Abstract
This study catalogs production of industrially important enzymes and changes in transcript expression caused by 2-deoxy D-glucose (2-DG) treatment in Arthrinium malaysianum cultures. Carbon Catabolite Repression (CCR) induced by 2-DG in this species is cAMP independent unlike many other organisms. Higher levels of secreted endoglucanase (EG), β-glucosidase (BGL), β-xylosidase (BXL), and filter paper activity assay (FPase) enzymes under 2-DG treatment can be exploited for commercial purposes. An integrated RNA sequencing and quantitative proteomic analysis was performed to investigate the cellular response to 2-DG in A. malaysianum. Analysis of RNASeq data under 2-DG treated and control condition reveals that 56% of the unigenes do not have any known similarity to proteins in non-redundant database. Gene Ontology IDs were assigned to 36% of the transcripts (13260) and about 5207 (14%) were mapped to Kyoto Encyclopedia of Genes and Genomes pathway (KEGG). About 1711 genes encoding 2691 transcripts were differentially expressed in treated vs. control samples. Out of the 2691 differentially expressed transcripts, only 582 have any known function. The most up regulated genes belonged to Pentose Phosphate Pathways and carbohydrate degradation class as expected. In addition, genes involved in protein folding, binding, catalytic activity, DNA repair, and secondary metabolites were up-regulated under 2-DG treatment. Whereas genes encoding glycosylation pathways, growth, nutrient reservoir activity was repressed. Gene ontology analysis of the differentially expressed genes indicates metabolic process (35%) is the pre-dominant class followed by carbohydrate degradation (11%), protein folding, and trafficking (6.2%) and transport (5.3%) classes. Unlike other organisms, conventional unfolded protein response (UPR) was not activated in either control or treated conditions. Major enzymes secreted by A. malaysianum are those degrading plant polysaccharides, the most dominant ones being β-glucosidase, as demonstrated by the 2D gel analysis. A set of 7 differentially expressed mRNAs were validated by qPCR. Transmission electron microscopy analyses demonstrated that the 2-DG treated cell walls of hyphae showed significant differences in the cell-wall thickness. Overall 2-DG treatment in A. malaysianum induced secretion of large amount of commercially viable enzymes compared to other known species.
Highlights
Endophytes represent a promising group of organisms, as they are mostly untapped reservoirs of metabolic diversity
Of which most of them were related to metabolism (Hexokinase, Fructose bisphosphate, Isocitrate lyase, Transaldolase), protein folding (Protein disulfide isomerase, HAC) and cell wall degrading enzyme (β-glucosidase) were selected for validating the results obtained RNAseq analyses using Quantitative Real time PCR (qPCR) (Table 5)
This study showed that most of the gene related to metabolism and protein folding were found to be upregulated in 2-deoxy D-glucose (2-DG) treatment compared to the control, which showed no such up-regulation
Summary
Endophytes represent a promising group of organisms, as they are mostly untapped reservoirs of metabolic diversity. They are often able to degrade cellulose and produce an extraordinary diversity of metabolites (Gianoulis et al, 2012). Are endophytes grown in diverse ecological niches, known to produce bioactive compounds that have pharmacological and medicinal applications (Crous and Groenewald, 2013). The first Arthrinium sp., A. saccharicola was isolated from Saccharum officinarum and was later reported from various habitats such as plants, air and seawater (Miao et al, 2006). A. malaysianum is a known contaminant on Termitomyces cultures (Sawhasan et al, 2012) and in the present study, this organism is isolated from Termitomyces mycellial mats. Reports of several other endophytes belonging to Arthrinium sp. Papulacandins, with inhibitory and antifungal activity against bacteria, molds, yeasts, and fungi are some of the most commonly found secreted products from this group (Traxler et al, 1980; Larrondo et al, 1996; Aissaoui et al, 1998; Adelantado et al, 2005; Calvo et al, 2005)
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