Recombinant protein technology has facilitated high level production of proteins to try and meet the demand from industry. One major factor that has been reported to influence the scales of production in industrial settings is the relationship between host cell growth rate and protein production rate. Therefore, expression systems with fast growth and high protein production rates are favorable for high volume recombinant protein production. Filamentous fungi have over the years been used to produce a range of valuable metabolites including biologically active proteins and organic acids. Their ability to synthesize and secrete extracellular enzymes in large amounts made them the preferred host systems for heterologous protein production. In this study the CMW: Culture Collection of the Forestry and Agricultural Biotechnology Institute (FABI), University of Pretoria, South Africa, was screened using SDS-PAGE gels to identify a fungal isolate with the natural ability to secrete a highly expressed protein (HEP). The HEP was identified through liquid chromatography-mass spectrometry (LC-MS/MS) and the 330 bp gene encoding the HEP was isolated, cloned and transformed into E. coli cells. The gene sequence was further investigated using a modified SiteFinding-PCR chromosome walking technique, to acquire the full man gene sequence and to identify the upstream promoter and regulatory sequences flanking the gene in the 5 to 3 direction. In silico analysis of acquired nucleotide sequences in the 5 direction promoter region of the gene, predicted transcription initiation sites, transcription factor binding sites as well as TATA boxes that have been previously identified in the promoter region of other Ophiostoma proteins. The growth conditions of O. phasma 20676 were also optimized for high level protein production.