CHAIN has created a suite of underpinning technologies for microbiome therapy including;
- An advanced Clostridia-based gut delivery platform for bioactive products
- A diverse culture collection of Clostridia including commensal bacteria from the gut
- Access to synthetic biology tools for advanced metabolic engineering
- Superior chemical and peptide targets
Live Biotherapeutic Products (LBPs) are advanced treatments capitalising on recent scientific discoveries relating to gut bacteria, collectively known as the gut microbiome. Viable bacterial cells are ingested, either in tablet or capsule form, and multiply inside the gut conferring a health benefit or ‘probiotic effect’.
Unlike classical probiotics strains are not selected due to their ease of cultivation or their preference to grow in food products such as milk or yoghurt. LBP strains are selected for their natural ability to live inside the gut and specific functional properties contributing to overall health. LBPs are subject to the same rigorous double-blinded clinical trials that are required in the pharmaceutical industry, effectively defining these treatments as drugs rather than food supplements.
CHAIN’s lead production strain is a Clostridium species present in the human gut with a proven safety record for use as a probiotic and nutritional supplement. The strain has been engineered to produce specific natural chemicals and synthetic peptides.
Bacteria are cultivated until they produce spores, a robust but inert form of the bacterial cell. Spores are purified from bacterial culture and formulated into tablets. This is advantageous due to the natural resistance of spores to stomach acid. After ingestion, the spores pass through the stomach and into the gastrointestinal tract until they reach anaerobic conditions in the colon. Here, the spores germinate into vegetative cells which produce bioactive compounds that interact with cells of the gut, the immune system and other gut microbiota.
This concept is novel, simple, targeted and extremely cost-effective.
CHAIN has access to a diverse collection of Clostridium bacterial strains. These bacteria make attractive hosts for industrial biotechnology as well as higher value products for nutrition and healthcare markets.
These bacteria have several advantageous features including:
- Naturally present in the gut microbiome, helping maintain healthy gut function
- They possess unique fermentative biochemistry (i.e. broad substrate range), producing a range of reduced fermentation products such as alcohols, short chain fatty acids and other natural products including anti-microbials
- Robust, easy to cultivate and scale-up. Anaerobic fermentation provides unique process advantages
- Some strains are proven at industrial scale with a long track record for solvent production
Clostridium can now be engineered to add functionality and enhance productivity using CHAIN’s synthetic biology expertise and tools available in our labs at Imperial College, London and the University of Nottingham.
CHAIN has identified specific chiral chemicals (R)-3-hydroxybutyrate (R-3-HB) and (R)-1,3-butanediol (R-1,3-BDO) that are difficult to produce using chemical catalysis.
One key innovation involves augmenting metabolic pathways in the microbe to produce chemical intermediates in the correct enantiomeric form (proprietary Chiral Switch™ technology). The change in chirality from the (S)– to the (R)-form enables these chemicals to be incorporated into high value nutritional products rather than conventional lower-value chemical products such as polymers, plastics and coatings.
We have successfully engineered Clostridium bacteria to produce (R)-3-HB and (R)-1,3-BDO as new (and additional) fermentation products in the specific bioactive form.
Modular shuttle vectors allow a systematic approach to plasmid design and construction. The pMTL80000 series contains 18 modules flanked by four rare type II restriction sites. These Plasmids are utilised by CHAIN in its own research, and are available to other researchers in the field. A kit of all modules for the pMTL80000 series can be purchased exclusively via CHAIN.
Recombinant plasmids are usually constructed and cloned via E. coli bacteria before being transferred (‘shuttled’) into the production organism of choice. These shuttle plasmids therefore need to include all the features required for replication and maintenance in both hosts as well as an optional transfer function for a conjugation donor strain.
Choosing a combination of modules
Data on the practically-relevant functional properties of the Gram+ replicons and antibiotic markers used in the pMTL80000 series are described in the paper. Using these results and the pMTL80000 system, you can now quickly construct shuttle plasmids for particular hosts and applications using rationally-chosen combinations of modules. Use this handy sequence generator provided by the University of Nottingham to construct a FASTA seqeunce of your vector of interest. The annotated sequences are generated in Genbank (.gb) format. You can open these files using many molecular biology / bioinformatics tools such as GENtle, ApE and others, including some in this list. When viewing the sequences of pMTL80000 plasmids, set your software to show the following restriction sites: AscI, FseI, PmeI, SbfI, ApaI, NotI, NdeI and NheI.
Due to the standard construction, the sequence of pMTL80000 plasmids is precisely defined by the choice of modules, so can easily be determined automatically. Plasmid names are determined by the combination of modules: “pMTL8” followed by the numbers of the combination of modules used, in the correct order.
Components of the Modular Plasmid Kit
|Gram+ replicon||Marker||Gram– replicon||Application-specific|
|0. spacer||1. catP||1. p15a||0. spacer|
|2. pBP1||2. ermB||2. p15a + tra||1. MCS|
|3. pCB102||3. aad9||4. ColE1||2. Pthl + MCS|
|4. pCD6||4. tetA(P)||5. ColE1 + tra||3. Pfdx + MCS|
|5. pIM13||4. catP reporter|