Genetic diversity of degradation pathways for monocyclic aromatics hydrocarbons
Creators: name: Rasol, Aveen Hawal
Publisher: name: No Publisher Supplied
Year: 2024

description: Chemicals can be released into the environment from various industrial practices and later can be exposed to different biotic and abiotic weathering reactions, such as biochemical and physical modification. Synthetic chemicals are essential to our lives, but their continuous production and release are increasing. Chemical pollutants can alter the composition of the environment's water, air, and soil and cause toxicity, also influencing their fate and biodegradation. They are harmful not just to the environment but also to human health; they can reach the human body via inhalation, oral absorption, and ingestion and cause various diseases depending on the exposure duration and the intensity of pollutants. Many physical and chemical techniques have been used to reduce the release of various toxic chemical pollutants into the environment, including bioremediation. Bioremediation uses different microorganisms to break down environmental pollutants, which can be less expensive, can propagate and adapt quickly to different conditions, and requires simple growth conditions. Many microorganisms possess oxygenase enzymes that have a broad specificity against different types of chemical pollutants. Microbial oxygenases oxidize the substrate by transferring oxygen from molecular oxygen using FADH/NADH/NADPH as a co-substrate. They are divided into monooxygenases and dioxygenases based on the number of oxygen atoms used for oxygenation. The first goal of this work was to study the aerobic biodegradation of benzoate by Sphingomonas wittichii RW1. Benzoate is the simplest compound that is produced from industrial activities and is an intermediate in the biodegradation of many aromatic compounds. When benzoate is degraded aerobically, it will produce catechol as the last intermediate for the upper degradation pathway. Once catechol is produced, it will enter two different routes using two different ring cleavage enzymes, and both pathways are found in Sphingomonas wittichii RW1. The first goal of this study was to identify the role of the catA gene encoding catechol 1,2-dioxygenase in the ortho-pathway in benzoate metabolism. The catA gene was identified by the similarity of the encoded protein to known catechol 1,2-dioxygenase enzymes. A clone carrying a knockout of the catA gene was constructed in Sphingomonas wittichii RW1 and tested using 5 mM benzoate. The growth curve result showed that there was no difference between the wild type and the knockout strain. Next, the role of two different catechol 2,3-dioxygenase mutants in the meta-pathway of benzoate metabolism was identified. Construction of double and triple knockout strains was prepared by combining the knockout catA gene with the two previously prepared meta-cleavage strains. These two strains involved RW1ΔdbfB (Swit_4902) and RW1Δ3046 (Swit_3046), in addition to the double knockout strain RW1ΔdbfBΔ3046. Growth curve results showed that the double mutant strain ΔcatAΔ3046 and the triple mutant strain ΔcatAΔdbfBΔ3046 did not grow on 5 mM benzoate. Results also indicated that mutant strain ΔcatAΔ3046 was able to grow when complemented by either the cloned catA gene or the cloned 3046 gene. In addition, results also showed that the double mutant strain ΔcatAΔdbfB grew well with benzoate, indicating that dbfB is not involved in metabolizing catechol. Growth curve results showed that catechol can be metabolized through the ortho pathway with the catA gene or the meta pathway with the 3046 gene. The second goal of this study was to analyze the genome of new Burkholderia strains that were isolated in our lab by a visiting Fulbright professor Habiba Atta. These strains were collected from a contaminated wetland area near an oil refinery in Delta state, south-south region, Nigeria. The strains were tested on some selected aromatic substrates such as o-xylene, m-xylene, p-xylene, ethylbenzene, dibenzofuran, biphenyl, naphthalene, toluene, o-cresol, m-cresol, p-cresol, and phenol. The growth tests showed a variation of the growth on different aromatic substrates suggesting that these strains do not have the same degradation pathways towards these substrates. The initial screening involved checking the existence of the three essential genes in Burkholderia strains genomes, the xylM, xylE, and tmo. The xylM is a gene for the toluene monooxygenase enzyme that attacks the methyl group, a gene for the TOL plasmid pathway, xylE is a gene for the ring cleavage enzyme, and tmo is a gene for the toluene monooxygenase enzyme. Furthermore, whole genome sequencing was performed to analyze the genome of the Burkholderia strains and verify the presence of these essential genes for aerobic aromatic degradation. The analysis showed that all strains had a highly similar but not identical phenol/toluene dmp/tmo operon, except strain 20Lm, which was missing the genes for the initial phenol hydroxylase. The strains 26mm, 5mx, and 20Lm have genes for a TOL plasmid type pathway, with 26mm and 20Lm having nearly identical operons but 5mx having more distantly related genes in a different arrangement. Furthermore, strain 7to did not have genes for a TOL plasmid pathway, and strain 12mnd may have genes for a TOL plasmid pathway. The variation showed in the genetic analysis of the isolated strains explained the evolution that happened with time, even though they were isolated from one location. The third goal of this study was to identify which aromatic degradation pathway(s) our Nigerian strains are using. The strain 12mnd was chosen for cloning the initial monooxygenase and later in testing the monooxygenase enzyme activity using three different chemicals: o-cresol, p-cresol, or m-cresol. Following the cloning of the monooxygenase enzyme, heterologous gene expression was performed, and the results indicated that the biotransformation media changed from noncolored solutions to dark brown with o-cresol and m-cresol, and light brown with p-cresol reflecting the oxidation of all three cresols to methylcatechol and confirming the catalytic activity of the monooxygenase enzyme in these cells. Furthermore, a clone carrying the ring cleavage enzyme gene was constructed, and the gene expression of monooxygenase and ring cleavage enzymes against the same three chemicals was performed, the analysis showed that the biotransformation media changed faster from noncolored solutions to a darker color with o-cresol but lighter with m-cresol and p-cresol explaining that once catechol was formed, it was metabolized through the ring cleavage enzyme to the meta-cleavage product. In addition, plasposon mutagenesis was used to identify which aromatic degradation pathway(s) the isolated Nigerian strains are using. The strain 26mm was used as a test strain since it has genes for both the monooxygenase and TOL-type pathways. In addition, the strain showed the best and the fastest growth in 24 hours among the other strains. Rescue cloning, which involves digestion of the total genomic DNA, self-ligation, and transformation into highly competent E. coli cells, was used. Subsequently, the transposon was isolated and used in a sequence reaction with designed primers facing outward from the transposon, and the strains were tested after the transposon mutagenesis on MSB media supplemented with m-xylene. Results showed two strains only lost the ability to grow on m-xylene. Further sequencing and genomic analysis were performed for the two mutant strains, which showed a deletion in the mutant strains of degradation genes in addition to a transposon insertion, and this deletion prevented the mutant strains from growing on m-xylene. Also, the analysis showed that neither of the transposon insertions in the mutants were in genes involved in the degradation of aromatic hydrocarbons. The results also showed that the wild type of strain 26mm metabolizes m-xylene through a TOL plasmid-type pathway and additionally contains a phenol/cresol degradation pathway. By inference, the other Nigerian Burkholderia strains metabolize monocyclic compounds in the same way.