Team:DTU-Denmark/Human Practices
To ensure that humans have a world in which they may thrive for many years to come, we need to create novel solutions to the problems facing the world of today. To buck the trend of a rapidly changing climate, significant changes must be made in many aspects of daily life. A major concern is our current consumption of resources. While every person has an individual responsibility to make sustainable choices, these could be facilitated by ensuring our everyday products are produced in a sustainable manner. This does not currently happen to a sufficient extent leading to a world filled with non-sustainable consumption.
As a team we therefore wanted to ensure that our solution could contribute to increasing the feasibility of sustainable production practices. After two weeks of initial brainstorming, we came up with several different problems for which we wanted to try to explore solutions. To determine which project would be most appropriate for our team, we decided to use two main parameters to score our project ideas.
The first parameter was industrial relevance which is important to us as aspiring engineers as we strive to create something that could be adopted by industry and impact production practices. The idea of improving Aspergillus niger as a cell factory stood out in this respect for several reasons. One key reason was that A. niger is a well established organism in industry so any improvements we could create could potentially be integrated into existing processes. Another reason was that effectivising the cell factory would decrease cost of some processes, allowing the replacement of unsustainable processes with sustainable production in A. niger.
The second parameter was potential for positive impact on the world. To evaluate the potential benefits of different ideas, we used the UN sustainable development goals (SDGs) as a metric (Refugees, 2020). Improving filamentous fungi cell factories could potentially have a positive influence on a number of the SDGs, depending on which specific processes are targeted. We specifically identified five SDGs which we could influence by engineering A. niger, two directly and three indirectly.
Directly impacted SDGs
The twelfth goal focuses on minimization of waste, increasing recycling and creating more sustainable production methods. As stated, our project could impact this by enabling the replacement of older production methods with processes based around A. niger. This would allow increased use of renewable resources in production decreasing reliance on fossil fuels and similar resources.
Goal 8: Decent work and economic growth.
The eighth goal is centered on economic development measured by GDP growth, employment and education. Our solution would likely lead to an increase in sustainable production driving a growth in GDP as well as creating new production processes and thereby more employment opportunities.
Indirectly impacted SDGs
The third goal focuses on health both as freedom from disease and mental and physical well-being. By increasing the efficiency of A. niger cell factories the cost of producing generic biological drugs can be reduced. A drop in price could be expected to make the drugs more accessible to people around the world thereby promoting well-being for all.
Goal 6: Clean water and sanitation for all.
The sixth goal is centered around ensuring that everyone around the world has access to clean water. Improving A. niger would allow for more processes to be made in a sustainable manner. This would minimize the amount of chemicals being released into the ground water from chemical processes thereby working to ensure safe drinking water around the world.
Based on the above, we felt confident that our project idea could have a positive impact on the world and we were eager to start working.
However, creating a successful project is not just a matter of considering your own motivations and who you think it will benefit. It is also important to consult stakeholders who can help the project succeed. We therefore highly encourage you to look at our integrated human practices page to see how we involved industry and leading scientists in making our project a reality.
Integrated Human Practices
Consultation with external stakeholders played a crucial role in identifying and shaping our project. To create an industrially relevant solution, we had to acquire insights from multiple stakeholders from different areas.. First we contacted stakeholders in industry who could help us identify current problems that biotech companies face when working with A. niger. This was instrumental in shaping our engineering efforts. Then we got into contact with leading scientists who could help us make crucial design decisions in order to create a viable solution. Finally, in order to bridge the gap between science and industry, it was important for us to finish up our project by taking our findings and solution to the industry so it might be implemented. Through this approach we hoped to make the greatest possible impact.
2020 presented not only the iGEM community but the entire world with a difficult global challenge: A pandemic in the form of Covid-19. This pandemic has affected everyone around the globe and we have all had to change the way we do work and keep each other safe. We therefore decided to keep all interaction with industry online in order to comply with social distancing and to help keep everyone safe. Furthermore, we limited the number of people who could be in the lab at any time to decrease the risk of spreading virus. We are aware that these measures affect what can be achieved in the lab but we believe that the safety and wellbeing of everyone should be the first priority in times like these.
To ensure that we were still able to gain new insights through all of these online meetings with stakeholders we decided to set three simple goals for each meeting:
All three items were documented for each meeting and are presented in the form of a description, a contribution, and an outcome in the timeline below, where you can follow the process through the whole project.
Associate Manager - Novo Nordisk A/S
Mhairi Workman is an associate manager at Novo Nordisk A/S, an industry leading pharmaceutical company within the field of insulin production. The company has the world's largest production facilities of insulin located in Kalundborg, Denmark, and many decades of experience with working with fermentation on an industrial scale. Furthermore, Mhairi Workman previously worked as an associate professor at DTU within the field of systems biology and would therefore be a perfect starting point for getting industry insights into the challenges of A. niger cell factories.
Contribution:
We discussed in depth the many aspects of A. niger morphology and learned that different morphologies are favorable depending on which product the cell factory produces. This means that even though one morphology might decrease viscosity and thereby lower the energy requirements, it might easily come at the cost of lower product yields thereby canceling out any reduction in production costs.
Outcome:
After our talk with Mhairi, we adjusted our goal from creating one optimal morphology for all processes involving A. niger to focusing on creating a solution which would improve protein production. We therefore came up with the idea of focusing on hyper branching and creating a library of signal peptides to aid in the secretion of proteins from hyphal tips.
Kim Hansen
Senior science manager Novozymes A/S
Description:
Novozymes A/S is an industry leader in the production of industrial enzymes and biopharmaceutical ingredients having a 48% global share of the enzyme market. It is helping lead the field in development of new bioproducts which can be produced in industrial scale cell factories. As a senior science manager within the company he has specialised in the field of taking novel lab scale innovations into the large scale fermentation facilities. It was therefore important to get his perspective on our possible lab solutions to optimize their chance of working in large scale fermentation setups.
Contribution:
Kim Hansen was instrumental in giving us insight into the difficulties of taking something from the lab scale to an industrial setup. Especially when it comes to morphology, the behaviour of a filamentous fungus such as A. niger changes significantly when you scale up the fermentation setup. Furthermore he also provided key insights into certain engineering pitfalls that we should avoid for our engineering solution. This included avoiding modifying the fermentation medium to obtain a given morphology because this would likely increase the operating costs of running the fermentations or of the downstream processing. Furthermore, he stressed the importance of avoiding that any genetic engineering relied on antibiotic resistance due to safety concerns and legislative constraints in the industry.
Outcome:
Kim Hansen’s feedback led us to decide to focus our engineering efforts on a solution that would not require the introduction of resistance genes through genetic engineering, or change the fermentation media composition. Additionally, we made it a goal that our solution should be scalable to ensure that it could have a real world impact.
Professor - DTU Bioengineering
Description:
Professor Frisvad has been a leading researcher within the field of mycology for many years with a special focus on filamentous fungi. He has helped describe and discover multiple new species of filamentous fungi where morphological characterisation plays a key role. Furthermore he is an expert within the genus Aspergillus. He was therefore the perfect candidate to help us gain an in-depth understanding in the world of filamentous fungi.
Contribution:
Professor Frisvad contributed by educating us on the intricate structures that filamentous fungi form which are very different from what is otherwise observed in the microbiological world. This allowed people on our team that had never heard or worked with fungi before to understand the organism better.
Outcome:
From the information provided we could clearly identify areas where we had to study and learn more about filamentous fungi to complete the project.
Dr. Edyta Szewczyk
Senior Scientist in Biology - Bolt Threads
Description:
With a background as a fungal geneticist, Dr. Edyta is working as a strain engineer at Bolt threads. As a company Bolt threads strive to produce sustainable biomaterials to replace current unsustainable materials. One such example is a leather substitute based on mycelia formed by filamentous fungi with the commercial name Mylo™. The company therefore built up a lot of experience in modifying the morphology of filamentous fungi.
Contribution:
Dr. Edyta stressed that the need for a solution to control the morphology of filamentous fungi such as A. niger has existed for many decades and that no universal solution has been found yet. This reinforced our belief that our idea could have a real impact. Based on her extensive experience in working with filamentous fungi she provided knowledge on where we might get relevant information on genetic engineering targets and on solutions that other people have previously tried.
Outcome:
Our discussion with Dr. Edyta increased our incentive to create an industrially relevant solution, avoiding the pitfall of creating something that would work only on a small scale. We immediately began to follow up on all the sources of information that Dr. Edyta had provided on working with A. niger and filamentous fungi morphology. This helped immensely in expanding our knowledge within the topic.
Science Manager and Project Leader - Novozymes A/S
Description:
Kenneth Jensen is a science manager and project leader in the protein engineering department at Novozymes. We wanted to talk to him to gather information about relevant proteins as well as growth and measurement methods to make our project as industrially relevant as possible.
Contribution:
We discussed which protein would be interesting to produce, weighing the advantages and disadvantages of glucoamylase vs red fluorescent protein. Kenneth also emphasized that extracellularly secreted product is very industrially interesting as it simplifies downstream processing. Therefore the focus on good signal peptides could be interesting. When discussing which growth experiments to perform, Kenneth mentioned that shake flasks are difficult to get homogenous conditions in and data from these is therefore not as relevant as other growth methods, such as biolector or bioreactor growth.
Outcome:
After this meeting, we decided to use glucoamylase as our production protein instead of red fluorescent protein as it is more industrially relevant. Further, we decided to focus on growth in biolector rather than shake flasks as they are not representative of growth patterns at larger scales.
José Luis Martinez Ruiz
Associate Professor - DTU Bioengineering
Description:
José is the manager of the DTU Fermentation Core, a state of the art university facility that investigates microbial physiology of cells and other aspects related to fermentation technology. We wanted to talk to him to get his thoughts on aspects to consider when engineering filamentous fungi and how they act in bioreactor growth.
Contribution:
José mentioned several different aspects in filamentous fungi morphology engineering that would be interesting to achieve and consider. He mentioned that in a filamentous morphology there is a larger surface area per volume of cell compared with pelleted growth and that this will generally result in larger secretion. Therefore, when engineering to decrease the level of filamentation, considerations should be made to try and keep the surface to volume ratio large so as not to negatively impact production too much. José emphasized that the risk when trying to engineer morphology was that we would most likely lose some other positive aspects such as productivity or growth rate. However, it might still be advantageous overall as complicated growth and difficulties with cleaning are large issues in industry.
Outcome:
Talking to José made us think about the different aspects of fungal morphology that we considered changing and what we were trying to achieve. Additionally, further consideration was given to that fact that certain changes might have unwanted consequences and that the balancing act between advantages and disadvantages should be kept in mind when designing and assessing the mutants.
Head of fermentation pilot plant - Novozymes A/S
Description:
As in most biotech companies, the final step before setting up a new large industrial process will be testing the process in a pilot plant. Novozymes is no exception to this and we were therefore very eager to get into contact with Morten, the head of the Novozymes pilot plant. He is an expert within the field and has unique knowledge on what works beyond a small bioreactor.
Contribution:
Morten emphasized that high viscosity is a major problem in industry when working with filamentous fungi but that making the fungi grow as a small dispersed pellet would not be the best solution either as oxygen transfer might then be limited. Furthermore, any tools that we could develop to help predict morphology would be of value to industry and could help make better decisions before entering the pilot plant scale for new products.
Outcome:
After talking with Morten we decided that it was very important that our simulation models would have the best predictive abilities possible. This way they would be more likely to be used in industry.
Jakob Blæsbjerg Hoof
Associate Professor - DTU Bioengineering
Description:
Jakob is an associate Professor at DTU Bioengineering in the Department of Biotechnology and Biomedicine. He is the co-author of ‘Fungal Genomics: methods and protocols’ being one of the experts in genome editing of filamentous fungi.
Contribution:
Jakob suggested analyzing our selected signal peptides under different conditions, since these can heavily influence the secretion profile. He commented on how cloning 10 different signal peptides into the same protein would give very valuable data, and suggested we look for similarities in the signal peptides to understand what common motifs make a signal peptide a good secreting sequence.
Outcome:
After the meeting, the idea of creating a bioinformatic program to create synthetic peptides was considered. A meeting with drylab was arranged and a model for the design of the synthetic signal peptides was started.
Associate Professor - DTU Bioengineering
Contribution:
We decided to contact José again in order to gain insights into what types of bioreactors we should run and how they should be set up. In an ideal world he suggested that we would run both batch and fed-batch fermentations of all of our engineered strains. Due to the time constraints of iGem he also suggested we prioritize batch fermentations as it would be the most informative setup to run if we only had time for one. Furthermore, he recommended that we not use dry weight samples to determine growth rates due to the lack of homogeneity in filamentous fermentations but instead used the CO2 concentration measured in the off-gas of the bioreactors.
Outcome:
We decided to only do batch fermentations because we would not have unlimited time to use the bioreactors. Additionally, dry weight measurements were dropped in favor of CO2 measurements to determine growth rates.
Kenneth Jensen follow up meeting
Science Manager and Project Leader - Novozymes A/S
Contribution:
Kenneth gave valuable feedback on what kind of data we should produce for all of our engineered strains of A. niger to determine which of our engineered strains would be most industrially relevant. He also helped us set up a meeting where we would present all of our findings to Novozymes A/S with the hope of getting our findings out into industry.
Outcome:
Given this unique opportunity we decided to make a large presentation that we could take to Novozymes A/S, the largest enzyme producer, with the hope of actually making an impact in the industry.
Senior Bioinformatician - Novozymes A/S
Description:
As it is not that easy to find bioinformaticians working within the field of fungi, we were pleased to hear about Dennis’ engagement. Dennis is senior bioinformatician at Novozymes A/S who works with machine and deep learning and both protein secretion and fungal morphology are everyday topics in Dennis’ work. Thus he was a perfect source to discuss our drylab work with.
Contribution:
Dennis was very interested in all parts of our drylab work and had many insights with respect to how we could make our models even more relevant to industry and more user-friendly, so that companies such as Novozymes A/S could use them. He also contributed with ideas of future improvements to our models as well as ideas for benchmarking.
Outcome:
Dennis made the excellent point regarding our models that if they were collected in easy-to-use packages that require little preparation to run, then there is no reason for the industry not to try them out. Naturally, this whetted our appetite and encouraged us to improve our code and the user-friendliness of our models.
19. October 2020
Just before the wiki freeze, we gave a presentation of our engineering solutions and software tools to representatives from the Novozymes pilot plant and strain construction team. They were very excited about our genetic engineering solution. With this we hope that our results can help improve the industry and have some of the desired effect that we have been striving towards.
References
-
Refugees, U. (2020). Transforming our world : the 2030 Agenda for Sustainable Development. Retrieved 27 October 2020, A/RES/70/1, from https://www.refworld.org/docid/57b6e3e44.html