For the experts

The first Aspergillus oryzae production strains developed by Novozymes were made by transforming an A. oryzae type strain with a DNA construct that directed efficient production of a product gene (Christensen, 1988).

These strains represented a substantial improvement on existing technology and were the beginning of our journey to continuously design and construct superior recombinant production strains and optimize the fermentation processes.

At Novozymes we screen enzymes of all classes from natural diversity and we generate in vitro diversity by, for example, site-directed mutagenesis or shuffling of homologous genes. When an enzyme has been screened or designed for an application, a production strain is developed.

If it is an enzyme selected from natural diversity, the production strain could simply be the native microorganism that produces this enzyme. But mostly the product yields are very limited and the microorganism produces unwanted side activities. Sometimes the microorganism just cannot grow in modern production facilities.

At Novozymes we solve these problems using recombinant gene technology. We design and construct superior recombinant production strains based on our vast experience of classical microbiology and fermentation technology.

A good recombinant production strain is characterized by several different properties:

  • Versatility: Novozymes’ capability to produce the huge diversity of enzymes means that our host strains must be able to produce an array of different enzymes
  • Safe: Meaning it is nonpathogenic toward humans, animals, and plants, and that it has no adverse effects on the environment
  • Good protein secretion: The fermentation yield of the enzyme product must be very high to ensure an economically and environmentally sustainable production process. Furthermore, the product purity and quality of the final product are highly dependent on the fermentation yield
  • Lack of side activities: The strain should not produce unwanted side activities that can negatively influence the product stability or hamper the product purity
  • Amenable to an industrial fermentation process: It must have characteristics that make it suitable for growth in a stainless steel fermentor. The strain must grow submersed to an appropriate biomass at an appropriate temperature, and the morphology of the culture must allow efficient stirring and aeration

Host strain development

When Novozymes first faced the challenge of producing recombinant enzymes, the company was already a very experienced enzyme producer. So we had a very solid basis for choosing microorganisms that could be used as host strains for recombinant production from the criteria listed above.

Novozymes has developed both bacterial and fungal expression systems, and we continue to improve these systems.

Novozymes has developed a number of different bacterial hosts that have demonstrated well-documented safe use for over 30 years in production. The hosts are derived from species like Bacillus subtilis, Bacillus licheniformis, and Bacillus clausii. The genus Bacillus is known to have a very high potential for secreting enzymes into the medium in a pure form (Zukowski, 1992) and is an ideal organism for enzyme production on a large scale. Tools for genetic engineering of our Bacillus hosts have been developed, patented, and implemented for industrial application over the years. Significant improvements of the wild-type host strains have been introduced as a result of either classical mutagenesis or specific changes imposed by use of recombinant techniques. These alterations cover, for example, disruption of genes coding for unwanted enzyme activities like amylases and proteases, and genes that are responsible for initiation of sporulation.

Novozymes has developed fungal host cells from strains of Aspergillus oryzae, Aspergillus niger, Fusarium venenatum, and Trichoderma reesei. The Aspergillus and Trichoderma strains have an extended history of safe use (Barbesgaard, 1992) and are both generally regarded as safe (GRAS). The strain of Fusarium venenatum also has a history of safe use since it is used for single-cell protein for human consumption in the United Kingdom (Royer, 1995).

A. niger and A. oryzae both secrete substantial amounts of amylases and amyloglucosidase and are thus very efficient enzyme producers. The extended history of safe use and the ability to produce huge amounts of extracellular protein encouraged Novozymes to develop A. oryzae as our favorite fungal recombinant expression system.

When producing an enzyme product, the native amylases and amyloglucosidases are undesirable side activities. Some of the genes encoding these enzymes have been deleted by gene disruption from our A. oryzae and A. niger host strains. The protease levels are also very high in these strains. Apart from being undesirable side activities, proteases can also degrade the enzyme products that we wish to produce. Genes encoding protease and protease regulators have therefore been disrupted.

A number of unwanted metabolites have also been removed. For example, A. niger produces rather high amounts of oxalic acid. Oxalic acid forms precipitates with calcium that cause problems in product recovery and formulation. Also, the oxalic acid production is a waste of the carbohydrate used in the medium. Finally, the presence of oxalic acid in the final product cannot be tolerated. Novozymes has solved this problem by disrupting a gene crucial for oxalic acid formation so that our current A. niger host strain does not produce oxalic acid (Pedersen, 2000).

Expression vector improvement

In addition to an optimal host strain, an optimal expression vector is required for an optimal production strain. At Novozymes our expression vectors are plasmids or fragments of plasmids that are integrated into the genome of the host organisms in one copy or in multiple copies. The genetic stability of integrated vectors has proven to be superior to that of episomal plasmids.

In principle, an expression vector holds a selection marker, an expression cassette, and, in the case of bacterial vectors, a replication function that determines the copy number of the vector in a given host. The expression cassette consists of a strong promoter, the product gene coding for the enzyme of choice, and a transcriptional terminator. The efficiency of the expression cassette is a result of a complex interplay between the promoter efficiency, the structural gene, and the host strain. The nature of the product gene determines the choice of host, promoter, and the vector used for chromosomal integration.

The promoter of choice in Aspergillus is an amylase promoter. This was even used in the very first recombinant A. oryzae production strain launched in 1988. Since then we have gained in-depth knowledge of the transcription factors acting on this promoter and its regulation (Petersen, 1999). This knowledge has enabled us to isolate variants of the promoter that are even stronger promoters. For many Bacillus production strains, amylase promoters and selected strong variants thereof are used for the production of many different enzyme products.

Selection of high-yielding transformants

In our fungal systems the expression vectors integrate by ectopic multicopy integration. This means that different transformants have the vector integrated at different loci in the genome and at different copy numbers, leading to different levels of protein production.

Some loci in the genome are hotspots for expression, whereas others are relatively silent in terms of expression. So the site of integration is important. Even though no strict correlation between copy number and expression exists, investigations of strains having the expression plasmid integrated in the same locus but at different copy numbers have shown that higher copy numbers result in higher expression levels. So a population of transformants shows a quite large diversity in expressing the product gene.
In order to select the transformants with the highest expression level they are fermented in different systems (various scales and media) and the product yields are assayed.

Selection of mutants

When an optimal transformant is selected as described in the previous section, the fermentation yield can be increased by classical mutagenesis. In this process Novozymes uses the classical microbial skills that the company completely relied on before gene technology was introduced.
The selected transformant is subjected to chemical mutagenesis, UV, or radioactive irradiation. Mutated progeny is screened for yield increase in very much the same way as the transformants were originally screened.

Barbesgaard, P et al. (1992), Appl Microbiol Biotechnol, 36:569–572.
Christensen, T et al. (1988), BioTechnology, 6, 1419–1422.
Pedersen, H et al. (2000), Metab. Eng., 2:34–41
Petersen, KL et al. (1999), Mol Gen Genet, 262:668–676.
Royer, J et al.
(1995), Bio/Technology, 13:1479–1483
Zukowski, MM (1992), Biology of Bacilli: Applications to Industry. Butterworth-Heinemann, Boston: 311–337