General Introduction: Fungal Secondary Metabolites Gene Clusters: Strategies to Activate the Biosynthesis of Non-expressed and Novel Compounds

Secondary metabolites of fungal species are produced by biosynthetic genes clusters. Polyketide synthase (PKS), non-ribosomal peptide synthetase (NRPS), or hybrid NRPS-PKS hybrids (HPN) are a part of these clusters and usually catalyze the main backbone structures of many natural compounds. Since their production is no absolutely required for the survival of the organism, their expression and regulation are most often related to particular environmental conditions. The activation strategies, with the support of genome sequences, range from molecular and epigenetic modifications to modifications of natural habitats such as microorganism co-interactions or nutrients accessibility. Examples of successfully applied techniques for awaking the silent gene clusters are broadly discussed, including their advantages and limitations. 1 1.1. Penicillium chrysogenum 3 1.1 Penicillium chrysogenum The filamentous fungus Penicillium chrysogenum is a major industrial producer of the β-lactam antibiotic penicillin. Penicillin was discovered in by Alexander Fleming in 1928, and since then it has found its application as antimicrobial agent in human health care. The current industrial high β-lactam yielding strains emerged from classical strain improvement programs over the last years (Berg et al. [2008]). In 2008, the genome (32.19 Mb) was sequenced, revealing an unexploited reservoir of nonribosomal peptide synthetases and polyketide synthases genes encoding potential bioactive compounds (Berg et al. [2008]). 1.2 Fungal Megasynthases A large number of natural products known as secondary metabolites require sophisticated multi-domain enzymes for their biosynthesis: Examples of these multifunctional megasynthases include nonribosomal peptide synthetases (NRPS), polyketide synthases (PKS) and the NRPS-PKS hybrids (HPN). They constitute one of the largest protein complexes within microbial cells. The study on fungal biosynthetic gene cluster studies has lagged behind as compared to the bacterial enzymes, which have been studied extensively. Despite the large size of fungal genes, which contain introns, and challenges in cloning, several analysis techniques used on bacteria can still be applied to fungi. 1.2. 1 NonRibosomal Peptide Synthetase (NRPS) In addition to protein amino acids, NRPSs can utilize a variety of nonprotein amino acids to generate peptides, which are different from linear mRNA-directed sequences of ribosomal derived sequences polypeptides (Döhren [2004]). In contrast to bacteria in which several NRPSs biosynthetic genes are organized in an operon, fungi may use a single NRPS gene to synthesize the product (Weber et al. [1994]). However, the size of fungal NRPS genes is often much larger than that of the bacterial genes (Finking and Marahiel [2004]). Mechanisms of NRPS biosynthesis are classified into three categories: (i) linear the number and sequence of the modules in the NRPS matches the number and order of amino acids in the peptide, (ii) iterative the modules or domains of the synthetase are used more than once to synthetize the peptide, and (iii) nonlinear the arrangement of the modules does not match the sequence of amino acids (Cane and Walsh [1999]). Fungal NRPS pathways tend to be linear (Figure 1.1A) although iterative pathways are suggested for the synthesis of siderophores, as discussed in Chapter 5 of this thesis. The three basic enzymatic functions at the core of each NRPS module are adenylation, thiolation, and condensation (Figure 1.1A). The first structure of 1 4 1. General introduction eukaryotic adenylation domain of the siderophore-synthesizing NRPS (SidN) of the endophytic fungus Neotyphodium lolii, was identified to specifically accommodate N’-cis-anhydromevalonyl-N’-hydroxy-L-ornithine (cis-AMHO) in its binding pocket (Lee et al. [2010]). Structural insights in the A domain of the fungal cis-AMHO activating domain (Hur et al. [2012]), and the bacterial phenylalanine-activating domain (PheA) of the gramicidin S (Conti et al. [1997]) and 2,3-dihydroxybenzoic acidactivating domain (DhbE) of the bacillibactin (May et al. [2002]) demonstrate the presence of large N-terminal domain and a small C-terminal domain, with the active site located at the junction of these two subdomains (Hur et al. [2012]). The initial determinant of substrate selectivity, the adenylation domain (A), recognizes and activates their cognate’s amino acids in a double mode process: while ATP is consumed the cognate amino acid is selectively bound and converted into an aminoacyl adenylate intermediate. The activated substrate is transferred to the thiol group of the 4’-phosphopantetheine on the Peptidyl Carrier Protein (T) domain and covalently bound through a thioester linkage (Stachelhaus et al. [1995]). The T domain mediates the transfer of amino acids or peptidyl substrates through the catalytic step of NRP synthesis (Linne et al. [2001]; Finking and Marahiel [2004]). Prior to NRP biosynthesis, T domains are post-translational modified (Figure 1.2) by Phosphopantetheinyl Transferases (PPTases), which is essential for converting the inactive apo-T to the active holo-T. The 4’PP cofactor which is bonded as a thioester is transferred to a conserved serine residue of the T domain by the PPTase in an Mgdependent reaction. The 4’PP cofactor acts as a flexible arm to allow the bound amino acyl and peptidyl substrate to be shuttle between the Ts of each module during the peptide elongation process Finking and Marahiel [2004]; Hur et al. [2012]). The regeneration of the functional 4’-phosphopantethine arm of misprimed T is catalyzed by a type II thioesterases (TEII), which is encoded by a distinct gene associated with the NRPS cluster (Schwarzer et al. [2002]). Conversion from the apo to holo form is performed by 4’PP transferases (Figure 1.2), as shown for the PPTase from B. subtilis (Sfp) group, can load both CoA and acyl-CoA derivatives as 4’PP donors (Weinreb et al. [1998]). Acyl-CoA or any incorrect amino acid loaded on the T results in misprimed NRPSs. This commonly occurring mispriming of the T requires a TEII restoring activity. TEII recognizes and hydrolyses the T bound stacked aminoacyl and peptidyl substrates (Schwarzer et al. [2002]). The cleaning activity after the misaminoacylation prevents further processing of wrong loaded amino acid and prevents cleavage of the correctly growing peptide (Schwarzer et al. [2002]). Next to the cleanup function, the TEII enzyme plays a critical role in the deblocking after T mispriming (Schwarzer et al. [2002]) (Figure 1.2). 1 1.2. Fungal Megasynthases 5 T C A T T A C C A TE A) NRPS