Fungal Molecular Ecology & Biotechnology

Screening of a soil metatranscriptomic library by functional complementation of Saccharomyces cerevisiae mutants.

Introduction

Aim of this study was to detect functional transcripts in a soil metatranscriptomic library to complement yeast mutants, here his3- and pho5-. Using this “functional” approach we are able to produce and characterize important fungal enzymes, which are potentially involved in soil/biogeochemical processes (i.e. phosphatase in P cycle).

Methods
pTEF-MF-SfiI
We used extracted RNA from an organic horizon of a forest soil. cDNA was synthezised using SMART technology (Clontech), the amplified cDNA contained SfiI A/B sites on the transcript ends to allow a directional ligation into a modified yeast expression vector (pTEF-MF-SfiI A/B, see picture).

The vector was first transformed in E. coli, and positiv transformants were pooled (~13,000). From this pool, vectors including the soil inserts were extracted (big plasmid prep) and used for transformation of yeast mutants.

 

 

 


Results and Discussion

Four candidates complementing the pho5- mutation were found.

Fig_phosphatase_plate_test_color

The candidates were sequences and one phosphatase gene (acid phosphatase/phytase), probably belonging to a soil basidiomycete was characterized.

Phosphatase_Tree

The full annotated gene:

final_gene_characterisation

 

Literature

Kellner H., Luis P., Portetelle D., Vandenbol M. (2011): Screening of a soil metatranscriptomic library by functional complementation of Saccharomyces cerevisiae mutants. Microbiological Research 166: 360-368

 

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