Metabolic Engineering to Develop a Pathway for the Selective Cleavage of Carbon-Nitrogen Bonds

摘要

The objective of the project was to develop a biochemical pathway for the selective cleavage of C-N bonds in molecules found in petroleum. Specifically, the development of a novel biochemical pathway for the selective cleavage of C-N bonds in carbazole was the focus of research in this project. The cleavage of the first C-N bond in carbazole is accomplished by the enzyme carbazole dioxygenase, that catalyzes the conversion of carbazole to 2-aminobiphenyl-2,3-diol. The genes encoding carbazole dioxygenase were cloned from Sphingomonas sp. GTIN11 and from Pseudomonas resinovorans CA10. Obtaining an enzyme capable of selectively cleaving the C-N bond in 2-aminobiphenyl-2,3-diol was the focus of much of the research in this project, however; no suitable enzyme was found. Project accomplishments included expressing the genes for carbazole dioxygenase in Rhodococcus erythropolis and Escherichia coli, development of gene expression vectors for Rhodococcus, and isolation of a Pseudomonas sp. strain GTIN-G4 that has the novel biochemical ability to replace one of the nitrogen-associated hydrogen atoms in 2-aminobiphenyl with formaldehyde. Rhodococcus cultures are capable of metabolizing a wide range of substrates, including hydrophobic substrates like petroleum, and may find widespread use in the development of biotechnology processes in the future. The results of this project will directly benefit the development of future biotechnology processes/projects employing Rhodococcus hosts. Three manuscripts were prepared and submitted for publication based on the data obtained in this project: (1) ''Formylation of 2-aminobiphenyl by Pseudomonas sp. strain GTIN-G4''; (2) ''Screening and Analysis of DNA Fragments that Show Promoter Activities in Rhodococcus erythropolis''; and (3) ''Microbial Biocatalyst Developments to Upgrade Fossil Fuels''.