15 NSFBIO - Synthetic Biology for Lignin Utilization

Our current reliance on fossil fuels is unsustainable and there is a clear need to find alternative sources of renewable fuels and chemicals to meet the needs of an expanding global population. A vast amount of work has been done in developing the technologies to turn waste plant material (biomass) into sugar that can be fermented to produce bioethanol for our cars, trains and planes. This relies on enzymes, like those used in washing detergents, to break down the plant material so that that it can be converted into biofuels - a direct alternative to fossil fuels. Although the technology now exists to do this, it is simply too expensive. So how can we make it cheaper?

Plant cell walls contain cellulose and it is this polymer that can be broken down into sugars to make fuels. However, up to one-third of plant material is made up of a sticky brown compound called lignin. This is currently vastly underused in commercial plants and it is mainly burned to generate power. This is a huge waste, and one of the reasons that biofuels are currently so expensive. Lignin is actually a very valuable compound, and with the right enzymes, can be turned into useful products such as chemicals, plastics and even carbon-fibre. Not only would these materials be renewable and sustainable, their high value would make biofuels cheaper.

As usual, nature already has the answer. We have found microbes that can live on lignin as a food source and have evolved powerful enzymes break this resistant compound apart. We plan to research these enzymes and link them together in new systems to make them efficient. We will use genetics to help us evolve better enzymes, work out their 3D structures and use advanced computer models to work out the best combinations. If we succeed, we have the potential to make biofuel production commercially viable and create a new range of plant-based products. The result will be transportation fuels that don't rely on dwindling fossil fuel reserves, are sustainable and are kinder to the environment.

The microbial production of fuels and chemicals from renewable feedstock is a grand challenge for synthetic biology. To date, no microbial chassis has been developed for lignin utilization despite the success of similar approaches with sugars. Although lignin is an abundant, energy-dense polymer that makes up ~30% of plant biomass, it is recalcitrant to degradation. In contrast to cellulose, lignin cannot be readily cleaved into homogenous subunits because it is composed of diverse phenyl-propanoid compounds connected by non-uniform chemical linkages. This complexity makes the targeted degradation of lignin a daunting challenge and results in the dramatic, wasteful underutilization of lignin as a feedstock. In nature, the complete degradation of lignin involves microbial consortia. Although no single organism encodes all the enzymes needed for efficient lignin catabolism, natural metabolic pathways provide a rich catalytic toolbox.

We have assembled a multi-disciplinary team to engineer the first lignin-degrading chassis using a bacterium, Acinetobacter baylyi ADP1. Utilising the unique genetic system of ADP1, we will evolve efficient catabolic devices to expand the degradation of mixtures of lignin-derived aromatic compounds. To complement the microbial genetics, a combination of GC-MS metabalomic and metabolic flux methods will be used to quantify intermediates in the lignin catabolic pathway and in silico metabolic modelling will allow us to target enzymatic bottlenecks and improve lignin catabolism. These specific enzymes will enter our structural biology and protein engineering platform, where we will fully characterise and adapt them with the goal of constructing superior enzyme 'machines' for the efficient conversion of lignin to desired products. These engineered enzymes will then be incorporated back into ADP1 and multiple iterative cycles will allow us to continually improve the efficiency of the system.

The aim of this research is to produce enzymes with enhanced lignin degrading activities that can be used directly in the production of fine chemicals from waste material to support commercially viable processes. Crucially, this technology uses waste biomass and therefore does not compete with food crops, making it a sustainable alternative to fossil-based transportation fuels and provide a new and sustainable source of high-value chemicals. Our main objectives directly address several BBSRC strategic priority areas:

Synthetic biology:
Adopting an enzyme engineering approach, informed by metabolomics and driven by genetic evolution, to generate enhanced enzymes and novel metabolic pathways.

Bioenergy:
Addressing a key economic bottleneck in 2nd Generation bioethanol production and sustainable fine chemicals.

Industrial biotechnology:
The newly developed recombinant enzymes may find application in other industrial sectors such as paper and textile manufacturing and biological washing detergents. New enzymes have the potential to aid in the manufacture of natural products and drugs that remain difficult to synthesise.

Increased international collaboration:
Capitalising on the world-class infrastructure between the 3 NSF-funded groups in the USA, the DoE-funded facility at NREL and the biophysics and structural biology expertise in the UK.

Public engagement:
For public engagement, previous work in related areas has already proved to be particularly attractive to the media and has headlined national TV news, newspapers and in excess of 20 websites worldwide (Google: 'gribble mcgeehan'). Bodies such as the BBSRC, STFC have also covered this work together with large facilities such as the Diamond Light Source, UK and the National Science Foundation, USA.