Design

Here we describe the procedure that we used in the design of the different modules of our system: molecules production, regulation by optogenetics, and coculture process and fermentation with an overview of our experimental plan.

Overview

We created a system for the production of nutrient-enriched yeast to supplement the food supply of astronauts during long-duration space missions. The system has been designed to work inside a spaceship or a space station. In order to use the fewest resources possible, we designed a synthetic symbiosis between Saccharomyces cerevisiae and Clostridium ljungdahlii. The yeast will be engineered to produce β-carotene which can be cleaved by the intestinal enzyme β,β-carotene 15,15’-monooxygenase into two molecules of vitamin A. We also planned to engineer the yeast to produce either a flavor of lemon or a flavor of sweet rose. The choice of the flavor can be decided by the astronauts and it is inducible with optogenetics.

Click on the light to change the flavor of the yeast!

Module 1: Production of nutrients and flavors

First, yeast is a microorganism known to be nutritive. It naturally produces vitamin B which is essential for humans but it does not produce vitamin A nor vitamin D. We hesitated for a long time about which vitamin to produce. Vitamin D was too complicated to synthesize with all the different modules we planned. So, as a proof of concept for yeast-based vitamin production, we will produce β-carotene which is a precursor of vitamin A that humans can assimilate.

Secondly, nutrient intake and taste are two major aspects of nutrition that should not be separated. Once in space, astronauts undergo noticeable changes in taste, which causes them to eat too much sugar, salt or spices. This is why we aim to add strong flavors to the yeast and give astronauts the choice between three of them. We had a long reflection about which flavors we would choose. Each flavor had to be given by only one molecule and by the addition of only one enzyme to avoid too many cloning experiments. We thought about sour, spicy, meaty and sweet flavors. We even thought of Ghrelin, a peptide that increases appetite, as loss of appetite is a problem frequently encountered by astronauts in space. In the end, we chose raw yeast, lemon and sweet rose flavors because these three flavors - or rather the molecules that induce them - come from the same metabolic pathway which is easier to engineer.

β-carotene

The β-carotene-producing enzymatic system from Xanthophyllomyces dendrorhous has already been expressed in Saccharomyces cerevisiae (Rabeharindranto et al. 2019). This enzymatic system is composed of two enzymes: CrtI and the multidomain protein CrtYB. Low yield is the main problem for the production of β-carotene in yeast. This is due to a low precursor flow that leads to the production of coproducts by CrtYB and CrtI (figure 1).

Fig. 1: From Rabeharindranto et al. 2019. Presumed biosynthetic pathway for the synthesis of β-carotene in X. dendrorhous (Verdoes et al., 1999). The bifunctional lycopene cyclase/phytoene synthase (CrtYB) enzyme is depicted in orange, the phytoene desaturase enzyme (CrtI) is depicted in red. Orange or red boxes represent the localization of the modifications introduced by the CrtYB or CrtI enzymes respectively.

First, CrtYB is a transmembrane protein while CrtI is a cytosolic protein. This spatial separation of CrtYB and CrtI leads to a long metabolite transfer between the enzymes. According to Rabeharindranto et al. 2019, the fusion of CrtI and CrtYB leads to a better production yield of β-carotene.

The design of the plasmid containing CrtYB(ek)I is inspired from this publication. Our design differs from it in the choice of the promoter. The promoter TDH1 has been chosen instead of Gal1/10 since we planned to use the latter for regulating other genes. We kept the artificial linker named “ek” since it is the linker which leads to a better β-carotene production (Rabeharindranto et al. 2019). We chose the CYC1 terminator (tCYC1) as a strong terminator.

Fig. 2: CrtYB(ek)I-pUC19. The integrative locus used in yeast is HO. The selective marker used is URA3. The genes coding for CrtYB and CrtI come from X. dendrorhous and are fused using the artificial linker ek. We use the TDH1 promoter and the tCYC1 terminator. The plasmid chassis for this construction and all our others is the pUC19.

On the other hand, β-carotene derives from the mevalonate pathway and so do limonene and geraniol (figure 3). We therefore decided to enhance the mevalonate pathway and the production of GGPP. According to Rabeharindranto et al. 2019, the enhancement of the mevalonate pathway can be achieved by overexpressing the gene HMG1 and CrtE. Our design here again differs from this publication in the choice of the promoter since we used the bidirectional TDH3-TEF1 promoter to obtain the same level of expression for both genes and avoid recombination due to gene homology. The terminators also have to be different, this is why we chose the strong terminator tCYC1 and tPGK. We thus created the plasmids containing a truncated version of HMG1 gene from S. cerevisiae and the CrtE gene from X. dendrorhous as on figure 4.

Fig. 3: Metabolic pathway of β-carotene, limonene and geraniol from Acetyl-CoA in engineered yeast.

Fig. 4: HMG1-CrtE-pUC19. The integrative locus used is DPP. The selective locus used is HIS. The genes coding for the truncated version of HMG1 comes from S. cerevisiae. The genes coding for CrtE comes from X. dendrorhous. We use the bidirectional TDH3-TEF1 promoter and the terminators tCYC1 and tPGK.

How will we test it?

The β-carotene production should color the yeast in orange. We will therefore compare the color of BY4241 wild-type strain, the strain BY4241 containing the CrtYB(ek)I insert, and the strain BY4241 containing the CrtYB(ek)I and HMG1-CrtE inserts.

The pool of GGPP will be analyzed by GC/MS and we will compare the GGPP pool of the strain BY4241 and the strain BY4241 containing the HMG1-CrtE insert. For further analysis, β-carotene could also be detected by Mass Spectrometry.

What is in the iGEM registry?

There are already several parts on the registry coding for CrtE, CrtI and CrtYB for the production of lycopene or of β-carotene but not all of them come from X. dendrorhous. The parts BBa_K530000, BBa_K530001 and BBa_K530002 are respectively for CrtYB, CrtE and CrtI from X. dendrorhous. Nevertheless, there are no fusion proteins to enhance the production of β-carotene.

The part BBa_K2733006 is a truncated version of HMG1 for the improvement of limonene production and the part BBa_K849000 is also a truncated version of HMG1 but for the improvement of β-carotene production. Our aim is to characterize how the addition of the truncated HMG1 impacts the pool of GGPP since the other parts did not describe this specific effect.

How we plan to integrate the regulation of the flavor production

We wanted the production of β-carotene to be constitutive unlike the production of the lemon and sweet rose flavor. We also wanted the astronauts to choose the flavor using optogenetic systems. We chose optogenetics because it is easy to use and does not need to add any chemicals to the culture. We therefore constructed the plasmids for geraniol/brazzein and limonene production in order to add an optogenetic regulation system. The plasmids for geraniol/brazzein and limonene production are designed as below.

The promoter is surrounded by two enzymatic restriction sites (XmaI and SalI) and two other enzyme restriction sites (XbaI and BamHI) are located after the coding sequence. XbaI and BamHI restriction sites will be used to add the gene coding for a light sensitive transcription factor and XmaI and SalI restriction sites will be used to replace the promoter by the promoter which is regulated by the transcription factor added.

We chose these enzymes because their restriction sites are not in the coding sequence of geraniol synthase, brazzein and limonene synthase. We also chose them to respect the submission standard.

Geraniol and Brazzein

This module of aims to add a sweet rose flavor to yeast. The taste of rose was given by geraniol and the sweet taste was given by brazzein which is a small peptide.

Geraniol is produced from geranyl-PP by the geraniol synthase of Catharanthus roseus (Jiang et al. 2017). The brazzein gene comes from the artificial sequence LG263246.1 (Kong et al. 2016).

We used a double promoter in order to have the same level of expression for both genes. The terminators also have to be different to avoid homologous recombination, this is why we chose the strong terminator tCYC1 and tPGK.

Fig. 5: brazzein-CrGES-pUC19. The integrative locus used is yprcΔ15. The selective marker used is LEU2. The gene coding for geraniol synthase (CrGES) comes from C. roseus. The gene coding for brazzein comes from an artificial sequence (LG263246.1). We used the terminators tCYC1 and tPGK. In the original construction, the promoter Gal 1/10 is used to be compatible with the red regulation but it can be replaced by the promoter C120 to be compatible with the blue regulation.

How will we test it?

The geraniol will be detected by UV with a C18 RP fusion HPLC column at 30°C and with a mix of 70% of water and 30% of acetonitrile as eluent. The geraniol can also be detected by the smell of the culture.

The brazzein could be detected by Western blot since we are obviously not allowed to taste it.

What is in the iGEM registry?

The only team which worked with brazzein is iGEM Harvard 2010. They posted the part BBa_K382020 which is coding for an optimized brazzein in E. coli. The protein sequence is the same as ours except that ours lacks the amino acid D after the first methionine.

Five teams worked on the geraniol production but essentially in E. coli and the genes used did not come from C. roseus (BBa_K727006, BBa_K1653027, BBa_K1138000, BBa_K2281001 and BBa_K2753003).

Limonene

This part of our design aims to add a lemon flavor to the yeast. This flavor is given by the limonene which is synthesized from geranyl-PP by limonene synthase (Jongedijk et al. 2016). We used the sequence from the part of the iGEM 2012 team Munich which comes from Citrus limon.

We designed the plasmid below in order to produce and regulate the production of limonene. YPRctau3 is used as the integrative locus and MET17 is the selective marker. The gene coding for limonene synthase comes from C. limon. In the original construction, the promoter Gal1/10 is used to be compatible with the red regulation but it can be replaced by the promoter C120 to be compatible with the blue regulation. The strong terminator tPGK was used.

Fig. 6: LimoneneSynthase-pUC19. The integrative locus used is YPRctau3. The selective marker used is MET17. The gene coding for limonene synthase comes from C. limon. We used the terminator tPGK and the promoter Gal1/10.

How will we test it?

The limonene will be detected by GC/MS. The limonene can also be detected by the smell of the culture.

What is in the iGEM registry?

We used the sequence of the part BBa K801061 but there are other parts available for limonene synthase in the registry. Some teams confirmed the integration of the limonene synthase by the smell of the yeast culture and other teams confirmed it by analysis with GC/MS. Here, we are not looking for validating this part but proving the feasibility of choosing flavors via changes in light wavelengths.

Module 2: Regulation of the production

Our aim is to add lemon and sweet rose flavors to the yeast and to give the choice to astronauts between either raw yeast, lemon or sweet rose taste. This is why we thought of using optogenetic systems to regulate the production of limonene, geraniol and brazzein using light. Optogenetic systems do not involve the addition of chemicals in the reactor which is an advantage for cultivation in space.

There are various optogenetic systems that have been tested for the yeast but they are not all optimal and some work far less than others. Gilles Truan, a researcher at the Toulouse Biotechnology Institute, already tried a few of them and advised us about the ones to avoid and the ones that may work. On his advice we chose the EL222 blue system and avoided the PhyA/Pif3 red system. Our second choice stopped on the PhyA/FHY1 red system because it does not need the addition of a chromophore contrary to most other red optogenetic systems.

Red optogenetic system, PhyA-FHY1

Fig. 7: Red optogenetic system, PhyA-FHY1. PhyA: phytochrome A; FHY1: Far-red elongated HYpocotyl 1; GBD: Gal4 DNA binding domain; GAD: Gal4 activation domain; GOI: gene of interest.

This system allows activating the transcription of the promoter Gal1/10 by exposing the yeast to a red light (660nm). It is based on the two proteins PhyA and FHY1. PhyA is linked to a chromophore which, under the action of wavelength 660 nm, changes the configuration of PhyA. This change of configuration allows PhyA and FHY1 to interact. This interaction is reversible under far-red light (740 nm) or after certain period of time in the dark.

To use these proteins for gene regulation, we planned to fuse the Gal4 DNA binding domain (GBD) with PhyA and the Gal4 activation domain (GAD) with FHY1. When red light is applied, PhyA and FHY1 interact together and bring the GBD and the GAD domains closer, enabling the activation of the promoter Gal 1/10 (Hiltbrunner et al. 2005).

This system has been proven without needing an exogenous addition of the chromophore in the yeast (Sorokina et al. 2009).

pGBKT7 and pGADT7 AD are a pair of plasmids used for two hybrid assays using the transcription factor Gal4. We designed the plasmid below with sequences of genes fusion from this pair of plasmids. We added the sequence of PhyA and FHY1 respectively in the MCS of pGBKT7 and of pGADT7 AD using the online cloning tool Benchling.

Fig. 8: PhyA-FHY1-pUC19. The genes coding for PhyA and FHY1 come from Arabidopsis thaliana and it is between the XbaI and BamHI restriction sites in order to be integrated in the Limonene Synthase-pUC19 or the brazzein-CrGES-pUC19.

How will we test it?

We will insert the PhyA-FHY1 insert in the Limonene Synthase-pUC19 or in the brazzein-CrGES-pUC19 and transform the yeast with the resulting insert. One petri dish will be stored in the dark and the other will be stored in a drawer and exposed to red LED light. Then, we will compare the production of limonene or geraniol by GC/MS or HPLC analysis of the supernatant.

What is in the iGEM registry?

There are very few parts about red optogenetic systems (team Valencia UPV 2017 with the system PhyB/Pif6). Only one team dealt with the system PhyA/FHY1: Freiburg 2007. Nevertheless, their parts are not characterized. So we aim to bring new information and results about the system PhyA/FHY1.

Blue optogenetic system, EL222

Fig. 9: Blue optogenetic system, EL222. GOI: gene of interest.

When exposing yeast to a blue light, this system enables transcription activation of the gene positioned downstream the artificial promoter C120. It is based on the EL222 bacterial transcription factor from Erythrobacter litoralis. EL222 has the capacity not only to sense blue light (450nm) but also to self-dimerize upon light stimulation due to its light oxygen and voltage (LOV) domain. EL222 has a DNA binding domain and the artificial promoter C120 contains 5 repeated binding sites for the fixation of EL222 and one sequence of minimal promoter.

EL222 cannot be used alone as a transcription factor in the yeast. So, we used a gene which is the fusion between a Nuclear Localisation Site (EL222) and a herpes simplex virus (HSV) virion protein which acts as a transcription factor (VP16) (Zhao et al. 2018). We got this gene and the sequence of the promoter C120 in a plasmid from Sylvain Pouzet, a PhD Student at the Laboratoire Matière et Systèmes Complexes at the University Paris Diderot.

Our goal is to regulate the levels of expression of two genes simultaneously, so we created a bidirectional C120 promoter. We designed the plasmid containing NLS-EL222 and the dual promoter C120 as below:

Fig. 10: EL222-pUC19. The gene coding for NLS-EL222-vp16 is between XbaI and BamHI restriction sites in order to be integrated in the Limonene Synthase-pUC19 or the brazzein-CrGES-pUC19. We used the terminator ADH1 and the promoter pPGK1 for the gene NLS-EL222-vp16. The promoter C120 is between the restriction sites of XmaI and SalI in order to replace another promoter by this one. We added the GFP after pC120 to test it before its integration in the yeast.

How will we test it?

We will use the GFP fluorescent reporter to test the system by subcloning it between the XbaI and BamHI restriction sites.

We will insert the EL222 insert in the Limonene Synthase-pUC19 or in the brazzein-CrGES-pUC19 and transform the yeast with the resulting construction. One petri dish will be stored in the dark and the other will be stored in a drawer under a blue LED light. Then we will compare the production of limonene or geraniol by GC/MS or HPLC analysis of the supernatant.

What is in the iGEM registry?

Numerous teams worked on optogenetics. We can cite Heidelberg 2017 and IONIS Paris 2015. Nevertheless, BostonU 2018 is the only team to have used NLS-EL222-vp16 as a blue optogenetic system. Their parts are not characterized and we will aim to do it.

To put it in a nutshell, here is a summary of all the cloning and integration in the yeast that we planned:

Figure 11: Illustration of the integrations we planned to do in the yeast.

Module 3: Coculture

The coculture is one of the central elements of our project. Its rationale is that Saccharomyces cerevisiae is edible but cannot grow from the resources available from the spaceship (CO₂ and H₂), while Clostridium ljungdahlii can grow from these minimal resources but is not edible. Since Clostridium ljungdahlii produces ethanol and acetate that can be used by the yeast (Martin et al. 2016), we designed a synthetic coculture with both microorganisms so that the the yeast can indirectly grow from the spaceship minimal resources.

Supply of nutrients

The electrolysis

In our culture system, CO₂ comes from the environment of the spaceship and from the yeast. O₂ and H₂ come from the electrolysis of water which is already used for the conversion of water into O₂ while H₂ is currently discarded. We therefore decided to valorize this compound.

The supply of nitrogen

Urine contains salt and ammonium but it can also contains toxic compounds and its composition differs from one person to another. Treatment of urine is required to provide the salts and nitrogen contained in urine to the coculture.

The medium and culture conditions

The range of temperature for the yeast is [25;37] °C and the range of temperature for the bacteria is [30;40] °C. This is why we fixed the coculture temperature at 33 °C. The optimal pH of S. cerevisiae is 5.5 and C. ljungdahlii can grow at this pH.

We elaborated a medium for the coculture based on the media used for Clostridium ljungdahlii and Saccharomyces cerevisiae [21],[22]. To design it, we received help from Jason M. Whitham. All the compounds used in S. cerevisiae media are approximately in the same quantity in the media used for C. ljungdahlii. The medium is therefore composed of YNB (what the yeast needs) and we added everything else the bacteria needs. Here is how we elaborated the first coculture medium:

Dissolve ingredients of the pre-medium, warm the solution if necessary to dissolve YNB. Add 10 mL of the trace element solution which should be at pH=4. Sparge medium with 80% N₂ and 20% CO₂ gas mixture for several minutes to make it anoxic and adjust pH to 5.5. Autoclave at 121 °C for 15 min. Add 10 mL of vitamins (sterilized by filtration) from sterile stock solutions prepared under 100% N₂ gas atmosphere. Adjust pH of complete medium to 7 by adding the appropriate volume of NaOH (at 1 mM). Before yeast inoculation, be sure to have a pO₂ at 5%.

How will we test it?

First, we will perform batch cultures of Saccharomyces cerevisiae on various mixtures of ethanol and acetate at 33°C and pH 5.5 to collect data such as growth rates and substrate consumption rates. These data will be used to implement a model destined to assert the feasibility of the project. We will choose the best ethanol/acetate ratio according to this model. Then, we will set up a chemostat with a pO₂ below 8%, to mimic the condition of the coculture.

Next, we will perform batch cultures of Clostridium ljungdahlii on CO₂ and H₂ at 33°C and pH 5.5 to collect data such as growth rates and acetate and ethanol production rates. H₂ will come from an electrolyzer. At the end of the batch, we will remove the bacterium cells from the medium and inoculate it with S. cerevisiae in order to know if C. ljungdahlii produces any toxic compounds for the yeast.

The reactor

Homogeneity of the medium

The system consists of two reactors, one for C. ljungdahlii and one for S. cerevisiae. The medium is circulating between both reactors via two channels through membranes that prevent contamination of the yeast compartiment. We do not want the yeast to be in contact with the bacteria since yeast have to be harvested without any contamination to be consumed by the astronauts. To prevent the formation of microbial cake on the membranes, the medium flow will be reversed sequentially.

System of induction

The yeast compartment is protected from external light and contains red and blue leds.

Supply of gases

Gases input into our system is a major issue for several reasons:

the dissolution of CO₂ and H₂ are the bottleneck of C. ljungdahlii growth.
the dissolution of O₂ is the bottleneck of S. cerevisiae growth on ethanol and acetate.
a gas phase in a liquid reactor in space is not safe and would limit the transfer because of the surface of the gas bubbles.
the apparent concentration of dioxygen must be below 8% in order to not hinder C. ljungdahlii growth.

Because of all these constraints, we decided to supply the gases using hollow fibers membranes (Anggraini et al. 2019). Hollow fiber membranes will enable a better dissolution of gases and thus avoid a gas phase.

Using the work done in the implementation page about the specifications, we solved the main technical issues and designed a 3D-model of the coculture for space application. Our experiments are designed to prove the feasibility of this device prototype.

How will we test it?

To prove that the coculture is possible, we first thought of two glass reactors of 500ml linked by two tubes as shown below. We did not have the possibility to use hollow fiber membranes during the summer and simply transfered gases by bubbling them in the reactor through tubes.

Fig. 12: First draft of the experimental coculture system.

The problem is that we cannot control the pO₂ with this system, but it still needs to remain below 8%. So, we designed another system using a 2L reactor for S. cerevisiae in which the dissolution of O₂ can be monitored with a computer.

Fig. 13: Second draft of the experimental coculture system.

After checking the material that we had in the lab, we realized that we did not have pumps that were accurate enough to be used as we had planned. The alternative was to design a system with one tube and the direction of medium circulation will be reversed sequentially.

Fig. 14: Final draft of the experimental coculture system.

What is in the iGEM registry?

In fact, this section of our project does not provide any “part” as the iGEM competition intends. However, we have designed a system to grow together two species originating from totally different environments (i.e., a synthetic consortium). The use of such biological elements to create a non-natural system is also an exciting part of synthetic biology.

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