Metaproteogenomics-guided enzyme discovery: Targeted identification of novel proteases in microbial communities

Abstract

Linköping Studies in Science and Technology. Dissertation No. 1932 Electronic publication: http://www.ep.liu.sePrinted by LiU-Tryck, Linköping, Sweden, 2018 If we knew what it was we were doing, it would not be called research, would it. Albert Einstein Till Ludvig V AbstractIndustrial biotechnology is a large and growing industry as it is part of establishing a "greener" and more sustainable bioeconomy-based society. Using enzymes as biocatalysts is a viable alternative to chemicals and energy intense industrial processes and is en route to a more sustainable industry. Enzymes have been used in different areas for ages and are today used in many industrial processes such as biofuels production, food industry, tanning, chemical synthesis, pharmaceuticals etc. Enzymes are today a billion-dollar industry in itself and the demand for novel catalysts for various present and future processes of renewable resources are high and perfectly in line with converting to a more sustainable society.Most enzymes used in industry today have been identified from isolated and pure cultured microorganisms with identified desirable traits and enzymatic capacities. However, it is known that less than 1% of all microorganisms can be can be obtained in pure cultures. Thus, if we were to rely solely on pure culturing, this would leave the 99% of the microorganisms that constitute the "microbial dark matter" uninvestigated for their potential in coding for and producing valuable novel enzymes. Therefore, to investigate these "unculturable" microorganisms for novel and valuable enzymes, pure-culture independent methods are needed.During the last two decades there has been a fast and extensive development in techniques and methods applicable for this purpose. Especially important has been the advancements made in mass spectrometry for protein identification and next generation sequencing of DNA. With these technical developments new research fields of proteomics and genomics have been developed, by which the complete protein complement of cells (the proteome) and all genes (the genome) of organisms can be investigated. When these techniques are applied to microbial communities these fields of research are known as meta-proteomics and meta-genomics.However, when applied to complex microbial communities, difficulties different from those encountered in their original usage for analysis of single multicellular organisms or cell linages arises, and when used independently both methods have their own limitations and bottlenecks. In addition, both metaproteomics and metagenomics are largely non-targeting techniques. Thus, if the purpose is still to -somewhat contradictory -use these non-targeting methods for targeted identification of novel enzymes with VI certain desired activities and properties from within microbial communities, special measures need to be taken.The work presented in this thesis describes the development of a method that combines metaproteomics and metagenomics (i.e. metaproteogenomics) for the targeted discovery of no...

Dynamics of a Perturbed Microbial Community during Thermophilic Anaerobic Digestion of Chemically Defined Soluble Organic Compounds

“…Furthermore, operational conditions for thermophilic AD of the chemically defined substrate in a laboratory continuous stirred tank reactor (CSTR) were optimized (i.e., substrate loading rate and retention time) to ensure a constant conversion rate of the substrate to biogas. As a result, a stable thermophilic AD process was operated for several years with constant biogas production and substrate-conversion rates, implying the establishment of a thermophilic microbial culture at a metabolic steady state [12]. In an attempt to reproduce the thermophilic AD process in a new reactor by using the aforementioned microbial culture, we faced difficulties to stabilize the process.…”