Do not judge a bacterial species by its headlines: there is more to Clostridium than food poisoning and botulism.

Do not judge a bacterial species by its headlines: there is more to Clostridium than food poisoning and botulism. Many Clostridium species are non-pathogenic and have been industry champions for decades, used for the large-scale production of essential chemical commodities. Now, they have a brand-new talent: introducing Biocleave, who are transforming Clostridium into the new recombinant protein expression system.

Not all Clostridium species are harmful. Whilst Clostridium botulinum, Clostridium tetani and Clostridium perfringens give these Gram-positive anaerobes a deadly reputation, many species in this large family are not harmful and are commonly found in soil. Clostridium acetobutylicum and its close relatives are known as solventogenic bacteria, fermentation specialists that metabolise a wide range of carbon sources into solvents and organic acids. Clostridium species have been important industrial allies for over 100 years, especially for bio-acetone and specialty chemical production…but what about recombinant protein expression?

Commercial recombinant protein production is usually reserved for a handful of expression hosts, namely Escherichia coli and Bacillus species, yeasts, insect, CHO and HEK cells. However, whilst providing an extensive protein catalogue between them, each system has critical limitations, which means that some proteins are very difficult to express and purify into a usable product.

This Industry Spotlight showcases the recent research achievements of Biocleave, a small group of scientists based in Oxfordshire who are harnessing and re-branding Clostridium bacteria to provide recombinant proteins to researchers across academia and industry.

How Clostridium-based protein expression can facilitate research

By identifying protein targets that are inaccessible using conventional methods, Biocleave offers an alternative solution to provide high-quality recombinant proteins. This is the first time that Clostridium species have been engineered for this commercial application.

With thousands of recombinant protein products on the market, scientists rely on Research Use Only (RUO) proteins to conduct their experiments. Examples of where they play a life-saving role in research include the development of vaccines and identifying new therapeutics for diseases such as cancer and dementia. Recombinant proteins are needed for immunoassays, activity assays and structural biology studies; from these experiments, diseases can be characterised in more detail and novel prevention and treatment solutions can be developed.

The requirement for recombinant proteins does not stop here. Used within agritech, they can provide solutions that will help to improve food safety and security, within the flavour and fragrances sector they can limit the dependence on food sources for fragrances, and in the growing industrial biocatalysis market, they can be used in the synthesis of drug substances, and other fine and bulk chemicals.

A major problem is accessibility – obtaining recombinant proteins can be very difficult, with some being commercially unavailable or in limited supply. They may be hard to generate using conventional hosts, resulting in scientists spending days, weeks or potentially months in the lab just trying to produce proteins required for their experiments. Where supplies are sparse, recombinant proteins can carry a hefty price tag of up to £200,000 per milligram! If you are a researcher with a multitude of experiments to complete, each requiring milligrams of protein, the extortionate cost of some recombinant proteins is very unlikely to be covered by your consumables budget. The worst outcome is that research projects can be changed or abandoned, whereas, had the protein been available, the scientists could have progressed to make groundbreaking discoveries.

Research is tough, and time is precious. Nobody wants to waste laboratory hours trying to express protein with disappointing results or break the bank sourcing recombinant proteins. That is where Biocleave come in, using proprietary microbial engineering and fermentation processes to make challenging recombinant protein targets accessible to researchers, allowing advancement of their research and the development of new technologies.

This new application of Clostridium species provides an innovative approach to expressing proteins that until now have proved very challenging to source and use in research. Currently, no other production platform uses Clostridium species for producing recombinant proteins, representing huge progress in both clostridial and broader applied microbiology research.

How it works: the advantages of Clostridium expression

Despite being a new company, formed in 2020, Biocleave evolved from Green Biologics Ltd where the team had been working with Clostridium species for over 15 years. Previously focused on solvent and specialty chemical production, Green Biologics’ patented CRISPR-based CLEAVETM technology had already been used to generate new clostridial strains with stable SNPs, insertions and deletions to the Clostridium genome. CLEAVETM exploits the CRISPR-Cas machinery already present in the bacteria as a powerful selection tool for creating precise modifications. Now, Biocleave has further developed its proprietary toolkit to transform expression vectors directly into Clostridium, fast-tracking recombinant strain generation. These methods enable production of proteins that were previously impossible to produce in E. coli due to toxicity effects, and creates a straightforward and efficient protein expression process.

Why use Clostridium for protein expression?

Using Clostridium has the benefits of bacterial expression; Clostridium species have a fast doubling time, and compared with mammalian and insect cultures, growth media requirements and fermentation set-up are straightforward. However, unlike E. coli, Clostridium is a Gram-positive expression system, meaning there are no lipopolysaccharide endotoxins on the bacterial surface. Endotoxins are harmful contaminants that can disrupt results and need to be removed from crude recombinant protein prior to sale. Endotoxins are not present in Clostridium-generated recombinant proteins and so the endotoxin removal step is not required, resulting in fewer losses in protein product and higher final yields. Biocleave has shown that endotoxin levels in their protein samples are less than 0.005 endotoxin units (EU) per microgram.

In addition, E. coli can ‘reject’ expression of certain recombinant genes if the resulting recombinant protein is toxic to E. coli. Clostridium provides alternative expression strategies and has evolved a secretion mechanism, which offers the potential for secretion of recombinant proteins directly into the fermentation media.

Biocleave’s product pipeline

This year, Biocleave launched its first product, the Tetanus Toxin Light Chain (the zinc protease half of the notorious neurotoxin produced by C. tetani that causes lethal ‘lockjaw’). The Light Chain on its own is non-toxic, as it cannot penetrate the neuronal cells where its target is located without its partner in crime, the Heavy Chain. That said, the recombinant light chain is essential for characterising how the tetanus toxin works, for example by conducting activity assays and structural studies. Biocleave’s expression system provides a safe source of the active toxin fragment using C. tetani’s non-pathogenic cousin as a host!

Bacterial proteins are not the only proteins in the clostridial pipeline. Biocleave’s targets also include poly(ADP)-ribosyltransferases, commonly known as PARPs. PARPs are human proteins with critical roles in the DNA damage response and consequently are implicated in diseases such as cancer. Although PARP inhibitor therapeutics are now available for cancer treatment, recombinant PARP proteins are still in high demand due to the need for further research in this area.

Also in the pipeline are terpene synthase enzymes. Terpenes are isoprene-derived, volatile compounds and are highly sought after in the flavour and fragrances industry, forming key components in perfumes, cosmetics and cleaning materials. Providing recombinant terpene synthases could provide a more sustainable alternative to sourcing large quantities of flavour and fragrance molecules from plants (which can include food crops) and is unaffected by seasonal variations in yields and pricing.

In addition, terpenes have been found to act as semiochemicals; insect- or plant-derived chemicals that affect insect behaviour and which can act as attractants or repellents. When used in field trials, terpenes have been shown to deter pests away from crops, avoiding crop damage without killing the insects outright. This contrasts with the use of conventional insecticides, which are indiscriminate and not only kill the target insect pests but also vital pollinator species. Using recombinant enzymes to produce terpenes for new pest management systems will help to reduce crop damage and improve yields whilst preserving insect and pollinator biodiversity, which is also essential for maintaining food security.

 #Watch this space!

The development of Biocleave’s Clostridium-based protein expression platform and the release of its first products represents a key milestone in applied microbiology. Not only is Biocleave using Clostridium for a never-used-before application but it is also addressing unmet needs that are relevant to some of the Society for Applied Microbiology’s Priority Areas, such as Food Safety and Security and Future Applications of microbes.

Find out more about Biocleave’s progress on our new website where you can browse products and get in touch with the team. Biocleave also has active Twitter and LinkedIn accounts where you can catch up with the latest news. If you are struggling to access a troublesome protein, perhaps our Clostridium-based system could be the answer!