We belong to the KTH Royal Institute of Technology in Stockholm. KTH is very firmly an engineering school, and as a result, much of our research has an applied focus. We want to find new, more efficient ways of breaking down complex natural biomass into its component molecular parts. And we want to find efficient new ways of using those molecular components to make fuel or materials, for example. But we believe that even applied research like this needs to be built on a strong foundational core of basic science. This is why we are also investigating how biomass is produced by organisms in the first place, while also studying the basic microbial physiology of biomass-degrading environments like the soil. Below you can see a summary of each of our main research areas, and some recent relevant papers from the group – more to come soon, we hope!
Basic bacterial investigations
Much of the research that has led to the applications outlined below began with semi-fortuitous discoveries made in Lauren‘s personal mission to understand everything about her favourite bacterium, Chitinophaga pinensis. She started working with the species back in her first post-doc year at KTH, and continues to be fascinated by the weird and wonderful new CAZymes and related domains to be found hiding in its genome. We want to understand the means by which C. pinensis and related environmental (i.e. non-gut dwelling) species are able to comprehensively deconstruct plant and microbial biomass, and so we are attempting to characterise as many C. pinensis-encode CAZymes as we can. Some of these turn out to have really interesting activities that we can use for industrial processes such as the bioenergy and biomaterials projects outlined below. Some are capable of antagonising living fungi and oomycetes, so they have important implications for biocontrol. Soon we will begin an investigation of a new industrial microbial ecosystem, seeking species and enzymes that are hardy and resistant to inhibitors, metals, and unpleasant temperatures.
Polysaccharide degradation by the Bacteroidetes — mechanisms and nomenclature. McKee LS, La Rosa SL, Westereng B, Eijsink VGH, Pope PB, Larsbrink J. Environmental Microbiology Reports (2021)
Focussed metabolism of β-glucans by the soil Bacteroidetes Chitinophaga pinensis. McKee LS, Martínez-Abad A, Ruthes AC, Brumer H. Applied and Environmental Microbiology 85 (2019) e02231-18.
Proteomic insights into mannan degradation and protein secretion by the forest floor bacterium Chitinophaga pinensis. Larsbrink J, Tuveng TR, Pope PB, Bulone V, Eijsink VGH, Brumer H, McKee LS. Journal of Proteomics 156 (2017) 63-74.
Biofuel and bioproduction – detailed characterisation of CAZyme activities
The fractionation of lignocellulosic biomass is a key step in efficient production of ethanol fuel from this renewable energy resource. Biomass hydrolysis is a key bottleneck in biofuel production, and enzymatic hydrolysis is considered the most sustainable hydrolysis method, but efficiency can be low at the high processing temperatures required for substrate solubility, partly due to protein instability. This low efficiency means that enzyme loading needs to be very high, increasing the sales price of bioethanol. We have recently discovered that a new type of carbohydrate-binding domain, uncharacterised until our current projects, can confer stability benefits onto biomass-fractionating CAZymes, and may improve overall enzyme efficiency by altering substrate-binding behaviours. We have characterised just a few of these domains so far, but He Li is now driving this project as part of her post-doctoral work. In summer 2021 she was joined by research intern Elin Wedin Kvick. Then in spring 2022, Master’s thesis students Lova Sandin and Vasiliki Makrygianni made some important contributions. And in spring 2022, Rasmus Gustafsson joined this initiative to characterise some more examples, helping us to get a view of how these domains impact CAZymes from a wide range of families.
Some of the CAZymes we have discovered in our various discovery projects can be used to help release economically valuable oligosaccharides from biomass, and this is another focus area for the group now. Working with KTH colleagues like biorefinery experts Amparo Jimenez Quero and Francisco Vilaplana, we are optimising ways of releasing and then purifying such oligos, and are even beginning to explore the possibility of commercialising these techniques in the future. In 2021-22, Master’s thesis students Wilhelm Sundewall and Kasane Suzuki helped us screen different types of biomass, while Tom Svesse helped us develop some purification processes.
Multiple enzymatic approaches to hydrolysis of fungal β-glucans by the soil bacterium Chitinophaga pinensis. Lu Z, Rämgård C, Ergenlioglu I, Sandin L, Hammar H, Andersson H, King K, Inman AR, Bulone V, McKee LS. The FEBS Journal (2023)
Growth of Chitinophaga pinensis on plant cell wall glycans and characterisation of a glycoside hydrolase family 27 β-L-arabinopyranosidase implicated in arabinogalactan utilisation. McKee LS, Brumer H. PLOSONE 10 (2015) e0139932
Biocontrol
Biological control is the use of living organisms to control pests and pathogens, instead of using chemical pesticides. This can for example mean that animals are used to control insect populations, or that microbes are used to kill insects or other pathogenic microbes. We are currently investigating whether Chitinophaga pinensis is capable of inhibiting the growth of pathogenic oomycetes that cause disease in pea, pepper, and potato plants. C. pinensis has an amazing ability to sense complex carbohydrates in its local environment, and respond to this by changing the types of enzymes it secretes. We think that some species might be able to sense the carbohydrates found in pathogen cell walls, and respond by secreting enzymes or other molecules that can attack the pathogen. Three fantastic Master’s thesis students have worked on various aspects of this C. pinensis project: Zijia Lu, Lovisa Brandt, and Yi-Hsuan Lee. In similar projects, we looked at the soil bacterium Bacillus subtilis natto, which showed interesting responses when fed with carbohydrates derived from fungal cell walls. Master’s thesis students Anna Schönbichler and Amrutha Seshadri worked on the Bacillus project.
McKee LS. One soil bacterium performs two important jobs to help us produce healthy food. Frontiers for Young Minds (October 2020) (This article is part of the Research Topic Tiny Microbes, Big Yields: The Future of Food and Agriculture)
Schönbichler AS, Díaz-Moreno SM, Srivastava V, McKee LS. Frontiers in Microbiology 11(2020) 521. Exploring the potential for fungal antagonism and cell wall attack by Bacillus subtilis natto. (This article is part of the Research Topic Advanced Microbial Biotechnologies For Sustainable Agriculture, which you can download as a complete e-book)
Biosynthesis
If you want to find new enzyme tools to break down biomass and separate its components, you first need to understand what the cell wall looks like! For plants, especially trees, there are still some important unknowns. Lignin is a naturally abundant organic polymer that makes up 20-30% of plant biomass. It gives strength that supports vascular plants, and it allows the transfer of water through the plant. The structure of lignin depends on the plant species, tissue, and age, among other factors, and it is also produced in response to certain kinds of stress. On top of these hugely important biological roles, lignin is a highly valuable molecule in industry, as it can be used to make thermoset plastics and conducting materials, for example. But before it can be used, it needs to be extracted from biomass (such as wood), and this can be extremely challenging to achieve without drastically altering the structure of native lignin, because the cell wall matrix in which lignin is embedded is so complex and inter-connected.
In fact, typical lignin extraction methods cause such substantial disruption to the native lignin structure that there remains controversy about whether certain molecular structures often detected in lignin are even really naturally occurring! Having better knowledge about the real structure of lignin in wood will allow us to design better biorefineries to take advantage of lignin at scale, and will also give us a much better understanding of the natural world around us. As part of her PhD, Ioanna Sapouna has designed new methods for mild extraction of lignin, and investigated how the lignin she extracts differs from that obtained by conventional methods (hint: it looks and smells a lot better!). She is also developing a series of biomimetic experiments to replicate the process of natural lignin biosynthesis, so she can see what lignin looks like without ever having to deal with interference from the rest of the cell wall! In 2022, Ioanna was joined by Master’s thesis student Emelie Heidling, who optimised a green lignin extraction procedure for hardwood. The overall goal for Ioanna’s thesis is to show that mild biomass extractions to release intact lignins can yield valuable molecules for materials design, and she has even made her own lignin-based materials.
Sapouna I, Lawoko M. Deciphering lignin heterogeneity in ball milled softwood: unravelling the synergy between the supramolecular cell wall structure and molecular events. Green Chemistry 23 (2021) 3348-64.
Biomaterials
A hydrogel is a solid material made of a cross-linked network of long polymeric molecules. Many hydrogels contain at least 90% water. Hydrogels are extremely useful and versatile materials, as they are strong, flexible, highly absorbent, and adaptable to different applications. Nowadays they are used widely in cosmetic products, as their high water content makes them ideal for moisturising gels and facemasks. Hydrogels are also widely used in medical and pharmaceutical products. This includes tablets, liquid-containing capsules, and implants for slow release of drug molecules. Although they are increasingly prevalent, the current standard industrial processes for manufacturing hydrogels use harmful chemicals, and are often made from fossil-derived polymers, which is obviously not sustainable. A more sustainable approach is to use non fossil-based natural biopolymers. It is possible to use polysaccharides in hydrogel production, which means we can use renewable resources, but polysaccharide hydrogel manufacture is still usually not environmentally safe. The polysaccharides need to be extensively modified using harsh chemicals, and hazardous solvents and reactive chemical cross-linkers are also used, adding to the expensive waste-disposal burden on the manufacturer, ultimately increasing costs for the consumer. We are developing a new method of producing hydrogels from sustainably sourced polysaccharides, using non-catalytic proteins to cross-link the polymer. This work is being driven by Mengshu Hao as part of her post-doctoral work. In summer 2021 she was joined by research intern Srijani Saha. And in 2021-22, Erasmus student Beatriz Monteiro Coelho helped advance our understanding of the gel system and the different proteins that can function within it.
Stockholm CAZyme Lab 