Genetic Modification Of Strain Rhodococcus Erythropolis XP

Microbial treatment of petroleum has been a promising method to remove organic sulfur and nitrogen, and is considered as a compliment to the conventional hydrodesulfurization (HDS) method. One of the challenges that are facing this technique is to develop biocatalysts with application potential. To this end, it requires that the biocatalysts should have high and stable activity, a broad substrate spectrum to cope with the complex components of petroleum, cost-effective preparation method, as well as regeneration ability. Strain Rhodococcus erythropolis XP is capable of specifically removing the sulfur atom from organic sulfur-containing compounds such as dibenzothiophene, resulting in the production of sulfite or sulfate. Therefore, the emission of sulfur dioxide during the combustion of fossil fuels will be reduced, and in turn less acid rain will be formed.The carbazole 1,9a-dioxygenase gene (car A) from strain Pseudomonas sp. 4-9 has also been cloned and expressed. It is composed of genes encoding terminal oxygenase, ferredoxin and ferredoxin reductase, which showed 99%, 100% and 100% similarities to those of the model strain P. resinovorans CA10, respectively. It was reported in 1993 that the product of carbazole catalyzed by car A was 2-aminobiphenyl-2,3-diol; however, this result has not yet been confirmed by high resolution mass spectrometry. By over-expressing car A on different expression vectors as biocatalyst, the reputative product can be accumulated for further identification.Based on the principle of site-specific homologous recombination, a plasmid vector designated as pK18mobsacB-rrp-carA-rdna has been constructed to integrate the gene of interest (carbazole 1,9a-dioxygenase gene) into the chromosome of strain XP, without introducing any selectable marker gene. This integration enables strain XP to degrade both dibenzothiophene and carbazole with better stability than employing shuttle vectors. Organic nitrogen-containing compounds (e.g. carbazole) in petroleum have previously been reported to poison catalysts during the process of hydrodesulfurization. Therefore, it is necessary to remove them before performing HDS.Since oxygenation is the first step of both biodesulfurization and biodenitrogenation, and the oxygen atom is from molecular oxygen, enhancing the cellular concentration of oxygen or the speed of oxygen transportation may boost the reactions. The aerobic strain Vitreoscilla sp. has been found to express bacterial hemoglobin (VHb) under hypoxic conditions. This protein functions in a similar way as human hemoglobin. By cloning and expressing the Vitreoscilla hemoglobin gene vhb on an Escherichia coli-Rhodococcus shuttle vector in R. erythropolis XP, the desulfurization activity of strain XP is expected to increase. Likewise, on the basis of the vector pK18mobascB, another vector can be constructed to integrate the vhb gene into the chromosome of strain XP to stably express the VHb gene.