Forbidden FRUITS - stable microbial production strategies for non-native compounds

Presented by Team Amsterdam 2020

Dennis Dekker, Samira van Den Bogaard, Robin Laird, Diana Victoria Ramírez López, Joris Visser, Mingdong Liu and Kelly Klomp


Genetically engineered cellular systems can be used to produce industrially valuable compounds in a sustainable way. A challenge is that it is more beneficial for cells to use their resources exclusively for growth, resulting in a loss of production ability. Therefore, we have developed Forbidden FRUITS, a pipeline that can solve this problem by calculating and optimizing engineering strategies to couple a product forming pathway to microbial growth. Multiple databases, constraint-based programming and gene-protein-reaction associations are used to devise suitable strategies. These strategies are then optimized using pathfinding methods and sequence optimization. As proof-of-principle, we applied Forbidden FRUITS to salicylic acid, lactate and mannitol production in Synechocystis PCC6803, lactate in Synechococcus UTEX 2973 and salicylic acid in Escherichia Coli. Forbidden FRUITS is shown to be flexible and allow for the fast development of stable production strains, making the full-potential of biotechnology evermore attainable.


  1. Changing the way we produce things such that we have a sustainable tomorrow.
  2. Generating genetic engineering strategies for stable production of any compound in any microbe.
  3. Writing a modular and extensible algorithm to generate these genetic strategies with a predicted growth rate and yield.
  4. Verifying growth-coupled genetic strategies created by Forbidden FRUITS in the lab using widely studied microorganisms.

Green bar down

Just a green bar, no forbidden FRUITS hidden here...

Motivation, Stakeholders and Approach

Our motivation for iGEM? It's gonna be legen — wait for it — dary.

In this section, we will sketch the storyline of how we started with our project, developed and designed it and hope to revolutionize biotechnology.

Why cell factories? And what is a current problem?

Humanity shares the collective responsibility to reduce the ecological footprint as much as possible. For many years we have been taking our natural resources for granted, and consumption has been growing with the atmosphere being filled up with CO2. The conversion of CO2 into products is the foundation of the bio-based economy, which has the potential to replace the use of oils. Right now, we see that the industry is growing and has diversified by using a variety of microorganisms to produce compounds, which are called “cell factories”.
A major problem with cell factories is the genetic instability of engineered strains that eventually leads to phenotypic instability. This basically means that genetic changes leading to production are not essential and cost energy for microorganisms. Over time, evolution will enter the stage and a faster-growing non-producing microorganism will outcompete the producing microorganisms.

Stakeholder needs and perspectives

From our industrial, academic, governmental and public engagement we learned that there are a wide variety of needs that arise when sketching the problem.
  • Academic - Could use clear easy-to-use tools that generate genetic strategies to couple growth to production and use components within the strategy.
  • Industry - Mostly in need of optimal growth rate together with optimal production rate. Regarding the product, secondary metabolites are more economically feasible. Additionally, the industry needs more public acceptance and change of GMO legislation in order to be interested.
  • Governmental - Risk assessment of genetic strategies for GMOs is a big need and could help change legislation.
  • Public - Needs transparency and information in order to become more accepting towards genetic modification.


Forbidden FRUITS was born with the purpose of coupling production to growth in order to overcome genetic and phenotypic strain instability.

The project was formulated under the idea that, given a target compound, it is possible to find a set of reactions that can be added to a metabolic network of a microorganism in a growth-coupled fashion.

This means that, in principle, Forbidden FRUITS, would be able to identify a genetic engineering strategy to stably produce any compound in any microorganisms for which a metabolic model exists.

Based on our stakeholder’s needs we decided to follow a modular and extendable approach for the development of the algorithm. This means that each of the team members took care of one part of the algorithm making sure that in the end each part could be merged into one pipeline, thus creating Forbidden FRUITS.

Green bar down 2

Another green bar, do you see any FRUITS?

Forbidden FRUITS pipeline

You have found yourself a pipeline for forbidden FRUITS, can you follow the trail and explore what it does?


Combines information from multiple online databases.
  • Exploiting complementary information from online databases.
  • A bigger and more complete dataset leads to the predictions of better strategies.

In our project we combined BiGG, KEGG and MetaNetX (see image below).


Provide the metabolic network of the microbe of choice and provide the name of the desired product. In the image below, we explain the concept with a toy example and a real life example.

Gene-Protein-Reaction-Associations (GPRA)

Transforms the metabolic network provided in the model with associations between genes, proteins and reactions obtained from the database.
  • Linking reactions in the network directly to gene knockouts.
  • The information about isozymes and promiscuous genes improves the feasibility of the generated design.


Find a path relating our product to the network using network and database.
  • Any path relating the product to native metabolites is possible.

Below, we see a toy example (on the left) and a real life example (on the right) of how we can find such path.

Cheap Lunch

Finds indirect paths coupling product formation to growth.
  • Expand selection of compounds which can be stably produced.
  • ‘There is no such thing as a free lunch’: there is a cost attached to forming a product using multiple reactions. The cheap lunch strategy finder finds the path which is the most feasible, e.g. has the ‘cheapest’ cost

In the image below, we illustrate how we can expand a current to create secondary metabolites.

Proof of Concept

How do we prove our concept?

We implemented Forbidden FRUITS strategies for different compounds into different microorganisms. We want to demonstrate two concepts:
  1. Indicating the generalizability of different compounds within a species.
    1. Salicylic Acid, lactate and mannitol in Synechocystis PCC6803.
  2. Indicating the generalizability of one compound across different species.
    1. Salicylic acid production in Synechocystis PCC6803 and Escherichia coli.
    2. Lactate production in Synechocystis PCC6803 and Synechococcus UTEX 2973.

Why these organisms?

  • The cyanobacteria Synechocystis PCC6803 and Synechococcus UTEX 2973 are interesting due to their ability to sustainably produce compounds directly from CO2.
  • Escherichia coli is a widely used bacterium, which is important to make to ensure a broad reach to Forbidden FRUITS strategies.

Why these products?

  • Salicylic Acid and Lactate production prove the ”plug and play” principle of the pipeline. The metabolic network of the host forms the basis, which is extended by production pathways to create a cell factory.
  • Mannitol production proves our “cheap lunch” strategy because the production pathway to make mannitol contains multiple non-native reactions which are not directly linked to growth in our engineered strain.


Returns all feasible combinations of knock-ins and knockouts.
In the table below, we want to show how we visually depict the output strategies. It consists of knockouts, knockins + additional supplement for the organism and we would predict the productivity and growth rate based on available knowledge.

Then the user determines the best strategies based on:
  • Experience with gene knockouts.
  • Availability of readily available knockout strains.
  • Costs of medium supplements.
  • Other factors.

Future Optimization steps

Improvement of strategies generated by Forbidden FRUITS.
  1. Optimization using Downstream Metabolite Information.
    • Product yield can be improved by redirecting flux in the network.
    • Create a chassis more relevant for the industry.

  2. Sequence optimization to optimize the amino acid and nucleotide sequence of knock-ins to better fit the host organism.


Combines the network and the path ensuring that the production pathway is the only way through which biomass can be formed.
  • Uses linear program to calculate the minimal number of modifications in an efficient and precise manner.
  • Adds ‘faucet’ and ‘sinks’ to add or remove flux, respectively.
  • Because of these faucets and sinks, the plumber can be used in the design of chassis dependent on a specific metabolite.


Resolves each faucet and sink individually by finding multiple non-native reactions which could replace the faucet or sink.

Hardware and Implementation

Knowledge is not power, the implementation of knowledge is power...

In the following sections we introduce our hardware, which we will use to test our engineered microbes, and the implementation of Forbidden FRUITS in the real world.


A cultivation setup that allows the selection of individual light settings for real-life screening of a large set of samples in a well plate.


We address the need for a high-throughput platform for the culture of phototrophs.
  • Explore their biological diversity.
  • Screen and select cell factories.
  • Evaluate their commercial potential.

We can control the light intensity of each well depending on real-time optical density (OD) measurements of the culture in it.
  • Constant amount of photons per cell.
  • Simultaneous testing of new strains (e.g. our proof of concept) in different conditions and in parallel with many replicates.

Four 7-day long experiments to observe the growth characteristics of Synechocystis in this new apparatus.

Our 24-well cultivator allowed us to study the effect of carbon limitation and light limitation on Synechocystis.


To explore how we would implement Forbidden FRUITS, we engaged with three societal sectors outside academia: the general public, industry, and the government.


  • We conducted a consumer survey to explore what students in Amsterdam think of genetically modified organisms and how they would spend their money.
    • We conducted a consumer survey to explore what students in Amsterdam think of genetically modified organisms and how they would spend their money.
    • Subjects were more accepting of genetic engineering use in industrial settings, and less so with personal care and food products.
  • We hosted an ethical discussion at CRISPRcon 2020 to explore the moral implications related to the expanded engineering potential Forbidden FRUITS offers and to create an open space for attendees to ask questions or express fears.
    • Takeaway: the onus of ethical reflection should fall on all parties, including scientists, business people, and legislators.
    • It is also important to engage people from different disciplines and backgrounds to more robustly build an ethical framework for technological implementation.


  • We conducted interviews with academics and market research with Dutch biotech companies, whom we interviewed as potential clients and users of Forbidden FRUITS.
    • Takeaways: there is commercial interest in using Forbidden FRUITS through both a metabolic engineering consultation service and a microbial strain development service.
    • However, the greatest hurdle facing these organizations is the strict regulation of genetic engineering in industries like food production.


  • We held a brainstorm and subsequent presentation with the RIVM--the Dutch Ministry of Public Health--to explore the ways in which Forbidden FRUITS could aid risk assessment. Through this collaboration, we planned a number of adjustments to our existing pipeline, like the flagging of hazardous byproducts, so researchers using Forbidden FRUITS could more easily include safety in their strategy selection.

Acknowledgements and Education

Gaining knowledge was our first step to wisdom. Sharing it was our first step to humanity...

In this section we thank our supervisors for all the knowledge they provided us with and want to elaborate about how we tried to share this information with the general public.


As a team we accomplished a lot, but we like to take a moment of gratitude to thank our supervisors from the Molecular Microbial Physiology group from the Swammerdam Institute for Life Sciences.
  • We thank Dr. Filipe Branco dos Santos for his organizing talent, his advice and critical questions.
  • We want to thank Dr. Wei Du for his supervision within the wetlab project and his point of view about different strategies for different organisms.
  • A special thank to MSc. Max Guillaume for organising lectures about linear programming and Forbidden FRUITS, supervising many wet and dry lab projects and most of all for his patience.
  • A word of gratitude to MSc. Wenyang Wu for the supervision in the lab and his humor.
  • We are grateful for the help offered by MSc. Joeri Jongbloets, especially for his programming lectures, supervision in the lab, help with structured programming and his accurate/fruity comments.
  • And last but not least we thank MSc. Hugo Pineda Hernandez for the supervision in the lab and providing us with music when working late in the lab. Due to their assistance we were able to achieve and learn a lot in this short period of time!

Additional thanks to:
Prof. Dr. Stanley Brul and Dr. Ivo Huijbers (University of Amsterdam), Prof. Dr. Davide Ianuzzi and Prof. Dr. Bas Teusink (Vrije Universiteit), Genootschap t.b.v Natuur, Geneeskunde en Heelkunde (GNGH), Jasper Buikx (Coordinator), Ruben Janssen (Education) & Ruben Theunissen (Content Marketeer) from Micropia, Prof. Dr. Jeroen Hugenholtz (Wageningen University), Dr. Harald Ruijssenaars (Corbion), Dr. Tjeerd van Rij (DSM), iGEM team Eindhoven, Just One Giant Lab (JOGL), Peter van der Sijde and Research Ethics Review Committee (BETHCIE), Dr. Brett G. Olivier (VU), Nicole van ’t Wout Hofland (Editor of, Joana Lima Alves, Photanol B.V., and all previous iGEM teams - for setting a solid base on which we could build!

Our sponsors:
  • University of Amsterdam
  • Vrije Universiteit Amsterdam
  • DemonstratorLab
  • Amsterdams Universiteitsfonds
  • ACE Incubator
  • Snapgene
  • Benchling
  • Integrated DNA Technologies (IDT)
  • Lions Printing
  • Sponsors through crowdfunding


We explored the opinions and feelings that people with different backgrounds had about genetic modification through:
  • Online workshops about genetic modification in biotechnology.
  • Blog about the iGEM experience of Kelly
  • Crowdfunding campaign: Weekly newsletter
  • Workshops at Micropia (museum of microbes)
  • Ethical engagement at CRISPRcon 2020


We aimed to explain the importance of biotechnology for a sustainable future and to inspire people to learn more about it.

Online workshops about genetic modification in biotechnology

A series of 3 workshops that you can find in our YouTube channel.
  1. Genetic modification: the how to's and why's.
  2. Genetically modified organisms today.
  3. Exploring the potential of (genetically modified) microbes.

Blog about the iGEM experience of Kelly

Kelly wrote three blog posts about biotechnology and our project for the Dutch website

Crowdfunding campaign - Weekly newsletter

We sent a weekly update of our project to every person who donated to our campaign.
  • Fun facts on biology, biotechnology and genetic modification.
  • Sharing knowledge with different people coming from different backgrounds.

Workshops at Micropia (Microbe Museum)

Together with Micropia, we set up experiments to show the public about how bacteria can be used in biotechnology.

  • Sensory experiments that actively included the audience.
  • Hands on explanation about cyanobacterial cell factories.
  • More than 250 visitors.
  • Open to the public for over 2 weeks!

Ethical engagement at CRISPRcon 2020

Our team hosted the discussion “The Ethical Implications of Creating Anything in Anything” to broadly discuss the ethics behind Forbidden FRUITS and its potential to produce any compound in any microbe in a growth-coupled manner.