Exported proteins of Neurospora crassa: 1-glucoamylase

Polypeptide were exracted from the culture supernatants of Neurospora crassa growing in a variety of media. The polypeptides were analyzed by quantitative SDS-PAGE and the single polypeptide produced in the largest amount under the conditions tested was of molecular weight 69,000 and was referred to as p69. Synthesis of p69 was induced by starch and maltose, and studies on a partially purified preparation established that it was a glucoamylase. The sequence of the N-terminal 30 amino acids of p69 showed that this was related to sequence of analyses from Aspergillus spp. A synthetic oligonucleotide probe was synthesized and used to search for the corresponding gene(s) in Neurospora. A unique region was identified and mapped in a clone from a genomic library. The implications of the work for the development of Neurospora export and expression systems are discussed. © 1989

Establishment of Neurospora crassa as a host for heterologous protein production using a human antibody fragment as a model product

Background: Filamentous fungi are commonly used as production hosts for bulk enzymes in biotechnological applications. Their robust and quick growth combined with their ability to secrete large amounts of protein directly into the culture medium makes fungi appealing organisms for the generation of novel production systems. The red bread mold Neurospora crassa has long been established as a model system in basic research. It can be very easily genetically manipulated and a wealth of molecular tools and mutants are available. In addition, N. crassa is very fast growing and non-toxic. All of these features point to a high but so far untapped potential of this fungus for biotechnological applications. In this study, we used genetic engineering and bioprocess development in a design-build-test-cycle process to establish N. crassa as a production host for heterologous proteins. Results: The human antibody fragment HT186-D11 was fused to a truncated version of the endogenous enzyme glucoamylase (GLA-1), which served as a carrier protein to achieve secretion into the culture medium. A modular expression cassette was constructed and tested under the control of different promoters. Protease activity was identified as a major limitation of the production strain, and the effects of different mutations causing protease deficiencies were compared. Furthermore, a parallel bioreactor system (1 L) was employed to develop and optimize a production process, including the comparison of different culture media and cultivation parameters. After successful optimization of the production strain and the cultivation conditions an exemplary scale up to a 10 L stirred tank reactor was performed. Conclusions: The data of this study indicate that N. crassa is suited for the production and secretion of heterologous proteins. Controlling expression by the optimized promoter Pccg Inr in a fourfold protease deletion strain resulted in the successful secretion of the heterologous product with estimated yields of 3 mg/L of the fusion protein. The fungus could easily be cultivated in bioreactors and a first scale-up was successful. The system holds therefore much potential, warranting further efforts in optimization