Comparative proteome profiling of fusarium chlamydosporum and elucidation of pigment biosynthetic pathway under nitrogen stress

Elsevier

Available online 20 February 2023, 104851

Journal of ProteomicsAuthor links open overlay panel, Highlights•

Comparison of the proteome profile of F. chlamydosporum in two different culture media with varying Nitrogen concentrations.

Protein extraction and digestion performed followed by non-gel based label free proteomics using LC-MS/MS analysis coupled to SWATH MS.

Protein identification processed with Protein pilot 5.0; advanced peak identification and profiling by Peak view 2.2 and relative protein quantification analyzed using Marker view (1.2.1, AB Sciex), bioinformatics analysis was performed by Gene Ontology, UniProt KB and DAVID functional annotation.

Protein profile of F. chlamydosporum in Nitrogen rich culture medium revealed the presence of proteins regulated for terpenoid and carotenoid production via Mevalonate pathway; proteome profiling in Nitrogen scarce medium accounted for the expression of proteins in polyketide and fatty acid synthesis.

Elucidation of the biosynthesis pathway of two pigments (carotenoids and polyketides) were possible using the proteome profiling and expression analysis.

Abstract

The main aim of this study was to understand the protein expression of F. chlamydosporum in two different medium composition in varying concentrations of nitrogen. The interesting phenomenon of producing diverse pigments by a single strain in different concentrations of Nitrogen made us further to explore the difference in the protein expression of the fungus when grown in these two media. For this, we had adopted non-gel-based method of protein separation by LC-MS/MS analysis followed by label free identification of proteins by SWATH analysis. The molecular and biological functions of each protein and their Gene Ontology annotations were analyzed by UniProt KB and KEGG pathway; the secondary metabolite pathways and the carbohydrate metabolic pathways were analyzed by DAVID bioinformatics tool. The positively regulated proteins biologically functioned for the secondary metabolite production in optimized medium were Diphosphomevalonate decarboxylase (terpenoid backbone biosynthesis), Phytoene synthase (carotenoid biosynthesis), 6,7-dimethyl-8-ribityllumazine synthase (riboflavin biosynthesis). The main characteristic change observed was that proteins related to carotenoid biosynthesis and terpenoid synthesis were not regulated in nitrogen limited medium. Except for the protein 6,7-dimethyl-8-ribityllumazine synthase, all the enzymes related to fatty acid biosynthesis and polyketide chain elongation were up regulated. Apart from the proteins related to secondary metabolite production, two novel proteins were found to be up regulated in Nitrogen Limited Medium; C-fem protein responsible for fungal pathogenesis and DAO domain containing protein which functions as a neuromodulator and catalyzes the synthesis of dopamine.

Significance

This particular strain of F. chlamydosporum of immense genetic and biochemical diversity represents an interesting example of a microorganism which can produce a variety of bioactive compounds and this can be exploited in various industries. The production of carotenoids and polyketides by this fungus when grown in the same media with different concentrations of Nitrogen has been published by us following which we analyzed the proteome sequence of the fungus in varying Nutrient conditions. Following the proteome analysis and expression, we could derive the pathway leading to the biosynthesis of varying secondary metabolites by the fungus which has not been published or studied so far.

Keywords

Fusarium chlamydosporum

SWATH MS

Mevalonate pathway

Polyketide synthase

Gene ontology

UniProt KB

AbbreviationsNLM

Nitrogen limited medium

SWATH - MS

Sequential Window Acquisition of all Theoretical Mass Spectra

KEGG

Kyoto Encyclopedia of Genes and Genomes

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