In vitro effects of sterculic acid on lipid biosynthesis in Rhodococcus opacus strain PD630 and isolation of mutants defective in fatty acid desaturation

The in vivo effects of sterculic acid methyl ester on triacylglycerol fatty acid composition in the oleaginous, hydrocarbon-degrading bacterium R. opacus strain PD630 was investigated. Sterculic acid, a cyclopropene fatty acid and an inhibitor of the stearoyl-CoA desaturase system, strongly inhibited the synthesis of monoenic fatty acids, of saturated fatty acids with more than 16 carbon atoms and of odd-numbered fatty acids when added to the culture medium. In addition, chemical mutagenesis and the application of the penicillin enrichment technique provided mutants, which were more or less completely impaired in the desaturation of long-chain fatty acids and exhibited in some cases a similar fatty acid composition like the wild-type in the presence of sterculic acid methyl ester. The implications of these findings for fatty acid metabolism in R. opacus strain PD630 are discussed.     

Saccharification of Cellulose by Recombinant Rhodococcus opacus PD630 Strains

The noncellulolytic actinomycete Rhodococcus opacus strain PD630 is the model oleaginous prokaryote with regard to the accumulation and biosynthesis of lipids, which serve as carbon and energy storage compounds and can account for as much as 87% of the dry mass of the cell in this strain. In order to establish cellulose degradation in R. opacus PD630, we engineered strains that episomally expressed six different cellulase genes from Cellulomonas fimi ATCC 484 (cenABC, cex, cbhA) and Thermobifida fusca DSM43792 (cel6A), thereby enabling R. opacus PD630 to degrade cellulosic substrates to cellobiose. Of all the enzymes tested, five exhibited a cellulase activity toward carboxymethyl cellulose (CMC) and/or microcrystalline cellulose (MCC) as high as 0.313 ± 0.01 U · ml⁻¹, but recombinant strains also hydrolyzed cotton, birch cellulose, copy paper, and wheat straw. Cocultivations of recombinant strains expressing different cellulase genes with MCC as the substrate were carried out to identify an appropriate set of cellulases for efficient hydrolysis of cellulose by R. opacus. Based on these experiments, the multicellulase gene expression plasmid pCellulose was constructed, which enabled R. opacus PD630 to hydrolyze as much as 9.3% ± 0.6% (wt/vol) of the cellulose provided. For the direct production of lipids from birch cellulose, a two-step cocultivation experiment was carried out. In the first step, 20% (wt/vol) of the substrate was hydrolyzed by recombinant strains expressing the whole set of cellulase genes. The second step was performed by a recombinant cellobiose-utilizing strain of R. opacus PD630, which accumulated 15.1% (wt/wt) fatty acids from the cellobiose formed in the first step.     

Preferential oxidative dehalogenation upon conversion of 2-halophenols by Rhodococcus opacus 1G

The regiospecificity of hydroxylation of C2-halogenated phenols by Rhodococcus opacus 1G was investigated. Oxidative defluorination at the C2 position ortho with respect to the hydroxyl moiety was preferred over hydroxylation at the non-fluorinated C6 position for all 2-fluorophenol compounds studied. Initial hydroxylation of 2,3, 5-trichlorophenol resulted in the exclusive formation of 3, 5-dichlorocatechol. These results indicate that, in contrast to all other phenol ortho-hydroxylases studied so far, phenol hydroxylase from R. opacus 1G is capable of catalyzing preferential oxidative defluorination but also oxidative dechlorination.     

Glycogenformation by Rhodococcus species and the effect of inhibition of lipid biosynthesis on glycogen accumulation in Rhodococcus opacus PD630

Members of the genus Rhodococcus were investigated for their ability to produce glycogen during cultivation on gluconate or glucose. Strains belonging to Rhodococcus ruber, Rhodococcus opacus, Rhodococcus fascians, Rhodococcus erythropolis and Rhodococcus equi were able to produce glycogen up to 0.2–5.6% of cellular dry weight (CDW). The glycogen content varied from 0.8% to 3.2% of CDW in cells of R. opacus PD630, which is a well-known oleaginous bacterium, during the exponential growth phase, when cultivated on diverse carbon sources. Maltose and pyruvate promoted glycogen accumulation by cells of strain PD630 to a greater extent than glucose, gluconate, lactose, sucrose or acetate. This strain was able to produce triacylglycerols, polyhydroxyalkanoates and glycogen as storage compounds during growth on gluconate, although triacylglycerols were always the main product under the conditions of this study. Cerulenin, an inhibitor of de novo fatty acid synthesis, inhibited the accumulation of triacylglycerols from gluconate and increased the content of polyhydroxyalkanoates (from 2.0% to 4.2%, CDW) and glycogen (from 0.1% to 3.0%, CDW). An increase of the polyhydroxyalkanoates and glycogen content was also observed in two mutants of R. opacus PD630, which produced reduced amounts of triacylglycerols during cultivation of cells on gluconate.     

Accumulation and mobilization of storage lipids by Rhodococcus opacus PD630 and Rhodococcus ruber NCIMB 40126

The time course of the accumulation of triacylglycerols (TAGs) in Rhodococcus opacus PD630 or of TAGs plus polyhydroxyalkanoates (PHA) in Rhodococcus ruber NCIMB 40126 with gluconate or glucose as carbon source, respectively, was studied. In addition, we examined the mobilization of these storage compounds in the absence of a carbon source. R. opacus accumulated TAGs only after the exhaustion of ammonium in the medium, and, with a fixed concentration of the carbon source, the amounts of TAGs in the cells increased with decreasing concentrations of ammonium in the medium. When these cells were incubated in the absence of an additional carbon source, about 90% of these TAGs were mobilized and used as endogenous carbon source, particularly if ammonium was available. R. ruber accumulated a copolyester consisting of 3-hydroxybutyrate and 3-hydroxyvalerate already during the early exponential growth phase, whereas TAGs were synthesized and accumulated mainly during the late exponential and stationary growth phases. In the stationary growth phase, synthesis of TAGs continued, whereas PHA was partially mobilized. In the absence of an additional carbon source but in the presence of ammonium, mobilization of TAGs started first and was then paralleled by the mobilization of PHA, resulting in an approximately 90% and 80% decrease of these storage compounds, respectively. During the accumulation phase, interesting shifts in the composition of the two storage compounds occurred, indicating that the substrates of the PHA synthase and the TAG synthesizing enzymes were provided to varying extents, depending on whether the cells were in the early or late exponential or in the stationary growth phase. Received: 12 January 2000 / Received revision: 22 February 2000 / Accepted: 25 February 2000     

Location, catalytic activity, and subunit composition of NAD-reducing hydrogenases of some Alcaligenes strains and Rhodococcus opacus MR22

Six new strains of Alcaligenes enriched for and isolated as nickel-resistant bacteria resemble Alcaligenes eutrophus H16 and contain both an NAD-reducing, tetrameric soluble hydrogenase and a membrane-bound hydrogenase. None of the soluble hydrogenases share with the Rhodococcus opacus MR11 enzyme tetramer the property of being cleaved easily into two dimeric moieties [a hydrogenase (βδ) and an NADH:acceptor oxidoreductase (αγ)], in the absence of nickel or at low ionic strength. The soluble hydrogenase of the newly isolated strain MR22 of R. opacus equalled that of strain MR11. The absence of a membrane-bound hydrogenase in Alcaligenes denitrificans strain 4a-2 and in Alcaligenes ruhlandii was confirmed. Received: 14 May 1996 / Accepted: 7 November 1996     

Mechanism of the oxidative stress-mediated increase in lipid accumulation by the bacterium,R. opacus PD630: Experimental analysis and genome-scale metabolic modeling

Appropriate species of oleaginous bacteria, with their high growth rates and lipid accumulation capabilities, can be good contenders for industrial triacylglycerol (TAG) production, compared to microalgae. Further, oxidative stress (OS) can be used to significantly increase TAG yields in oleaginous microbes, but the mechanism is unexplored. In a first, this study explored the mechanism behind OS-mediated increase in TAG accumulation by the bacterium, Rhodococccus opacus PD630, through experimental analysis and metabolic modelling. Two mechanisms that could increase acetyl-CoA (TAG-precursor) levels were hypothesized based on literature information. One was OS-mediated inactivation of the aconitase (TCA cycle), and another was the inactivation of the triosephosphate isomerase (TPI; glycolysis). The results negated the involvement of aconitase in increased acetyl-CoA levels. Analysis of the metabolic model showed that inactivation of TPI, re-routed the flux through the pentose phosphate pathway (PPP), supplying both NADPH and acetyl-CoA for TAG synthesis. Additionally, inactivation of TPI increased TAG flux by 143%, whereas, inactivating both TPI and aconitase, increased it by 152%. We present experimental evidence for OS-mediated decrease in TPI activity and increase in activity of glucose-6-phosphate dehydrogenase (PPP enzyme). The findings indicate that increased flux through PPP can be explored to improve TAG accumulation on a large-scale.     

Degradation of pyridine by Arthrobacter crystallopoietes and Rhodococcus opocus strains

Abstract Two pyridine-degrading microorganisms Arthrobacter crystallopoietes (VKM Ac-1334D) and Rhodococcus opacus (VKM Ac-1333D) were isolated from soil. The Gas chromatography-mass spectroscopy analysis showed that the former species formed 3-hydroxypyridine, 2,3- and 2,6-dihydroxypyridines during its growth in media containing pyridine, while the latter formed 2-hydroxy- and 2,6-dihydroxypyridines as degradation intermediates. Products of the pyridine ring cleavage (5-amino-2-oxo-4-pentenoic acid and 3-pentenoic acid monoamide) were also detected.     

Specificity of catechol ortho-cleavage during para-toluate degradation by Rhodococcus opacus 1cp

Degradation of para-toluate by Rhodococcus opacus 1cp was investigated. Activities of the key enzymes of this process, catechol 1,2-dioxygenase and muconate cycloisomerase, are detected in this microorganism. Growth on p-toluate was accompanied by induction of two catechol 1,2-dioxygenases. The substrate specificity and physicochemical properties of one enzyme are identical to those of chlorocatechol 1,2-dioxygenase; induction of the latter enzyme was observed during R. opacus 1cp growth on 4-chlorophenol. The other enzyme isolated from the biomass grown on p-toluate exhibited lower rate of chlorinated substrate cleavage compared to the catechol substrate. However, this enzyme is not identical to the catechol 1,2-dioxygenase cloned in this strain within the benzoate catabolism operon. This supports the hypothesis on the existence of multiple forms of dioxygenases as adaptive reactions of microorganisms in response to environmental stress.     

Regioselective oxidation of xylene isomers by Rhodococcus sp. strain DK17

Rhodococcus sp. strain DK17 is able to utilize a variety of monocyclic aromatic hydrocarbons, including benzene, phenol, toluene, and o-xylene, as growth substrates. Although DK17 is unable to grow on m- and p-xylene, this strain could transform these two xylene isomers to some extent after induction by o-xylene. The major accumulating compounds formed during the degradation of m- and p-xylene by DK17 were isolated by high-pressure liquid chromatography and identified by gas chromatography-mass spectrometric and (1)H nuclear magnetic resonance spectral techniques. Both xylene isomers were transformed to dihydroxylated compounds by what must be two successive hydroxylation events: m-xylene was converted to 2,4-dimethylresorcinol and p-xylene was converted to 2,5-dimethylhydroquinone. The rigorous structural identification of 2,4-dimethylresorcinol and 2,5-dimethylhydroquinone demonstrates that DK17 can perform distinct regioselective hydroxylations depending on the position of the substituent groups on the aromatic ring.     

Structural analysis of an extracellular polysaccharide produced by Rhodococcus rhodochrous strain S-2

A possibility has been suggested of applying the EPS produced by Rhodococcus rhodochrous strain S-2 (S-2 EPS) to the bioremediation of oil-contaminated environments, because its addition, together with minerals, to oil-contaminated seawater resulted in emulsification of the oil, increased the degradation of polyaromatic hydrocarbons (PAH) of the oil, and led to the dominance of PAH-degrading marine bacteria. To understand the underlying principles of these phenomena, we determined the chemical structure of the sugar chain of S-2 EPS. The EPS was found to be composed of D-galactose, D-mannose, D-glucose, and D-glucuronic acid, in a molar ratio of 1:1:1:1. In addition, 0.8% (w/w) of octadecanoic acid and 2.7% (w/w) of hexadecanoic acid were also contained in its structure. By 1H and 13C NMR spectroscopy, including 2D DQF-COSY, TOCSY, HMQC, HMBC, and NOESY experiments, as well as chemical and enzymatic analyses, the polysaccharide was shown to consist of tetrasaccharide repeating units with the following structure: (see formula in text).     

Improvement of phytoremediation of an aged petroleum hydrocarbon-contaminated soil by Rhodococcus erythropolis CD 106 strain

The aim of this study was to assess the impact of soil inoculation with the Rhodococcus erythropolis CD 106 strain on the effectiveness of the phytoremediation of an aged hydrocarbon-contaminated [approx. 1% total petroleum hydrocarbon (TPH)] soil using ryegrass (Lolium perenne). The introduction of CD 106 into the soil significantly increased the biomass of ryegrass and the removal of hydrocarbons in planted soil. The fresh weight of the shoots and roots of plants inoculated with CD 106 increased by 49% and 30%, respectively. After 210 days of the experiment, the concentration of TPH was reduced by 31.2%, whereas in the planted, non-inoculated soil, it was reduced by 16.8%. By contrast, the concentration of petroleum hydrocarbon decreased by 18.7% in non-planted soil bioaugmented with the CD 106 strain. The rifampicin-resistant CD 106 strain survived after inoculation into soil and was detected in the soil during the entire experimental period, but the number of CD 106 cells decreased constantly during the enhanced phytoremediation and bioaugmentation experiments.

The plant growth-promoting and hydrocarbon-degrading properties of CD 106, which are connected with its long-term survival and limited impact on autochthonous microflora, make this strain a good candidate for improving the phytoremediation efficiency of soil contaminated with hydrocarbons.     

Isolation and identification of berberine and berberrubine metabolites by berberine-utilizing bacterium Rhodococcus sp. strain BD7100

Based on the finding of a novel berberine (BBR)-utilizing bacterium, Rhodococcus sp. strain BD7100, we investigated the degradation of BBR and its analog berberrubine (BRU). Resting cells of BD7100 demethylenated BBR and BRU, yielding benzeneacetic acid analogs. Isolation of benzeneacetic acid analogs suggested that BD7100 degraded the isoquinoline ring of the protoberberine skeleton. This work represents the first report of cleavage of protoberberine skeleton by a microorganism.     

Inhibition of diethyl ether degradation in Rhodococcus sp. strain DEE5151 by glutaraldehyde and ethyl vinyl ether

Alkyl ether-degrading Rhodococcus sp. strain DEE5151, isolated from activated sewage sludge, has an activity for the oxidation of a variety of alkyl ethers, aralkyl ethers and dibenzyl ether. The whole cell activity for diethyl ether oxidation was effectively inhibited by 2,3-dihydrofurane, ethyl vinyl ether and glutaraldehyde. Glutaraldehyde of less than 30 microM inhibited the activity by a competitive manner with the inhibition constant, K(I) of 7.07+/-1.36 microM. The inhibition type became mixed at higher glutaraldehyde concentrations >30 microM, probably due to the inactivation of the cell activity by the Schiff-base formation. Structurally analogous ethyl vinyl ether inhibited the diethyl ether oxidation activity in a mixed manner with decreasing the apparent maximum oxidation rate, v(max)(app), and increasing the apparent Michaelis-Menten constant, K(M)(app). The mixed type inhibition by ethyl vinyl ether seemed to be introduced not only by the structure similarity with diethyl ether, but also by the reactivity of the vinyl ether with cellular components in the whole cell system.     

Co-fermentation of lignocellulose-based glucose and inhibitory compounds for lipid synthesis by Rhodococcus jostii RHA1

The recalcitrant nature of lignocellulosic biomass entails pretreatment during which multiple byproducts (e.g., weak acids, furan derivatives, lignin-derived compounds) are generated. Such byproducts are generally inhibitory to fuel-producing microorganisms. In this study, lignin-derived monomers and acetate were co-fermented with glucose by Rhodococcus jostii RHA1 for lipid synthesis. The ability of R. jostii RHA1 to utilize acetate and representative lignin-derived monomers, namely p-coumaric acid, ferulic acid, 4-hydroxylic acid, and vanillic acid, were tested. The experimental results showed that R. jostii RHA1 utilized individual lignin monomers in varying degrees. The mixtures of inhibitory compounds at different levels showed higher toxicity than individual compounds, indicating synergistic effects of these monomers. When the mixture contained lower levels of glucose (5 g/L or below), adaptive-evolved (AE) R. jostii RHA1 utilized such inhibitory mixtures better for lipid synthesis. When the glucose levels were increased to 20 g/L or above, adaption evolution appeared to shorten the lag phase of co-fermentation but not necessarily enhance lipid production. This study demonstrated that R. jostii RHA1 was capable of utilizing commonly unfavorable carbon sources for lipid synthesis, which would also serve as a means to in situ detoxify inhibitory compounds.     

A sensitive, viable-colony staining method using Nile red for direct screening of bacteria that accumulate polyhydroxyalkanoic acids and other lipid storage compounds

The oxazine dye Nile blue A and its fluorescent oxazone form, Nile red, were used to develop a simple and highly sensitive staining method to detect poly(3-hydroxybutyric acid) and other polyhydroxyalkanoic acids (PHAs) directly in growing bacterial colonies. In contrast to previously described methods, these dyes were directly included in the medium at concentrations of only 0.5 μg/ml, and growth of the cells occurred in the presence of the dyes. This allowed an estimation of the presence of PHAs in viable colonies at any time during the growth experiment and a powerful discrimination between PHA-negative and PHA-positive strains. The presence of Nile red or Nile blue A did not affect growth of the bacteria. This viable-colony staining method was in particular applicable to gram-negative bacteria such as Azotobacter vinelandii, Escherichia coli, Pseudomonas putida, and Ralstonia eutropha. It was less suitable for discriminating between PHA-negative and PHA-positive strains of gram-positive bacteria such as Bacillus megaterium or Rhodococcus ruber, but it could also be used to discriminate between wax-ester- and triacylglycerol-negative and -positive strains of Acinetobacter calcoaceticus or Rhodococcus opacus. The potential of this new method and its application to further investigations of PHA synthases and PHA biosynthesis pathways are discussed. Received: 12 August 1998 / Accepted: 11 November 1998