The AMP-Activated Protein Kinase Homolog Snf1 Concerts Carbon Utilization, Conidia Production and the Biosynthesis of Secondary Metabolites in the Taxol-Producer Pestalotiopsis microspora

Abstract

Highly conserved, the Snf1/AMPK is a central regulator of carbon metabolism and energy production in the eukaryotes. However, its function in filamentous fungi has not been well established. In this study, we reported functional characterization of Snf1/AMPK in the growth, development and secondary metabolism in the filamentous fungus Pestalotiopsis microspora. By deletion of the yeast SNF1 homolog, we found that it regulated the utilization of carbon sources, e.g., sucrose, demonstrating a conserved function of this kinase in filamentous fungus. Importantly, several novel functions of SNF1 were unraveled. For instance, the deletion strain displayed remarkable retardation in vegetative growth and pigmentation and produced a diminished number of conidia, even in the presence of the primary carbon source glucose. Deletion of the gene caused damages in the cell wall as shown by its hypersensitivities to Calcofluor white and Congo red, suggesting a critical role of Snf1 in maintaining cell wall integrity. Furthermore, the mutant strain Δsnf1 was hypersensitive to stress, e.g., osmotic pressure (1 M sorbitol), drug G418 and heat shock, though the mechanism remains to be illustrated. Significantly, disruption of the gene altered the production of secondary metabolites. By high-performance liquid chromatography (HPLC) profiling, we found that Δsnf1 barely produced secondary metabolites, e.g., the known product pestalotiollide B. This study suggests that Snf1 is a key regulator in filamentous fungus Pestalotiopsis microspora concerting carbon metabolism and the filamentous growth, conidiation, cell wall integrity, stress tolerance and the biosynthesis of secondary metabolites.

Involvement of PaSNF1 in Fungal Development, Sterigmatocystin Biosynthesis, and Lignocellulosic Degradation in the Filamentous Fungus Podospora anserina

“…A comparable function for SNF1 in the C. neoformans JEC21 has been reported (Yang et al, 2010). Our data clearly demonstrated the crucial roles of PaSNF1 in the tolerance of fungus to heat shock; the mycelium of the PaSNF1 mutant was sensitive to heat shock of 45 • C. This function of SNF1 has been previously reported in other fungi, such as C. neoformans and Pestalotiopsis microspora (Yang et al, 2010;Wang et al, 2018). Supplementary Table S1).…”

“…The PaSNF1 protein consisted of a serine/threonine (Ser/Thr) kinase catalytic domain (KD) (residues 67-318) and a two CTD (residues 532-628 and 633-704), showing the same structure with the SNF1s identified in many other organisms (Zhang et al, 2013;Islam et al, 2017). In addition, the PaSNF1 protein generally shared a high sequence identity with the SNF1s of other organisms, especially the catalytic domain (Cziferszky et al, 2003;Ospina-Giraldo et al, 2003;Wang et al, 2014Wang et al, , 2018. Phylogenetic analysis of SNF1 homologous proteins from filamentous fungi and yeasts formed a separate cluster.…”

“…The ability to form the perithecia and ascospores was affected in GzSNF1 deletion mutants (Lee et al, 2009), conidiation was also affected by the loss of PdSNF (Zhang et al, 2013), and the ablation of LmSNF1 would result in impaired sporulation and spore germination (Feng et al, 2014). In MoSNF1 mutant, the sporulation ability was abolished (Yi et al, 2008), and the PmSNF1 also played a critical role in conidiation and vegetative growth in Pestalotiopsis microspora NK17 (Wang et al, 2018). Deletion of SNF1 in Fusarium graminearum led to defects in vegetative growth and sexual reproduction (Yu et al, 2014).…”

“…In yeast, the SNF1 protein kinase has been necessary for its response to various environmental stresses such as sodium and lithium salts, alkaline pH, heat shock, and hyperosmolarity (Hong and Carlson, 2007). Treatment with high concentrations of salts (0.8 M of KCl and 1 M of NaCl), 1 M of sorbitol, and 2 mM of oxidant H 2 O 2 could dramatically inhibit vegetative growth of the PmSNF1 mutant strain (Wang et al, 2018). However, the response of the SNF1 mutant of Cryptococcus neoformans to osmotic, salt, and oxidative stresses was not significantly different from that of the WT (Hu et al, 2008).…”

“…Maximum likelihood method was applied to analyze the similarities among above 11 proteins, and a phylogenetic tree was constructed (Figure 1). The predicted amino acid sequence of PaSNF1 shared an identity of 75.64% with NcSNF1 of N. crassa (CAD70761), 64.67% with HjSNF1 of H. jecorina (AF291845) (Cziferszky et al, 2003), 67.73% with FoSNF1 of Fusarium oxysporum (AAN32715) (Ospina-Giraldo et al, 2003), 66.06% with BbSNF1 of Beauveria bassiana (EJP66968) (Wang et al, 2014), 63.41% with PmSNF1 of Pestalotiopsis microspora (AUW40141) (Wang et al, 2018), 57.53% with MgSNF1 of Magnaporthe grisea (ABF48563) (Yi et al, 2008), 57.94% with SsSNF1 of Sclerotinia sclerotiorum (CAB40826) (Vacher et al, 2003), 44.35% with PdSNF1 of Penicillium digitatum (AFS18464) (Zhang et al, 2013), and 43.43% with CcSNF1 of Cochliobolus carbonum (AAD43341) (Tonukari et al, 2000). PaSNF1 protein showed lower homology with SNF1 protein from S. cerevisiae, which was 42%.…”