Introduction

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We come from a technical university in a small country, where innovation is going to be our livelihood, so we have throughout our studies been encouraged to think outside the box and strive to make the impossible possible. But since we are just a group of young, blue eyed students coming from the comfort of the university, we wanted to investigate how our idea would fit into real life settings and explore the viability of basing industrial biotechnological processes on waste streams.

Throughout our project, from the initial development of the idea to finalizing the lab work, we have been in close contact and collaboration with the industry. We started out searching for abundant waste streams in Denmark by contacting and visiting local factories, then we presented our idea to biotech manufacturing companies and all together the feedback made us more aware of the challenges our technology is facing and it ultimately led us to a more feasible product.

Investigating local industrial waste streams

Denmark is a country known for having the happiest people, the royal family and bicycles. We are also home to the world’s biggest pork exporter and you have probably tasted a beer from Carlsberg or played with Lego’s at some point of your life. We started of with the textbook example of an abundant industrial waste stream on the rise - glycerol from biodiesel production. *Numbers on biodiesel and increasing amounts of glycerol*

We came in contact with local biodiesel producers, Perstorp and Emmelev A/S, to investigate their waste streams with the perception that glycerol was an abundant waste product from their productions and thereby a problem, we could help them get rid of. Surprisingly, we quickly learned that glycerol is far from being waste, but actually already is a valuable byproduct used in the chemical industry. This made us go back to the drawing board. We turned to a local brewery, a canola oil producer and a company processing household waste. One true waste source we came across was the press cake sediment from cold pressed canola oil production.

Implementation of substrates from waste streams in the biotech manufacturing industry

The second part of our integrated human practices consisted of interviews and feedback from two of the biggest biotech companies in Denmark, Novo Nordisk A/S and Novozymes A/S.

Interview with Novo Nordisk A/S

Novo Nordisk develops and produces more than 50% of the insulin worldwide along with growth factors and medicine for hemophilia. To get the perspective of Novo Nordisk on how the increasing demand for insulin and higher demand of crops/edible carbon sources will require a more sustainable technology, we went to visit the Novo Nordisk Science Park in Måløv, Denmark *Insert photo*, to interview Nina Gunnarsson and Kjeld Olesen, who both are researchers in the Strain Development department in Novo Nordisk. With more than five years of employment at the company, they provided us with some insights on how they currently produce insulin, what they value in a production organism and what it would take for them to apply our technology. Click here to read the full interview.

Novo Nordisk currently produces recombinant insulin in the conventional baker's yeast, Saccharomyces cerevisiae, by growing it on different refined sugars. “We use a very little amount of sugars compared to what is consumed for food, so right now that sort of sustainability is not that a big of a concern for us. We are more focused on our obligation to provide sufficient amounts of insulin to the patients,” said Kjeld Olesen, when we asked them if changing their substrate into a less competitive and sustainable one was something they were considering. Nina Gunnarsson added: “We obviously have a big focus on having the most optimal processes with the highest possible yield with the minimum environmental impact.” And the most optimal process is for Novo Nordisk is one, where the demand for purity is met. Nina and Kjeld explained to us that purity in regards to avoiding degradative proteins and post-translational modification, especially O-glycosylation, is very important, but the purity of the entire process is essential to obtain the high level of quality required for a therapeutic protein.

We ultimately asked Nina and Kjeld from Novo Nordisk, if they believed changing their insulin production to Yarrowia lipolytica growing on abundant waste streams would be an option: “Well, it would be a huge project! Because the product quality is so important, it would be a very long and complicated changeover, which will include a new factory and new experts, so it would have to be a very good business case,” Nina replied and Kjeld followed: “A stabile, continued supply of substrates is very important. The content of the substrate cannot vary either.” And in conclusion, Nina suggested we should consider applying our technology for something other than therapeutic compounds: “Maybe this would be more applicable for production of other things than therapeutics - such as bulk chemicals, enzymes or food additives, that do not have the same quality and purity requirements.”

Summary of the main points from the Novo Nordisk interview:

• Purity and quality of the product along with a stable supply chain and production is the most important aspects when producing therapeutics.
• Triple bottom line mindset (economic, social and environmental sustainability) and a strong focus on the obligation of meeting the insulin demand.
• Novo Nordisk sees our technology as more applicable for production of less regulated compounds such as bulk chemicals, enzymes or food additives.

Interview with Novozymes

With this in mind, we turned to another major league player in the biotechnological industry - Novozymes A/S. Novozymes has a very diverse production line and produces enzymes for industries ranging from agriculture, household care and food to bioenergy and wastewater solutions. Furthermore, they are a top 10 company on the Fortune Magazine’s Change the World list, so we thought - who else would be more likely to seize our idea and technology?

“I’m not saying that it IS a challenge, but it could be a challenge. If you cannot get the dirt out of the fermentor, then you need to use organic solvents and it might also have other consequences. But in general, I would say yes! There is a lot of available fat sources that could be used. But we need to know the consequences.” was what Gernot J. Abel from the Research and Technology department at Novozymes in Bagsværd, Denmark, said, when we asked him, whether Novozymes would consider switching to Y. lipolytica as production organism in case our molecular toolbox is up and running, and then base the production on waste streams such as glycerols, fatty acids and media containing other complex carbohydrates. “The more you can do with one single strain, the better. But if Yarrowia is excellent making more homogeneous proteins, then that would be a selling point. Get rid of your waste, so less waste and more value. If you can cut production costs 10% down and use different waste streams, then cool!”

Novozymes currently base their production on easy accessible proteins and carbohydrates such as sugars and starch and as cheap as possible, but Gernot J. Abel is open to the idea of using waste streams as substrates. However, he also made us aware of some the main points of the Novozymes strategy, which our technology must accommodate in order to be viable. Gernot stated that having a process, where the fermentor is cleanable and the fermentation controllable is essential: “One question is if we can make these cell factories cheaper… It has to be controlled, it needs to be clean and temperature controllable. You have to bring in all the nutrients. Cell factories is our heart and our production system, so it’s extremely important.”

Like Novo Nordisk, Novozymes have one main goal and that is to deliver, what the customers require both in regards to meeting the demand for quality and quantity. “Novozymes is about manufacturing. Our R&D research is important in developing but in the end of the day, you have to deliver to the market place and be able to send enzymes and ship them to the customers... For us it’s more important that our enzymes can do their job. We keep trying to optimize our enzymes to perform better. That is where our focus is compared to changing our production host.”

Summary of the main points from the Novozymes interview:

• The process should be cleanable and controllable.
• Production on waste streams should be cheaper compared to traditional production.
• Quality and quantity of the product is the most important.

Exploring the possibilities

One of the key features of using Y. lipolytica as a chassis is its diversity both in regards to substrate demands and to production of desired compounds. We aimed to make the expression system modular and in compliance with the iGEM Biobrick standards, so the production would not only be limited to one product, and with the inputs from Novo Nordisk and Novozymes in mind, we began to think about other possibilities of our system. Searching the registry, we came across biobricks for production of the food additive beta-carotene in yeast. However, we had to improve these biobricks to make them applicable for in-frame fusion...