Towards developing a new host-vector system for high-level protein expression

Abstract

Filamentous fungi are critical for the production of many commercial enzymes and organic compounds. Due to their decomposer lifestyle, they have a natural potential for large scale production of proteins, many of these extracellular. The concept of this project was to exploit the protein synthesis and secretory capability of a large number of indigenous fungi as the basis to generate a new fungal expression platform. Filamentous fungi were obtained from the culture collection (CMW) of the Forestry and Agricultural Biotechnology Institute (FABI) University of Pretoria, South Africa. Screening of the isolates for extracellular protein expression in malt extract liquid medium shake flasks, led to the identification of a Clonostachys rosea 17970 isolate after internal transcribed spacer (ITS) sequencing and morphological studies. A ~35 kDa protein secreted at high levels was identified through LC-MS/MS as a cuticle-degrading serine protease (Cdsp). The optimum pH and temperature for protein production were found to be 9.0 and 32°C respectively, after 96 h of incubation on malt extract liquid media. Designed primers were used for the PCR amplification of an internal fragment of the cdsp gene from chromosomal DNA. Sequence data was used for further PCR-amplification of the up and downstream regions through chromosome walking using Inverse and SiteFinding PCR technology. Analysis of the assembled cdsp gene fragment (2,923 bp) revealed that the gene harbours a 5 upstream region (1,141 bp), coding region (1,300 bp) and 3 downstream region (509 bp). The coding region contains three introns. Reverse transcriptase-PCR of the coding region revealed a 1,140 bp open reading frame that encodes Cdsp. The putative cdsp signal peptide, promoter and terminator regions were identified in silico and are predicted to be important elements for the construction of an expression vector. Overall, these results demonstrate that C. rosea 17970 could be a good source for extracellular protein production and is a potential candidate for development as a new host-vector system based on its secretory capability and identified regulatory regions via in silico analysis.