Team:Nottingham/Design

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Project Design

Aims

To make a safe biotherapeutic strain of bacteria able to produce DBHB which could be easily administered to patients we set the following objectives:

  1. Selection of a suitable bacterial strain for the biotherapeutic.

  2. Selection and engineering of synthetic pathways to produce DBHB.

  3. Selection and engineering of a biosafety strategy.

  4. Develop an administration method for delivery of our biotherapeutic.

Selection of a suitable biotherapeutic strain

The first decision for our project was to select a suitable organism which would be basis for NeuroTone. The genus Clostridia presents a promising source of gut based therapeutics and as our supervisors in the SBRC have a wealth of knowledge of working with the Clostridia, we narrowed down our decision to a species from this genus.

The team worked to come up with a list of potential Clostridium species to research further, looking into the genomes for genes beneficial or detrimental to our project. As Clostridia are anaerobic spore formers, they are ideally suited to life in the human gut, a number of Clostridia already live there!

We immediately eliminated certain Clostridia from our list, although quite suited to life in the gut, their pathogenicity was unfortunately a deal breaker; C. difficile, C. tetani, C. perfringens and C. botulinum for example! We eventually arrived at C. acetobutylicum, C. butyricum and C.  sporogenes.

All three of these strains are non-pathogenic and have some of the genes which could be beneficial for ketone production, as well as other desirable characteristics for our project.


Clostridium acetobutylicum is a species of industrial relevance in solvent production, harbouring the genes required for the Acetone-Butanol-Ethanol (ABE) fermentation pathway, which we are aiming to use as a basis for ketone production [1]. However, as C. acetobutylicum has not been used outside of its industrial setting, along with the possible complications of solvent production in the human gut, we decided not to go with this organism.

Clostridium butyricum has proven success in the probiotic industry, it is used to promote gut health and protection against pathogens [2]. This species is also a native of the human gut. This bacterium is capable of producing butyric acid, with pathway components which could be manipulated to produce our therapeutic ketone, DBHB.

Clostridium sporogenes has not been used as a probiotic, but work has been carried out to use this organism as an anticancer therapy in the human body [3][4]. It is capable of colonising the human gut, but is not a native commensal [5]. The 2019 iGEM Nottingham team have successfully introduced a synthetic pathway to produce acetone in C. sporogenes, the precursor, acetoacetate, could also be used to produce DBHB in our project.

We ultimately chose to go with C. sporogenes, and there were a number of features which influenced our decision:

  1. Familiarity: The SBRC has a lot of experience and success working with C. sporogenes, especially in the iGEM competition!
  2. Spore Production: As the name suggests, C. sporogenes has a reputation for making a lot of spores, which is important in the manufacturing process for a biotherapeutic, and fortunately, our supervisor Raquel has a wealth of knowledge on the sporulation process in this species.
  3. Indolepropionic Acid (IPA) Production: C. sporogenes already produces IPA, a potent antioxidant which has been shown to have neuroprotective effects, which would complement our project aims [6].
  4. A Novel Gut-based Biotherapeutic: C. sporogenes has not been used in this capacity before and it has great potential as a biotherapeutic in the human gut [7].

For all of these reasons, we selected C. sporogenes (strain NCIMB 10696) as the organism for NeuroTone [8].

Selection of suitable DBHB production pathways

To ensure the success of genetically engineering C. sporogenes to produce DBHB, both the modellers and our DBHB subgroup helped to form structural models with the following aims in mind:

  1. Identify the metabolic pathways in C. sporogenes that can be manipulated to produce DBHB.

  2. Identify the best pathway to implement (e.g. the most thermodynamically feasible and efficient in anaerobic conditions, DBHB yield).

When constructing possible DBHB pathways we concluded the most viable route to producing these ketones using the components of the Acetone-Butanol-Ethanol (ABE) fermentation pathway (see Figure 1) [9]. From within this pathway the DBHB subgroup hypothesised synthetic pathways to produce DBHB, both utilising acetoacetyl-CoA. One manipulating the acetoacetate pathway and the other using 3-hydroxybutyrate-CoA (3HB-CoA) as an alternative intermediate. The genes required for these two pathways were assembled into synthetic gene operons. The dry lab team then modelled these two pathways to identify the most suitable pathway for DBHB production.

ABE-fermentation
Figure 1. The ABE Fermentation Pathway: Showing the microbial conversion of sugars into the acids acetate and butyrate and the solvents acetone, ethanol and butanol. The Thiolase (thl) and CoA-Transferase subunit (ctfA/B) genes are highlighted as potentially useful for our synthetic ketone production operon(KPO).

Since acetoacetate (the first ketone body produced in the fasting state) is produced as an intermediate product we thought it was the best place to start. Inspired by the Nottingham’s 2019 iGEM team’s acetone production operon we utilised the first two enzymes to produce acetoacetate: using the thl, ctfA and ctfB genes from Clostridium acetobutylicum. But instead of producing acetone we added an alternative enzyme to convert the acetoacetate to DBHB. This final gene required is beta-hydroxybutyrate dehydrogenase (bdhA), taken from Streptococcus dysgalactiae subsp. equisimilis, a Gram-positive coccal bacteria commonly found as a commensal in the gut. This pathway was then designated as DBHBA.

acetoacetate-operon
Figure 2. Our synthetic DBHAA ketone production operon based off the acetoacetate pathway of the ABE fermentation.

Acetoacetyl-CoA can also be metabolised to produce DBHB by a series of alternative reactions that don’t rely on acetoacetate as a metabolic intermediate. For this pathway, called DBHBB, we produced a three gene operon with each enzyme taken from different bacteria. The first enzyme, as in the acetoacetate pathway, is thiolase encoded by the thl gene taken from C. acetobutylicum which was suggested by our modelling advisor. The acetoacetyl-CoA produced by the thiolase can then be converted to 3HB-CoA by acetoacetyl-CoA reductase (encoded by phbB) from Cupriavidus necator. The final enzyme in the pathway, thioesterase II (encoded by tesB), from Escherichia coli forms the desired DBHB from 3HB-CoA. These enzymes were taken from pre-existing genetically engineered pathways that have been successfully implemented in previously published experimental studies [10].

butyrate-operon
Figure 3. The DBHBB synthetic operon as an alternative production operon based on the conversion of 3HB-CoA.

Engineering of C. sporogenes for DBHB production

Testing our synthetic pathways using shuttle vectors

To determine if the production of therapeutic levels DBHB is achievable via either proposed synthetic pathway in C. sporogenes, shuttle vectors carrying each operon were designed. This would help inform the decision of which pathway to implement in our final production strain. The chosen pathway would ultimately be integrated into the chromosome of C. sporogenes for more stable expression in the biotherapeutic strain.


Shuttle vectors allow you to transfer a plasmid containing our genes of interest into the desired host, in our case C. sporogenes. The cells that contain this plasmid are typically selected using an antibiotic resistance marker present on the shuttle plasmid. By adding the respective antibiotic into the growth medium, this would select the cells with successful integration of the plasmids. The vectors are usually constructed and stored in E. coli and then transferred or ’shuttled’ into the organism of interest. Therefore, the shuttle plasmid must be compatible for replication in both construction strain and final recipient strain. The plasmids are generally ‘shuttled’ by conjugation, therefore the plasmid must contain conjugal transfer genes.
For our project we utilised the pMTL80000 modular plasmids designed for use in Clostridial species. We choose to use the pMTL82151, displayed in Figure 4 below, in our shuttle vector experiments for DBHB production [11].

Shuttle-vectors
Figure 4. pMTL82151 vector backbone used for expression of our synthetic DBHB pathways in C. sporogenes. Features annotated include a plasmid transfer module (traj), gram positive replicon (pBP1), Gram negative replicon (ColE1), Chloramphenicol/thiamphenicol antibiotic resistance marker (catP) and a Multiple Cloning Site (were our pathway will be inserted) flanked by strong terminators.

DBHB shuttle vector variants

Both the acetate and hydroxybutyrate-CoA pathways will be assembled into the pMTL82151 shuttle vector by HiFi assembly, with four variant promoter regions upstream of the ketone production operon (KPO).

  1. KPO with Pfdx - constitutive expression using the native fdx promoter as a positive control.
  2. KPO with PX - no promoter producing no expression of KPO genes as a negative control.
  3. KPO with Pntnh - expression of KPO genes dependent upon sigma factor botR expression.
  4. KPO with Plac - expression of KPO genes dependent upon presence of the inducer molecule; lactose.

These four promoter variants were chosen to create strains able to produce the most desirable levels of DBHB. Each promoter will enable a different DBHB production profile.


This promoter is taken from the C. sporogenes genome and controls the expression of the ferredoxin gene (fdx). This promoter is known as a constitutive promoter because it controls the expression of an essential gene which is always ‘switched on’ and will ensure a high expression level of the KPO genes. This promoter variant will act as our positive control and should theoretically produce the highest levels of DBHB.

The ntnh gene (non-toxic non hemagglutinin) and its native promoter (Pntnh) is encoded in the botulinum neurotoxin cluster in C. botulinum.. An alternative sigma factor (botR) is also located in the neurotoxin cluster and is responsible for transcriptionally activating Pntnh [12].
The ntnh promoter module we used consisted of the PbotR, botR and Pntnh sequence, all of which are found next to one another natively. This ultimately allows expression of the KPO from the Pntnh. This promoter module was used by the 2019 Nottingham iGEM team and demonstrated that the expression from it in C. sporogenes showed late growth phase expression, peaking after 48 hours of growth. This promoter would act as an extra control for production of DBHB, tied to the growth of the bacterial culture, allowing the culture to establish in the gut before production of DBHB.

We wanted to include an inducible promoter system that would act as yet another way of controlling the production of DBHB if required. This lactose-inducible system was taken from another clostridia – Clostridium perfringens. In the presence of lactose the transcriptional regulator, bgaR allows expression from the divergently transcribed promoter bgaL [13].

We will then test for DBHB production using gas chromatography in these strains harbouring the shuttle plasmids at several time points across the growth of the culture. This will help inform which pathway we want to take to the next step - chromosomal integration. In this project, the modelling will inform which pathway we ultimately choose to proceed with.

Genomic integration of the synthetic pathway

Once the DBDHA KPO was predicted by the modelling team to be the most favourable pathway we proceeded to design plasmid vectors to integrate this pathway into the C. sporogenes chromosome. Integration into the chromosome will allow stable expression of the pathway in our therapeutic strain without the need to maintain a plasmid vector by antibiotic. The operon was designed with strong bidirectional terminators upstream (Tfad) and downstream (Tfdx) to isolate from chromosomal read through (Figure 5).

integration_DBHBa
Figure 5. Vectors to insert the DBHBA pathway, preceded by one of the three promotor variants, into the C. sporogenes chromosome at the pyrE locus were designed. Integration relies on homologous recombination between designed homology arms of the editing cassette.

We chose to integrate our KPO into the pyre locus of the C. sporogenes genome. The pyrE locus is a tried and tested region of the genome for insertion of heterologous DNA in Clostridia, as demonstrated in the SBRC and by the 2019 Nottingham iGEM team [14][15][16]. We designed our homology arms to simultaneously remove the pyrE gene and replace it with our KPO. To integrate our operon, we decided to use the RiboCas editing tool developed within the SBRC [17].

RiboCas is a novel plasmid tool developed for clostridia allowing CRISPR/Cas9-mediated genomic integrations. The system relies on homologous recombination-based replacement of the chromosomal region with the vector’s editing cassette (for us our KPO). The vector contains a CRISPR associated protein 9 (Cas9) which acts as a pair of molecular scissors and is directed by a designed RNA guide. This guide RNA is designed to be complementary to the region being deleted (in our case pyrE). Only cells in which the desired recombination event occurs will evade cutting by the directed Cas9 and so acts a selection for only the edited cells. In RiboCas the cas9 gene is tightly controlled by a theophylline induced riboswitch. This means less toxicity issues are encountered in cloning and time can be allowed for the required recombination events to occur before cleavage.

Analysis of our therapeutic production strain

Once the DBHBA operon with the three promotor variants are integrated, will we once again test for DBHB production using gas chromatography. This should mirror the results seen in the shuttle vector experiments. Growth and spore production assays shall be carried out to ensure our integration has not had any unintended impact the cells. All promotor variants would be carried forward until a decision on which would be best suited for our project in vivo was reached.

Selection and engineering of a biosafety strategy

As our project involves the delivery of genetically engineered microorganisms to the human gut, we needed to design a biosafety mechanism to prevent our engineered C. sporogenes strain escaping from the gut into the environment. Inclusion of a biosafety mechanism in our biotherapeutic strain of bacteria would help address concerns surrounding the biocontainment of synthetic organisms. The chosen mechanism could also allow the treatment to be stopped if the patient was no longer willing to use the biotherapeutic, or if their medical status changed.
Biosafety mechanisms include kill switches, addiction systems, and self-destroying modalities [18]. The simplest kill switches involve deleting a gene that encodes a critical molecule for the organism’s survival. The critical molecule can be provided in the environment so that the researchers have control over the organism’s survival. Following this there are various options, but each must be tailored to each individual project (for example, it would be useless to employ a lactose-based mechanism in an environment that is rich in lactose).


There is no ‘gold-standard’ that we could implement in our project due to a lack of current products that deliberately use genetically modified clostridia in the human gut. Our team explored three kill switch mechanisms during the selection process; lactose, synthetic amino acid and tetracycline.
The lac repressor is a regulatory protein associated with the lac operon system. It is encoded by the lacI gene and prevents the transcription of the lac genes by binding to the operator. This presents a physical barrier to RNA polymerase so that expression cannot occur. The lac repressor is inactivated by the presence of lactose leading to a question of validity if this approach is implemented in our bacteria. The potential presence of lactose in the human gut would prevent repression of our target genes and render this method useless if implemented.
The synthetic amino acid route would involve genetically reprogramming the bacteria to rely upon the supplementation of a synthetic amino acid for cells to grow. This could be achieved by reassigning UAG stop codons to instead incorporate a non-standard synthetic amino acid [19]. Introducing UAG codons into essential target genes would lead to a dependence on this synthetic amino acid. To implement this strategy, we would also need to modify all existing UAG stop codons in C. sporogenes to an alternative stop codon and introduce an engineered aminoacyl-tRNA snythetase. Analysis of the C. sporogenes genome suggested that this may be more time consuming than initially hoped and ultimately proved much too large a challenge, one that could be made into a whole project on its own!
The third mechanism involved use of a tetracycline repression system and was ultimately the one we chose.

To implement the tetracycline repression system, we would genetically engineer C. sporogenes so that a TetR sequence is found upstream of our target genes. This sequence consists of the tetR gene, encoding the repressor TetR, and two divergent promoter sequences, one which expresses tetR and another controlling target gene expression [20]. In the absence of tetracycline, TetR binds to operator sequences within these promoters to prevent expression of our target genes. When tetracycline is present, TetR is induced and detaches from the DNA allowing our target genes to be expressed (see Figure 6). If our biotherapeutic was to enter industrial production anhydrotetracycline, which also induces TetR, would be supplied in the production culture to allow expression of our target genes, but outside of this factory environment (and not in the presence of anhydrotetracycline) target genes will not be expressed.

tet-promoter1 tet-promoter2
Figure 6. Diagram to show how the Tet repressor system we plan to implement as a biosafety control for our biotherapeutic strain works. In the top panel, anhydrotetracycline (ATC) is absent (as in the gut) and the tet repressor (TetR) binds to the tet promotor upstream of the target gene and no expression of the target gene occurs. However, when ATC is present in the production process (as shown in the in bottom panel) it binds to tetR preventing it binding to the tet promotor and expression of the target gene can occur – in this case spoIVA.

We decided to target sporulation genes with our tetracycline repression system due to the ease of control they provide. We wanted a mechanism that would prevent the survival of our bacteria outside of the human gut. In order to survive outside of this environment, clostridia would have to sporulate due to their anaerobic nature. Therefore, repressing sporulation genes would prevent the survival of C. sporogenes outside of our target environment.
As we moved to a sporulation method of control, we needed to identify some key sporulation genes to target in C. sporogenes. Luckily one of our supervisors, Raquel, is an expert in C. sporogenes sporulation and provided some transposon-directed insertion site sequencing (TraDIS) library data on the essentiality of various genes for sporulation. We went through this list and found genes with low transposon insertion indexes which indicated they were important in the sporulation process. Mutants that had these genes deleted rarely underwent sporulation.
To further narrow these targets down, we researched these genes further, noting and eliminating those with interactions not directly related to sporulation. Most were relatively under researched but had general functions relating to sporulation such as peptidoglycan remodelling or forespore engulfment. We then used Snapgene to locate these genes within the C. sporogenes genome. We wanted our targets to be relatively far apart in the genome to prevent one gene transfer event from wiping them all out and rendering our control method useless. This led to the suggestion of three gene targets: SpoIID, SpoIVA and SpoIIIAA. In our final strain we decided on targeting all three of these target genes to multiply the potential effectiveness and robustness of our mechanism (Figure 7). With this strategy, should a mutagenesis event occur which knocks out the repression of one target gene then there are still other genes repressed that would continue to prevent sporulation.

tet-promoter2
Figure 7. To increase the effectiveness of the mechanism all three of the target sporulation genes identified would be engineered to be proceeded by a tet promotor and tet repressor.

Human practices has intimately shaped our work in the for development of a control mechanism from the beginning of our project. First, we attended the meeting with Dr Green in July where we were advised that our biotherapeutic colonies should die out over time and should need to be repeatedly replaced with new doses of spores. We could achieve this by using a dead man’s handle. This would mean providing a nutrient vital to our biotherapeutic’s survival with the dose of spores. Over time our biotherapeutic colony would run out of this nutrient and would die off. We researched various ways of achieving this and were impressed with the synthetic amino acid method. After further research into the synthetic amino acid system, we decided that this would not be within the scope of our project. Hundreds of C. sporogenes genes used the UAG stop codon and it would be very difficult to affect that number of genes without seriously affecting the organism. Also, it is very difficult to reduce the system to only target a few genes (We aimed to target 4-5 genes for the control mechanism).

Develop an administration method for delivery of our biotherapeutic

When drugs are targeted to the human body, they must take a “route of administration”. This is the path by which a substance is introduced into the body, and it can be affected by several factors: target site, desired effects of the drug and certain properties of the drug.

In the case of NeuroTone, since our project was based around the gut-brain axis, we decided to follow a new range of treatments based on the gut microbiome and go down the oral route of administration. Several different aspects were considered for the formulation of our product, and these are described below.


We wanted NeuroTone to target the brain. However, we also wanted to push some boundaries and show that it is possible to make use of a new but highly touted area of the body [21] - the human gut microbiome.
With exciting steps being made in the study of the gut microbiome, the gut-brain axis and ketones, we felt that we weren’t simply trying something new - this could be a method of treatment that could revolutionize long-term medicine.

There were a few requirements that we identified as crucial to shaping the route of administration. Our treatment should be safe, non-invasive, long-lasting and require low maintenance.

The most important requirement for our treatment was safety. The therapeutic should not cause any harm to the patient and it should not trigger an immune response. We thought that the best way to demonstrate the safety of our product was to have a history of its use in humans. If nothing similar had ever been done before, we wanted to ensure we followed every step to properly test the therapeutic and demonstrate that it is safe.

Due to the nature of the diseases we are targeting, we felt that our treatment would be better accepted if it was non-invasive. Many treatments can require injections of drugs intravenously, intramuscularly, or subcutaneously, and these can be painful and hard to carry out by an untrained person. Therefore, we decided to avoid this route and focus our efforts on a patch/diffusion or enteral delivery method - to be swallowed into the gastrointestinal tract.

We learned from an insightful meeting with Ms. Cook that elderly patients strongly disliked having to take regular medications, and that they would sometimes try to avoid them on certain days. This meeting guided us towards a long-term and low maintenance treatment that would benefit both the patients and the carers.

As previously mentioned, C. sporogenes produces a high number of spores which could can easily be administered orally, in a capsule or tablet. The highly resistant spores could survive the oxygen, acidity, bile salts and other adverse conditions found in the digestive system and then are able to germinate in the anaerobic environment of the gut to produce the DHDB [22][23][24]. As the bacteria has been found to colonise the human gut the strain is expected to reproduce and be metabolically active in the intestine [5]. This means that the DBHB can be produced over a period of time and that NeuroTone should require fewer administrations than traditional exogenous ketones to maintain therapeutic levels of DBHB.

After researching preceding work like ours, we came across many examples of spore probiotics. LactoSpore® [25], using the spore-forming bacteria Bacillus coagulans, is a product that has been certified as GRAS (Generally Regarded As Safe) by the FDA (Food and Drug Administration) that proves its safety in humans in clinical trials. It is to be used as a food ingredient and once consumed will safely colonize in the human gut and produce lactic acid - beneficial for the microbial environment in the gastrointestinal tract [26]. Another well-known example of delivering microorganisms to the gut is the drink Yakult [27]. This drink contains Lactobacillus paracasei Shirota. Recently, the use of Clostridium strains as probiotics has also been explored [28][7]. Some probiotics are sold as supplements, but LactoSpore®, for instance, was submitted to clinical trials. We decided we wanted NeuroTone to be a biotherapeutic subject to clinical trials to test its efficacy and safety. This decision was influenced by our meeting with Dr Edward Green from Chain Biotech.

After investigating other probiotics, we realised we had a few options: we could administer our spores as part of a drink (yogurt, for instance), as a sachet or as a capsule. We decided to use a capsule, because the formulation is simpler and there is a reduced risk that one of its components could trigger premature germination of the spores. Moreover, since NeuroTone is a biotherapeutic, we felt that a capsule would be more appropriate for a medical setting.

The spores are the most important component of the capsule, so we had to figure out a way to produce high amounts in an industrial scale. In the lab, C. sporogenes spores are typically produced by starving the cells, heat treating the samples to kill vegetative cells (the spores survive because they are sturdy) and purifying the spores by eliminating vegetative cell debris. A protocol for spore production and purification at a lab scale can be found here. We wondered if we could make this process even more efficient, and one of the options that occurred to us was to try different media. C. sporogenes is usually grown in TYG (tryptone, yeast extract and sodium thioglycolate), but examples of sporulation media can be found in the literature. Sporulation media usually contain limited nutrients to trigger sporulation by starvation or simulate environments in which bacteria are known to sporulate. More information on this can be found here.

If we could go into the lab, we would perform sporulation assays to compare the media and determine which one would allow higher spore yields, while also keeping in mind the price of the media and the amount of time required to produce the spores. Despite not being able to test the industrial scale production of spores, even if we had access to the lab, we still wondered what it would look like. Based on the manufacturing process of LactoSpore®, we tried to adapt an industrial protocol for C. sporogenes.

Once the protocols were determined, we had to decide how many spores to include in our capsule. According to the literature, spore-based probiotics typically contain 105 – 109 spores and capsules commonly contain 109 spores [29]. We expect that this amount would also allow germination and outgrowth of C. sporogenes in the gut, so we decided that each NeuroTone capsule should contain 109 spores. During a discussion with the rest of team, we decided that we should start with a low amount of spores in order to allow a gradual and non-disruptive establishment of the culture in the gut. This decision was based on results from our modelling team. Therefore, we would create capsules with 2 different spore doses: 105 for initial doses and 109 afterwards.

What about the rest of the capsule? A previous study has shown that sodium alginate/cellulose nanofiber gel macrospheres are able to protect the probiotics from the acidity of the stomach, and then release the bacteria in the gut due to a change in pH [30]. We expect that the highly resistant spores should survive those conditions anyway, but the extra protection and the fact that the spores would only be released in the gut seemed appealing to us.
Other examples of ingredients that can be found in spore probiotics are:

spore-ingredients
Table 1. Commercially available spore-based probiotics and their compositions

From these examples, it appears that cellulose is the major component of the capsule itself, while hydroxypropyl methylcellulose, microcrystalline cellulose and magnesium stearate are used as excipients [31][32][33].

If we had access to the lab, we would perform germination assays to test if any of these components would trigger germination of C. sporogenes spores.

Since we didn’t have access to the lab, we looked at the germinant receptors present in C. sporogenes to try and predict which germinants usually trigger germination in this strain:

spore-ingredients
Table 2. Germination receptors present in C. sporogenes

We know that GerA receptors recognise L-alanine, while GerK and GerB recognise AGFK, a combination of L-asparagine, D-glucose, D-fructose, and K+ [34]. C. sporogenes appears to have GerA and GerK receptors, so any of these germinants could trigger germination. Although none of these is present in the proposed components of the capsule, there are uncharacterised germination proteins in C. sporogenes that could be triggered by other nutrients/environmental factors.

The selected formulation of NeuroTone.

In conclusion, NeuroTone would consist of a sodium alginate/cellulose capsule, containing 105 or 109 C. sporogenes spores engineered to produce DBHB, which would be stabilized by hydroxypropyl methylcellulose, microcrystalline cellulose and/or magnesium stearate (depending on results from the germination assays).

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