Searching for treasure in the ‘dark matter’ of microbial genes
Biomass such as agricultural residue, forestry products and food waste is an important resource in the EU, with an annual supply of nearly 1 billion tonnes expected by 2030. Much of this is used as an energy source – either fermented into fuel or burned directly. The EU-funded BHIVE project sought ways to extract greater value from this feedstock by mapping the ‘dark matter’ of microbial genomes. “The goal was to delve deeper into gene sequences with no known function, with particular attention on those likely to transform biomass into sought-after products,” explains project coordinator Emma Master. The function of the majority of the genes identified so far through genome sequencing remains unknown. “That’s a treasure trove of biocatalysts that can be developed for various industrial sectors,” adds Master. Her team at Aalto University in Finland studied common sequences found across multiple microbial genomes, hoping to find novel enzymes which can transform lignocellulose, the fibrous material which makes up the bulk of plant dry matter.
Value-added product
In northern countries such as Finland, biomass takes a long time to grow, and is difficult to process. This makes it compelling to convert biomass into higher-value products. “Many biocatalysts developed for this conversion are focused on deconstruction – turning cellulose into sugars for fermentation to commodity chemicals and fuels,” says Master. “We were focused on biocatalysts that upgrade, not degrade.” The enzymes would be added to cellulose fibres from pulped trees, for example, to functionalise that material. To explore the uncharacterised genomic space of plants, Master and her team had to develop new techniques. “In genomics, you get what you screen for. We can only discover things we know how to observe,” she adds. “If you don’t know what the function is, how do you shed light on that?” Instead, the team started from the types of enzymatic activity they were interested in, and worked backwards to build a screen that would identify catalysts capable of such reactions. To make matters more difficult, enzyme screens are typically carried out in solution, but lignocellulose is not soluble. “We had to rethink how to look for that activity, and develop fluorescent labelling and mass spectrometer methods that allowed us to look for changes in these insoluble substrates,” notes Master. The team examined thousands of genes, and narrowed these down to several hundred which were screened for expression. “We used a guilt by association approach – clues in the genome that helped us home in on sequences likely to be important for lignocellulose functionalisation,” she explains. Ultimately, they characterised close to 100 proteins.
New and improved
Several of the newly discovered enzymes held particular interest. Among them was an aminating enzyme that can add amine function into polysaccharides. “This is useful for making antimicrobial materials and chelating polymers,” notes Master. “Plants don’t produce polysaccharides with amines in large amounts, but they are useful for textile applications.” Another enzyme was found to alter the structure of cellulose fibre, changing its porosity and flexibility, useful functions for producing textiles like rayon. “The process to make rayon is not sustainable, and there is a lot of interest in a more sustainable route to the textile, replacing cotton with wood,” remarks Master. Work on the enzymes is now continuing at Aalto University through a collaboration with researchers in Spain and Sweden and an industry partner. “We couldn’t have done this without the ERC funding,” says Master. “It’s been transformative, and has opened new avenues for us, and for Europe, in biocatalysts and biomaterials engineering.”
Keywords
BHIVE, enzyme, biocatalyst, genomics, wood, pulp, cellulose, upgrade, transform, biomaterials, engineering
