Penicillium oxalicum S-adenosylmethionine synthetase is essential for the viability of fungal cells and the expression of genes encoding cellulolytic enzymes
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.
Section snippets
Fungal strains, media, and phenotypic analysis
The wild type (WT) strain P. oxalicum 114-2 (CGMCC 5302) and the mutants were cultivated on 10 % wheat bran extract agar slants at 30 °C for five days. For phenotypic analysis, 1 μL of conidial suspension was spotted onto normal potato dextrose agar (PDA) and Vogel’s minimal medium (VMM) agar added with 2 % glucose (VMMG) at 30 °C for five days. The strains were also cultivated on VMMG agar added with 2M sorbitol or 0.05 % Congo red. To rescue the phenotypes of the heterokaryon, 0.2 mg/mL of
Identification of AdoMet synthetase in P. oxalicum
We performed a reciprocal BLASTP search using the sequence of S. cerevisiae SAM1 and SAM2 as queries to find AdoMet synthetase ortholog in P. oxalicum genome. Only one ortholog, PDE_07230, was identified within the P. oxalicum genome and named as PoSasA (AdoMet synthetase). PoSasA was phylogenetically conserved in many filamentous fungi, such as Aspergillus, Trichoderma, Neurospora, and Fusarium. PoSasA shared the highest identity of 91.79 % with the ortholog in P. chrysogenum; 89.5 % identity
Discussion
PoSasA was the only one AdoMet synthetase found in P. oxalicum. We could not obtain absolutely PosasA deletion strain but only obtained heterokaryon cells (HKsasA), in which PosasA expression decreased compared the WT strain. The absence of PoSasA was lethal for P. oxalicum as AdoMet was the main methyl donor and essential in numerous biochemical metabolic reactions (Luo et al., 2008).
The HKsasA-1 strain displayed slightly smaller colony diameter and a pink colony. This phenotype reminded us of
Funding
This work was supported by the National Natural Sciences Foundation of China (Grant No. 32070077, 31971387), the Shandong Provincial Natural Science Foundation, China (Grant No. ZR2019MC007), the National Key Research and Development Project of China (Grant No. 2018YFA0900500); and the Major Basic Research Project of Natural Science Foundation of Shandong Province, China (Grant No. ZR2019ZD19).
Declaration of competing interest
The authors declare that they have no conflict of interest.
Acknowledgements
We thank Senior Engineer Xuezhi Li and Dr. Jingyao Qu of the State Key Laboratory of Microbial Technology, Shandong University, for helping subcellular localization observation and the MS data analysis.
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