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).
Production of Glycolipids
The kinetics of glycolipids production has considerable variations among various systems. Only a few generalizations can be made, by grouping kinetic parameters into the following types: (1) growth-associated production, (2) production under growth-limiting conditions, (3) production by resting or immobilized cells, and (4) production with precursor supplementation (Desai and Banat 1997; Santos et al. 2016).
Examples for the growth-associated production (1) can be the synthesis of rhamnolipids by some Pseudomonas spp strains. The biosurfactant production is in direct relation to growth and substrate utilization. The excreted emulsan-like substance accumulates on the cell surface during the exponential phase of growth (Desai and Banat 1997).
The production under growth-limiting conditions (2) is characterized by an accentuated increase in the biosurfactant level as a result of the limitation of one or more medium components. An example is the production of rhamnolipids by Pseudomonas aeruginosa, particularly when the cells become limited for nitrogen or iron. The limitation results in an overproduction of biosurfactant when the culture reaches the stationary phase of growth (Desai and Banat 1997; Kumari et al. 2010).
Production by resting or immobilized cells (3), describes a biosurfactant production with no cell multiplication; whereby the cells nevertheless continue to utilize the carbon source for biosurfactant synthesis. For example the gram-positive bacterium Arthobacter crystallopoietes was shown to directly produce trehalose from maltose by resting cell reaction (Desai and Banat 1997; Seo and Shin 2011).
Santos et al. (2016) report that the addition of biosurfactant precursors (4) to the growth medium can cause qualitative and quantitative changes in the final product (Ashby et al. 2008; Santos et al. 2016).
Furthermore different factors, such as the carbon source, nitrogen source or environmental conditions can influence the glycolipids production and will be focus on next points in the case study of trehalose lipids.
1.4 Production of Trehalose Lipids
Trehalose lipids are the basic component of the cell wall glycolipids in Mycobacteria and Corynebacteria and are known to be produced by Gram-positive bacteria, as Actinomycetales such as Mycobacterium, Nocardia or Corynebacterium and they differ in the structure, size and degree of saturation (Cappelletti et al. 2020; Franzetti et al. 2010).
Rhodococcus erythropolis DSM43215 was reported for the first time as a producer of trehalose lipids with chain length (C20–C90) of the esterified fatty acids in 1982 (Kretschmer et al. 1982) and in 1983. These trehalose lipids were characterized as trehalose-6-monocorynomycolates, trehalose 6,6´-diacylates and trehalose-6-acylates (Kretschmer et al. 1982). A non-ionic trehalose lipid, consisting of one major and ten minor components was produced using Rhodococcus strain H13-A (Bryant 1990). Other types of trehalose lipids, including mono-, di- and tri-corynomycolates, mono-, di-, tetra-, hexa- and octa-acylated derivatives of trehalose, trehalose tetraesters and succinoyl trehalose lipids were produced, in the following years, using R. erythropolis and R. ruber (Esders and Light 1972; Uchida et al. 1989; White et al. 2013). The large-scale production of trehalose lipids is very challenging. The effective use of biosurfactants is limited by the high cost of production and complex downstream processing (Franzetti et al. 2010). In addition, when Rhodococcus strains are used for this purpose, the major problem is the fact that trehalose lipids are associated with the cell walls leading to an increase in the costs of downstream processing and recovery (Espuny et al. 1996). Furthermore, several studies have shown that the production of trehalose lipids can either be extracellular, or cell-bound, depending on the growth conditions. Experiments presented that R. erythropolis ATCC 4277 was able to produce extracellular trehalose lipids, which were all released into the medium, using glycerol as the sole carbon source, while the production was partially cell-bound when cells were grown on n-hexadecane (Ciapina et al. 2006; Franzetti et al. 2010).
The yields of trehalose lipids appear to be very low compared to sophorolipids, rhamnolipids and mannosylerythritol lipids. They are often bound to cell surfaces, which reduces the production yield and increases downstream costs. Three basic strategies have been adopted to make the fermentation process cost-competitive: (i) using cheap and waste substrates, (ii) development of efficient bioprocesses, such as optimization of fermentative conditions, (iii) development of overproducing mutant or recombinant strains (Franzetti et al. 2010; Uchida et al. 1989). One study has shown that a high phosphate buffer concentration and neutral pH conditions optimize the production of succinoyl trehalose lipids in R. erythropols SD-74 up to 40 g L-1 (Franzetti et al. 2010; Uchida et al. 1989).
In the bioproduction of trehalose lipids many factors need to be considered. Firstly, the microorganism must be carefully chosen to produce the trehalose lipid required, with special attention to the purpose of its application. The microbial production can be influenced by different factors such as media composition (e.g., carbon and nitrogen sources, salt composition or use of extract in culture broth), bioreactor and environmental conditions (e.g. temperature, pH, oxygen, speed). As well as sophorolipids, for trehalose lipids, there is the possibility of further chemical modifications to obtain novel analogues with diverse properties.
1.4.1 Microorganisms
Trehalose lipids are made up of a disaccharide, trehalose, linked by an ester bond to a-branched b-hydroxy fatty acids (Lang and Philp 1998). The a-branched b-hydroxy fatty acids are connected at the C6 and C60 of the carbohydrate structure in the case of the trehalose dimycolates and at C6 for the monomycolates; other structure types have also been reported (Lang and Philp 1998). The production of trehalose lipids is associated with most species of Mycobacterium, Rhodococcus and Corynebacterium.
Trehalose lipids are usually produced by Gram-positive bacteria. Many studies display use of a different microorganisms, such as Arthrobacter, Rhodococcus, Gordonia (Lang and Philp 1998), Mycobacterium, Nocardia and Corynebacterium, which allowed trehalose lipids which differ in structure, size, degree of saturation and mycolate group (Franzetti et al. 2010).
Nevertheless, some of the producing bacteria are pathogenic mycobacteria (including