Applied Microbiology: Open Access
Open Access
ISSN: 2471-9315
+44 1300 500008
ISSN: 2471-9315
+44 1300 500008
Matthias Moerch and Angel Angelov
Wolfgang Liebl Technische Universit�¤t M�¼nchen, Munich, Germany
Posters & Accepted Abstracts: Appli Microbiol Open Access
The constant depletion of fossil energy sources and the raising demand for more eco-friendly alternatives challenges science
to develop new technologies. The utilization of plant biomass is a promising substitute for conventional systems by being
a rather inexpensive energy carrier and of high sustainability at the same time. Its availability through cultivation of energy
crops and by usage of agricultural/forestry waste account for great industrial value. In particular, the valorisation of waste by
converting it to fermentable substrates has the advantage not to threaten food security. Since plants themselves consist primarily
of lignocellulosic fibres (a mixture of cellulose, lignin and hemicellulose), the first step in the process of fuel production is the
hydrolysis of this bigger structures into smaller, more soluble sugars. Its depolymerization into glucose monomers is often
facilitated by using cellulases � an enzyme group consisting of endoglucanases (EC 3.2.1.4), cellobiohydrolases (EC 3.2.1.91)
and �²-glucosidases (EC 3.2.1.21) which work synergistically together. With increasing market share of new bio fuels, the future
demand for (novel) cellulases � which today already make up 8 % of worldwide industrial enzyme demands � will therefor
rise as well constantly. In this work we developed a system to select for thermostable glycoside hydrolase enzymes (GH)
using the extremely thermophilic bacterium Thermus thermophilus. Unlike mesophilic bacteria (e.g. E. coli), T. thermophilus
provides an overall more suitable enzymatic background and thereby greater potential to express thermostable recombinant
proteins properly. This increases the probability of detecting novel thermostable cellulases when transformed in this organism;
compared to the commonly used host E. coli. As an advantage, growth-based selection approaches already result in favourable
enzyme variants, compared to traditional screening methods which require testing of every single clone. In order to obtain a
GH-negative strain, we constructed a T. thermophilus knock-out strain which lacks four glycosidases. As confirmed by paranitrophenol
(pNP) enzyme assays and incubation of cell extract with X-Gal and X-Glu, these deletions reduced the hosts
ability to cleave �²-glycosidic and �²- galactosidic bonds to a minimum. Without these GHs, the knock out strain is not able to
grow in minimal medium. Complementation with the hosts own �²-glucosidase via the shuttle vector pMK18 re-established
growth of the knock out strain. For purpose of following system verification, cglT � a glycosyl hydrolase belonging to GH
family 1 from the thermophilic bacterium Thermoanaerobacter brockii � was transformed in the T. thermophilus knock-out
strain. This novel approach of complementation-based selection in an extreme thermophilic organism is a promising tool to
look through big meta genomes or mutagenesis libraries, selecting for enzyme variants of higher thermostability and/or other
substrate specificity in a highly efficient manner.
Recent Publications:
1. Angelov A.*, Pham VTT.*, Ã?Â?belacker M., Brady S., Leis B., Pill N., Brolle J., Mechelke M., Moerch M., Henrissat B.,
Liebl W. A metagenome-derived thermostable Ã?Â?-glucanase with an unusual module architecture which defines the
new glycoside hydrolase family GH148. Sci Rep. 2017 Dec 11;7(1):17306 *These authors contributed equally to this
work.
2. Leis B.*, Held C.*, Bergkemper F., Dennemarck K., Steinbauer R., Reiter A., Mechelke M., Moerch M., Graubner S.,
Liebl W., Schwarz WH., Zverlov VV. Comparative characterization of all cellulosomal cellulases from Clostridium
thermocellum reveals high diversity in endoglucanase product formation essential for complex activity. Biotechnol
Biofuels. 2017 Oct 23;10:240 *These authors contributed equally to this work.
Matthias Moerch has completed his Masters in Molecular Biotechnology at the Technical University of Munich in 2014, comparing different protein digestion methods for shotgun proteomics in his Master’s Thesis. Since 2015 he pursues his doctorate at the Department of Microbiology at the Technical University of Munich establishing the extreme thermophilic bacterium Thermus thermophilus as an alternative host for metagenome analysis.
E-mail: matthias.moerch@tum.de