A wide variety of biological activity can be found in the realm of secondary metabolites. The tripyrrole prodigiosin illustrates this perfectly with activities ranging from antimicrobial to immunosuppressive and anticancer. Microorganisms producing the red pigment prodigiosin were first isolated more than 150 years ago. Yet, its structure was only elucidated in the 1960s and the biosynthetic pathway remained mainly unknown until the 2000s. This secondary metabolite results from a bifurcated pathway where monopyrrole 2-methyl-3-pentyl-1H-pyrrole (MAP) and bipyrrole 4-methoxy-1H,1'H-2,2'-bipyrrole (MBC) are produced independently before condensation. As MBC is an intermediate in the synthesis of other natural products, the enzymes involved in its formation have been well characterised. In contrast, the three enzymes – PigD, PigE and PigB – involved in the formation of MAP are specific to the biosynthesis of prodigiosin and less is known about them. This thesis focuses on the latter two enzymes. PigE was first described as a transaminase catalysing the transformation of 3-acetyloctanal into 3-acetyloctylamine (which cyclises to form dihydroMAP) and this activity has been confirmed by feeding intermediates to various gene-knockout strains. However, in vitro studies have demonstrated that 3-acetyloctanal could not be the product of PigD. In addition, bioinformatics analysis of its amino acid sequence showed that PigE has two domains: a transaminase Cterminal moiety and an unspecified N-terminal one, which we propose is a thioester reductase that converts a 3-acetyloctanoyl thioester to 3-acetyloctanal. Attempted chemical complementation of a pigD-knockout strain of Serratia using synthetic thioester, carboxylic acid and aldehyde substrates showed that both the thioester and the aldehyde can be used for pigment production, indicating that a thioester reductase is involved in prodigiosin biosynthetic pathway. Furthermore, the PigE protein was expressed in a heterogeneous host, purified and submitted to a number of activity and kinetic assays, which demonstrated that it can reduce a 3-acetyloctanoyl thioester substrate. The oxidation of dihydroMAP to MAP had previously been shown to be catalysed by an FADdependent oxidase PigB. The kinetics of HapB, a homologue of PigB had been studied by a previous group member. To take this project further we studied the substrate flexibility of the enzyme and used it to form new analogues of prodigiosin by mutasynthesis. Ten analogues of dihydroMAP with modifications either in the C2 or C3 positions were synthesised. Both extensions and truncations in the length of the chain at the C3 position could be accepted, whereas alkyl chains longer than 3 carbons on the C2 position could not be accommodated. Similar results were found in vivo when the analogues were fed to a pigD-knockout strain of Serratia, showing that PigB and the condensing enzyme PigC shared similar flexibility. Eight analogues of prodigiosin were hence obtained and their antimicrobial activities against Gram-positive and Gram-negative bacteria were assessed.