Penicillium oxalicum S-adenosylmethionine synthetase is essential for the viability of fungal cells and the expression of genes encoding cellulolytic enzymes

Abstract

As the universal methyl donor for methylation reactions, S-adenosylmethionine (AdoMet) plays an indispensable role in most cellular metabolic processes. AdoMet is synthesized by AdoMet synthetase. We identified the only one AdoMet synthetase (PoSasA) in filamentous fungus Penicillium oxalicum. PoSasA was widely distributed in mycelium at different growth stages. The absence of PoSasA was lethal for P. oxalicum. The misregulation of the PoSasA encoding gene affected the synthesis of extracellular cellulolytic enzymes. The expression levels of cellobiohydrolase encoding gene cbh1/cel7A, β-1-4 endoglucanase eg1/cel7B, and xylanase encoding gene xyn10A were remarkably downregulated as a result of decreased PosasA gene expression. The production of extracellular cellulases and hemicellulases was also reduced. By contrast, the overexpression of PosasA improved the production of extracellular cellulases and hemicellulases. A total of 133 putative interacting proteins with PoSasA were identified using tandem affinity purification and mass spectrometry. The results of functional enrichment on these proteins showed that they were mainly related to ATP binding, magnesium ion binding, and ATP synthetase activity. Several methyltransferases were also observed among these proteins. These results were consistent with the intrinsic feature of AdoMet synthetase. This work reveals the indispensable role of PoSasA in various biological processes.

Introduction

In prokaryotes and eukaryotes, S-adenosylmethionine (AdoMet, also known as SAM and SAMe) is the universal methyl donor for methylation reactions and plays an indispensable role in most cellular metabolic processes. AdoMet is widely recognized as a critical intermediary metabolite to control central metabolism (Kim et al., 1995). AdoMet serves as a methyl donor for lipids, nucleic acids, and proteins and as a precursor molecule in pathways such as transmethylation, aminopropylation, and transsulfuration (Lieber and Packer, 2002). For example, AdoMet participates in contributing three methyl groups in the synthesis of phosphatidylcholine from phosphatidylethanolamine (Malakar et al., 2006). It is also a core inhibitor of the methionine biosynthetic pathway in Escherichia coli (Yocum et al., 1996).

The formation of AdoMet is catalyzed by AdoMet synthetase (EC 2.5.1.6), which is also known as methionine adenosyltransferase (MAT) (Graham et al., 2000). AdoMet synthetase catalyzes the biosynthesis of AdoMet by reacting methionine (a non-polar amino acid) and ATP (the primary currency of energy). With the degradation of MgPPPi to MgPPi and Pi, the complete tripolyphosphate chain and MgATP enable a displacement reaction to take place in order to produce AdoMet and magnesium tripolyphosphate as enzyme-binding intermediates (Markham et al., 1987). The comparison of protein sequences of different AdoMet synthetases from bacteria, fungi, archaea, and mammals shows that they have an evolutionarily conserved core structure, including the motifs related to the binding of methionine, ATP, and pyrophosphate (Sanchez-Perez et al., 2004), as well as active-site catalytic amino acids (Reczkowski et al., 1998).

Different species have different numbers of AdoMet synthetase encoding genes. Mammalian cells possess three AdoMet synthetases, MAT1A, MAT2A, and MAT2B, with different functions and distinct expression profiles (Majano et al., 2001). Saccharomyces cerevisiae possesses two distinct AdoMet synthetases (SAM1 and SAM2), which share partial sequences and overlapping functions. However, the expression of SAM1 and SAM2 is regulated differently: the regulation of SAM1 expression is identical to that of other genes implicated in AdoMet metabolism, whereas the regulation of SAM2 is subject to inositol–choline regulation similar to genes encoding enzymes involved in phospholipid biosynthesis (Thomas & Surdin-Kerjan 1991; Kodaki et al., 2003). S. cerevisiae SAM1 and SAM2 also have functional differentiation: losses of SAM1 and SAM2 have different effects on S. cerevisiae genome stability (Hoffert et al., 2019). Although the functions of AdoMet and AdoMet synthetases have been described in S. cerevisiae and humans, few studies have reported about their functions in filamentous fungi. Except for the discovery that Aspergillus nidulans AdoMet synthetase (AnSasA) can regulate sporogenesis and the production of secondary metabolites (Gerke et al., 2012), and variation in AdoMet levels in Neurospora crassa modifies the flux of AdoMet-dependent metabolic pathways (Mautino et al., 1996), reports on the biological functions of AdoMet synthetase in other filamentous fungi are lacking.

Fungi play a major role in the global carbon cycle because of their ability to utilize plant biomass by producing a broad range of degrading enzymes (Benocci et al., 2017). Penicillium is a group (genus) of mold found worldwide. Ascomycetous fungi contain more than 350 species. Penicillium species such as Penicillium camemberti and Penicillium chrysogenum have been applied in food processing and medical industries, respectively (van den Berg et al., 2008; Bodinaku et al., 2019). Penicillium oxalicum can produce various extracellular glycoside hydrolases (GHs) including typical amylase, cellulase, hemicellulase, and other carbohydrate-active enzymes (Qu et al., 1991; dos Santos Costa et al., 2016). Thus, it has been used in the enzymatic degradation of plant cell wall polysaccharides in various biotechnological processes, such as the production of food, pulp, and bioethanol (Yan et al., 2017).

The synthesis of extracellular GHs of ascomycetous fungi is mainly controlled at the transcription level. Several key transcription factors such as AmyR, Cre1/CreA, and Clr2/ClrB play essential roles in regulating GHs encoding gene expression (Li et al., 2015). However, an increasing number of reports showed that the methylation of histones is also involved in GHs encoding gene expression (Karimi-Aghcheh et al., 2013; Vu et al., 2013; Li et al., 2019a, 2019b). For example, the putative methyltransferase Trichoderma reesei Lae1, carrying a conserved AdoMet-binding site, is a positive regulator of the synthesis of at least 40 GHs (Seiboth et al., 2012). The methylation of histone 3 lysine 4 (H3K4) is an active marker, whereas that of histone 3 lysine 36 (H3K36) is a repression marker for the expression of GHs encoding genes in P. oxalicum (Li et al., 2019b). As many methyltransferases are AdoMet-dependent and AdoMet synthetase participates in histone methylation processing (Hayashi et al., 2018), we investigated AdoMet synthetase in P. oxalicum and found that it is essential for the viability of fungal cells and the expression of GHs encoding genes, specifically cellulolytic enzyme encoding genes.