mono mycolate; TDM: trehalose dimycolate.

The third reaction consists of an extracellular transportation of TMM via an ABC-transporter.

In the fourth reaction trehalose dimycolates(TDM) (Figure 1.5) and arabinogalactan-mycolate are formed from TMM through an extracellular mycolyltransferase (Ag85/Fbp/PS1) (Franzetti et al. 2010; Takayama et al. 2005).

The key reaction for synthesis of the final resulting sugar residue, trehalose-6-phosphate, is catalyzed by a trehalose-6-phosphate synthetase (TPS) which links the two D-glycopyranosyl units at C1 and C10. UDP-glucose and glucose-6-phosphate act as the immediate precursors. In fact, the trehalose moiety and the fatty (mycolic) acid moiety of trehaloselipid molecules are synthesized independently and are subsequently etherified. Trehalose monomycolate was claimed to be an intermediate of TDM biosynthesis. The formation of the mycolate is considered to be a Claisen-type condensation of two fatty acids, a carboxylated acyl-coenzyme A and an activated acyl chain to yield a 3-oxo intermediate, which would then be reduced to form mycolic acid (Kuyukina and Ivshina 2010)

The additional reactions involved in the synthesis of THL have been elucidated for TDMs in which the production happens in the final stages of the synthesis of the cell wall. In this phase, the newly synthesized mycolic acids are transported and attached to the peptidoglycan-arabinogalactan complex of the cell wall, followed by the formation of TDM which occurs by four different reactions.

The biosynthesis proceeds when the first reaction occurs through the transmission of the mycolyl group to D-mannopyranosyl-1-phosphoheptaprenol by a proposed cytoplasmic mycolyltransferase I to form 6-O-mycolyl-b-D-mannopyranosyl-1-phosphoheptaprenol (Myc-PL) (Figure 1.6). For the second reaction the mycolyl group is transferred to trehalose 6-phosphate by a membrane-associated mycolyltransferase II to form trehalose mono mycolate (TMM)-phosphate and, after dephosphorylation, results in the formation of TMM. The third reaction happens when TMM is transported outside the cell by an ABC transporter. A rapid and efficient transfer of TMM from the inside to the outside of the cell is necessary for the synthesis of cell wall arabinogalactan-mycolate and TDM. The fourth and last reaction occurs by the action of the extracellular mycolyltransferase called Ag85/Fbp/PS1, the final products of the cell wall arabinogalactan-mycolate and TDM are formed from TMM (Franzetti et al. 2010).

Figure 1.6 Scheme of trehalose-mono- and -dimycolate synthesis from n-alkanes (adapted from Kuyukina and Ivshina 2010).