Sweet spots for fungal lignocellulose degradation; elucidating the enzymatic mechanism underpinning interaction of Aspergillus niger with wheat straw

As efficient degraders of dead plant biomass, fungi are able to produce enzymes that can break down the complex of polysaccharides and lignin (together lignocellulose) comprising the plant cell wall, forming more simple sugars. This ability is exploited in biotechnology to release sugars from renewable resources such as wheat straw, which would otherwise be agricultural waste. These sugars are subsequently used to produce biofuels and high-value chemicals.

In the last decade, our understanding of how fungi behave when encountering lignocellulose, and which enzymes they produce upon doing so has increased dramatically, especially for industrially important fungi such as Aspergillus niger. However, we have very limited understanding of how the activity of the fungal enzymes affects lignocellulose. This interdisciplinary project aims to study the biochemical mechanism underpinning the degradative effect of A. niger and its enzymes on a complex lignocellulose substrate, using state-of-the-art techniques novel to this field.

When A. niger grows on lignocellulose, genes encoding polysaccharide-degradative enzymes are switched on consecutively. This suggests that the fungus sequentially secretes degradative enzymes, which deconstruct the lignocellulose to transiently expose individual polysaccharides. Via enzymatic degradation of these polysaccharides, soluble sugars are released that can act as signalling molecules to switch on expression of genes, resulting in sequential gene expression. This project investigates both the time-staged deconstruction of polysaccharides on the surface of the model lignocellulose wheat straw, as well as the degradative capacity of the enabling enzymatic machinery.

State-of-the-art tools conventionally applied for the characterisation of material surfaces will be used here to show how polysaccharides are accessed by and exposed to fungal degradative enzymes. To characterise the fungal enzymes, a method that has recently been developed by us will be expanded to allow informative and fast screening of many different enzyme activities simultaneously. This method will be applied to characterise the activities of degradative enzymes produced by A. niger on wheat straw.

This research will enhance our understanding of how the fungal enzymatic machinery interacts with and deconstructs lignocellulose, a prerequisite for exploitation of fungi as enzyme cell factories. Understanding gained by studying the regulatory and enzymatic aspects of A. niger lignocellulose deconstruction, as well as the broadly applicable tool set, can be applied to understand other uncharacterised species of fungi.

Research will be executed by Dr Jolanda van Munster, a microbiologist and enzymologist, based in the Chemical Biology research group of Professor Sabine Flitsch, in the Manchester Institute of Biotechnology of the University of Manchester. The surface analysis experiments will be done in the laboratory of Paul Knox, Professor of Plant Cell Biology in the Centre for Plant Sciences at the University of Leeds, and in the laboratory of Emma Master, Associate Professor in the Chemical Engineering & Applied Chemistry Department, at the University of Toronto, Canada.

Fungi secrete enzymes to efficiently degrade complex plant lignocellulose to simple sugars. This ability is exploited in biotechnology, which uses the enzymes to produce sugars from renewables for subsequent conversion to biofuels and high-value chemicals. We have very limited understanding of how activity of such enzymes affects the actual substrate. This interdisciplinary project aims to study the biochemical mechanism underpinning the degradative effect of Aspergillus niger and its enzymes on a complex lignocellulose substrate using state-of-the-art techniques novel to this field.

The induction of carbohydrate active enzyme-encoding genes in A. niger growing on lignocellulose is sequential, suggesting sequential enzyme secretion and lignocellulose deconstruction hallmarked by transient exposure of individual polysaccharides on its surface. Thus, polysaccharide degradation likely involves the ordered release of soluble sugars that then act as inducers of successive gene expression. This project will investigate both the time-staged deconstruction of polysaccharides on the surface of the model lignocellulose wheat straw, as well as the degradative capacity of the enzymatic machinery enabling this degradation.

Surface characterisation tools will be used to show how polysaccharides are accessed by and exposed to fungal degradative enzymes. Lignin and polysaccharides will be localised with Time-of-Flight Secondary Ion Mass Spectrometry (with Prof. Master, Univ. of Toronto) and immunohistochemistry (with Prof. Knox, Univ. of Leeds). In the Chemical Biology group of Prof. Flitsch (Univ. of Manchester) mass-spectrometry compatible arrays with plant oligosaccharides will be developed and used to characterise the substrate and product specificities of secreted enzyme activities.

This research will enhance our understanding of how the fungal enzymatic machinery interacts with and deconstructs lignocellulose, a prerequisite for improving their exploitation.

The outcomes of the proposed research will have impact in the private sector, the public sector and on the general public.

The private sector:
The enzymatic production of high-value compounds from renewable resources such as lignocellulose is typically the remit of the private sector. The proposed research develops tools and detailed knowledge and understanding that are all applicable to this area of biotechnology. The outcomes of this research project are therefore highly relevant to the private sector, especially for companies involved in production of hydrolytic enzymes for lignocellulose degradation in second generation biofuel production (i.e. Novozymes, Dupont, DSM) and for those producing fungal enzymes for food, feed and pharmaceutical applications (i.e. AB enzymes, the Kerry Group, Chr. Hansen).

In detail, carbohydrate array can be applied as basis for enzyme activity screenings that need to ascertain substrate and product specificities, and as such they are valuable for commercial parties that need to identify novel or complementary enzyme activities that degrade or modify plant polysaccharides. Importantly, the conversion of genomics data into novel enzymes that perform well in an industrial setting is poor. Development of high-throughput screens that detect activity on complex lignocellulose substrates instead of model substrates is potentially a game changer. The application of ToF-SIMS for this purpose holds great promise. Furthermore, knowledge on the limitations of lignocellulose degradation underpins improvement of enzyme mixtures by identifying areas of improvement. Importantly, discovery of any novel or key enzyme activities is of value in commercial applications for lignocellulose degradation or modification novel type of enzyme activity can have a tremendous impact on the efficiency and costs of such enzyme mixtures in commercial applications as exemplified by the lytic polysaccharide monooxygenases. Finally, the outcomes from this research can identify potential new sources for sugars that induce expression of enzyme encoding genes, an area of high interest to the identified private sector beneficiaries.

The public sector
To proposed research project has benefits for secondary and higher education institutions and their students. The academic network generated by the PI between the higher education institutions will benefit collaboration between the researchers and potentially increase interdisciplinary funding applications, which in turn benefit their academic institutions through increased excellent research output for the REF2020. Students from secondary education and higher education can benefit through being inspired for a career in science and/or develop knowledge during project related outreach activities and placements.

The general public:
The research outcomes as well as the developed tools contribute strongly to improvement of enzymatic conversion of renewable resources to generate future fuels, packaging material and other high value compounds. This development underpins the switch from an oil-based to a bio-based economy and will have a high impact on the general public through contribution to a cleaner, healthier environment with reduced pollution and reduced carbon emissions, to the development of economic activities and business and the creation of jobs.

Industrial uptake and application of research outcomes may be constrained by public acceptance, (i.e. in the use of heterologously produced enzymes for food applications), which is generally improved after knowledge increase. Therefore, engagement activities are aimed at increasing knowledge and awareness of the public with regard to the broader topics of the bio-based economy and the enzymatic conversion of renewables to high-value compounds, as well as more specifically the outcomes and potential application of the proposed research programme.