Discussion of the use of gene technology has been a central element of our communication activities with various stakeholder groups for many years. It has also been an important part of our annual environmental reports for the past five years. This year the gene technology section focuses on safety in the environment, and on use of genetically modified organisms
(GMOs) in food production and antibiotic resistance markers.

Novo Nordisk is a pioneer in gene technology. For more than 15 years, recombinant technologies have been at the core of our innovations. In 1986 we started producing human insulin with the help of genetically modified yeast cells. Shortly afterwards recombinant human growth hormone was introduced on the market, and in 1988 we were the first company in the world to produce a fat-degrading detergent enzyme using a genetically modified micro-fungus. Since then, a steady flow of products satisfying real needs has been developed and marketed, and today the

majority of our products are made using gene technology.

Documenting safety
From the beginning the use of gene technology was accompanied by stringent safety legislation, where Denmark in 1986 was the first country in the world to adopt specific legislation on genetic engineering and the environment.

Since all our production strains are characterized as safe, i.e. presenting no risk to human health or the environment, the environmental authorities have approved the release of a limited number of GMOs from our production processes.

One of the concerns often raised in the public debate is the possibility of GMOs surviving outside the production plant and of them disturbing the ecological balance. To date, there have been no examples of environmental damage due to fermentation-based use of GMOs. We are aware that we must continue to document an excellent safety record in order to achieve public acceptance.

Production organisms not established in the environment
In 1997, we published the results of a field study of the retrievability of two genetically modified production strains in the environment around our Kalundborg plant. The outcome of the study was that no viable production organisms could be identified in the collected samples. An article on the study can be viewed on our website.

In 1998, a similar evaluation was performed on the possible presence of two important enzyme production strains in the vicinity of our production site in Franklinton, North Carolina.

The isolation procedure was based on selective media and the detection of enzyme activity. The final identification was performed using the PCR technique. 130 soil samples from 26 different locations around the Franklinton facility were evaluated. No production strains could be isolated from the samples.

The use of modern biotechnology in food
One area where the use of gene technology arouses much debate is its application to food products. After the introduction of genetically modified plant materials such as soybeans and maize in Europe, the discussion has centred especially on the issue of labelling and the consumer's right to choose.

A growing number of our enzymes for food manufacturing are produced using GMOs. During the production process the enzyme product is recovered from the GMOs and the nutrient broth. It is further purified and does not contain GMOs.

Enzymes can be used to produce top-quality foods with a reduced need for additives. They can also increase productivity and cost-effectiveness, and reduce the environmental impact of food processing. Food enzymes are normally used as processing aids in the manufacture of food or food ingredients and have no function in the final consumer product.

We believe that improving the basic knowledge of genetic modification and food production processes will lead to greater understanding and eventually wider public acceptance of the use of genetic engineering.

Our approach is based on transparency, and we always inform both our customers and other interested parties about the techniques and processes used to make our products. We state openly when our enzyme products are made using gene technology and a full list is available on our website.

Agricultural products for fermentation
Maize and soybean meal are among the raw materials used in our enzyme fermentation process. We use only agricultural products that are safe, including genetically modified soya and maize approved for food and feed.

Antibiotic resistance markers
Novo Nordisk makes use of different antibiotic resistance genes as selection markers in the development of its production organisms. The marker genes are used in the laboratory for the selection of the recombinant host organism in the construction and maintenance phases. Antibiotics are not used in large-scale production. The possibility of gene transfer has been minimized in our gene constructions and our use of GMOs is not expected to have any impact on the environment.

However, we recognize the increasing public concern related to the increase in antibiotic resistance. Over the coming years we will develop other selection methods. This will happen when the use of alternative markers can be incorporated into new high-yielding strains and cell lines. We have already developed yeast and fungal strains without antibiotic resistance markers. Upon approval from the authorities, these new strains will be introduced in our production.

In 1998, an EU project on "Antibiotic resistance genes in the

environment: A comprehensive, muliti-phasic survey of prevalence and transfer" was initiated with the participation of Novo Nordisk.

Enzymes are found almost everywhere. For example, many of Novo Nordisk's enzymes have originated from soil samples. Soil contains millions of microorganisms that produce enzymes.

A long wait
The step from identifying an interesting enzyme to producing it commercially can be a long one. The story of phytases illustrates the challenge biotechnology companies face when developing products from microorganisms.

Phytases were first discovered in rice bran extract in 1907, but it was not until 1962 that the American company International Minerals and Chemicals (IMC) made the first commercial attempt to develop a phytase. After screening over 2,000 microorganisms, an organism taken from the soil in a flowerpot was found to produce the highest yields of phytase. It has later been identified as the fungus Aspergillus niger. Despite investing many years of research, IMC had to abandon the project because yields were too low to make production feasible.

Why modify microorganisms?
It was not until 1973-75 that a new technique - genetic engineering - brought a revolution to experimental biology. Genetic studies began on phytases in 1984 and the first commercial phytases appeared on the market in the mid-1990s. They were made with the help of genetically modified organisms.As demonstrated by electrophoresis analysis, microorganisms produce many different proteins. Each peak on the diagram represents a different enzyme protein. For a particular industrial application only one of these enzymes is usually of interest, such as a phytase. When the wild-type microorganism is fermented industrially, however, it will produce numerous other enzymes that are of no commercial value. Yields of phytase will also be very low.

Recombinant DNA technology allows the gene expressing the phytase to be transferred into another microorganism. In the case of our phytase, the gene was inserted into Aspergillus oryzae, which is a fungus known for its ability to produce high yields of enzymes. As a result, a pure phytase could be produced because there are fewer by-products and virtually no unwanted enzymes. Yields of the phytase increased hundred-fold compared to using the wild-type microorganism.

Apart from making commercial production feasible, resources can be saved. One study based on another enzyme was published in the 1995 Novo Nordisk Environmental Report. It showed savings of 41% in raw materials, 47% in water, 48% in steam and 49% in electricity based on a comparison of producing the same enzyme from a wild-type strain or by the use of a GMO.

Phytases are added to pig and poultry feed to release phosphate trapped in plants. It would otherwise pass right through these animals without being metabolized. The phosphate would then end up in the manure,

where it represents a potential source of pollution. Excessive phosphorus in water courses and the sea can lead to eutrophication.