Metagenomics for new enzyme discovery and industrial biocatalysis

The aim of the proposed research is to find new enzymes that have potential uses in industry by searching for the genes for these enzymes in the DNA extracted directly from soil, compost or other environments. Enzymes are very useful in biocatalysis which is a sustainable method of making chemicals in industry. If enzymes are used the eventual industrial process can be cleaner and greener as it avoids the use of toxic reagents such as metals needed for many chemical catalysis steps, and often uses water-based systems. Biocatalysis can also replace several steps in a chemical process with one enzyme step due to their selectivity and this has a major effect of saving money and time in the overall process for making high value chemicals such as bioactive compounds in the fine chemical and pharmaceutical industry.

We will use a technique called metagenomics to find new enzymes for biocatalysis. Many enzymes are derived from microbial sources and these would normally be found by growing bacteria on agar plates and analysing the enzymes they contain using special assays. However, several years ago scientists studying soil microorganisms found that there was a very large difference between the numbers of bacteria they could grow from a soil sample compared with the numbers they could identify by analysing the DNA from the same quantity of soil. These DNA techniques showed that there were over 1,000 times more bacteria in the soil than can be grown on agar plates. So by using plating and growth techniques to find bacteria for biocatalytic enzymes were are missing over 99.9% of the potential enzymes.

A technique called metagenomics was developed by several researchers which started with the extraction of DNA directly from a soil sample and this DNA would potentially contain all the genes of the bacteria including the genes from bacteria that cannot be grown in the laboratory. We will use this metagenomic technique to isolate DNA from soils and other environmental samples. The metagenomic DNA will be sequenced and potential genes for biocatalysis will be searched for using computer based techniques to analyse the metagenome. When we find what could be useful genes we will amplify the gene from a sample of the metagenomic DNA and put the amplified gene into a laboratory bacterium that we can grow in large amounts and test the activity of the new biocatalytic enzyme. We call this overall method Functional Metagenomics.

The new biocatalysts will be tested in collaboration with researchers at Almac who use enzymes and chemistry to make pharmaceutical compounds. We will test the range of reactions the new biocatalysts can perform and test the chemicals made.

A new concept called enrichment metagenomics will also be investigated where we will enrich for bacteria able to use a specific compound before doing the metagenomics. This has the potential to increase the number of bacteria with the desired biocatalytic enzyme.

Another new concept called cDNA metagenomics will be tested where we extract messenger RNA from the sample and convert this into what is known as cDNA. This technique will allow us to look for genes from the microorganisms such as soil fungi that have introns in their DNA. This could enable us to find a hitherto unaccessed pool of new enzymes for biocatalysis.

Biocatalysis is a powerful tool in the manufacture of fine chemicals and pharmaceutical compounds. Industry needs new classes of enzymes such as transaminases, reductases and oxygenases for biocatalysis and new ways of finding such enzymes. We have been working on ways of mining microbial metagenomes for genes encoding enzymes of use in biocatalysis. The metagenome comprises all the DNA from an environment such as a soil sample. Metagenomics allows us to access all of the bacteria that exist in the environment as studies show that we can only culture less than 0.1% of the organisms from soils and less than 0.01% of the organisms in sea water so we cannot ever access all the diversity in the environment by plating in the lab.

We will use next generation DNA sequencing to sequence the metagenomes from e.g. soils, mud, and composts and assemble contiguous DNA fragments comprising of at least gene sized double stranded DNA segments. This contig library can be searched for any enzyme type by BLAST or Pfam searches and the enzyme coding regions can be easily amplified by PCR from the metagenomic DNA. We will collaborate with the participating company Almac in the screening of the cloned and expressed enzyme libraries and develop some new two-step enzyme cascades using the pool of new enzymes.

We will also test 2 new hypotheses in this work, one is the use of enrichment metagenomics where we will add a particular compound to an environmental sample to enrich for microorganisms that might transform the compound. The we will apply our functional metagenomic methods to the isolation of enzyme genes and by sequencing the metagenome before and after the enrichment we can establish if we have enriched for the target genes. The other is using mRNA extracted from the metagenome and converting this to cDNA. This can potentially access fungal enzymes that would be missed in our normal functional metagenomics pipeline due to the presence of introns in the fungal genome.

The project will have impact in each of the areas defined by the BBSRC: economy, society, knowledge and people. In terms of economic impact the benefits for the partner company Almac will arise from access to project deliverables (new methodologies to biocatalysts, new biocatalysts and synthetic routes), resulting in new market opportunities in the UK and overseas. The data will also be made available (after IP protection) to the wider academic and industrial community via publication and dissemination at meetings.

The societal impact will arise from the development of methods to identify new enzymes and the use of these enzymes in the UK that will lead to job creation and inward R&D investment at Almac and elsewhere. The use of biocatalysis in the chemical and pharmaceutical industry is growing, but is limited by the number of available enzymes: this project will overcome this problem and may result in the wider use of biocatalysis by companies. Beneficiaries of this work therefore extends beyond the immediate participants. It will benefit the UK economy by sustaining high-level research and the growth potential, and will aid in the delivery of new jobs. Successfully developed technology and improved enzymes will be promoted widely within the pharmaceutical and fine chemical industries, in addition to internal use, such that take-up could result in improved manufacturing processes for these customers, with accompanying cost benefit. There is significant competition from Asia for simple chemistry services, and for Almac and other companies to be competitive they must now tackle higher value products and services using differentiating technologies such as biocatalysis. Successful completion of this programme of work will help support the growth and financial stability of Northern Ireland's knowledge economy. The enchanced products and services offering will bring direct revenues to Almac Sciences and UCL, leading to business growth and stability. This in turn will lead to job security leading to wider benefits for the UK.

By identifying new enzymes and methods for sustainable synthesis, these resources will assist the UK government meet its targets on greenhouse gas emissions and help mitigate the negative environmental effects of global warming. In addition, if new or existing chemicals and medicines are made more sustainably and cheaply this will impact positively on the general public. The impact of knowledge generation from the research will arise from publication of new scientific advances and public outreach activities. Finally the impact on people will arise from training and career development of the PDRAs and provision of skilled scientists in the industrial biotechnology sector.