13 ERA IB: Integrated Process and Cell Refactoring Systems (IPCRES) for Enhanced Industrial Biotechnology.

Valorisation of low-value waste glycerol could transform the economics of biodiesel production.Key challenges for achieving this with biocatalysis are the rate of glycerol conversion to products, integrating bioprocess steps and tolerance of crude feedstock contaminants by host strains.

We are industrial and academic bioprocess, cell-engineering, omics and bio-systems specialists. We will port pathways into Saccharomyces cerevisiae and Pichia pastoris for production of two valuable compounds from glycerol: chiral amino-alcohols (CAA) and 1,2-propanediol (PDO).

Cells will be characterised with high throughput micro-scale process techniques and at scale. Transcriptomic and metabolomic data will be combined with process insights to dictate refactoring of both pathways and host 'chassis'. These steps will be repeated for second-iteration cells, driving better performance. New strains, processes and integrated methods will be of use to biodiesel and Industrial Biotechnology sectors.

An key 'biorefinery' goal for biodiesel production is valorisation of the low-value glycerol by-product, potentially transforming process economics. E. coli is only modestly effective as a glycerol whole cell biocatalyst and is susceptible to contaminant bacteriophage and competing bacteria. Challenges for bio-conversion of crude glycerol into valuable products are:

- identification of heterologous pathways that efficiently convert glycerol and transferring them to robust, tractable host organisms for valuable product formation
- tolerance of multiple crude glycerol contaminants - consolidating processing of crude feedstock, biocatalysis and product recovery

We are industrial and academic bioprocess, cell-engineering, omics and bio-systems specialists and will address these challenges through integration of our capabilities. We will firstly establish yeast 'chassis' for glycerol conversion.
We will engineer Saccharomyces cerevisiae, a model organism with myriad recombinant DNA tools, and Pichia pastoris which grows rapidly to high biomass (600g/L) on glycerol. We will port pathways into both species for i) chiral amino-alcohol (CAA) and ii) 1,2-propanediol (PDO) production.
CAAs are synthesis intermediates for medicines such as Darunavir and PDO is used worldwide as an aircraft de-icing fluid. E. coli platforms for production of these compounds typically achieve 20% yield and conventional chemo-catalysis remains the only viable option for their production.
First generation engineered strains will be characterised with high throughput micro-scale process techniques, transcriptomics and metabolomics. New insights will be tested at scale. Process and 'omic' data will dictate refactoring of both the heterologous pathways and the host 'chassis' using advanced bio-systems approaches and novel cloning techniques. Next-generation strains will be tested as above to drive better performance.

UK Bioscience:
The project will deliver a positive environmental impact since it promotes the use of green, renewable, clean enzyme catalysis in medicinal and process chemistry, thereby lowering costs, reducing waste and increasing productivity. This work proposes Synthetic Biology approaches as an alternative to chemo-catalysis at extreme pH which is energy and resource intensive, toxic and non-renewable.

Key beneficiaries in the short term will be industrialists seeking to develop low environmental impact routes to synthesis chiral compounds with anti-tumour, anti-microbial, anti-inflammatory, anti-HIV, and analgesic properties. For these reasons this proposal has significant commercial interest. Beneficiaries therefore include the pharmaceutical sector, the medical and life science research sectors and ultimately the UK public, if outputs from this research programme lead to new drugs and therapies, or sustainable syntheses that have less environmental impact.

The centre of the fine chemicals industry has been gradually migrating east to China and India over the last 30 years. The fine chemicals sector in the UK has very much retracted in this time, whilst many UK-based big-pharma companies source chemical intermediates from China and India and often the provenance of the material with respect to the sustainability and environmental impact of manufacture is unknown. The fundamental studies proposed here will help re-establish a knowledge-based bio-economy (KBBE) in the UK in which basic science has been harnessed to produce fine chemical and chiral intermediates in a way that is profitable, sustainable and in which the UK has a commercial advantage in terms of technical know-how.

Third Sector:
In the longer term this work will increase large pharmaceutical companies' ability to make discounted medicines available to developing countries by lowering their production costs. This will enable these companies to fulfil their stated commitments to the WHO and Global Pharma Health Fund to promote international development and protect vulnerable populations.

General Public:
This proposal will help improve public and media perception of Synthetic Biology by providing an intuitive practical application of this new discipline which remains widely unknown in the UK. The longer the absence of practical applications for Synthetic Biology, the more newsworthy it will be to label it as either over-hyped or dangerous (or both, as public opinions can often be contradictory). The practical achievements and benefits of this work will be communicated through a range of events and media in order to positively impact sentiment toward Synthetic Biology across a range of general publics.

Training:
The UK has a recognised skills shortage in Industrial Biotechnology as evidenced by the government's 2009 Industrial Biotechnology Innovation and Growth Team report. Delivering effective technical and career-progression training for the PDRA is a critical impact of this proposal. In addition to classical and 'omic' molecular cell biology bench skills, the PDRA will acquire deep learning of Industrial Biotechnology activities such as bioreactor cultivation and biocatalysis analytics. Valuable 'soft' skills in multidisciplinary collaboration and research management will be gained by learning from and with chemical engineering, biochemical engineering and systems/mathematical biology researchers. The PDRA will also develop presentation and communication skills and assist in the training of junior laboratory members. Report writing, IT skills, multitasking and public engagement activities will also be key to the role.