Scaling Up Production of Recombinant DNA Products Using Filamentous Fungi as Hosts

Traditionally, filamentous fungi have been used as producers of antibiotics, bulk enzymes, and specialty chemicals, examples of which are indicated in Table 1. More recently, due to advances in the molecular biology of filamentous fungi, these systems are now being considered as suitable alternatives to Escherichia coli and Saccharomyces cerevisiae for the industrial production of recombinant proteins. Their value as production hosts for modern biotechnology applications is derived from the experience gained from a well-cataloged biological history, and some of these factors are listed in Table 2. In terms of their use as producers of heterologous proteins, several criteria are important:

It is possible to generate a high expression level of the recombinant protein.

In vivo modification of the gene products is possible to produce molecules with similar physical and biological properties as the native molecules. Of the factors listed in Table 2, secretion (and secretory mechanisms) is presently an issue that is receiving considerable attention, since it avoids several problems (identified in Table 3) that make the production of heterologous proteins as much an art as a science.

Table 1 Examples of Products Traditionally Made Using Filamentous Fungi

Product

Species

Penicillin G

Penicillium chrysogenum

Cephalosporin C

Acremonitalicum chrysogenum

Gritalicseofulvin

Penicillium patulum

Citric acid

Aspergillus niger

Gluconic acid

Aspergillus niger

Itaconic acid

Aspergillus terreus

Proteases

Aspergillus oryzae

Amyloglucosidase

Aspergillus niger

Alkaloids

Claviceps purpurea

Steroid hydroxylations

Coniothyrium hellbori

Table 2 Advantages of the <italic>Aspergilli</italic> as Expression/Secretion Hosts

Eukaryotic host

Good protein secretor

F.D.A. approved (A. niger)

Accepted by industry (organic acid and industrial enzyme production)

Rapid growth on simple, inexpensive media

Well characterized genetically (A. nidulans)

- available promoters with well-defined regulation

- many mutants

Mitotically stable integrated transformants

Table 3 Biological Advantages of Extracellular Secretion as a Method of Protein Production

Avoids intracellular accumulation of toxic levels of product

Potentially avoids intracellular hydrolysis or undesirable modification of product

Takes advantage of posttranslational events

— glycosylation

— protein folding

— other modifications

Potential use of directed endoproteolysis