Essential Cofactors in Anaerobic Microbial Consortia Used for Bioremediation: Biosynthesis, Function and Regeneration
Year: 2018
Type: dissertation
Abstract: Most microorganisms in nature live in communities and have developed tightly coordinated metabolism via metabolite exchanges. Cofactors are “helper molecules” in all cells, modulating the activity of many enzymes or serving as the electron shuttle. However, biosynthesis of cofactors, such as cobamides, is sometimes accomplished by only one specific phylogenetic group of microorganisms. Therefore, cofactors and their producers play pivotal roles in the functionality, metabolic rates, and population structure of a microbial community. This thesis focuses on three types of cofactors: (i) cobamides, (ii) NADPH, and (iii) prenylated flavin mononucleotide (prFMN) that are involved in anaerobic bioremediation of chlorinated solvents and aromatic pollutants. Cobamides are a family of cobalt-containing tetrapyrroles involved in biochemical reactions including methyltransfer reactions, isomerizations, and reductive dehalogenation. This thesis reports the identification of a functional cobamide prosthetic group in tetrachloroethene dehalogenases (PceA) of Desulfitobacterium hafniense and Geobacter lovleyi using a tiered blue-native polyacrylamide gel electrophoresis (BN-PAGE) and liquid chromatography-mass spectroscopy (LC-MS) method. The metabolic annotations of Dehalobacter restrictus (Dhb), a model organism for bioremediation of chlorinated solvents, were experimentally verified. The verified annotations were written into a constraint-based metabolic model, which identified that NADPH regeneration and de novo serine biosynthesis could be bottlenecks in Dhb metabolism. Using an integrated computational/experimental approach, the stringent nutrient requirements of Dhb were characterized. Further experimental analysis on the Dhb-enriched ACT-3 consortium has revealed an interspecies malate-pyruvate shuttle system between Dhb and its syntrophic partner. prFMN is a newly identified cofactor of UbiD reversible aromatic decarboxylases that are involved in ubiquinone biosynthesis, biological decomposition of lignin monomers, and anaerobic biodegradation of aromatic pollutants. We discovered that in Escherichia coli, dimethylallyl-monophosphate (DMAP), the prenyl donor of prFMN, can be produced from either prenol phosphorylation or from dimethylallyl-pyrophosphate dephosphorylation. In conclusion, this thesis reports biosynthesis and function of new cofactors as well as new mechanisms for reconciling cofactor regeneration in anaerobic microbial communities. Realizing these metabolic interdependencies generates opportunities to manipulate the microbiomes for better outcomes in bioremediation and industrial biotechnology. Interestingly, the identified interspecies cofactor exchange mechanism also provides insights into how life gradually evolved into complexity.
Source: TSpace (University of Toronto)
Author: Po‐Hsiang Wang
Language: English
Subfield: Pollution
Field: Environmental Science
Domain: Physical Sciences
Open Access status: green
OpenAlex ID: W2950730966