Exploration and Production Technologies
Metabolic Engineering to Develop a Pathway for the Selective Cleavage of Carbon-Nitrogen

DE-FC26-02NT15382

Project Goal
The goal of this project is to develop biochemical pathways for the selective cleavage of carbon-nitrogen bonds in molecules found in petroleum to mitigate the deleterious effects of nitrogen in refining processes.

Performer
Gas Technology Institute (GTI)
Des Plaines, IL

Project Results
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.

Benefits
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.

Background
In North America alone over 3 trillion barrels of known petroleum resources are largely untapped or underutilized because of their high sulfur/nitrogen/metals content and attendant viscosity problems. Nitrogen in petroleum contributes to air pollution and decreases refinery efficiency by poisoning catalysts, while it is difficult to remove organically bound nitrogen without destroying the calorific value of the fuel.

The selective removal of nitrogen from petroleum is a relatively neglected topic in comparison with sulfur removal. Moreover, most metals in oil are associated with nitrogen compounds, and nitrogen compounds contribute to the instability of petroleum byproducts. The selective removal of nitrogen from oil would be highly desirable, but effective processes are not currently available. The selective removal of sulfur from dibenzothiophene and from petroleum by biochemical reactions performed by microorganisms has been demonstrated. Biorefining can also potentially be used to remove nitrogen and metals from petroleum, but so far this area of research has received very little attention. A complete biochemical pathway for the selective cleavage of both carbon-nitrogen bonds in the typical petroleum compound carbazole is currently unknown. However, metabolic engineering could potentially allow such a biochemical pathway to be created and that was the goal of this project.

Project Summary
The development of biocatalysts with improved ability to cleave carbon-nitrogen bonds was addressed by cloning the genes for carbazole dioxygenase, aniline dioxygenase, and other enzymes. The removal of nitrogen from aromatic compounds like carbazole requires the cleavage of two carbon-nitrogen bonds. The cleavage of the first carbon-nitrogen bond is accomplished by the enzyme carbazole dioxygenase while an enzyme capable of selectively cleaving the second carbon-nitrogen bond has not yet been identified. The enzyme carbazole dioxygenase is encoded for by three genes: carAa, carAc, and carAd. The carAacd genes have been cloned and sequenced from several different microbial cultures including Sphingomonas sp. GTIN11 and Pseudomonas resinovorans CA10. Enrichment culture experiments and directed evolution experiments were performed to obtain an enzyme that can selectively cleave the second carbon-nitrogen bond in carbazole.

Enrichment culture experiments designed to isolate a culture capable of cleaving the carbon-nitrogen bond in 2-aminobiphenyl, and thus being capable of providing an enzyme for the cleavage of the second carbon-nitrogen bond in carbazole, resulted in the isolation of a unique microbial culture Pseudomonas sp. GTIN-G4. Pseudomonas sp. GTIN-G4 is capable of metabolizing 2-aminobiphenyl and related compounds, but does not appear to be capable of cleaving the carbon-nitrogen bond. Instead it has the unprecedented ability to modify 2-aminobiphenyl by replacing a hydrogen bound to the nitrogen atom with a formaldehyde group. Other enrichment culture experiments resulted in the isolation of numerous bacterial cultures capable of metabolizing a variety of organonitrogen compounds such as 2-aminobiphenyl, aniline, 4,4’-azodianiline, benzamide, carbazole, methylbenzylamine, piperidine, pyridine, pyrolidine, quinoline, quinazoline, ortho-, meta-, and para-toluidine, and triazine. However, none of these cultures had the ability to selectively cleave the C-N bond in 2-aminobiphenyl or related molecules.

Directed evolution experiments were performed by scientists at the University of Illinois at Chicago. Genes that encode the benzamide amidase from Rhodococcus sp. MP50, the melamine deaminase from Pseudomonas sp. NRRL B12227, and the aniline dioxygenase from Acinetobacter strain YAA were all included in directed evolution experiments designed to obtain derivative enzymes with altered substrate ranges. No enzyme capable of selectively cleaving the C-N bond in 2-aminobiphenyl was obtained.

Research performed in this project also included gene expression studies in Rhodococcus erythropolis. The genes that encode carbazole dioxygenase from Sphingomonas sp. GTIN11 and Pseudomonas resinovorans CA10 (carAacd), aniline dioxygenase from Acinetobacter sp. YAA (atdA1, atdA2, atdA3, atdA4, and atdA5), and resistance to the antibiotic kanamycin were cloned and introduced into Rhodococcus erythropolis, a bacterial host that tolerates exposure to petroleum. It was hoped that by simultaneously expressing the genes for carbazole dioxygenase and aniline dioxygenase in the same bacterial host then enrichment culture experiments employing carbazole as a sole nitrogen source may result in the isolation of derivative cultures with altered abilities to cleave C-N bonds. However, no culture capable of selectively cleaving both C-N bonds in carbazole was obtained. Because no gene encoding an enzyme that could selectively cleave the C-N bond in 2-aminobiphenyl was available, it was not possible to construct a new metabolic pathway for the selective removal of nitrogen from carbazole.

In the course of gene expression studies an increased understanding of gene transcription in Rhodococcus erythropolis was developed. Chromosomal DNA fragments were screened for promoter activity, transcription initiation sites were mapped, and promoter regions were examined to detect conserved sequences. No DNA sequences that resemble the two conserved hexanucleotide sequences typically found in E. coli promoters were present in DNA fragments that functioned as promoters in R. erythropolis. However, possible conserved DNA sequence motifs were observed and synthetic DNA fragments were constructed to test the importance of these motifs for promoter activity in both E. coli and R. erythropolis. A DNA sequence about 10 bp upstream from the transcriptional initiation site was demonstrated to be important for promoter activity in R. erythropolis. This sequence differs significantly from typical E. coli promoters. This information will aid in our understanding of gene regulation/expression in the genus Rhodococcus.

Current Status (June 2006)
The project has been completed. It will be necessary to identify an enzyme capable of selectively cleaving the C-N bond in 2-aminobiphenyl before a biochemical pathway for the selective removal of nitrogen can be constructed.

Project Start: October 1, 2002
Project End: March 31, 2006

Anticipated DOE Contribution: $867,103
Performer Contribution: $216,776 (20% of total)

Contact Information
NETL - Jim Barnes (jim.barnes@netl.doe.gov or 918-699-2076)
Gas Technology Institute - John Kilbane (john.kilbane@gastechnology.org or 847-768-0723)

Publications 
Kilbane II, J. J. Microbial Biocatalyst Developments to Upgrade Fossil Fuels. Current Opinion in Biotechnology 17:305-314, 2006.

Kilbane II, J. J., and J. Robbins, Formulation of 2-aminobiphenyl by Pseudomonas sp. strain GTIN-G4. Submitted to Biochemical and Biophysical Research Communications.

Kilbane II, J. J., and J. Robbins, Screening and Analysis of DNA Fragments that Show Promoter Activities in Rhodococcus erythropolis. Manuscript in preparation.

Kilbane II, J. J., Applications of Biotechnology to the Energy Industry. Oil Asia Journal, May 2003, pp. 19-25.

Kilbane II, J. J., Bioprocessing of Fossil Fuels: Current Status and Future Possibilities. PETROTECH 2003, New Delhi, India, January 9-12, 2003.

Kilbane II, J. J., Recent Technical Advances in the Bioprocessing of Fossil Fuels. PETROTECH 2003, New Delhi, India, January 9-12, 2003.

Kilbane II, J. J., Biorefining Research at GTI, TATA Energy Research Institute, New Delhi, India, January 9, 2003.

Annual Reports October 2003 through September 2005 and the Final Report are available by calling NETL at 918-699-2000.


Carbazole degradation pathways. The top pathway illustrates the existing carbazole degradation pathway that results in overall degradation, whereas the bottom pathway illustrates the potential pathway for the selective removal of nitrogen from carbazole that was a goal in this project.

 
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