**4. NRPS and PKS clusters as gene sources of antimicrobial secondary metabolites**

Non-ribosomal peptide synthetases (NRPS) and polyketide synthetases (PKS) metabolic pathways encompass a cluster of multi domain subunits, where each subunit performs a separate enzymatic activity. The coordinated activity of these multi domain units in a mega synthetases complex performs the synthesis of Non ribosomal peptides (NRPs) and three different Polyketides (PKs)-secondary metabolites that exhibit clinically valuable biological activities as anti-microbial, anti-fungal, anti-tumour, anti-parasitic, and immunosuppressive agents [30]. The NRPs biosynthesis on NRPS enzyme complex is done through ordered arrangement and addition of amino acid monomers whereas the PKs biosynthesis on PKS enzyme complex follows the sequential addition of 2C ketide unit derived from thioester of acetate precursors or other short chain carboxylic acids [31]. These enzyme clusters are either modular (NRPS and modular type I PKS) or iterative (iterative type I PKS, type II PKS and type III PKS). In case of NRPS and modular type I PKS, each module is designed to hold an obligatory or a minimal core domain. The minimal core domain in NRPS module consists of an Adenylation domain (A) - for selective activation of amino acid from a pool of precursor amino acids, Condensation domain (C) for peptide bond formation and chain elongation Thiolation/Peptidyl carrier protein (T/PCP) domain with a phosphopantetheine group that transfer the starter monomer units or an extender growing chain to different catalytic sites in a mega enzyme complex. Likewise a modular type I PKS obligatory or a minimal core domain includes an Acyl transferase domain (AT) for starter/extender unit loading of acyl-CoA on acyl carrier protein (ACP) and a Ketoacyl synthase domain (KS) for condensation and decarboxylation of acyl CoA starter or extender units. In both cases of NRPs and PKs biosynthesis, the Thioesterase domain (TE) catalyses the release of full length NRPs and PKs [32–36]. There are few starter or extender units for biosynthesis of PKs however a larger pool of about 50 different amino acid precursors- natural or unnatural act as starter or extender units for biosynthesis of NRPs. Thus though the substrate specificity for PKS is not a complex process, the prediction of substrate specificity for NRPS is a challenging task [31]. The corresponding modules in NRPS and modular PKS are held together by short peptide chains called linkers that establish functional communication between modules [32]. In addition to core domains of NRPS and PKS, some non obligatory but essential auxiliary domains can be loaded mostly on elongation modules. These auxiliary domains include ketoreductase (KR), dehydratase (DH), or enoylacyl reductase (ER) enzymatic domains for partial and/or complete reduction of keto groups. These ketide chain length modifications enhance the structural complexity and increase diversity of mature PKs [37]. The auxiliary domains loaded on the modules of NRPS include cyclization of peptide chain into thiazoline or oxazoline rings, oxidation of thiazolines and oxazolines to thiazoles and oxazoles, reduction into thiazolidines and oxazolidines, amino acid epimerization into D isomers. Other processing modification of final NRPS chain peptide includes acylation, glycosylation, hydroxylation and halogenations [38–39]. Notably, it is reported that actinobacteria have a higher number of these biosynthetic genes [40]. These genes upon translation

form modular NRPS and PKS, non modular iterative PKS and type III PKS. The modular genetic engineering of NRPS and PKS and biochemical and bioinformatic investigation of iterative PKS to unlock and discovery more iterative enzymes complexes of relative function are gaining attention. Addition or deletion of whole modules in an enzyme complex or most importantly an auxiliary domain addition or deletion in a module alters the chain length and modify the enzyme complex. This if executed successfully may give rise to diverse novel secondary metabolites, many of which could work as potential antimicrobials. Amalgamation of NRPS and PKS to form a Hybrid NRPS-PKS synthesised secondary metabolite are also successfully engineered [41–43].

The antiSMASH (antibiotics and secondary metabolites analysis shell) database is a handy tool in secondary metabolite gene cluster prediction analysis of bacterial genomes, it can however also be used against fungal and plant complete or draft genomes. Genome mining by antiSMASH gives an overview of the antimicrobial potential of different gene clusters along the genomic stretch of a given query organism (e.g. NRPS, different types of PKS, hybrid NRPS-PKS, lanthipeptides, siderophores, ectoines and terpenes). The antiSMASH results depict the type of gene cluster to which query is most similar to along with the percentage similarity. It searches a query sequence against the MIBiG database of different characterised gene clusters, selects the best possible hit, determines the start and stop origins or cluster coordinates along the genome length and percentage statistics of top hit to the query sequence.

## **5. Insect microbiome: symbiotic actinomycetota as antimicrobial sources**

The mechanism of defensive symbiosis is employed by insects and this association with antimicrobial producing bacterial symbionts is critical for insect survival. Until recently soil microbiome was considered the only rich source of actinobacteria. Metagenomic analysis for actinobacteria from soil, fresh water, oceanic and insect associated microbiome revealed that the number of streptomyces reads per megabase (rpM) to be 172.72 rpM, 47.49 rpM, 24.65 rpM, 129.32 rpM- suggesting that insect microbiome also serve as the rich source of actinobacteria. Further when compared to other sources, the insect associated streptomyces exhibit higher inhibition against gram positive, gram negative and fungal microorganisms and insect streptomyces are inhibitory against antimicrobial resistant pathogens more than the soil streptomyces. Antimicrobial defensive symbiosis is shown in wasps, beetles, fungus growing ants where actinobacteria live in symbiosis with these insects; produce several antibacterial, antifungal and antimalarial substances akin to that used in human system. A discovery lead by Marc G. Chevrette et al. and published in 2019 exploited the insect microbiome diversity for antimicrobial detection. The studies described Cyphomycin-a new antimicrobial molecule against MDR pathogens. Genomic and metagenomic revelations show that the streptomyces from insect micro biota have immense potential to synthesise bioactive metabolites. The inhibitory secretions by Actinomycetota stop the spread of pathogenic microorganisms in insects and help them successfully flourish different microbe dwelling habitats [44]. Despite the above mentioned habitat sources for these predominant antimicrobial producing microorganisms, actinobacteria have also been found to grow in other extreme habitats like hyper saline and hyper alkaline marine and terrestrial regions, hyper arid deserts, volcanoes and glaciers. But for the sake of brevity we have limited our discussions to only the sources highlighted in this chapter. Current and future research on all extreme sources will delve deep into the

bioactivity evaluation of these extremophilic actinobacteria and pave way for isolation and characterisation of new drugs from these still to be believed as golden drug reserves for next generation antibiotic discovery [45–48].
