Enhancing cobalamin (vitamin B12) production in E. coli to address demand and global security

Lead Research Organisation: University of Kent
Department Name: Sch of Biosciences

Abstract

Anemia is a public health condition of epidemic proportions, affecting 30% of the worlds' population (2 billion people). The highest prevalence is in preschool-age children and pregnant women. The condition results in ill-health (weakness, cognitive-development), premature death and reduction in work capacity of entire populations, bringing serious economic consequences to countries and continents. Although anaemia has been recognized as a public health problem for many years, little progress has been reported and the global prevalence of anaemia remains unacceptably high. WHO and UNICEF emphasize the urgent need to combat anaemia and stress the importance of recognizing its multifactorial etiology for developing effective control programmes. Iron deficiency accounts for around a third of anaemia cases, but significantly vitamin B12 deficiency has been identified as one of the major components of pernicious (or non-iron deficient) anaemia, which is further associated with spina bifida, higher levels of brain atrophy in the elderly and is linked with the development of cardiovascular disease and diabetes. A world-wide B12-supplementation programme would have a significant effect but this has so far not been attempted because the current price of vitamin B12, which is also referred to as cobalamin, is prohibitively expensive, especially for developing countries where it is needed most. Moreover, around 90% of B12 is made in China which raises nutrition security issues in the event of conflict or natural disasters. Not only is the nutrient important for human health it is also important as a supplement for animal feeds and for certain industrial processes such as the biological production of methionine and 1,3-propanediol.

Vitamin B12 is one of the few vitamins and nutrients that is produced through fermentation. It is also one of the lowest yielding processes with the nutrient being produced only at around 400 mg/L of culture. This low yield is coupled with a strain that grows very slowly making the overall production very inefficient. In this project we will apply a combination of rational and random screens to develop a high-yielding producer of B12 in E. coli, building on the pathway knowledge of the Warren group and the world-leading expertise of DuPont/Tate & Lyle in strain development and optimisation. The project will result in the generation of a strain that will produce B12 on the g/L scale, ensuring a ready supply of the nutrient for commercial and humanitarian requirements.

This project will provide basic and fundamental insights into the control and regulation of the B12 pathway. Our work will help identify some of the key bottlenecks in the biosynthesis, which is one of the most complex found in biological systems, and develop methods to overcome these issues. The project aims to reduce of the complexity in the pathway by using biosynthetic enzymes that lack inhibitory constraints. The pathway will be engineered into E. coli using gene editing technology to allow for optimal expression of the relevant genes. The strain will be further engineered to allow for the cellular procurement of the various building blocks that are required in the construction of the molecule. This project therefore looks to apply synthetic biology approaches to a current real-world problem and in so-doing will deliver a strain that will have enormous benefit to both industry and the nutrition markets.

For this LINK project, DuPont/Tate & Lyle will provide a total of £470k, including a £100k cash investment, which will go towards funding a full-time technician.

Technical Summary

This project aims to apply synthetic biology and metabolic engineering strategies to one of the most complex biochemical pathways found in nature in order to address a current need for a cheaper and more reliable source of vitamin B12, the so-called anti-pernicious anaemia factor. Vitamin B12, cobalamin, is a cobalt-containing modified tetrapyrrole that acts as a coenzyme and cofactor for a number of key biological processes. It is unique among the vitamins in that it is made solely by prokaryotes but is retained by many eukaryotes as a nutrient because of the greatly improved rates of reaction observed with B12-dependent enzymes in comparison to B12-independent processes. Vitamin B12 is a structurally complex molecule and this is reflected in an equally complex biochemical pathway. The Warren group have worked for many years on the biochemistry of the pathway and are recognised for their many contributions to this area. DuPont/Tate & Lyle are pioneers in the area of bio-based renewables, especially with the production of 1,3-propanediol, where they have successfully developed a system that is much more efficient that the petro-chemical-derived synthesis. However, the bio-based production of propandiol, called BioPDO, requires vitamin B12 as a key ingredient in their fermentation process and they wish to develop a strain that will act as a reliable and comparatively cheap source of the nutrient. This joint project between the Warren lab and DuPont/Tate & Lyle therefore presents a project that will provide new key fundamental insights into the operations of the B12 biosynthetic pathway and at the same time lead to the construction of a strong B12-overproduction strain. By utilising the respective strengths in both partners, cobalamin biosynthesis with strain development and optimisation, the project will yield a strain that can be used to produce B12 on the g/L scale. The project will keep the UK at the cutting edge of research in the area of metabolic engineering.

Planned Impact

The research described in this application will have a major impact on several areas of science, including synthetic biology and metabolic engineering. The research relates to how cells can be engineered to help in the overproduction of a complex small molecule, vitamin B12. This approach will be applicable to a broad range of other natural products. With an increase in the interest of secondary metabolites such an approach is likely to prove popular with chemical biologists and medicinal chemists alike. Specifically, this research will generate a safe, reliable and affordable source of the nutrient for both industry and humanitarian causes. A cheap source of B12 could potentially improve the lives of tens of millions of people worldwide if it is employed in a supplementation programme.

The research falls well within the remit of synthetic biology and is therefore addressing a key priority area. In this respect the project applies the engineering paradigm of modularity and abstraction to metabolism. In essence, the project employs the transfer and redesign of existing, natural biological systems for useful purposes. The research also has the potential to engineer improvements in existing biological products and especially improve our understanding of biological systems through researching the role of modularity.

The beneficiaries of this research will be researchers in academia and industry who are interested in synthetic biology and its applications. There is a current strong interest in this area and science needs to put forward a strong representation in terms of the positive contributions that it can make. The research will not only provide essential information about how pathways and enzymes can be investigated and modified, but it will also provide greater insight into the provision and procurement of pathway substrates. It will demonstrate how cells can be engineered to resource their nutrient components to allow for fast and efficient synthesis. We will ensure that our findings are widely disseminated through oral communication, research publications and reviews, and press releases.

The Kent group is heavily involved in outreach programmes, through interactions with local schools and community groups. Kent is a member of the Authentic Biology Project, which is funded by a Wellcome Trust society award to bring real research into schools. Regular talks and demonstrations are given through organized events during science week and at other times by direct invitation from schools and societies, ensuring there is good dissemination with the general public on a range of important issues.

The skills acquired by those involved in this project include not only a wide range of important biological techniques ranging from microbiology and recombinant DNA technology through to strain development and optimisation, but also to provide the chance to interact with a leading company involved in the area of renewables. The knowledge and techniques will provide those employed with skills that can be used across education and industry. The intellectual property resulting from this project will be protected and used via the Innovation and Enterprise Office. The research will be published in high impact journals and oral communications given at international conferences. Using the infrastructure of the new Centre for Industrial Biotechnology within the University of Kent, the research will be brought to the attention of many other leading industrial companies.

Publications

10 25 50
 
Description We have developed a recombinant bacterial strain that is capable of producing high levels of vitamin B12, which is the most expensive of all the nutrients to produce. Moreover, the structure of the molecule is too complex to allow for a chemical synthesis and hence production of B12 is restricted to fermentation with specific bacteria. The strain we have developed is an extremely good producer and we are currently investigating the use of this strain in pilot scale-up experiments. As a result of this engineering process we have learnt a great deal about the biochemical pathway and how the flux through the pathway can be further improved.
Exploitation Route Our major industrial sponsors are keen to see this strain developed to allow for cheaper production of the nutrient, given that the price of the vitamin has fluctuated significantly over the past few years. They are also keen to see more production facilities opened to help improve global nutrient security, given that the vast majority of the nutrient is produced in China.
Sectors Chemicals,Healthcare,Manufacturing, including Industrial Biotechology

 
Description There is extremely strong interest in the production of vitamin B12, as it is an expensive nutrient to produce. The research associated with this project is at the cutting edge of metabolic engineering and synthetic biology in the design of modular and abstractable circuits and as such appeals to basic scientists trying to understand the nature of fundamental processes within the cell, and applied biotechnologists who are looking for novel findings for exploitation. We have been working with both DuPont and BASF to improve the yield of the nutrient from bacterial sources, research that also can be applied to other complex biochemical pathways. Our approach continues to provide new insights into the biosynthesis of B12 and how nature is able to coordinate the resourcing of minerals for biosynthesis. Our strain development is already at a good stage to initiate pilot-level studies and to start thinking about the problems of down-stream processing.
First Year Of Impact 2019
Sector Chemicals,Healthcare,Manufacturing, including Industrial Biotechology
Impact Types Societal,Economic

 
Description Committee on Toxicology regarding folate fortification
Geographic Reach National 
Policy Influence Type Participation in a advisory committee
 
Description Collaboration with DuPont via Dr Jim Zahn 
Organisation BASF
Country Germany 
Sector Private 
PI Contribution The collaboration involves the development of a bacterial strain that overproduces vitamin B12. Both DuPont and BASF have vast experience in the commercialisation of overproduction strains, especially from the fermentation standpoint.
Collaborator Contribution Our partners are providing us with help and assistance in the fermentation of bacteria for the enhanced production of a key commodity chemical.
Impact The major outcome has been the development of a strain of bacteria that produces a large amount of the nutrient.
Start Year 2018
 
Description Collaboration with DuPont via Dr Jim Zahn 
Organisation DuPont
Country United States 
Sector Private 
PI Contribution The collaboration involves the development of a bacterial strain that overproduces vitamin B12. Both DuPont and BASF have vast experience in the commercialisation of overproduction strains, especially from the fermentation standpoint.
Collaborator Contribution Our partners are providing us with help and assistance in the fermentation of bacteria for the enhanced production of a key commodity chemical.
Impact The major outcome has been the development of a strain of bacteria that produces a large amount of the nutrient.
Start Year 2018
 
Description Authentic Biology - Research in Schools Project 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Schools
Results and Impact As a result of his popular work of the madness of King George, Warren was invited to give numerous outreach presentations, many in local secondary schools. This led to an important initiative which blurs the divide between schools and University. Authentic Biology has involved setting up a research project in a school, supported by the University of Kent Biosciences Department, that successfully proved that A-level school children can engage in genuine academic research in molecular biology and protein biochemistry. More than 500 students have taken part in this project, learning and applying skills to investigate human myelin basic protein (www.mbp-squared.org). To demonstrate that the concept that real scientific research can be carried out in schools further, Warren was Co-I on a Wellcome Trust society award called Authentic Biology that has now brought real research into schools in London, Bristol, Sheffield and Winchester. In Canterbury the project has resulted in a doubling of the number of students doing A level biology. But there are wider benefits. The idea of the teacher as a part time researcher has had an impact on teacher retention and recruitment. Within the University framework researchers are involving the school student body in their research programme and the postgraduate scientists are gaining a range of skills in the process.

To demonstrate that the concept that real scientific research can be carried out in schools further, Warren was Co-I on a Wellcome Trust society award called Authentic Biology that has now brought real research into schools in London, Bristol, Sheffield and Winchester. In Canterbury the project has resulted in a doubling of the number of students doing A level biology. But there are wider benefits. The idea of the teacher as a part time researcher has had an impact on teacher retention and recruitment. Within the University framework researchers are involving the school student body in their research programme and the postgraduate scientists are gaining a range of skills in the process.
Year(s) Of Engagement Activity 2010,2011,2012,2013,2014
URL http://www.mbp-squared.org
 
Description TV work and outreach talks in school and to the general public 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Media (as a channel to the public)
Results and Impact Warren pioneered the area of scientific historiography by investigating the madness of King George III, and provided strong evidence that this was likely caused by variegate porphyria, exacerbated by the inadvertent administration of arsenic by his physicians. This research follows on from the idea proposed in 1969 that the episodic madness suffered by King George III (1738-1820) resulted from an acute hereditary porphyria, variegate porphyria, caused by deficiency of protoporphyrinogen oxidase. The diagnosis was based on the historical archive and a contentious claim that living members of the House of Hanover were affected with the condition. Warren re-examined this medical evidence and uncovered new historical material that suggested that porphyria did indeed exist in the Royal Houses of Europe. He reported the analysis of hair obtained from George III. Although no genomic DNA could be obtained, metal analysis revealed high concentrations of arsenic. Since arsenic interferes with haem metabolism, it is thought that this may have contributed to the King's unusually severe and prolonged bouts of illness. To substantiate this claim he identified sources of arsenic in the context of the medication George III received from his physicians. These stories were developed as documentaries for television and have been broadcast on both Channel 4 (Purple Secret, 1998) and BBC1 (Medical Mysteries, 2005), and were also written up as a general interest book called Purple Secret.

Warren is asked regularly to give similar talks to schools
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,
URL http://news.bbc.co.uk/1/hi/health/3889903.stm