Bioengineering of complex metabolic pathways

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.

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.