Team:DTU-Denmark/Human Practices

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Introduction

Resource scarcity and global warming are currently the two major global problems. With the fast-paced industrial growth that our society has experienced in the last years, the environment is degrading at a tremendous pace, stressing that the implementation of sustainable technologies is the only long-term solution to this issue. With our project, we faced the challenge to counteract these problems by targeting the Sustainable Development Goals, among others, 12: Climate Change, and 13: Responsible Consumption and Production (Fig. 1). Through the assimilation of methane in yeast we want to provide sustainable alternatives to harmful methane disposal methods used nowadays, thus actively contributing to the fight against climate change. Furthermore, we want to increase the market value of emitted methane by engineering our yeast to convert it to an industrially relevant protein of interest. This in turn could encourage the development of more efficient methane-capturing techniques and promote further development of solutions promoting a circular economy. To make all this possible, with our project PHEAST we have created a toolbox to genetically engineer K. phaffii into a methane-consuming cell-factory. Although the intended use of our genetic tools is to embrace science for a better world, we are aware that with every technological advancement comes the risk of misuse. To look into the dual-use potential of our platform, we explored the possible impact of our project - positive as well as negative. For this reason, we have engaged with relevant stakeholders from industry, politics, and academia to further discuss the matter.

Figure 1: Sustainable Development Goals PHEAST lies its focus on.

Integrated Human Practices Sustainable Development Goals

UN Sustainable Development Goals

Out of the 17 SDGs which UN has emphasized on, our project PHEAST positively impacts 7 SDGs. The idea of our project stems from the UN SDG 13 which is to immediately act upon climate change, and we chose to tackle this by developing the cell-factory and utilizing a GHG, methane, as a carbon source to produce products of interest. Through the methane-consuming yeast cell-factory, it will be possible to provide nutrition to the ever-increasing global population, move away from fossil fuels to bio-based energy generation and support newer and attractive industrial implementation for energy production. Through our project, we can move towards a circular economy and produce valuable products. With the current human activities which are constantly increasing the GHG in the environment, we are losing biodiversity at a faster rate than we should, and this project provides scope to revert to the rich biodiversity the earth possesses. Most importantly, it is essential to get the project accessible to the general public, and to do this we were constantly engaged in collaborations and communication with relevant people and understand its potential of ending up in the market. Read more on our Sustainable Development Goals page.

Integrated Human Practices

Figure 2: Overview of the different stakeholders we engaged with during our integrated Human Practices activities.

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In this overview, the major outputs of the different stakeholder interviews (Fig 2) can be seen. We divided the outputs in those which concern the synthtic biology part of the project (blue) and those concerned with the fermentation set-up and operation (red).

Click on the major outputs to read more about the interviews!

Kenneth Jensen

PhD, Protein Engineering, Novozymes

Outcome: Due to Kenneth's advice, we dismissed the idea of co-culturing two K.phaffii strains and instead engineered one strain so that it is able to do both conversions steps. Furthermore, thanks to his input, we decided to use a mix of both native and strong consecutive promoters and our idea got confirmed to use native signal peptides of the pMMO. From his industrial point of view, he was able to also provide valuable input in the later set-up of the fermentation which we will consider in the continuation of the project.

Interview on the 01.06.2021
Description: Novozymes A/S is a global biotechnology company and an industry leader in the production of industrial enzymes and biopharmaceutical ingredients with 48% global share of the enzyme market. For their production of industrial enzymes they use fermentation processes with differnet cell factories. Kenneth Jensen is a science manager and project leader in the protein engineering departmnet at Novozymes. With his experience he is able to provide us valuable feedback for our project from an industrial pointt of view.

Contribution: The interview with Kenneth was very positive, he liked our idea and admitted that it is realistic to accomplish our goals in the given time horizon, which is especially important at the start of such a project However, he saw possible problems with the insertion of pMMO into the membrane. Therefore, he suggested and reinforced our idea to use native signal peptides to ensure that the pMMO goes into the right membrane. To prove that, instead of doing a GFP fusion he proposed to use fluorophores, with subsequent ultracentrifugation and confocal microscopy to confirm the right integration of pMMO. About co-culturing, he concluded not to do a co-culture as in general the ratios between the organisms are hard to balance and outcompetition of one of the organism might occur. Once the design and build work has finished and fermentation can start, Kenneth suggested using fed-batch and supplementing the fermentation with iron, which is needed for hemoglobin, as it might be tricky to keep in solution. He also proposed to use many small fermenters to faster find the optimal fermentation parameters. He also expressed Novozyme's interest in our project if we can make K. phaffii grow on methane. It is really great to see that already at such an early stage of our project we could spark the interest of a big industrial player.

Irini Angelidaki

Professor for Bio Conversion, Department of Chemical and Biochemical Engineering, DTU

Outcome: We learned from Irini that a project like this should be as simple and as economical as possible to find industrial application. Irini pointed out possible bottlenecks of our project and reminded us to turn the focus on the methane to methanol transition step. Thanks to her we also planned to do a techno-economic analysis to estimate the commercial feasibility of the project. We also looked into literature on maintaining sterility during methane fermentation processes which we planned to apply once our K. phaffii strain is developed.

Interview on the 01.06.2021
Description: Irini Angelidakis research group of Bio Conversions works in the fields of biofuel production, anaerobic process optimization and development of sustainable solutions for organic waste. We contacted Irini as she works in the field of circular economy and especially because she worked with methane before. After obtaining a first industrial perspective on the early stages of our project, we also wanted to get feedback from academia.

Contribution: Irini pointed out possible pitfalls of the project. She mentioned that our project might has too many elements for the short duration. She suggested focusing either on protein production or methane assimilation. In her opinion, we should focus on methane to methanol even if methane is quite complicated to work with. Irini put special attention on the difficulty of maintaining a sterile process during the fermentation process. She even introduced us to her current research where we learned how methane can be used to produce single cell protein.

Nikolaus Sonnenschein

Asscoiate professor in the Department of Biotechnology and Biomedicine, DTU and Senior Researcher, Novo Nordisk Foundation Center for Biosustainability, DTU

Outcome: Nikolaus was a vital contact when introducing us to the world of Metabolic Engineering. It was for that reason we decided to use GSMs for PHEAST. After that, he provided very valuable input throughout the project for model development and results interpretation. Moreover, from his recommendations to provide quality data to our model, we performed a methanol kill curve in our strain to consider the methanol toxicity in our model.

Interview on the 09.06.2021 and 27.08.2021
Description: Nikolaus Sonnenschein has wide experience in the field of Metabolic Engineering, and Genome-Scale Metabolic Models (GSMs) are a great part of his research and expertise. We contacted him as GSMs seemed to perfectly fit our modeling purpose and we wanted to get the opinion from an expert to verify this. He also has broad experience with COBRA (COnstraint-Based Reconstruction and Analysis) toolbox, a software widely used to work with GSMs.

Contribution: Working with GSM might be quite cumbersome if one does not have previous experience with this type of model. Hence, during our two meetings with Nikolaus, we had the opportunity to get to know more about GSMs, COBRA toolbox and its potential applications to our project from an expert in the matter. His literature recommendations were very useful and we used them as great guidance for our first steps.
Besides, during our model development, we were able to discuss our result interpretations with him and learned about some specific methods that turned out to help us in simplifying our genetic engineering software tool. He also provided valuable recommendations on which experiments to perform in cooperation with our lab work team to improve our system knowledge and improve our predictions.

Ib Christensen and Subir Kumar Nandy

Senior Executive Advisor, Principal Scientist, Unibio A/S

Outcome: Unibio reassured us in our choice of host-organism and the use of pMMO (as opposed to sMMO). For the future fermentation setup, we will consider safety regulations to deal with the oxygen methane mixture as well as consider only applying pure methane to guarantee that no by-products are formed. Byproducts could have grave consequences on both the fermentation process and the final product quality. On the basis of collaboration, Unibio offered that we could use their lab-scale U-loop reactor to do our fermentation experiments.

Interview on the 20.07.2021
Description: Unibio is a company which produces single cell protein in a methane fermentation using the bacterium Methylococcus capsulatus, therefore they have great knowledge within working with MMOs. We contacted them to hear about their experience and about a possible collaboration.

Contribution: As Unibio works with prokaryotes the main thing they could contribute to our project was the fermentation setup. Unibio utilizes a specific type of reactor, namely the U-loop reactor, for their fermentations as these allow for high utilization of the gases in the reactor [1]. This is important as methane has low water solubility and therefore is hard to supply in sufficiently large amounts for large scale fermentations. Their work proved to us that methane indeed could be used as a feedstock in commercial fermentation processes. In order to reach high productivity, we would need to supply the process with a lot of oxygen, which in turn could become an explosion hazard, which of course needs to be considered in a fermentation setting.
Unibio does not utilize GMOs in their production as their end product is mainly sold to food and feed industries, and EU legislation has been constraining them. One of the prokaryotes Unibio works with is M. capsulatus, we asked them about their opinions on us working with implementation of pMMO, to which they replied that pMMO is the most efficient of the MMOs, but that it is not very specific and can produce toxic byproducts, especially if the methane is supplied in biogas or just a mixture of different gases.

Eric Allain

Group Leader at Novozymes North America

Outcome: We followed Eric's advice and looked for transcriptomic data to obtain promoter strength. We also went through the information provided by Eric and got very interested in implementing GECKO and FBA into our GSM.

Interview on the 22.07.2021
Description: Kenneth Jensen recommended his colleague Eric, who works at Novozymes North America, as he is an expert in yeast metabolism and modelling of metabolic networks. We mainly focused the interview on obtaining feedback on our models, the GSM as well as the promoter kinetics model.

Contribution: To optimize several reactions in the GSM at once he suggested a reverse approach, first put in what we think we would need and then let the model simulate the growth. Furthermore, we should try to optimize for maximum ATP production to get around the biomass equation constraint. Here, the enzyme constrained method GECKO from Benjamin Sanchez could help. As a second point he mentioned using dynamic Flux Balance Analysis and he consulted us on how we can connect it to the GSM to model the whole process. This could also help with our promoter kinetics model. For it, he suggested searching for transcriptomic data to obtain promoter strength as we don't have the resources to determine the promoter strength experimentally.

Benjamin Sanchez

Research Scientist, Christian Hansen

Outcome: In conclusion, although GECKO is already showing great improvement for GSMs, due to time constraints and in accordance with Benjamín's opinion, we had to compromise on the idea of implementing GECKO in our GSM. However, with this meeting we confirmed our work as he agreed on the way we specified the constraints for pMMO based on literature. It is a more efficient approach, that is already providing valuable data to our model.

Interview on the 03.08.2021
Description: Benjamín Sanchez has been also involved in GSM study for almost 10 years and he was part of the development of a new method for GSMs which improved them by including enzyme dynamics. We got to know about GECKO and its potential after the meeting with Eric Allain, who was also working with it. After studying the content of the literature and seeing the great potential this new implementation could potentially have to our model, we decided to contact Benjamín, one of the authors.

Contribution: GSMs are a wide field in continuous development and improvement. In these late years, many new methods and approaches to these models have been constructed. GECKO is a new method to work with GSMs including enzymes’ kinetics constraining the metabolic fluxes. As one of his developers, Benjamín provided a lot of great input to our model and confirmed our reasoning behind different model assumptions based on literature research such as methane turnover as well as pMMO expression. He also mentioned the process we followed was completely fair and very similar to the idea behind GECKO. All in all, having a meeting with an expert in the matter was very productive for us as this is a field not so well-known and therefore the resources available are limited.

Jens Preben Morth

PhD, Protein Engineering, Novozymes

Outcome: Preben gave us many things to consider regarding the part of our project that focuses on constructing a methane consuming cell factory. As a result of this interview, we amped up the lab work on implementing pMMO, we allocated half of our lab work team to solely work on pMMO integration. We wanted to make a large library of different constructs with the pMMO’s genes, however with the constraint of time we had to compromise on this. Additionally, as Preben advised us to do, we looked into the “Why K. phaffii?” as we needed to be able to support our choice of host organism, therefore we turned to modelling to see if we could predict whether our K. phaffii strain could be as efficient as M. capsulatus.

Interview on the 10.08.2021
Description: From our previous interviews we determined that the pMMO integration is the crucial point of our project focusing on building a methane-consuming cell factory. As recommended by Unibio, we contacted Jens Preben Morth as he worked on pMMO and had consulted Unibio for their knowledge about pMMO in M. capsulatus for his work on structural analysis of pMMO.

Contribution: Jens Preben Morth recommended us to focus on the pMMO expression, as opposed to protein expression, as this is the main part of creating a methane-consuming cell factory. Preben advised us to not genetically engineer the protein but rather leave it as native as possible, as it might just work as it is. He also said that there are no real possibilities to influence whether the pMMO goes into the membrane. To guarantee the right integration, we should make as many different constructs as possible. and check for efficiency after successful integration. To enable the three subunits to work together, he proposed to take the entire operon from the bacteria, optimize it for yeast, and put it into the yeast genome. However, we should check if the size of the resulting insert causes a problem for the yeast cell. Additionally, he mentioned that putting in more than one copy of each subunit gene does not cause a significant increase in expression as it lowers the efficiency. Preben also mentioned that if the cloning doesn't work we could isolate pMMO from M. capsulatus directly as it is highly expressed. A final comment from Preben regarded the economical aspects of our project, where he mentioned that using the native bacteria is cheaper than genetically engineering another organism.

Alexandre Huchelmann and Ilaria Ciabattio

Civil Servants, EU Health and Food Safety

Outcome: Importantly for our project, we were informed that transgenic organisms were not at the table for being reassessed due to lack of evidence in terms of risk assessment. As we are dealing with a transgenic cell-factory this issue is highly relevant for us. Initially, we thought of applying the K. phaffii methane consuming strain in small-scale applications directly at sites of methane production, such as oil rigs or agricultural farms. Such an approach will not be applicable in Europe as the only way the technology could be applied even in the middle-term is in industrial, strictly contained settings. Such a setup is not realistic to envision at the aforementioned sites of methane production. Thus, we have incorporated the knowledge gained in our implementation and business plan. Accordingly, we now aim for an industrial application with methane from the gas network. We want to promote a solution in which methane can be captured at its origin and fed into the network, so that our system can also make use of it and represent a decisive piece in a circular economy.

Interview on the 13.09.2021
Description:
Synthetic biology research and its industrial implementations have met considerable political and legal hurdles, particularly in the EU. In our process we realised that we could develop the greatest technology, promising to significantly reduce atmospheric greenhouse gases but that it could never be implemented in an efficient way without change in GMO regulation in the EU and worldwide. Even if there is a legal possibility to put a synthetic biology into practice, its implementation is often not attractive to investors as the regulations are complicated and gives the potential product a bad image. Hence, we wanted to get an insight from EU officials about current state and progress of GMO regulation within the EU which has been getting attention recently with the publication of a study by the EU Commission on the way forward with GMO regulation. We conducted an interview with an EU parliamentarian to get the public representatives’ point of view as well as two civil servants from the EU department for Health and Food Safety as to get an opinion from the executive.

Contribution: Our interview with Alexandre Huchelmann and ILaria Ciabatti from the EU department of Health and Food Safety gave us valuable insights into the further process of taking action upon the EU study on new genomic techniques (NGTs) released in April (LINK). They commented that it is indeed taken as an incentive to propose a change of law to the EU parliament and Council by the Commission. Although the study suggests to legally categorize NGTs on the same basis as older ones such as modification by radiation, they emphasized that it was a process of a few years in which the public opinion plays a crucial role. We asked further about their take on the dilemma between a bottom-up approach which may prevent effective technological action on climate change and a fundamentally undemocratic top-down approach, they said that such a process can only be democratic in the EU while there is some room for the executive to put the topic on the agenda and fund respective projects of science and communication.

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References

[1] https://www.unibio.dk/technology/u-loop-fermentor/ 21.10.2021
References for pictures:
Kenneth Jensen: https://www.researchgate.net/profile/Kenneth-Jensen-4
Irini Angelidaki: https://www.energy-supply.dk/article/view/559167/dtuforsker_omdanner_affald_til_bioenergi
Nikolaus Sonneschein: https://orbit.dtu.dk/en/persons/nikolaus-sonnenschein
Ib Christensen: https://www.crunchbase.com/person/ib-christensen
Benjamin Sanchez: https://www.benjasanchez.com/
Jens Preben Morth: https://dagensmedicin.dk/lundbeckfonden-giver-25-mio-kr-til-jens-preben-morth/
Alexandre Huchelmann: https://www.linkedin.com/in/alexandre-huchelmann-aa270571/?originalSubdomain=be

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