Engineering of a xylose metabolic pathway in Rhodococcus strains

The two metabolically versatile actinobacteria Rhodococcus opacus PD630 and R. jostii RHA1 can efficiently convert diverse organic substrates into neutral lipids mainly consisting of triacylglycerol (TAG), the precursor of energy-rich hydrocarbon. Neither, however, is able to utilize xylose, the important component present in lignocellulosic biomass, as the carbon source for growth and lipid accumulation. In order to broaden their substrate utilization range, the metabolic pathway of d-xylose utilization was introduced into these two strains. This was accomplished by heterogenous expression of two well-selected genes, xylA, encoding xylose isomerase, and xylB, encoding xylulokinase from Streptomyces lividans TK23, under the control of the tac promoter with an Escherichia coli-Rhodococcus shuttle vector. The recombinant R. jostii RHA1 bearing xylA could grow on xylose as the sole carbon source, and additional expression of xylB further improved the biomass yield. The recombinant could consume both glucose and xylose in the sugar mixture, although xylose metabolism was still affected by the presence of glucose. The xylose metabolic pathway was also introduced into the high-lipid-producing strain R. opacus PD630 by expression of xylA and xylB. Under nitrogen-limited conditions, the fatty acid composition was determined, and lipid produced from xylose by recombinants of R. jostii RHA1 and R. opacus PD630 carrying xylA and xylB represented up to 52.5% and 68.3% of the cell dry weight (CDW), respectively. This work demonstrates that it is feasible to produce lipid from the sugars, including xylose, derived from renewable feedstock by genetic modification of rhodococcus strains.     

Functional annotation and characterization of 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1

The genome of Rhodococcus jostii RHA1 contains an unusually large number of oxygenase encoding genes. Many of these genes have yet an unknown function, implying that a notable part of the biochemical and catabolic biodiversity of this Gram-positive soil actinomycete is still elusive. Here we present a multiple sequence alignment and phylogenetic analysis of putative R. jostii RHA1 flavoprotein hydroxylases. Out of 18 candidate sequences, three hydroxylases are absent in other available Rhodococcus genomes. In addition, we report the biochemical characterization of 3-hydroxybenzoate 6-hydroxylase (3HB6H), a gentisate-producing enzyme originally mis-annotated as salicylate hydroxylase. R. jostii RHA1 3HB6H expressed in Escherichia coli is a homodimer with each 47kDa subunit containing a non-covalently bound FAD cofactor. The enzyme has a pH optimum around pH 8.3 and prefers NADH as external electron donor. 3HB6H is active with a series of 3-hydroxybenzoate analogues, bearing substituents in ortho- or meta-position of the aromatic ring. Gentisate, the physiological product, is a non-substrate effector of 3HB6H. This compound is not hydroxylated but strongly stimulates the NADH oxidase activity of the enzyme.     

High cell density cultivation of Rhodococcus opacus for lipid production at a pilot-plant scale

The triacylglycerol (TAG)-accumulating bacterium Rhodococcus opacus strain PD630 was investigated with respect to the fermentative production of TAGs consisting of an unusually high fraction of fatty acids with an odd-number of carbon atoms and unsaturated monoenic fatty acids from sugar beet molasses and sucrose. Fed-batch fermentations were optimized at the 30-1 scale in a stirred tank bioreactor at 30 degrees C using a mineral salts medium, which contained sugar beet molasses and sucrose as sole carbon sources. Approximately 37.5 g cell dry matter (CDM) per liter was the highest cell density that was obtained at that scale with a TAG content in the cells of 52%. This fermentative process was also applied to a 500-1 pilot-plant scale. Cell densities as high as 18.4 g CDM per liter were obtained, and 42% of the sucrose present in the medium was converted into cell mass which consisted of 38.4% TAGs.     

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.     

Identification of a toluene-degrading bacterium from a soil sample through H(2)(18)O DNA stable isotope probing

DNA stable isotope probing (DNA-SIP) with H(2)(18)O was used to identify a toluene-degrading bacterium in soil amended with 48 ppm toluene. After quantification of toluene degradation rates in soil, DNA was extracted from soil incubated with H(2)(18)O, H(2)(16)O, H(2)(16)O and 48 ppm toluene, or H(2)(18)O and 48 ppm toluene. A single DNA band formed along a cesium chloride gradient after isopycnic centrifugation of extracts from soils incubated with H(2)(16)O. With extracts from soils to which only H(2)(18)O was added, two distinct DNA bands formed, while three bands formed when DNA extracted from soil incubated with both H(2)(18)O and toluene was analyzed. We suggest that this third band formed because toluene does not contain any oxygen atoms and toluene-degrading organisms had to transfer oxygen atoms from H(2)(18)O into metabolic intermediates to form nucleic acids de novo. We extracted the third DNA band and amplified a large fraction of the bacterial 16S rRNA gene. Direct sequencing of the PCR product obtained from the labeled DNA, as well as cloned 16S rRNA amplicons, identified a known toluene degrader, Rhodococcus jostii RHA1. A toluene-degrading bacterial strain was subsequently isolated from soil and shown to be Rhodococcus jostii RHA1. Finally, quantitative real-time PCR analysis showed that the abundance of the 16S rRNA gene of Rhodococcus jostii RHA1 increased in soil after toluene exposure but not in soils from which toluene was withheld. This study indicates that H(2)(18)O DNA-SIP can be a useful method for identifying pollutant-degrading bacteria in soil.     

Adventures in Rhodococcus - from steroids to explosives

Rhodococcus is a genus of mycolic-acid-containing actinomycetes that utilize a remarkable variety of organic compounds as growth substrates. This degradation helps maintain the global carbon cycle and has increasing applications ranging from the biodegradation of pollutants to the biocatalytic production of drugs and hormones. We have been using Rhodococcus jostii RHA1 as a model organism to understand the catabolic versatility of Rhodococcus and related bacteria. Our approach is exemplified by the discovery of a cluster of genes specifying the catabolism of cholesterol. This degradation proceeds via β-oxidative degradation of the side chain and O2-dependent cleavage of steroid ring A in a process similar to bacterial degradation of aromatic compounds. The pathway is widespread in Actinobacteria and is critical to the pathogenesis of Mycobacterium tuberculosis, arguably the world's most successful pathogen. The close similarity of some of these enzymes with biphenyl- and polychlorinated-biphenyl-degrading enzymes that we have characterized is facilitating inhibitor design. Our studies in RHA1 have also provided important insights into a number of novel metalloenzymes and their biosynthesis, such as acetonitrile hydratase (ANHase), a cobalt-containing enzyme with no significant sequence identity with characterized nitrile hydratases. Molecular genetic and biochemical studies have identified AnhE as a dimeric metallochaperone that delivers cobalt to ANHase, enabling its maturation in vivo. Other metalloenzymes we are characterizing include N-acetylmuramic acid hydroxylase, which catalyzes an unusual hydroxylation of the rhodococcal and mycobacterial peptidoglycan, and 2 RHA1 dye-decolorizing peroxidases. Using molecular genetic and biochemical approaches, we have demonstrated that one of these enzymes is involved in the degradation of lignin. Overall, our studies are providing fundamental insights into a range of catabolic processes that have a wide variety of applications.     

pFiD188, the linear virulence plasmid of Rhodococcus fascians D188

Rhodococcus fascians is currently the only phytopathogen of which the virulence genes occur on a linear plasmid. To get insight into the origin of this replicon and into the virulence strategy of this broad-spectrum phytopathogen, the sequence of the linear plasmid of strain D188, pFiD188, was determined. Analysis of the 198,917 bp revealed four syntenic regions with linear plasmids of R. erythropolis, R. jostii, and R. opacus, suggesting a common origin of these replicons. Mutational analysis of pFi_086 and pFi_102, similar to cutinases and type IV peptidases, respectively, showed that conserved region R2 was involved in plasmid dispersal and pointed toward a novel function for actinobacterial cutinases in conjugation. Additionally, pFiD188 had three regions that were unique for R. fascians. Functional analysis of the stk and nrp loci of regions U2 and U3, respectively, indicated that their role in symptom development was limited compared with that of the previously identified fas, att, and hyp virulence loci situated in region U1. Thus, pFiD188 is a typical rhodococcal linear plasmid with a composite structure that encodes core functions involved in plasmid maintenance and accessory functions, some possibly acquired through horizontal gene transfer, implicated in virulence and the interaction with the host.     

Expression in Escherichia coli of biphenyl 2,3-dioxygenase genes from a Gram-positive polychlorinated biphenyl degrader, Rhodococcus jostii RHA1

Rhodococcus jostii RHA1 is a polychlorinated biphenyl degrader. Multi-component biphenyl 2,3-dioxygenase (BphA) genes of RHA1 encode large and small subunits of oxygenase component and ferredoxin and reductase components. They did not express enzyme activity in Escherichia coli. To obtain BphA activity in E. coli, hybrid BphA gene derivatives were constructed by replacing ferredoxin and/or reductase component genes of RHA1 with those of Pseudomonas pseudoalcaligenes KF707. The results obtained indicate a lack of catalytic activity of the RHA1 ferredoxin component gene, bphAc in E. coli. To determine the cause of inability of RHA1 bphAc to express in E. coli, the bphAc gene was introduced into Rosetta (DE3) pLacI, which has extra tRNA genes for rare codons in E. coli. The resulting strain abundantly produced the bphAc product, and showed activity. These results suggest that codon usage bias is involved in inability of RHA1 bphAc to express its catalytic activity in E. coli.     

Vanillin catabolism in Rhodococcus jostii RHA1

Genes encoding vanillin dehydrogenase (vdh) and vanillate O-demethylase (vanAB) were identified in Rhodococcus jostii RHA1 using gene disruption and enzyme activities. During growth on vanillin or vanillate, vanA was highly upregulated while vdh was not. This study contributes to our understanding of lignin degradation by RHA1 and other actinomycetes.     

Eukaryotic lipid body proteins in oleogenous actinomycetes and their targeting to intracellular triacylglycerol inclusions: Impact on models of lipid body biogenesis

Bacterial neutral lipid inclusions are structurally related to eukaryotic lipid bodies. These lipid inclusions are composed of a matrix of triacylglycerols (TAGs) or wax esters surrounded by a monolayer of phospholipids. Whereas the monolayers of lipid bodies from animal and plant cells harbor specific classes of proteins which are involved in the structure of the inclusions and lipid homoestasis, no such proteins are known to be associated with bacterial lipid inclusions. The present study was undertaken to reveal whether the mammalian lipid body proteins perilipin A, adipose differentiation-related protein, and tail-interacting protein of 47 kDa (TIP47), which comprise the so called PAT family proteins, and the maize (Zea mays L.) oleosin are targeted to prokaryotic TAG bodies in vivo. When fused to enhanced green fluorescent protein, all proteins except the oleosin were mainly located at the surfaces of lipid inclusions when heterologously expressed in the recombinant actinomycetes Rhodococcus opacus PD630 and Mycobacterium smegmatis mc(2)155. A more detailed intracellular distribution analysis of TIP47 in recombinant R. opacus cells by immunocytochemical labeling of ultrathin cryosections and freeze fracture replicas revealed a substantial amount of TIP47 protein also pervading the cores of the inclusions. We discuss the impact of these results on the current model of lipid body biogenesis in prokaryotes.     

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.     

Identification of DypB from Rhodococcus jostii RHA1 as a lignin peroxidase

Rhodococcus jostii RHA1, a polychlorinated biphenyl-degrading soil bacterium whose genome has been sequenced, shows lignin degrading activity in two recently developed spectrophotometric assays. Bioinformatic analysis reveals two unannotated peroxidase genes present in the genome of R. jostii RHA1 with sequence similarity to open reading frames in other lignin-degrading microbes. They are members of the Dyp peroxidase family and were annotated as DypA and DypB, on the basis of bioinformatic analysis. Assay of gene deletion mutants using a colorimetric lignin degradation assay reveals that a ΔdypB mutant shows greatly reduced lignin degradation activity, consistent with a role in lignin breakdown. Recombinant DypB protein shows activity in the colorimetric assay and shows Michaelis-Menten kinetic behavior using Kraft lignin as a substrate. DypB is activated by Mn(2+) by 5-23-fold using a range of assay substrates, and breakdown of wheat straw lignocellulose by recombinant DypB is observed over 24-48 h in the presence of 1 mM MnCl(2). Incubation of recombinant DypB with a β-aryl ether lignin model compound shows time-dependent turnover, giving vanillin as a product, indicating that C(α)-C(β) bond cleavage has taken place. This reaction is inhibited by addition of diaphorase, consistent with a radical mechanism for C-C bond cleavage. Stopped-flow kinetic analysis of the DypB-catalyzed reaction shows reaction between the intermediate compound I (397 nm) and either Mn(II) (k(obs) = 2.35 s(-1)) or the β-aryl ether (k(obs) = 3.10 s(-1)), in the latter case also showing a transient at 417 nm, consistent with a compound II intermediate. These results indicate that DypB has a significant role in lignin degradation in R. jostii RHA1, is able to oxidize both polymeric lignin and a lignin model compound, and appears to have both Mn(II) and lignin oxidation sites. This is the first detailed characterization of a recombinant bacterial lignin peroxidase.     

Expanding the set of rhodococcal Baeyer–Villiger monooxygenases by high-throughput cloning, expression and substrate screening

To expand the available set of Baeyer-Villiger monooxygenases (BVMOs), we have created expression constructs for producing 22 Type I BVMOs that are present in the genome of Rhodococcus jostii RHA1. Each BVMO has been probed with a large panel of potential substrates. Except for testing their substrate acceptance, also the enantioselectivity of some selected BVMOs was studied. The results provide insight into the biocatalytic potential of this collection of BVMOs and expand the biocatalytic repertoire known for BVMOs. This study also sheds light on the catalytic capacity of this large set of BVMOs that is present in this specific actinomycete. Furthermore, a comparative sequence analysis revealed a new BVMO-typifying sequence motif. This motif represents a useful tool for effective future genome mining efforts.     

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     

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.