Thermostabilization of desulfurization enzymes from Rhodococcos sp. IGTS8. Final technical report

The objective of this project was to develop thermophilic cultures capable of expressing the desulfurization (dsz) operon of Rhodococcus sp. IGTS8. The approaches taken in this project included the development of plasmid and integrative expression vectors that function well in Thermus thermophilus, the cloning of Rhodococcus dsz genes in Thermus expression vectors, and the isolation of bacterial cultures that express the dsz operon at thermophilic temperatures. This project has resulted in the development of plasmid and integrative expression vectors for use in T. thermophilus. The dsz genes have been expressed at moderately thermophilic temperatures (52 C) in Mycobacterium phlei and at temperatures as high as 72 C in T. thermophilus. The tools and methods developed in this project will be generally useful for the expression of heterologous genes in Thermus. Key developments in the project have been the isolation of a Mycobacterium phlei culture capable of expressing the desulfurization operon at 52 C, development of plasmid and integrative expression vectors for Thermus thermophilus, and the development of a host-vector system based on the malate dehydrogenase gene that allows plasmids to be stably maintained in T. thermophilus and provides a convenient reporter gene for the accurate quantification of gene expression. Publications have been prepared regarding each of these topics; these preprints are included

4p-inner-shell and double-excitation spectrum of Sr II

We report photoabsorption measurements from the 4p inner shell of singly ionized strontium in the spectral region between 26.0 and 37.4 eV. More than 60 levels of Sr II are reported which are interpreted as singly excited inner-shell transitions 4p65s 2S1∕2→4p45s(1P,3P)ns, md and doubly excited transitions 4p65s 2S1∕2→4p54d(1P,3P,3D)nl. Multiconfiguration Hartree-Fock calculations are performed in jj coupling and the levels are arranged into Rydberg series converging on seven limits, allowing the identification of two levels in Sr III

Biocatalytic Removal of Organic Sulfur from Coal

The objective is to characterize more completely the biochemical ability of the bacterium, Rhodococcus rhodochrous IGTS8, to cleave carbon-sulfur bonds with emphasis on data that will allow the development of a practical coal biodesulfurization process. Another approach for increasing the desulfurization activity of the IGTS8 cultures is to produce strains genetically that have higher activity. The goal of this part of research is to achieve strain improvement by introducing a stronger promoter using genetic engineering techniques. The promoter regulates the transcription of the genes for the desulfurization enzymes, and a stronger promoter, would up-regulate the expression of these genes, resulting in cells with higher desulfurization activity. Promoter probe vectors are used to identify and isolate promoters from a DNA library of the experimental organism. The major accomplishments have been to obtain high biodesulfurization activity in nonaqueous, media, especially using freeze-dried cells, and to have isolated strong promoters from R. rhodochrous IGTS8 which will be used to engineer the organism to produce strains with higher biocatalytic activity

Flow of Non-Newtonian Fluids in Tubes

The problem is to correlate existing non-Newtonian flow data to predict laminar, transitional, and turbulent flow. These correlations will be used to generate procedures for designing flow systems

METABOLIC ENGINEERING TO DEVELOP A PATHWAY FOR THE SELECTIVE CLEAVAGE OF CARBON-NITROGEN BONDS

The objective of the project is to develop biochemical pathways for the selective cleavage of C-N bonds in molecules found in petroleum. The initial phase of the project was focused on the isolation or development of an enzyme capable of cleaving the C-N bond in aromatic amides, specifically 2-aminobiphenyl. The objective of the second phase of the research will be to construct a biochemical pathway for the selective removal of nitrogen from carbazole by combining the carA genes from Sphingomonas sp. GTIN11 with the gene(s) encoding an appropriate deaminase. The objective of the final phase of the project will be to develop derivative C-N bond cleaving enzymes that have broader substrate ranges and to demonstrate the use of such strains to selectively remove nitrogen from petroleum. During the first year of the project (October, 2002-September, 2003) enrichment culture experiments resulted in the isolation of microbial cultures that utilize aromatic amides as sole nitrogen sources, several amidase genes were cloned and were included in directed evolution experiments to obtain derivatives that can cleave C-N bonds in aromatic amides, and the carA genes from Sphingomonas sp. GTIN11, and Pseudomonas resinovorans CA10 were cloned in vectors capable of replicating in Escherichia coli. During the second year of the project (October, 2003-September, 2004) enrichment culture experiments succeeded in isolating a mixed bacterial culture that can utilize 2-aminobiphenyl as a sole nitrogen source, directed evolution experiments were focused on the aniline dioxygenase enzyme that is capable of deaminating aniline, and expression vectors were constructed to enable the expression of genes encoding C-N bond cleaving enzymes in Rhodococcus hosts. The construction of a new metabolic pathway to selectively remove nitrogen from carbazole and other molecules typically found in petroleum should lead to the development of a process to improve oil refinery efficiency by reducing the poisoning, by nitrogen, of catalysts used in the hydrotreating and catalytic cracking of petroleum. Aromatic compounds such as carbazole are representative of the difficult-to-treat organonitrogen compounds most commonly encountered in petroleum. There are two C-N bonds in carbazole and the construction of a metabolic pathway for the removal of nitrogen from carbazole will require enzymes capable cleaving both C-N bonds. A multi-component enzyme, carbazole dioxygenase, which can selectively cleave the first C-N bond has been identified and the genes that encode this enzyme have been cloned, sequenced, and are being expressed in Rhodococcus erythropolis, a bacterial culture that tolerates exposure to petroleum. An enzyme capable of selectively cleaving the second C-N bond in carbazole has not yet been identified, but enrichment culture experiments have recently succeeded in isolating a bacterial culture that is a likely candidate and may possess a suitable enzyme. Research in the near future will verify if a suitable enzyme for the cleavage of the second C-N bond in carbazole has indeed been found, then the genes encoding a suitable enzyme will be identified, cloned, and sequenced. Ultimately genes encoding enzymes for selective cleavage of both C-N bonds in carbazole will be assembled into a new metabolic pathway and the ability of the resulting bacterial culture to remove nitrogen from petroleum will be determined

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

The objective of the project is to develop a biochemical pathway for the selective cleavage of C-N bonds in molecules found in petroleum. Specifically a novel biochemical pathway will be developed for the selective cleavage of C-N bonds in carbazole. 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. The selective cleavage of the second C-N bond has been challenging, and efforts to overcome that challenge have been the focus of recent research in this project. Enrichment culture experiments succeeded in isolating bacterial cultures that can metabolize 2-aminobiphenyl, but no enzyme capable of selectively cleaving the C-N bond in 2-aminobiphenyl has been identified. Aniline is very similar to the structure of 2-aminobiphenyl and aniline dioxygenase catalyzes the conversion of aniline to catechol and ammonia. For the remainder of the project the emphasis of research will be to simultaneously express the genes for carbazole dioxygenase and for aniline dioxygenase in the same bacterial host and then to select for derivative cultures capable of using carbazole as the sole source of nitrogen

PETROLEUM BIOREFINING FOR POLLUTION PREVENTION

The objective of this project was to isolate and characterize thermophilic bacterial cultures that can be used for the selective removal of nitrogen, sulfur, and/or metals in the biorefining of petroleum. The project was completed on schedule and no major difficulties were encountered. Significant progress was made on multiple topics relevant to the development of a petroleum biorefining process capable of operating at thermophilic temperatures. New cultures capable of selectively cleaving C-N or C-S bonds in molecules relevant to petroleum were obtained, and the genes encoding the enzymes for these unique biochemical reactions were cloned and sequenced. Genetic tools were developed that enable the use of Thermus thermophilus as a host to express any gene of interest, and information was obtained regarding the optimum conditions for the growth of T. thermophilus. The development of a practical biorefining process still requires further research and the future research needs identified in this project include the development of new enzymes and pathways for the selective cleavage of C-N or C-S bonds that have higher specific activities, increased substrate range, and are capable of functioning at thermophilic temperatures. Additionally, there is a need for process engineering research to determine the maximum yield of biomass and cloned gene products that can be obtained in fed-batch cultures using T. thermophilus, and to determine the best configuration for a process employing biocatalysts to treat petroleum

Biodesulfurization of water-soluble coal-derived material by Rhodococcus rhodochrous IGTS8

Rhodococcus rhodochrous IGTS8 was previously isolated because of its ability to use coal as its sole source of sulfur for growth. Subsequent growth studies have revealed that IGTS8 is capable of using a variety of organosulfur compounds as sources of sulfur but not carbon. In this paper, the ability of IGTS8 to selectively remove organic sulfur from water-soluble coal-derived material is investigated. The microbial removal of organic sulfur from coal requires microorganisms capable of cleaving carbonsulfur bonds and the accessibility of these bonds to microorganisms. The use of water-soluble coal-derived material effectively overcomes the problem of accessibility and allows the ability of microorganisms to cleave carbonsulfur bonds present in coal-derived material to be assessed directly. Three coals, two coal solubilization procedures, and two methods of biodesulfurization were examined. The results of these experiments reveal that the microbial removal of significant amounts of organic sulfur from watersoluble coal-derived material with treatment times as brief as 24 hours is possible. Moreover, the carbon content and calorific value of biotreated products are largely unaffected. Biotreatment does, however, result in increases in the hydrogen and nitrogen content and a decreased oxygen content of the coal-derived material. The aqueous supernatant obtained from biodesulfurization experiments does not contain sulfate, sulfite, or other forms of soluble sulfur at increased concentrations in comparison with control samples. Sulfur removed from water-soluble coal-derived material appears to be incorporated into biomass