**5.2 Cyanobactins biosynthesis**

Cyanobactin gene clusters are capable of encoding, two protease genes, A(Nterminal) and G(C-terminal) that are related to patA and patG genes from the patellamide biosynthetic pathway. A precursor peptide gene E being an homolog to patE which directly encodes cyanobactin structure that acts as a substrate for post translational modifications. Cyanobactin gene clusters may also encode homologs of PatD or PatF, denoted as D-protein and F-protein. Includes thiazoline/oxazoline dehydrogenases (responsible for the aromatization of the heterocycles to thiazoles and oxazoles, methyltransferases. This gene was proven to be essential for the synthesis of non-prenylated patellamides. Cyanobactins are classified into different groups based on a correspondence between genotypes and chemotypes.

## *5.2.1 Biosynthetic pathway*

Cyanobactin biosynthesis begins with the precursor E-peptide, which is composed of an N-terminal conserved leader sequence that is recognized by some of the modifying and cleaving enzymes. Cyanobactin genetic cluster may also employ more than one precursor peptide. Genetic cluster may contain upto10 precursor peptide gene. E-peptide that contains the enzyme recognition sequences, 1,4 hypervariable core regions may be present and dictate amino acid backbone of cyanobactins.

Cyanobactins are ribosomally synthesized and post-translationally modifies peptides produced in the ribosome. Biosynthetic enzymes for cyclic peptide synthesis are encoded in the *Prochloron* genome. Precursor peptides are posttranslationally modified by various enzymes adding the heterocycles derived from the cysteine, serine and threonine or isoprene units. Modifies peptides are cleaved from the precursor and cyclized to the natural products [39]. These products are capable of exhibiting combinatorial biosynthesis. Ribosomally synthesized and post translationally modified peptides combinatorial chemistry is made mainly because of the core peptide hypervariably, broad substrate [40] specificity, enzyme recognition sequences and modularity of post-translational elements. Many of these post-translational modifications are found in marine organisms. Mechanisms as well as the gene cluster involved in the formation of the thiazoline and oxazoline rings in cyanobactin are well studied. Patellamide pathway, coded by the pat gene cluster which is commomly expressed in *Prochloron* involves several enzymatic steps: Aminoacid heterocyclization, cleavage, peptide cleavage, peptide macrocyclization, heterocycle oxidation and epimerization. Some of the closely related products are also prenylated (**Figure 4(a**, **b**)).

In the presence of D-protein cyclodehydratase, heterocyclization of cysteins, serines or threonines will be directed by sequence recognition. A protease cleaves the precursor peptide RSII, leaving a free amine available for macrocyclization. G-protease splits the precursor peptide RSIII and causes the catalization of C-N macrocyclization. Other transformations may occur such as, prenylation of serine/ threonines and tyrosines/tryptophans residues catalyzed by the PatF class of prenyltransferases. Oxidation of heterocycles to oxazoles and thiazoles when oxidized, domain is present within the G gene or separate and geranylation [41].

#### *5.2.2 Heterocyclase*

Heterocyclase accompanies heterocyclization of cysteins, serine and threonine residues to thiazolines or oxazolines and eliminates water. Cyanobactins heterocyclases D has been studied in partellamide and trunkamide pathways. Heterocyclases D in both pathways is ATP dependent. An adenylase mechanism has been proposed for TruD, from trunkamide.

An adenylase mechanism has been proposed for TruD, from trunkamide pathway, whose crystal structure presents as the three, domain protein. Enzyme progreesivity requires the presence of a lead protein to be attached to the core, indicating that heterocyclization occurs before cleavage and macrocyclization of the precursor peptide.

**Figure 4.** *a)bistratamide M, b) bistratamide N produced from c) Trunkamide A.*

The sequence element present in the lead sequence is responsible for heterocyclization. Cyanobactin pathway encoding a heterocyclase modifies a oxidase domain responsible for oxidation of thiazolines and oxazoles to thiazoles and oxazoles.
