Bioengineering of complex metabolic pathways

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

Abstract

Vitamins are essential nutrients required by humans to complete their diet, and by definition are not made within their own body. Plants and vegetables are good sources of many vitamins. However, plants are missing one vitamin from their complement / vitamin B12. This is because plants neither make nor require vitamin B12 within their metabolism. In fact, vitamin B12 is unique among the vitamins in that it is the only vitamin whose synthesis is restricted solely to bacteria, as the ability to make this nutrient never successfully made the prokaryote to eukaryote transition. The consequence of this is that those on strictly vegetarian diets are prone to vitamin B12 deficiency - a state that is associated with a wide range of systems including megablastic anaemia, neurological disorders, and developmental problems in unborn babies. Vitamin B12 deficiency is also a problem in the elderly, where an increase in the level of B12 in the diet can alleviate the symptoms. There are thus strong medical reasons for increasing the levels of vitamin B12 in the diet. In this application we wish to explore the limitations and consequences of engineering complex metabolic pathways into different organisms, taking advantage of the latest developments and technologies in metabolic engineering. We plan to take the genetic software that allows bacteria to make vitamin B12 and transfer it into bacteria that are unable to make B12, yeast and a higher plant, thereby conferring upon these organisms the ability to make this essential nutrient. For bacteria, we wish to explore how the pathway can be enhanced for maximum vitamin production. Will increased levels of certain enzymes give increased metabolic flux, or will substrate bioavailabilty be limiting? These are questions that we will address in this application. For engineering into yeast, we have to look at a complex cloning procedure that ensures the genes have separate promoters and regulatory regions. For plants, we will take advantage of the fact that certain organelles within the plant, called plastids, have their own genetic material and in essence behave like bacteria within the plant. We will integrate the DNA from a bacterial species into the plastid and then monitor the level of the vitamin made during the growth of the plant. In fact, we will make a number of variants, of increasing complexity, to see how the plant is able to cope with this genetic modification. This application is aimed at increasing our understanding of how biochemical pathways operate, how they are controlled and how they can be engineered to enhance the metabolic ability of the host cell. The results of the project will provide knowledge that can be used to develop new technologies and products for agriculture and bioremediation. From medical, industrial and wealth creation strategic standpoints, this research programme closely follows the remit and aspirations of the BBSRC.

Technical Summary

The twenty first century is the age of metabolic engineering and synthetic biology, where advances in recombinant DNA technology will allow us to rewire and rewrite molecular circuits to help in the production of important metabolites. At present, we are restricted in our thinking about what metabolic engineering really is, and most projects tend to tinker with small pathways that are already present within the organism. However, we have the opportunity to apply our technology to much larger projects and we should be aiming to construct dedicated molecular cell factories or engineered life forms. To achieve this, though, we need to understand how best to organise molecular pathways, learn what the limitations are and optimise our engineering strategies. In this application we have outlined an highly ambitious project, in which we aim to engineer one of Nature's most complex biosynthetic pathways, that for vitamin B12, into a range of different organisms. This research builds on our previous research of characterisation of pathway enzymes and develops it into a new and innovative direction. The research contained within this proposal has the potential to open up dramatic and novel lines of work, and contribute to the sustainability of research progress in the area of metabolic engineering and synthetic biology. Moreover, we also see this as an important tool in studying genome-physiology relationships and as such contribute to our understanding of functional genomics. The research encompasses fundamental, strategic and applied areas of study and covers a broad range of biological and related disciplines, including biological chemistry, microbiology, flux analysis, nutrition and plant science. It will provide a strong training platform for all those employed on the grant and will serve to keep the UK at the cutting edge of metabolic engineering research.

Publications

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Description The fellowship has led to the setting up of a leading research centre in synthetic biology within the university, and has permitted an accelerated route for synthetic biology research. The fellowship has allowed the PI to engage with leading scientists at both a national and international level, who have combined their expertise to develop exciting programmes of innovative and interdisciplinary research. The fellowship has allowed the development of the PI's lab so that it acts as a superb environment in which early career researchers develop their skills in interdisciplinarity. The research undertaken as part of this fellowship has resulted in a significant number of high profile papers that have generated interest around the world.

the project allowed the cloning and engineering of the cobalamin biosynthetic pathway (or elements of it) into a range of organisms - including E. coli, B. megatherium and yeast. It also permitted the identification and characterisation of a number of the cobalamin biosynthetic pathway enzymes.
All the objectives from the original proposal were achieved. There were some minor variations but in essence the proposal expanded rather than contracted as we took advantage of a number of significant breakthroughs, especially in the area of metabolic engineering. The inclusion of a project dedicated LCMS was particularly important and allowed us to make some major advances not only on the biosynthesis of cobalamin but also in the discovery of a major new route for the synthesis of heme.
Exploitation Route The development of strategies for the generation of multi-gene constructs for the production of whole metabolic pathways has been adopted by a number of different research groups. The capacity to design and redesign biochemical pathways has huge potential, especially for the construction of analogues of important biological cofactors and prosthetic groups. The development of such such areas of research will have important implications for selective inhibitors of essential biological processes.
Sectors Chemicals,Education,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology

 
Description Our work on the biochemical engineering of the cobalamin biosynthetic pathway is being used by several research labs and several SMEs for the recombinant production of cobalamin. A number of different patents have been applied for relating to cobalamin biosynthesis. The movement of whole biochemical pathways from one organism to another is a key starting point for synthetic biology and our methodologies are being used in both academia and industry. The application of our approach to the combinatorial construction of vitamin variants is significant.
First Year Of Impact 2010
Sector Chemicals,Education,Government, Democracy and Justice,Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
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