Production of heterologous peptides

Details Production of heterologous peptides Production of heterologous peptides

1996-02-13

1998-11-10

Guzo, David

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving nucleic acid

435 691, 435 914, 435205, 4352544, 4353201, 536 231, 536 2433, C12Q 168, C12N 934, C12N 1581, C07H 2104

Patent

active

058341919

DESCRIPTION:

BRIEF SUMMARY
The invention relates to a method and recombinant means particularly, but not exclusively, expression cassettes and expression/export cassettes for the production of heterologous peptides. The method and means have particular application in the production of such peptides from the biotechnological exploitation of filamentous fungi and particularly Neurospora crassa.
The filamentous fungi secrete substantial amounts of protein, notably hydrolytic enzymes. Many of these enzymes are used in industrial processes such as the production of antibiotics and organic acids, the saccharification of starch, glucose isomerisation, the processing of wines and fruit juices and the degradation of cellulose and lignin (Bennett 1985; Bu'Lock and Kristiansen 1987). The promoter and signal sequences of the genes of such enzymes represent targets for manipulation for developing the filamentous fungi as hosts for heterologous gene expression. The potential for this technology has been reviewed with particular reference to the genus Aspergillus (Van den Hondel et al 1991).
The term heterologous gene expression is used in this document to mean the expression of genes not present or common in the host.
The genus Neurospora has several advantages for study with a view to its possible exploitation as a host for heterologous gene expression.
More specifically, the species Neurospora crassa is the most thoroughly studied and characterised of all the filamentous fungi (Reviewed by more genes cloned than any other species. It is extremely fast-growing, with simple growth requirement. It will grow on a wide range of carbon and nitrogen sources, and has a single complex growth requirement, for biotin. It will grow in liquid or on solid medium. It produces no toxic secondary metabolities, and in fact is a traditional oriental human food organism.
Neurospora in nature grows in a solid medium, and has efficient secreted enzyme systems for the utilisation of polysaccharide carbon sources. These include glucoamylase, the major exported protein when starch-induced. Secreted proteins in wide-type Neurospora reach levels of circa 1 g/l of spent medium, the glucoamylase, when starch-induced, accounts for circa 20% of the total. For glucoamylase, there is evidence for two regulatory components, carbon catabolite repression, and induction by the substrate or some partial hydrolysis product of such.
Glucoamylases have been cloned and characterised from several fungi: Aspergillus awamori (Nunberg et al 1984), A. awamonri var. kawachi (Hayashida et al 1989) A. niger (Boel et al 1984), A oryzae (Hata et al 1991), A. shirousami (Shibuya et al 1990), Humicola grisea var. thermoidea (Berka et al, personal communication), Rhizopus oryzae (Ashikari et al 1986), Saccharaomyces cerevisiae (Pardo et al 1988), S. diastaticus (Yamashita et al 1985), S. fibuligera (d, (Itoh et al 1987), and S. occidentalis (Dohmen et al 1990).
Glucoamylases (exo-1,4-x-D-glucan glucohydrolase, EC 3.2.1.3) are secreted in large amounts by a variety of filamentous fungi. They catalyse the removal of single glucose units from the non-reducing ends of starch and other poly- and oligo-saccharides. Their use in industrial processes includes the production of glucose syrups from starch (Kennedy et al 1988), and the fermentation of sake, (rice wine) in Japan. Heterologous expression systems in the above Aspergillus species of filamentous fungi commonly use their glucoamylase promoters to drive expression, their signal sequences to secrete foreign peptides, and their 3' flanking regions to direct termination (Archer et al 1990; Ward et at 1990, 1992).
Koh-Luar et al (1989) analysed culture supernatans of Neurospora crassa, growing on a variety of carbon sources, and showed that the protein present in the largest amount was a glucoamylase of approximately 69 kDa. This protein was purified and the N-terminal sequence of the glucoamylase determined.
The high expression and secretion properties of the Glucoamylase gene makes it an attractive candidate for use in heterologous gene expression. Th

REFERENCES:
patent: 5252726 (1993-10-01), Woldike
Koh-Luar et al. (1989) Enz. Microb. Technol. 11:692-5.
Stone et al. (1993) Curr. Genet. 24:205-11.

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