Isolation and characterization of a haloalkane halidohydrolase from Rhodococcus erythropolis Y2

Rhodococcus erythropolis strain Y2, isolated from soil by enrichment culture using 1-chlorobutane, was able to utilize a range of halogenated aliphatic compounds as sole sources of carbon and energy. The ability to utilize 1-chlorobutane was conferred by a single halidohydrolase-type haloalkane dehalogenase. The presence of the single enzyme in cell-free extracts was demonstrated by activity strain polyacrylamide gel electrophoresis. The purified enzyme was a monomeric protein with a relative molecular mass of 34 kDa and demonstrated activity against a broad range of haloalkanes, haloalcohols and haloethers. The highest activity was found towards alpha, omega disubstituted chloro- and bromo- C2-C6 alkanes and 4-chlorobutanol. The Km value of the enzyme for 1-chlorobutane was 0.26 mM. A comparison of the R. erythropolis Y2 haloalkane halidohydrolase with other haloalkane dehalogenases is discussed on the basis of biochemical properties and N-terminal amino acid sequence data.     

Cell-linked and extracellular cholesterol oxidase activities from Rhodococcus erythropolis. Isolation and physiological characterization

Rhodococcus erythropolis cells growing in a cholesterol-free glycerol-containing mineral medium displayed very low levels of a cell-wall-bound cholesterol oxidase activity. Addition of cholesterol induced a marked increase in the synthesis of this enzyme, which reached a maximum within 6 days and was subsequently followed by the appearance of extracellular cholesterol oxidase in the culture broth. Significant levels of induction were only achieved when cholesterol emulsified with Tween 80. The presence of chloramphenicol at the time of induction completely prevented the emergence of both enzymatic forms, suggesting the requirement of de novo protein synthesis. Upon transfer of cholesterol-growing cultures to fresh medium lacking cholesterol, the extracellular cholesterol oxidase was quickly erased, while the activity of the particulate enzyme decreased sharply. The electrophoretic pattern on native Western blotting as well as on sodium dodecyl sulphate/polyacrylamide gels, together with kinetic data, strongly support the idea that the particulate and extracellular cholesterol oxidases are two different forms of the same enzyme with an estimated molecular mass of 55 kDa. Received: 26 September 1996 / Received revision: 30 December 1996 / Accepted: 4 January 1997     

Rhodococcus erythropolis 手性醇脱氢酶的克隆表达及其性质

【目的】探讨红串红球菌中一种醇脱氢酶的性质及其对酮酯类及酮类底物的催化能力。【方法】从红串红球菌(Rhodococcus erythropolis ATCC 4277)中获取一段长度为1047 bp的醇脱氢酶(adh)基因,插入载体pET-22b(+)后,在大肠杆菌中进行重组表达。15℃的低温下用自诱导培养基诱导24 h,以苯乙酮为底物测定醇脱氢酶酶活。【结果】测得该诱导条件下重组菌体细胞破碎上清中醇脱氢酶酶活力为2.6 U/mg。经温度、pH耐受性等分析,发现该酶最适pH在6.0-6.5之间,耐受温度可以达到60℃,并且在该温度下保持5 h后,酶活也能保留80%。对于β酮酯类底物的催化反应,以对乙酰乙酸乙酯的催化能力最高。用4-氯乙酰乙酸乙酯(COBE)为底物进行全细胞水相催化反应,经手性液相色谱分析,发现在催化产物以R型4-氯-3羟基丁酸乙酯(CHBE)为主。【结论】该酶在酮酯类的底物转化方面有良好的开发潜力及应用前景。     

Biochemical identification and biophysical characterization of a channel-forming protein from Rhodococcus erythropolis

Organic solvent extracts of whole cells of the gram-positive bacterium Rhodococcus erythropolis contain a channel-forming protein. It was identified by lipid bilayer experiments and purified to homogeneity by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The pure protein had a rather low molecular mass of about 8.4 kDa, as judged by SDS-PAGE. SDS-resistant oligomers with a molecular mass of 67 kDa were also observed, suggesting that the channel is formed by a protein oligomer. The monomer was subjected to partial protein sequencing, and 45 amino acids were resolved. According to the partial sequence, the sequence has no significant homology to known protein sequences. To check whether the channel was indeed localized in the cell wall, the cell wall fraction was separated from the cytoplasmic membrane by sucrose step gradient centrifugation. The highest channel-forming activity was found in the cell wall fraction. The purified protein formed large ion-permeable channels in lipid bilayer membranes with a single-channel conductance of 6.0 nS in 1 M KCl. Zero-current membrane potential measurements with different salts suggested that the channel of R. erythropolis was highly cation selective because of negative charges localized at the channel mouth. The correction of single-channel conductance data for negatively charged point charges and the Renkin correction factor suggested that the diameter of the cell wall channel is about 2.0 nm. The channel-forming properties of the cell wall channel of R. erythropolis were compared with those of other members of the mycolata. These channels have common features because they form large, water-filled channels that contain net point charges.     

Identification and characterization of a transmissible linear plasmid from Rhodococcus erythropolis BD2 that encodes isopropylbenzene and trichloroethene catabolism.

Rhodococcus erythropolis BD2, which is able to utilize isopropylbenzene as a sole carbon and energy source, was shown to contain a conjugative linear plasmid, pBD2. The estimated size of pBD2 is 208 to 212 kb. Linear plasmid-deficient strains had lost both the isopropylbenzene degradation and trichloroethene degradation characteristics, as well as the arsenite resistance and mercury resistance phenotypes. Reintroduction of pBD2 restored all four characteristics. Conjugational transfer of pBD2 to a plasmidless mutant of strain BD2 and other R. erythropolis strains occurred at frequencies between 3.5 x 10(-5) and 2.6 x 10(-3) transconjugants per recipient. R. erythropolis BD2 degrades isopropylbenzene via 3-isopropylcatechol and 2-hydroxy-6-oxo-7-methylocta-2,4-dienoate. Both isopropylbenzene-oxidizing and meta-cleavage activities were shown to correspond with the presence of pBD2. Southern hybridizations with DNA encoding the toluene dioxygenase structural genes (todC1C2BA) from Pseudomonas putida F1 revealed homology to linear plasmid DNA. These results indicate that the isopropylbenzene degradation pathway encoded by linear plasmid pBD2 is initiated by an isopropylbenzene dioxygenase analogous to toluene dioxygenase.     

Isolation and Characterization of a Rolling-Circle-Type Plasmid from Rhodococcus erythropolis and Application of the Plasmid to Multiple-Recombinant-Protein Expression

We isolated, sequenced, and characterized the cryptic plasmid pRE8424 from Rhodococcus erythropolis DSM8424. Plasmid pRE8424 is a 5,987-bp circular plasmid; it carries six open reading frames and also contains cis-acting elements, specifically a single-stranded origin and a double-stranded origin, which are characteristic of rolling-circle-replication plasmids. Experiments with pRE8424 derivatives carrying a mutated single-stranded origin sequence showed that single-stranded DNA intermediates accumulated in the cells because of inefficient conversion from single-stranded DNA to double-stranded DNA. This result indicates that pRE8424 belongs to the pIJ101/pJV1 family of rolling-circle-replication plasmids. Expression vectors that are functional in several Rhodococcus species were constructed by use of the replication origin from pRE8424. We previously reported a cryptic plasmid, pRE2895, from R. erythropolis, which may replicate by a θ-type mechanism, like ColE2 plasmids. The new expression vectors originating from pRE8424 were compatible with those derived from pRE2895. Coexpression experiments with these compatible expression vectors indicated that the plasmids are suitable for the simultaneous expression of multiple recombinant proteins.     

Isolation and Characterization of Carbendazim-degrading Rhodococcus erythropolis djl-11

Carbendazim (methyl 1H-benzimidazol-2-yl carbamate) is one of the most widely used fungicides in agriculture worldwide, but has been reported to have adverse effects on animal health and ecosystem function. A highly efficient carbendazim-degrading bacterium (strain dj1-11) was isolated from carbendazim-contaminated soil samples via enrichment culture. Strain dj1-11 was identified as Rhodococcus erythropolis based on morphological, physiological and biochemical characters, including sequence analysis of the 16S rRNA gene. In vitro degradation of carbendazim (1000 mg·L⁻¹) by dj1-11 in minimal salts medium (MSM) was highly efficient, and with an average degradation rate of 333.33 mg·L⁻¹·d⁻¹ at 28°C. The optimal temperature range for carbendazim degradation by dj1-11 in MSM was 25–30°C. Whilst strain dj1-11 was capable of metabolizing cabendazim as the sole source of carbon and nitrogen, degradation was significantly (P<0.05) increased by addition of 12.5 mM NH₄NO₃. Changes in MSM pH (4–9), substitution of NH₄NO₃ with organic substrates as N and C sources or replacing Mg²⁺ with Mn²⁺, Zn²⁺ or Fe²⁺ did not significantly affect carbendazim degradation by dj1-11. During the degradation process, liquid chromatography-mass spectrometry (LC-MS) detected the metabolites 2-aminobenzimidazole and 2-hydroxybenzimidazole. A putative carbendazim-hydrolyzing esterase gene was cloned from chromosomal DNA of djl-11 and showed 99% sequence homology to the mheI carbendazim-hydrolyzing esterase gene from Nocardioides sp. SG-4G.     

Construction of Rhodococcus expression vectors and expression of the aminoalcohol dehydrogenase gene in Rhodococcus erythropolis

NADP⁺-dependent aminoalcohol dehydrogenase (AADH) of Rhodococcus erythropolis MAK154 produces double chiral aminoalcohols, which are used as pharmaceuticals. However, the genetic manipulation of Rhodococcus strains to increase their production of such industrially important enzymes is not well studied. Therefore, I aimed to construct Rhodococcus expression vectors, derived from the Rhodococcus–Escherichia coli shuttle vector pRET1102, to express aadh. The plasmid pRET1102 could be transformed into many actinomycete strains, including R. erythropolis. The transformation ef?ciency for a species closely related to R. erythropolis was higher than that for other actinomycete strains. Promoters of various strengths, hsp, 1200rep, and TRR, were obtained from Gram-positive bacteria. The activity of TRR was stronger than that of hsp and 1200rep. The aadh-expressing plasmid pRET1172 with TRR could be transformed into many actinomycete strains to increase their AADH production. The Rhodococcus expression vector, pRET11100, constructed by removing aadh from the pRET1172 plasmid may be useful for bioconversion.     

Sequence and structure analysis of cryptic plasmid pN30 from oil-oxidizing strain Rhodococcus erythropolis 30

Nucleotide sequence of cryptic plasmid pN30 from a Rhodococcus erythropolis 30 soil isolate was determined. Plasmid DNA consists of 5403 nucleotide pairs and contains about 62% GC pairs, which is typical of Rhodococcus DNA. No significant homology was determined between the pN30 DNA sequence and those of known plasmids. Computer-aided analysis of pN30 sequence revealed open reading frames that encode proteins strongly homologous to replicative proteins encoded by small cryptic plasmids of different actinomycetes.     

Genetic and biochemical characterization of a novel monoterpene epsilon-lactone hydrolase from Rhodococcus erythropolis DCL14

A monoterpene epsilon-lactone hydrolase (MLH) from Rhodococcus erythropolis DCL14, catalyzing the ring opening of lactones which are formed during degradation of several monocyclic monoterpenes, including carvone and menthol, was purified to apparent homogeneity. It is a monomeric enzyme of 31 kDa that is active with (4R)-4-isopropenyl-7-methyl-2-oxo-oxepanone and (6R)-6-isopropenyl-3-methyl-2-oxo-oxepanone, lactones derived from (4R)-dihydrocarvone, and 7-isopropyl-4-methyl-2-oxo-oxepanone, the lactone derived from menthone. Both enantiomers of 4-, 5-, 6-, and 7-methyl-2-oxo-oxepanone were converted at equal rates, suggesting that the enzyme is not stereoselective. Maximal enzyme activity was measured at pH 9.5 and 30 degrees C. Determination of the N-terminal amino acid sequence of purified MLH enabled cloning of the corresponding gene by a combination of PCR and colony screening. The gene, designated mlhB (monoterpene lactone hydrolysis), showed up to 43% similarity to members of the GDXG family of lipolytic enzymes. Sequencing of the adjacent regions revealed two other open reading frames, one encoding a protein with similarity to the short-chain dehydrogenase reductase family and the second encoding a protein with similarity to acyl coenzyme A dehydrogenases. Both enzymes are possibly also involved in the monoterpene degradation pathways of this microorganism.     

Isolation and characterization of a plasmid from Treponema denticola

Agarose gel electrophoresis of whole genomic DNA of the oral spirochaete Treponema denticola has revealed a plasmid-like fraction. Purification and restriction enzyme analysis has confirmed the presence of a 2.6-kb circular plasmid, which has been mapped for restriction sites and cloned into the Escherichia coli plasmid pUC18. Southern blot analysis of genomic T. denticola DNA, using the plasmid as a probe, has shown that the plasmid is present only as an extra-chromosomal element. No plasmid-coded recombinant gene product from a PstI insert in pUC18 has been detected in host cells of E. coli by SDS-PAGE or immunoblotting with polyclonal immune rabbit serum to T. denticola. The discovery of this plasmid may provide a useful tool in the application of new molecular approaches in spirochaetal biology.     

l-Pantoyl lactone dehydrogenase from Rhodococcus erythropolis: genetic analyses and application to the stereospecific oxidation of l-pantoyl lactone

The 1,2-propanediol (1,2-PD) inducible membrane-bound L-pantoyl lactone (L-PL) dehydrogenase (LPLDH) has been isolated from Rhodococcus erythropolis AKU2103 (Kataoka et al. in Eur J Biochem 204:799, 1992). Based on the N-terminal amino acid sequence of LPLDH and the highly conserved amino acid sequence in homology search results, the LPLDH gene (lpldh) was cloned. The gene consists of 1,179 bases and encodes a protein of 392 amino acid residues. The deduced amino acid sequence showed high similarity to the proteins of the FMN-dependent α-hydroxy acid dehydrogenase/oxidase family. The overexpression vector pKLPLDH containing lpldh with its upstream region (1,940 bp) was constructed and introduced into R. erythropolis AKU2103. The recombinant R. erythropolis AKU2103 harboring pKLPLDH showed six times higher LPLDH activity than the wild-type strain. Conversion of L-PL to ketopantoyl lactone was achieved with 92% or 80% conversion yield when the substrate concentration was 0.768 or 1.15 M, respectively. Stereoinversion of L-PL to D-PL was also carried out by using the combination of recombinant R. erythropolis AKU2103 harboring pKLPLDH and ketopantoic acid-reducing Escherichia coli.     

Cloning and heterologous expression of an enantioselective amidase from Rhodococcus erythropolis strain MP50

The gene for an enantioselective amidase was cloned from Rhodococcus erythropolis MP50, which utilizes various aromatic nitriles via a nitrile hydratase/amidase system as nitrogen sources. The gene encoded a protein of 525 amino acids which corresponded to a protein with a molecular mass of 55.5 kDa. The deduced complete amino acid sequence showed homology to other enantioselective amidases from different bacterial genera. The nucleotide sequence approximately 2.5 kb upstream and downstream of the amidase gene was determined, but no indications for a structural coupling of the amidase gene with the genes for a nitrile hydratase were found. The amidase gene was carried by an approximately 40-kb circular plasmid in R. erythropolis MP50. The amidase was heterologously expressed in Escherichia coli and shown to hydrolyze 2-phenylpropionamide, alpha-chlorophenylacetamide, and alpha-methoxyphenylacetamide with high enantioselectivity; mandeloamide and 2-methyl-3-phenylpropionamide were also converted, but only with reduced enantioselectivity. The recombinant E. coli strain which synthesized the amidase gene was shown to grow with organic amides as nitrogen sources. A comparison of the amidase activities observed with whole cells or cell extracts of the recombinant E. coli strain suggested that the transport of the amides into the cells becomes the rate-limiting step for amide hydrolysis in recombinant E. coli strains.     

Cloning of new acylamidase gene from Rhodococcus erythropolis and its expression in Escherichia coli

The gene for new Rhodococcus erythropolis TA37 acylamidase, which possesses unique substrate specificity, has been cloned and expressed in E. coli. Substrates for this enzyme are not only simple amides, such as acetamide and propionamide, but also N-substituted amides, such as 4′-nitroacetanilide. The 1431-bp gene was expressed in E. coli BL21 (DE3) cells on pET16b plasmid under the control of a promoter of the ? 10 gene from the T7 phage. The molecular mass of recombinant acylamidase in E. coli was 55 kDa, which corresponded to that of native acylamidase from Rhodococcus erythropolis TA37. Recombinant acylamidase was able to hydrolize N-substituted amides. A search of a nucleotide database and multiple alignment revealed that acylamidase belonged to the Amidase protein family PF01425, but its nucleotide and amino acid sequences differed significantly from those of the described amidases.     

Isolation and characterization of a linear plasmid from Streptomyces rimosus

Abstract A linear plasmid was isolated from a strain of Streptomyces rimosus . This plasmid was separated by agarose gel electrophoresis and its size, about 43 kb, determined both by this method and by electron microscopy. The cleavage pattern of the linear plasmid with 5 restriction endonucleases is given. A protein, which is removed by proteinase K, is probably associated to this plasmid. By ethidium bromides or acridine orange treatment we obtained mutants which had lost their aerial mycelium and their linear plasmid.     

Cloning, purification and characterization of two components of phenol hydroxylase from Rhodococcus erythropolis UPV-1

Phenol hydroxylase that catalyzes the conversion of phenol to catechol in Rhodococcus erythropolis UPV-1 was identified as a two-component flavin-dependent monooxygenase. The two proteins are encoded by the genes pheA1 and pheA2, located very closely in the genome. The sequenced pheA1 gene was composed of 1,629 bp encoding a protein of 542 amino acids, whereas the pheA2 gene consisted of 570 bp encoding a protein of 189 amino acids. The deduced amino acid sequences of both genes showed high homology with several two-component aromatic hydroxylases. The genes were cloned separately in cells of Escherichia coli M15 as hexahistidine-tagged proteins, and the recombinant proteins His₆PheA1 and His₆PheA2 were purified and its catalytic activity characterized. His₆PheA1 exists as a homotetramer of four identical subunits of 62 kDa that has no phenol hydroxylase activity on its own. His₆PheA2 is a homodimeric flavin reductase, consisting of two identical subunits of 22 kDa, that uses NAD(P)H in order to reduce flavin adenine dinucleotide (FAD), according to a random sequential kinetic mechanism. The reductase activity was strongly inhibited by thiol-blocking reagents. The hydroxylation of phenol in vitro requires the presence of both His₆PheA1 and His₆PheA2 components, in addition to NADH and FAD, but the physical interaction between the proteins is not necessary for the reaction.     

Cloning of amidase gene from Rhodococcus erythropolis and expression by distinct promoters in Bacillus subtilis

Amidase was a crucial enzyme responsible for the conversion of acrylamide to acrylic acid in Rhodococcus erythropolis. Its coding gene ami was amplified by PCR using the genomic DNA of R. erythropolis as template. Subsequently, it was ligated to expression plasmids and transformed in Escherichia coli and Bacillus subtilis. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis revealed that both recombinant E. coli BL21 (DE3) and B. subtilis generated amidase of 56 kDa. The expression mass and enzyme activity suggested that B. subtilis was more suitable as a host when ami gene was under the control of a powerful promoter. To further study the expression effect of different promoters in B. subtilis, five distinct promoters (sacB, amyE, p43, degQ, aprE) and their native signal peptide genes were employed to separately construct five different vectors harboring ami gene. Of the five novel vectors, the amyE promoter along with its native signal peptide gene was most effective. The maximum specific activity of amidase at pH 7.0 and 37 °C was about 8.7 U/mg and the conversion efficiency could approximately reach 90% within 6 h. This result indicated the expression difference of distinct promoters, which provided the basis for the forthcoming research.     

Functional expression of the particulate methane mono-oxygenase gene in recombinant Rhodococcus erythropolis

In order to construct an expression system for the particulate methane mono-oxygenase (pMMO) gene (pmo), the structural gene cluster pmoCAB amplified from Methylosinus trichosporium OB3b was inserted into a shuttle vector pBS305 under the control of a dsz promoter and transformed into Rhodococcus erythropolis LSSE8-1. A stable transformant was successfully obtained using ethane as the sole carbon source. Fluorescence in situ hybridization results showed that the dsz promoter allowed the pmo genes to be transcribed in the recombinant strain. The effects of Cu2+ and Zn2+ concentrations on cell growth and pMMO activity in ethane-containing medium were examined. It was discovered that 7.5 microM Cu2+ and 1.8 microM Zn2+ were suitable to achieve high cell concentration and pMMO activity, but the amount of methanol accumulated during methane oxidation by the recombinant strain was still low.