Prof. Martin Warren, Chief Scientific Officer at the Quadram Institute and a group leader at the University of East Anglia, has developed a cleaner, and more efficient method for biomanufacturing vitamin B₁₂ in response to commercial demand, environmental concerns and food security. To ensure future supply and stabilise price fluctuations the successful laboratory scale method is now being developed for larger commercial production.

Vitamin B₁₂, also known as cyanocobalamin, is an essential vitamin that is widely used in the medical and food industries. As the most chemically complex of all nutrients, it is not feasible to make vitamin B₁₂ by chemical synthesis, but it is one of the few nutrients that can be naturally produced by some bacteria during fermentation. However, prior to Prof. Warren’s recent discovery, the production process was inefficient, leading to harmful waste products that required costly treatment. Consequently, there are very few companies manufacturing vitamin B₁₂ and the end product is prohibitively expensive (it is currently priced at £20,000 per kg), particularly in developing nations.

The B12 market

The global vitamin B₁₂ market size was valued at USD 219.61 million in 2021 and is projected to reach USD 409.7 Million by 2027. The market is being driven by rising health consciousness among consumers and changing diets which have led to an increase in cases of deficiency.

Currently 90% of all manufactured vitamin B₁₂ is produced in China and the market price has varied significantly in recent years due to Covid-associated restrictions and lockdown policies; longer term geo-political issues remain a future concern. This has caused many problems in terms of maintaining a sustainable supply to worldwide commercial processes.

Current technology

During vitamin B₁₂ biomanufacturing, cobalt, which is central to the function of the vitamin, is inserted into the core of the vitamin B₁₂ molecule by a metal inserting enzyme. Ensuring that this enzyme is supplied with enough cobalt requires excess to be added to the growth medium. Cobalt that is not taken up by the bacteria results in waste effluent that contains potentially harmful levels of cobalt and requires specialist decontamination and disposal, which increases production costs. Cobalt is carcinogenic and long-term exposure is associated with cardiovascular problems, thyroid dysfunction, neurological issues and renal impairment.

Industrial production of vitamin B₁₂ is achieved through fermentation of selected microorganisms (7-10 days in huge vats >100,000L). The microbes most commonly used for vitamin B₁₂ production are Pseudomonas denitrificans (reclassified as Ensifer adhaerens) and Propionibacterium freudenreichii subsp. shermanii. After fermentation the nutrient is recovered from the culture medium via precipitation, chromatography and crystallisation. This process can take a further week or so before the nutrient is pure enough to be used in vitamin supplements or to fortify food products.

However, the strains that are used for commercial vitamin B₁₂ production have several shortcomings: long fermentation cycles; complex and expensive media requirements; and a lack of suitable genetic systems for strain engineering. Recently, engineers have shifted their attention to Escherichia coli (E. coli) as a platform for vitamin B₁₂ production.

The Discovery

E. coli does not normally produce B₁₂. However, during a collaborative BBSRC LINK grant, Prof. Warren and his team developed a recombinant E. coli bacterial strain that is capable of producing high levels of vitamin B₁₂. With further funding via a CASE studentship from BASF the cobalt uptake and delivery systems in this strain were improved. Using this new, adapted E. coli strain results in close to 100% incorporation of cobalt into the vitamin, thereby eliminating the creation of hazardous waste / trade effluent and making the manufacturing process more affordable and environmentally friendly.

Follow-up Collaboration

Dr Tessa Young, of the Department of Biosciences, Durham University, collaborated with Prof. Warren to further improve biosynthesis of vitamin B₁₂ by better understanding how enzymes obtain essential metals. To overcome the cobalt supply bottleneck, Dr Young and the Durham team created a ‘metalation calculator’ to optimise cobalt supply for B₁₂ manufacture.

“By understanding the mechanism that distributes vital metals, it has become possible to produce a calculator that industrial biotechnologists can use to optimise their manufacturing reactions. The calculator has been tested in the production of vitamin B₁₂ and we hope to see it adopted by biotechnology manufacturers to foster a more sustainable future.” Dr Young, Durham University.

The ability of the ‘metalation calculator’ to determine the metal requirements for producing B₁₂ on a large scale shows great promise, not only for manufacturing this supplement but also in wider sustainable manufacturing processes using biotechnology. 

Commercial impact

Dupont with Tate and Lyle in Tennessee, USA have developed an environmentally impactful example of biotechnology being used to replace a petroleum-based production process for manufacture of 1-3 propanediol (used in cosmetics, liquid detergents and industrial applications such as anti-freeze) with an E. coli-based bio fermentation process. Corn waste is used to create 1-3 propanediol on a huge scale but the large-scale fermentation facilities also require large quantities of vitamin B₁₂, which is a very expensive ingredient. E. coli-based production of 1,3-propanediol is great for the environment; it consumes 40% less energy and reduces greenhouse gas emissions by 20% compared with petroleum-based propanediol. However, rising costs in the vitamin B₁₂ market and lack of a steady reliable source of the nutrient has seen the price of vitamin B₁₂ vary from £3,000 to £20,000 a kg, resulting in concern over the viability of the process in the long-term.

The new E. coli strain producing vitamin B₁₂ that was developed by Prof. Warren has been successfully evaluated at the laboratory scale and has the capacity to produce enough B₁₂ to be added to the 1-3 propanediol production process. It now represents a stand-by option for the commercial collaborators in the case that costs of vitamin B₁₂ become prohibitive.

This work has attracted the interest of other commercial organisations involved in the production of sustainable nutrients. The Warren group are also developing rapid methods for isolation and extraction of vitamin B₁₂, which will further reduce manufacturing costs.

“It is brilliant to see how academics and industry experts can collaborate on research projects to develop new technologies, techniques and tools to improve manufacturing processes.” Prof. Warren, Quadram Institute.

Funding

This research was funded by The Royal Commission for the Exhibition of 1851 and the Biotechnology and Biological Sciences Research Council.

References

Young, T.R., Martini, M.A., Foster, A.W. et al. Calculating metalation in cells reveals CobW acquires Co^II for vitamin B₁₂ biosynthesis while related proteins prefer Zn^II. Nat Commun 12, 1195 (2021). https://doi.org/10.1038/s41467-021-21479-8