The enzymatic mechanism underlying fungal degradation of carbohydrates in plant lignocellulose

Lead Research Organisation: University of Manchester
Department Name: Chemistry

Abstract

This interdisciplinary project investigates the enzymatic mechanism underlying fungal degradation of complex carbohydrates in plant biomass.

As efficient degraders of dead plant biomass, fungi are able to produce enzymes that can break down the complex of polysaccharides and lignin in the plant cell wall. This ability is exploited in biotechnology to release sugars from renewable resources which are subsequently used to produce high-value chemicals and second-generation biofuels. Understanding how fungi degrade biomass is a prerequisite for their exploitation as cell factory for biofuel enzymes, and for development of novel enzymes for the engineering of complex plant-derived carbohydrates. The response of industrially important fungi to lignocellulose has been studied extensively with regard to gene expression and protein secretion (1,2). However, we have limited understanding of how fungi and their carbohydrate-active enzymes (CAZymes) affect the actual lignocellulose substrate, and the biochemical activity of a large proportion of plant degradative CAZymes has not been established experimentally.

This project investigates the enzymatic mechanism underlying fungal degradation of complex carbohydrates in plant biomass. We have recently established methods for the high-throughput informative screening of CAZyme activities on complex plant carbohydrates using glycan arrays coupled with mass spectrometry (3). In this project you will expand on these methods by using a combination of chemical and enzymatic synthesis to generate glycan arrays containing a defined set of enzyme substrates. These arrays will subsequently be applied to determine the substrate and product specificity of a subset of the CAZymes secreted by fungus Aspergillus niger during growth on agricultural waste. You will then investigate the localisation and effect of these enzymes on this plant substrate using fungal biology, molecular biology and surface characterisation tools.

Studentship Projects

Project Reference Relationship Related To Start End Student Name
EP/N509565/1 01/10/2016 30/09/2021
1902652 Studentship EP/N509565/1 18/09/2017 30/09/2021 Gregory Bulmer
 
Description We have cloned a variety of uncharacterised carbohydrate active enzyme (CAZyme) genes with predicted activity towards a variety of different components of the plant cell wall. After a variety of different expression systems we have now successfully expressed and begun to biochemically characterise some of these target genes with the aim of publication. One candidate represents a subfamily in which no fungal enzyme has currently being described.

Investigations into a promiscuous glycosyltransferase opened up a variety of new research paths due to significant new knowledge accquired. Here we found an enzyme capable of transferring several different monosaccharides onto a variety of acceptors such as sugars with tags enabling HPLC or MS analysis. Interestingly, the soluble enzyme was found to polymerase glucose and form oligosaccharides of Beta-1,4 linked glucose - homologous to those found in cellulose. This was confirmed via NMR analysis. Via polymerisation onto a glucose acceptor that can strongly ionise in a mass spectrometer (ITag-) we would we could sensitively detected a variety of soluble oligosaccharides. These could be used as substrates in which the degradation of the oligosacchides could be followed by incubation with hydrolytic and lytic polysaccharide monooxygenases (LPMOs) via analysis of the reaction products. This project was highly collaborative and developed a research network on both an intra- and inter- university level. This work is currently under journal submission.
Additionally this funding allowed participation in glycobiology/carbohydrate research which led to the papers Huang et al (2019) and Mattey et al (2019) mentioned in the research outputs.
Exploitation Route The glycosyltransferase findings will directly translate into new routes for further work. To follow from these results more research will be undertaken to understand how this enzyme can be integrated into technologies to create specific, bespoke glycans of industrial or medical relevance. Primarily this will be the trialling of production of high value glycans at a scale potentially of interest to industrial partners through innovative techniques to optimise product production by our described enzyme.

Due to the incomplete knowledge surrounding the CAZyme activities of the commercially relevant, degradative fungus Aspergillus niger work is further required to elucidate the degradative model. Our current characterisation of fungal CAZymes is ongoing and represents an opportunity to uncover new activities involved in the saccharification of plant lignocellulose. This offers an obvious route to both commercial and academic further work via the importance of this said degradation in the processing and ultilization of agricultural waste into high value chemicals for a bio based circular economy.
Sectors Agriculture, Food and Drink,Chemicals,Energy,Manufacturing, including Industrial Biotechology

URL https://www.biorxiv.org/content/10.1101/2020.02.14.949545v1
 
Description British Science Week Schools' Fair 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact To inspire pupils to go to University and study STEM subjects in the future and showcase courses and careers relating to science and engineering. The activities included making DNA plasmid bracelets to explain the structure of DNA and use of vectors to genetically modify cells, a protein translation puzzle with sweets to show how cells make useful medicines and a plush antibody / insulin-secreting cells for demonstration purposes. Overall the stand was well received with an overwhelming majority of visitors feeding back through a sticker chart that this was the first time they had spoken to someone working in biotechnology.
Year(s) Of Engagement Activity 2018
 
Description Headstart chemistry day 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Schools
Results and Impact Organised a whole day of a summer school in which around 30 students on a chemistry residential course visited the Manchester Institute of Biotechnology. Gave a lecture on Biotechnology, organised tours of the building and its facilities and delivered a variety of classroom based outreach activities.
Year(s) Of Engagement Activity 2019
 
Description Manchester Science Festival: Science Spectacular 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Schools
Results and Impact Present at the university's annual Science Spectacular event. Members of the general public were invited to interact with a stand on the science of sugars with fun activities for all ages, such as the Cell Invaders iPad game, glucose testing with enzymes and arts-and-crafts-style glycan building.
Year(s) Of Engagement Activity 2017
 
Description Museum Late (Museum of Science & Industry,) 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact The Museum of Science & Industry hosted a bee-themed late to coincide with the 'Bee In The City' public art event to showcase the bee sculpture hosted in the museum's courtyard. As such, the Manchester Institute of Biotechnology was represented at the event by an experiment using an enzyme which bees secrete into honey, giving it its antibacterial properties. This concept was used to introduce visitors to the idea of enzymes, what they do, where they are found and how they can be used to address global challenges in sustainable chemical manufacture and disease research.
Year(s) Of Engagement Activity 2018
 
Description New Scientist Live 2018 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Public/other audiences
Results and Impact Twenty two researchers manned the Manchester Institute of Biotechnology's stand, using fun, hands on activities about DNA, genetic modification and various flavours and fragrance compounds to tell the general public about the University's Industrial Biotechnology research beacon and synthetic biology research for more sustainable production of fine chemicals.
Year(s) Of Engagement Activity 2018
 
Description Polymer day tour 
Form Of Engagement Activity Participation in an open day or visit at my research institution
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Media (as a channel to the public)
Results and Impact Gave tour of School of Chemistry and its analytical equipment to a group of around 15 students.
Year(s) Of Engagement Activity 2019
 
Description STEM fair - Levenshulme High School 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Had a stall at their annual STEM fair to talk about non-medical biology related careers. Demonstrated an enzyme reaction and how it related to diabetes testing. Discussed University education with the students.
Year(s) Of Engagement Activity 2019
 
Description Science career panel 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Sat on a panel alongside researchers and those from industry to discuss our routes into STEM to an audience of around 40 A-level students.
Year(s) Of Engagement Activity 2019
 
Description Why study STEM talk 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Delivered a "Why study STEM" talk to a 6th form college in North Manchester, detailing the variety of courses offered.
Year(s) Of Engagement Activity 2019