18.11 Recombinant protein production by filamentous fungi

Fungal expression vectors have generally been constructed in Escherichia coli plasmids although other vehicles, like Agrobacterium and other plasmids, are becoming popular. Vectors used to transform fungi for protein (usually, but not exclusively, enzyme) production require (Fig. 24):

  • a strong constitutive or regulatable promoter sequences from the host or a related species;
  • secretion-facilitating signal sequences (upstream prepro sequences);
  • cloning sites for heterologous genes allowing incorporation of one or more copies of the ORF of the gene of interest;
  • downstream sequences for transcriptional termination and polyadenylation;
  • selectable marker(s) for screening transformants, auxotrophic, resistance or gain of function markers.
Diagrammatic representation of the general structure of fungal expression vectors
Fig. 24. Diagrammatic representation of the general structure of fungal expression vectors used to transform fungi for protein production.

Fig. 25 shows an example of plasmid pAW14S which was used for expressing the exlA gene of Aspergillus awamori in A. niger (Hessing et al., 1994). The filamentous fungus Aspergillus niger is used in industry for the production of fermented foods, organic acids, and enzymes, mainly because of its ability to secrete large amounts of protein (up to 30 g l-1) into the culture medium. For this reason alone this fungus is an attractive proposition for commercial use for production and secretion of homologous or heterologous proteins. The relevance of exlA is that the gene encodes a 1,4-β-endoxylanase and, commercially, fungal enzyme preparations with xylanolytic activity are used in baking. The DNA sequence of the pre(pro)xylanase gene (exlA) is inserted into the plasmid as shown in Fig. 25.

A map of plasmid pAW14S
Fig. 25. A map of plasmid pAW14S containing the cloned exlA gene of Aspergillus awamori, which encodes a 1,4-β-endoxylanase. The nature of the line showing the circumference of the circular plasmid shows the derivation of the corresponding DNA, and the arrows show the direction of transcription. The plasmid was based on the small (2686 bp) Escherichia coli plasmid pUC19; the ApR (also known as bla) gene from E. coli codes for β-lactamase and confers resistance to ampicillin on bacterial hosts. The amdS segment from Aspergillus nidulans confers, on transformed A. niger, the gain of function ability to use acetamide as a nitrogen or carbon source. The A. awamori exlA gene segment includes a characteristic pre(pro) leader signal sequence (shown as 'promoter region' ) and the termination and polyadenylation sequence (shown as 'termination region') either side of the protein coding region. Restriction enzyme sites are shown as S = SalI and E = EcoRI. Redrawn after Hessing et al., 1994.

Unless steps are taken to ensure that only homologous recombination occurs, transforming DNA in filamentous fungi generally integrates into the host chromosomes at more than one site. This means that most transformants contain more than one copy of the expression vector. Increased copy number is generally good for productivity (Table 16) although the relation between gene copy number and productivity is not always simple.

Table 16. Comparison of gene copy number and glucoamylase secreted by transformants of Aspergillus niger obtained using vectors containing one* or four† copies of the glaA gene

Strain

Number of strains studied

glaA copy number in transformants

Glucoamylase in culture medium

(mg l-1)

Parental

1

1

50

Transformants obtained using vector with one copy of glaA gene

8

2-60

25-425

Transformants obtained using vector with one copy of glaA gene

8

12-200

25-900

*plasmid shown in Fig. 26; † plasmid shown in Fig. 27

 

Plasmid vector pAB6-8
Plasmid (cosmid) vector pAB6-10
Fig. 26. Plasmid vector pAB6-8 with one copy of the glaA gene of Aspergillus niger. Restriction enzyme sites are shown as H= HindIII and E = EcoRI. Other abbreviations as in Fig. 25. Fig. 27. Plasmid (cosmid) vector pAB6-10 with four copies of the glaA gene of Aspergillus niger. Restriction enzyme sites are not shown; cos = the cohesive ends that emable bacteriophage packaging. Other abbreviations as in Fig. 25.

The amdS gene confers a useful phenotype on transformants which enables multicopy transformants to be identified easily by Petri dish growth tests. One copy of amdS is sufficient for growth on acetamide; several copies of amdS of the plasmid. gene are required for growth on acrylamide; and many copies of the amdS gene cause inhibition of growth of transformants when ω-amino acids (such as γ-aminobutyrate (GABA) or β-alanine) are used as sole nitrogen and carbon source.

The principal reasons for using filamentous fungi as hosts for recombinant protein production are:

  • their ability not only to produce but to secrete very high yields of protein into the medium;
  • the large knowledge base that exists for their cultivation under established fermenter conditions;
  • the similarity of fungal protein modification systems to those of mammalian cells;
  • so many have been used in foods and food production systems for many years that many fungi are Generally Recognised As Safe, the so-called GRAS status which is so important in having new products approved by regulatory authorities for general release to market.

Some examples of recombinant proteins successfully produced by filamentous fungi are:

  • xylanase, which is added to bread dough to improve production; 1,4-β-endoxylanase from Aspergillus awamori is produced in A. niger transformants containing multiple copies of the gene. The overproduced enzyme has the same biochemical properties as the original enzyme.
  • Phytase (Natuphos®; see http://www.natuphos.com/): phytate (myo-inositolhexakisphosphate) is a major phosphate reserve in plant seeds; in cereals and oilseeds, up to 90% of total phosphorus can be stored as phytate, which is almost indigestible to animals. Aspergillus niger produces a 3-phytase enzyme that initiates dephosphorylation at the 3-phosphate (IP3) position of inositolhexakisphosphate and efficiently breaks down phytate. In regions of high livestock density farmers use Natuphos as a tool to manage manure disposal and avoid overloading soil with high-phosphorus wastes. The enzyme is also useful as a feed supplement to release digestible phosphorus and other phytate-bound nutrients to monogastric animals such as pigs and poultry. Phytase produced by A. niger has high activity and is more thermostable than other microbial phytases. The enzyme is produced by an A. niger strain transformed with multiple copies of the phytase gene (phyA).
  • Lipase: triglyceride lipases are added to biological detergents to remove grease stains during the wash cycle. The lipase gene from the thermophilic Humicola (=Thermomyces) languinosus has been used to transform A. oryzae. The recombinant lipase differs from the native lipase in having greater glycosylation and better thermostability. Lipolase was first industrial enzyme made by a genetically engineered microorganism to be sold to the general public (‘The world’s first detergent lipase’ see http://www.novozymes.com/en/MainStructure/ProductsAndSolutions/Detergents/Laundry/Stain+Removal/Lipolase/Lipolase.htm). This example introduces the idea of making design changes to the recombinant protein. The enzyme Lipolase Ultra® gives enhanced fat removal at low wash temperatures as the result of changing just one amino acid in the protein; a negatively charged amino acid near the active site was replaced it with a neutral amino acid.
  • Chymosin is an aspartyl protein which cleaves casein in milk to promote clotting. Rennilase® (rennin) from Rhizomucor miehei has been used since 1969 as an alternative to bovine chymosin. To produce the recombinant enzyme a chymosin cDNA sequence was fused to the last codon of the Aspergillus awamori glucoamylase gene and the construct was used to transform A. awamori. Yield of the proteinase by the transformed strain was improved further by classical mutagenesis and selection, and by site-directed mutagenesis to introduce a glycosylation site. Final yield was in the g l-1 range, which is enough to make the process commercially viable.

For non-fungal proteins yields are generally much lower, in the μg l-1 to mg l-1 range. This is due to poor folding of the proteins in the endoplasmic reticulum which triggers a response known as the unfolded protein response (UPR) which leads to the removal of unfolded proteins through the ubiquitinisation route leading to degradation in the vacuole. That is, potential product is destroyed rather than being secreted. This response can be overcome by mutagenesis and strain selection, however to date this has only been done for the production of calf chymosin in A. niger, where a strain was mutated successively until the yield reached commercial levels.

Updated December 17, 2016