cAMP-PKA and HOG1 signaling pathways regulate liamocin production by different ways via the transcriptional activator Msn2 in Aureobasidium melanogenum

Highlights

• Liamocin synthesis is controlled by both cAMP-PKA and HOG1 signaling pathways via the Msn2.

• The cAMP-PKA signaling pathway controlled activity and subcellular localization of the Msn2.

• The HOG1 signaling pathway regulated only expression of the MSN2 gene.

Abstract

Liamocins, as the secondary metabolites synthesized and secreted by Aureobasidium spp., consist of a single mannitol or a single arabitol head group partially O-acylated with three 3,5-dihydroxydecanoic ester groups or directly esterified with three or four 3,5-dihydroxydecanoic ester tails. Very recently, the whole synthetic pathway of liamocins in A. melanogenum 6-1-2 has been elucidated. It was found that the promoter sequences of all the genes related to liamocin synthesis in A. melanogenum 6-1-2 had stress regulatory elements with core sequences of AGGGG or CCCCT. Therefore, expression of all the genes would be regulated by the Msn2. In this study, it was found that removal of the single one MSN2 gene in A. melanogenum 6-1-2 made the mutant decrease yield of extracellular liamocin by 92.28 %, while complementation of the MSN2 gene in the mutant rendered liamocin synthesis to be restored. When A. melanogenum 6-1-2 was cultured in the liamocin fermentation medium with high glucose and low nitrogen, the Msn2 was localized in the nucleus and positively regulated the expression of the genes related to liamocin biosynthesis. Furthermore, when the key BCY1 gene encoding regulatory subunit of the cAMP-PKA signaling pathway in A. melanogenum 6-1-2 was knocked out, the amount of extracellular liamocins synthesized by the mutant was decreased by 96.73 % and the Msn2 was localized in the cytoplasm. Similarly, when the key HOG1 gene in the HOG1 signaling pathway was deleted, liamocin biosynthesis in the knockout strain was decreased by 98.09 %. However, it was found that the Hog1 may be one part of the general transcription complex to regulate the transcription of the MSN2 gene, leading to the reduced Msn2 and liamocin synthesis in the mutant. In addition, the key TOR1 gene and SNF1 gene in the TOR1 signaling pathway and the SNF1 signaling pathway were not involved in the regulation of the Msn2 activity and liamocin synthesis. It was concluded that the transcriptional activator Msn2, the HOG1 signaling pathway and the cAMP-PKA signaling pathway were involved in the regulation of liamocin biosynthesis and production.

Introduction

Liamocins are synthesized and secreted by some strains of Aureobasidium spp. They consist of a single mannitol or a single arabitol head group partially O-acylated with many 3,5-dihydroxydecanoic ester groups or directly esterified with many 3,5-dihydroxydecanoic ester tails [[1], [2], [3]]. It has been well known that liamocins have many applications as they can display an antiproliferative effect against certain cancer cell lines, reduce surface tension as one kind of biosurfactants, are powerful selective antimicrobial molecules against Streptococcus agalactiae and Streptococcus uberis [2]. 5-Hydroxy-2-decenoic acid lactone released from liamocins also has many uses in food and pharmaceutical industries as it is used to flavor food and has high antimicrobial activity against fungi. Very recently, the whole synthetic pathway of liamocins in A. melanogenum 6-1-2 has been elucidated by Xue et al. [3]. In their pioneering research work, the key PKS1 gene responsible for biosynthesis of 3,5-dihydroxydecanoic acid of liamocin tails was found to be activated by the transcriptional activator Ga11. In addition, activity of the highly reducing polyketide synthase (HR-PKS) encoded by the key PKS1 gene was controlled by the phosphopantetheine transferase (PPTase) through post-translation modification (Fig. 1) [3]. However, it is still unknown how their biosynthesis is regulated by the fungal signaling pathways and transcriptional activators.

It has been well known that in fungi, “stress-responsive element” (STRE) in the promoters of many genes is a 5-bp sequence functional in both orientations (CCCCT or AGGGG) and two transcription factors, Msn2 and Msn4 in Saccharomyces cerevisiae, mediate the STRE-mediated gene expression [4]. The Msn2 usually contains two zinc finger motifs near the C terminus of the Cys₂His₂ type while the Msn4 bears 41 % identity in amino acid sequence to that of the Msn2. Of the two proteins, the Msn2 seems to play a more pronounced role in regulation of the relevant genes than the Msn4. Of the two genes, the expression of the MSN2 gene is constitutive under all the conditions, whereas the MSN4 gene expression is stress induced, and the induction is mediated by itself and the Msn2. The Msn2 and Msn4 each contain a nuclear localization signal (NLS) such as S288, S620, S625 and S633 near the C terminus so that the substitution of S288 with alanine or aspartate in the Msn2 leads to constitutive nuclear accumulation [4]. The cAMP-protein kinase A (PKA) signaling pathway, the nitrogen sensing TOR signaling pathway, high osmotic pressure sensing HOG1 signaling pathway and glucose repression SNF pathway have been described to play important regulatory roles in controlling Msn2/4 [5,6]. However, it is still completely unclear which signaling pathways will regulate liamocin biosynthesis through the transcriptional activators Msn2/4. It has been known that the central parts of the cAMP-PKA signaling pathway are Cyr1 (adenylate cyclase), TPK1-3 (protein kinase A, PKA), Bcy1 (regulatory subunit of PKA) and Msn2/4 [4] while the key components in the HOG1 signal pathway in fungi are the MAPKK Pbs2, the MAPK Hog1, transcriptional activators Sko1, Hot1 and Msn2 mediating the upregulation of nearly 600 genes [7]. In addition, the Tor1 is a key regulator in the nitrogen signaling pathway and the main components in the SNF pathway are Snf1 and Mig1 [6]. Therefore, the goals of this study were to know the regulatory roles of the different signaling pathways in liamocin biosynthesis in Aureobasidium melanogenum 6-1-2, a high liamocin producing yeast.