*2.1. Biosynthesis of Rapamycin*

Rapamycin is a 31-membered ring macrolide produced by *Streptomyces hygroscopicus* isolated firstly from the soil of Easter Island (Chile) in the South Pacific Ocean. It is a hydrophobic compound and known as an antifungal compound against *Candida albicans*, *Cryptococcus neoformans*, *Aspergillus fumigatus*, *Fusarium oxysporum*, and some pathogenic species from the genus *Penicillium*. The antifungal mechanism of this compound has been described by diffusing into the cell and binding to intracellular receptor immunophilin FKB12. The FKBP12-rapamycin complexes inhibit enzymes required for signal transduction and cell growth. These enzymes are TOR (target of rapamycin) kinases that are conserved and very important for cell cycle progression. Interestingly, it was also reported that rapamycin has not only antifungal activity, but also anticancer and immunosuppressant activity [8,27,38,39].

**Figure 3.** Some of the macrolides produced by *Streptomyces*.

Rapamycin is synthesized by type I PKS rapamycin synthase (RAPS) [40]. The rapamycin-PKS gene cluster (*rapPKS*) is 107.3 kb in size and has three remarkably large ORFs (open reading frames), *rapA*, *rapB*, and *rapC* which encode multifunctional protein RAPS1 (~900 kDa), RAPS2 (~1.07 MDa), and RAPS3 (~660 kDa), respectively. Protein RAPS1 comprises four modules for polyketide chain extension; protein RAPS2 contains six modules responsible for continuing the process of polyketide chain elongation until C-16; and RAPS3 possesses four modules that have a role in completing the polyketide fraction of the rapamycin molecule. Overall, these three giant proteins encompass 70 domains or enzymatic functions, and because of this, rapamycin PKSs are considered as the most complex multienzyme system discovered so far [26,27,34].

In rapamycin PKSs, there is a loading domain (LD) before module 1. In LD, there are three domains, i.e., coenzyme A ligase (CL), enoylreductase (ER), and acyl carrier protein (ACP) domain, which are considered to play a role in activating, reducing a free shikimic-acid-derived moiety starter unit, and finally passing it to the ketosynthase (KS) domain of the first module, respectively. The extender units required for producing rapamycin are malonyl-CoA and methylmalonyl-CoA. The mechanism of transferring from the last domain in rapamycin PKSs and cyclisation of polyketide molecule is assisted by pipecolate-incorporating enzyme (PIE), as depicted in Figure 4. This enzyme (170 kDa) is encoded by gene *rapP*, which is also located in the *rapPKS* gene cluster [26,27,34].


**Table 1.** Some polyketides produced by *Streptomyces* and their type I PKSs.

#### *2.2. Biosynthesis of Avermectin*

Avermectin is a 16-membered ring macrolide and one of the notable anthelmintic compounds produced by *Streptomyces avermitilis* [41,49]. The biosynthesis of avermectin involves type I PKSs (AVES1, AVES2, AVES3, and AVES4). AVES1 (414 kDa) contains one loading domain and two modules; AVES2 (666 kDa) consists of four modules; AVES 3 (575 kDa) comprises three modules; and AVES4 (510 kDa) has three modules. The process of avermectin biosynthesis includes assembling of the polyketide-derived initial aglycon (6, 8a-seco-6, 8a-deoxy-5-oxoavermectin aglycons) by AVES1–4, alteration of the initial aglycon to avermectin aglycons, and, as the last step, the glycosylation of avermectin aglycons to produce avermectins. The starter unit for avermectin biosynthesis is isobutyryl-CoA (derived from valine) or 2-methylbutyryl-CoA (derived from isoleucine), whereas the extender units involved in the production of avermectin are seven malonyl-CoAs (for acetate units) and five methylmalonyl-CoAs (for propionate units). The nucleotide sequence of the avermectin biosynthetic gene cluster comprises 18 ORFs spanning a distance of 82 kb, in which four large ORFs encode the avermectin polyketide synthase (AVES1, AVES2, AVES3, and AVES4) and some of the 14 ORFs encode polypeptides having important roles in avermectin biosynthesis [41].
