Rhodococcus strains

The biocatalytic differentiation of enantiotopic nitrile groups in prochiral or meso substrates has been studied by several research groups. For instance, the nitrilase-catalyzed desymmetrization of 3-hydroxyglutaronitrile [92,93] followed by an esterification provided ethyl-(Jl)-4-cyano-3-hydroxybutyrate, a useful intermediate in the synthesis of cholesterol-lowering dmg statins (Figure 6.32) [94,95]. The hydrolysis of prochiral a,a-disubstituted malononitriles by a Rhodococcus strain expressing nitrile hydratase/amidase activity resulted in the formation of (R)-a,a-disubstituted malo-namic acids (Figure 6.33) [96]. 

Bouchez M, D Blanchet, J-P Vandecasteele (1997) An interfacial uptake mechanism for the degradation of pyrene by a Rhodococcus strain. Microbiology (UK) 143 1087-1093. 

Shao ZQ, R Behki (1996) Characterization of the expression of the thcB gene, coding for a pesticide-degrading cytochrome P450 in Rhodococcus strains. Appl Environ Microbiol 62 403-407. 

Besse P, B Combourieu, G Boyse, M Sancelme, H de Wever, A-M Delort (2001) Long-range H- N heteronuclear shift correlation at natural abundance a tool to study benzothiazole biodegradation by two Rhodococcus strains. Appl Environ Microbiol 67 1412-1417. 

Bernhardt D, H Diekmann (1991) Degradation of dioxane, tetrahydrofuran and other cyclic ethers by an environmental Rhodococcus strain. Appl Microbiol Biotechnol 36 120-123. 

Koike, K., Takaiwa, M., Ara, K. et al. (2000) Production of isopropyl cti-6-hexadecenoate by regiospecific desaturation of isopropyl palmitate by a double mutant of a Rhodococcus strain. Bioscience, Biotechnology, and Biochemistry, 64 (2), 399 404. 

Table 2. Comparison of specific desulfurization rates of various Rhodococcus strains...

Other Rhodococcus strains similar to those described above in terms of the desulfurization ability have also been isolated [83], The purpose of identifying such Rhodococcus strains, in several cases, appears to be the development of in-house biocatalysts for BDS application. The specificity of the desulfurizing strains of organosulfur compounds in addition to DBT has also been studied (Table 3). 

Rhodococcus strain SY1 was reported to desulfurize dimethyl sulfide, dimethyl sulfoxide, and several alkyl sulfonates [41] in addition to DBT [78], Barium chloride has been used to precipitate sulfate and shown to alleviate sulfate repression partially. The authors proposed a tentative pathway for oxidative removal of sulfur from DBT and other organosulfur compounds. It should be noted that phenyl disulfide and thianaph-thene were not desulfurized by any of the Rhodococcus strains, but have been reported to be substrates of Gordonia CYKS2. 

Rhodococcus sp. Strain WU-K2R A Rhodococcus strain capable of sulfur-specific desulfurization of benzothiophene, naphthothiophene (NT), and some of their alkyl derivatives was reported [35]. The metabolites of BT desulfurization were BT sulfone, benzo[c][l,2]oxanthiin S-oxide, benzo[c][l,2]oxanthiin S,S-dioxide, o-hydroxystyrene, 2,(2 -hydroxyphenyl)ethan-l-al, and benzofuran. The NT metabolites were NT sulfone, 2 -hydroxynaphthyl ethene, and naphtho[2,l-b]furan [35], The exact biochemical pathway was not determined, however, part of the pathway for BT desulfurization was speculated to be similar to Paenibacillus All-2. 

Rhodococcus sp. Strain T09 A Rhodococcus strain T09 was isolated by enrichment on media-containing BT. The desulfurization mechanism of this organism was reported to be similar to Gordonia sp. 213E due to the observation of similar intermediates however, the substrate specificity was different. The strain T09 could use 2-methyl, 3-methyl and 5-methyl BT apart from BT as sole source of sulfur for growth, but not 7-methyl or ethyl derivatives. Additionally, it could also use methyl thiobenzothiazole, marcaptobenzothiazole, as well as benzene sulfide, benzene sulfonate, biphenyl sulfinate, dimethyl sulfate, dimethyl sulfone, dimethyl sulfide, methane sulfonic acid, thiophene, and taurine as sole sulfur sources. However, it could not grow on DBT or DBT sulfone. 

Recent work at Diversa [219] investigated use of alternate Rhodococcus strains as hosts for desulfurization genes. It was reported that R. opacus was the most suitable for the application. This was due to its ability to grow fast and use triglycerides as carbon source for production of reduced co-factors during the desulfurization process. 

Thus, several improvements have been made in the Rhodococcus strains to make desulfurization application possible or attractive however, the sulfur removal rate still remains the biggest bottleneck and no biocatalysts capable of rates needed for commercialization exist as of yet. 

An important parameter that has to be considered during desulfurization as well as for subsequent biocatalyst separation and recycle is the impact of the oil phase on the biocatalyst activity and half-life. Additionally, the effect of the biocatalyst on forma-tion/breakage of the oil-water emulsions is also important. The latter will be discussed in Section 2.3.3. It becomes important for lower boiling feedstocks such as gasoline, which offers the most toxic solvent environment for the biodesulfurization catalyst. The effect of solvents on biocatalysts has been investigated in very few reports. A study by the Monot group reported effect of two solvents on several Rhodococcus strains [254], The strains contacted with the solvents and their desulfurization activity, growth, and... 

The specificity of several of the enzymes identified in the 4S pathway of different organisms has been studied. In case of the DBT desulfurizing enzymes, little difference is expected in the specificity of the enzymes, say DszA, from different Rhodococcus strains found to date. This is essentially because the DNA sequence for the enzymes investigated so far has been the same. The difference in the specificity observed with whole cell assays is essentially due to the differences in substrate intake via the cell membrane and not necessarily due to a difference in the intrinsic enzyme specificity. It has been found that while isolated enzyme DszC (from KA2-5-1) can desulfurize up to 4,6 dipropyl DBT, whole cells cannot, indicating substrate transport as limiting factor. 

Engineered biocatalysts with altered specificity have been developed. A Rhodococcus strain capable of DBT as well as BT desulfurization has been developed by cloning dsz genes into a strain-containing BT desulfurization pathway. The variety of sulfur compounds in petroleum feedstocks and complexity of the problem may require use of a consortium rather than a single bacterial strain. Alternately, use of multiple bioreactors each with a single dominant strain may be employed to achieve maximum desulfurization [299],... 

Schwarz et al. in agreement with Shukla observed the formation of 2-Oxo-l, 2-dihydroquinoline, 8-hydroxy-2-oxo-l, 2-dihydroquinoline, 8-hydroxycoumarin, and 2,3-dihydroxy-phenylpropionic acid were found as intermediates of quinoline transformation by P. fluorescens 3 and P. putida 86 [325], They compared that metabolic pathway with the one obtained for Rhodococcus strain B1 (Fig. 22). This bacterium was unable to yield denitrogenated metabolites (i.e., 2-oxo-l, 2-dihydroquinoline, 6-hydroxy-2-oxo-l, 2-dihydroquinoline, and 5-hydroxy-6-(3-carboxy-3-oxopropenyl)-lH-2-pyridone). 

Figure 22. Proposed pathway for the transformation of quinoline by Rhodococcus strain Bl.

Tanaka, Y. Matsui, T. Konishi, J., et al., Biodesulfurization of benzothiophene and dibenzothiophene by a newly isolated Rhodococcus strain. Applied Microbiology and Biotechnology, 2002. 59(2-3) pp. 325-328. 

Further work at EniTecnologies was conducted with Rhodococcus strains. Rhodococ-cus was selected for its metabolical versatility, easy availability in soils and water, and remarkable solvent tolerance. Its capabilities for catalyzing diverse transformation reactions of crude oils, such as sulfur removal, alkanes and aromatics oxidation and catabolism caught their attention. Hence, genetic tools for the engineering of Rhodococcus strains have been applied to improve its biotransformation performance and its tolerance to certain common contaminants of the crude oil, such as cadmium. The development of active biomolecules led to the isolation and characterization of plasmid vectors and promoters. Strains have been constructed in which the careful over-expression of selected components of the desulfurization pathway leads to the enhancement of the sulfur removal activity in model systems. Rhodococcus, Gordona, and Nocardia were transformed in this way trying to improve their catalytic performance in BDS. In a... 

New cultures of Rhodococcus strains, useful for desulfurization of petroleum and its products, comprises high catalytic stability which allows culture recycling [86],... 

The patents, however, protected the microorganisms (biocatalysts/biocatalytic systems) [86,87] as well as process to use the microorganism [87], So far, there are no records of any other international protection. The patent reports new cultures of Rhodococcus strains, and a method to improve biocatalyst stability, which allows recycling. 

For many years the complete mineralization of atrazine by bacteria was considered to be possible only through the combined efforts of two or more bacteria (a bacterial consortium). For example, Behki and Khan (1986) indicated that the removal of the isopropyl group from atrazine by a Pseudomonas sp. results in a substrate (deisopropylat-razine) that a Rhodococcus strain is able to mineralize completely (Cook and Hutter, 1984). 

Genes encoding atrazine degradation activity from Rhodococcus strains have also been reported by Nagy et al. (1995a) and by Shao and Behki (1996). In Rhodococcus strain TE1, an atrA, gene-mediating A-dealkylation of atrazine has been cloned (Shao and Behki, 1995). 

Both alkyl side chains are necessary for activity of. v-triazinc dealkylating enzymes from Rhodococcus strain B-30 (Behki and Khan, 1994). 

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