In vivo alpha-olefin production: a sustainable hydrocarbon source

One of the main challenges our society faces is the dwindling level of oil reserves which we not only depend upon for transport fuels, but also plastics, lubricants and a wide range of petrochemicals. This application seeks to provide an answer through synthetic biology: making organisms produce "oil". Although the production of oil is mainly a biogenic process, the geochemical conversion of biological matter to oil occurs over thousands of years. Solutions that seek to reduce our dependency on fossil oil are therefore urgently needed. The direct production of hydrocarbon compounds by living organisms, bypassing the geochemical conversion of organic matter into oil, is an attractive process, but is unfortunately not part of the "mainstream" repertoire of biochemical reactions that would be required to make this an attractive sustainable alternative. Indeed, minor pathways or side-reactions resulting in the production of hydrocarbons such as alkenes or alkanes have only recently been documented. Unfortunately, these are not present in any organism to the scale and/or specificity that would support industrial application, let alone provide a valid alternative to fossil oil. However, the application of synthetic biology and metabolic engineering to modify these pathways is likely to result in innovative advances in this area.
To meet these challenges, we will combine state-of-the-art enzymology and laboratory evolution techniques with synthetic biology. The first challenge is enzymatic hydrocarbon production, a process that often starts with fatty acids. The enzymes involved in converting fatty acids to hydrocarbons have only very recently been identified and many remain unstudied. Furthermore, their properties (substrate and product specificity, stability and rate) are unlikely to support an industrial scale process. We will investigate the use of a wide range of enzymes using structure-based rational engineering and laboratory evolution, in order to create a comprehensive toolkit of catalysts that we will exploit for hydrocarbon production. Ultimately, we will attempt to integrate these with various components into a bacterial strain which can convert renewables into hydrocarbons, preferentially excreted to the outside environment, creating a sustainable process. This ambitious programme addresses an urgent industrial need for reducing our dependency on fossil oil. Through enzyme design and development of new pathways, it will generate "oil"-producing organisms, hence bypassing the need to drastically adapt oil-dependent processes while reducing the associated carbon footprint. Our project will focuses in particular on production of linear alpha-olefins, a high value, industrially crucial intermediate class of hydrocarbons that are key chemical intermediates in a variety of applications, such as flexible packaging, rigid packaging and pipes, synthetic lubricants used in passenger car, heavy duty motor and gear oils, surfactants, detergents, lubricant additives and paper sizing. At present, no "green" alpha-olefin production process is available, a situation which this application seeks to change.

We will produce new bacterial strains with the capability of manufacturing alpha-olefin compounds, which are valuable industrial products/intermediates. We will develop a synthetic biology programme in which we will engineer artificial pathways for alpha-olefin production, aiming for robust integration in the host metabolism. Our programme integrates synthetic biology with biocatalysis and analysis of enzyme structures and mechanisms. We have expressed and purified two key enzymes that each provide a different route to alpha-olefin production. None of these enzymes have been characterised in detail at the level of substrate biotransformation and kinetics, or structure determination (we have recently obtained the structure for one of them). In order for us to define precisely the reactions catalysed and engage in a rational laboratory evolution procedure aimed at improving alpha-olefin production (both in terms of rate and carbon chain length) will determine the exact mechanism and structure of each enzyme. We have developed rapid, plate based assays which we will use for screening of individual CASTing libraries of the various enzymes. Optimized variants will be expressed with additional components that link the final enzyme to host metabolism. Following the construction and demonstration of bona fide hydrocarbon production, we will alter bacterial host strains to enhance efflux and to improve production levels by using two-phase biotransformations. The programme builds on our expertise with enzyme systems, metabolic engineering and bacterial genetics and will offer a green and route to alpha-olefines, thereby circumventing the current industrial processes which is reliant on scarce natural resources (i.e. oil). The work programme is strategically important in the industrial biocatalysis area and maps directly onto the KBBE strategy.

Beneficiaries:
The outcomes this grant will impact 4 main beneficiaries: (i) The petrochemical industry - new biocatalytic/synthetic biology manufacturing processes for generating hydrocarbons such as linear alfa-olefins mitigate risk associated with the limited supply of reagents from natural resources. 'Natural synthesis' avoids use of toxic/non-renewable reagents with consequent environmental benefits and high acceptability for intermediate and end users. In addition, the tools and systems developed in this application can be further optimised for production of other petrochemical products. (ii) The manufacturing industry - the availability of 'green' products from the petrochemical industry will improve the renewable nature and C-footprint of manufacturing processes. In addition, the tools and systems developed in this application will impact widely in emerging biotechnology economies employing 'synthetic biology' to create rationally novel enzymes/pathways used in bulk/fine chemicals manufacture, food production and security. (iii) government policy makers - synthetic biology as outlined in this application is an emerging field that carries significant promise, assessing exactly how much scope there is for strategies as outlined in this proposal to provide alternatives to fossil oil is urgently needed. The degree of success/scope for translation to other areas will inform both government and industry future funding allocation and strategy. (iv) society in the wider sense - The projected depletion of the oil resources is a key concern to society. The impact of this event (and the inevitable lead up to it - i.e. unsustainable increase in oil prices) on our society cannot be underestimated. Credible alternatives need to be found, ideally providing "drop-in" replacements for existing petrochemical products so as to be directly compatible with existing infrastructure. This applications seeks to address a small part of this problem (replacing oil derived alpha-olefin production with a green alternative), but promises to have scope in providing similar answers to a wider range of petrochemicals, ultimately replacing fossil oil.

Exploitation: We anticipate that our newly designed bacterial strains/processes will have commercial impact. Our strategy for translating the technology is to establish IP protection in collaboration with our industrial sponsor and through UMIP (Manchester's IP office). We will communicate through networking events with external stakeholders (industry, other University groups, venture capital groups, policy groups).

Outreach: We anticipate wide interest in the progress and outcomes of this application. We will make use of the internet as the tool with the widest dissemination possibilities, with a range of www based communication tools: websites, podcase and blogs regularly updated and tailored to the various types of audience being targeted (scientific, industrial or general media). In addition, we will ensure direct channels of communication with the applicants and the research staff employed through representation at UK science fairs (Big Bang, Royal Society), engage with local schools and offer summer internships in the applicants' laboratories.