Efficient expression in <Emphasis Type="Italic">E. coli</Emphasis> of an enantioselective nitrile hydratase from <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

The genes encoding an enantioselective nitrile hydratase (NHase) from Rhodococcus erythropolis AJ270 have been cloned and an active NHase has been produced in Escherichia coli. Maximal activity was found when the genes encoding the α- and β-subunits were transcribed as one unit and the gene encoding the P44k activator protein as a separate ORF on a single replicon. Addition of n-butyric acid and FeSO₄ could improve NHase activity. Coexpression of the GroEL-GroES chaperone proteins increased activity in the absence of P44k protein but had no effect in the presence of P44k. The recombinant enzyme was highly enantioselective in the synthesis of S-(+)-3-benzoyloxy- 4-cyanobutyramide from the prochiral substrate 3-benzoyloxyglutaronitrile.     

<Emphasis Type="Bold">Characterization of nitrile hydratation catalysed by </Emphasis><Emphasis Type="BoldItalic">Nocardia</Emphasis><Emphasis Type="Bold"> sp. 108</Emphasis>

Abstract Nocardia sp. 108 exhibited strong acrylonitrile-hydrating activity and its nitrile hydratase was Co²⁺-dependent. Nocardia sp. 108 was active within a broad pH range from 6.0 to 10.0 at 30°C and thermostable at temperatures below 35°C, but became unstable at temperatures above 45°C. Furthermore, it was found that Nocardia sp. 108 can hydrate indole-3-acetonitrile, p-chlorobenzonitrile, p-hydroxybenzylcyanide, 3,4,5-trimethoxybenzonitrile, p-aminobenzonitrile, 3-cyanopyridine, o-chlorobenzonitrile to the corresponding amides and hence displayed a broad substrate specificity. The temperature and pH optima for these hydrations were 28°C and pH 7.0–7.5, respectively. At the observed concentrations, acrylonitrile was completely converted within 5 min, while 3,4,5-trimethoxybenzonitrile, p-aminobenzonitrile, indole-3-acetonitrile, p-chlorobenzonitrile were approximately 21.71, 8.98, 34.44, 93.10% hydrated. p-Chlorobenzonitrile appeared to be the preferred aromatic nitrile for Nocardia sp. 108.     

Bioconversion of butyronitrile to butyramide using whole cells of <Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> PA-34

Butyramide is an important chemical commodity, which is used for the synthesis of hydroxamic acids and electrorheological fluids and for the preparation of β-amodoorganotin compounds. The nitrile hydratase (Nhase) of Rhodococcus rhodochrous PA-34 catalyzed the conversion of butyronitrile to butyramide. The maximum Nhase activity [18 U/mg dry cell weight (dcw)] of whole cells of R. rhodochrous PA-34 was observed at pH 7.0 with 10% (v/v) butyronitrile and 1 mg cells (dcw)/ml reaction mixture at 10°C. The cells of R. rhodochrous PA-34 retained almost 50% activity when incubated for 1 h in the presence of 85% (v/v) butyronitrile. A yield of 597 g of butyramide (6.8 M) was obtained using 60% (v/v) butyronitrile, 1 g cells (dry weight) in a 1-l batch reaction at 10°C for 6 h.     

Extensive desulfurization of diesel by <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

The resting cells of a new isolate of Rhodococcus erythropolis FSD-2 were used to desulfurize diesel fuels. About 97% of the total sulfur content in the hydrodesulfurized diesel was removed by the two consecutive biodesulfurization (BDS) processes with the majority (∼94%) being removed in the first treatment, resulting in diesel with a sulfur content of 5.7 μg ml⁻¹.     

Potential of <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> as a bioremediation organism

Summary The capability of Rhodococcus erythropolis CCM 2595(ATCC 11048) to utilize phenol, pyrocatechol, resorcinol, p-nitrophenol, p-chlorophenol, hydroquinone and hydroxybenzoate, respectively, or as respective binary mixtures with phenol, was described. This capability was found to depend on the substrate and its initial concentration. Some monoaromatic compounds had a suppressive effect on the strain’s ability to utilize phenol in a binary mixture and easily utilizable monoaromatics were strong inducers of the phenol 2-monooxygenase (EC 1.14.13.7). The capacity of R. erythropolis to colonize a synthetic zeolite was demonstrated and the enhancement of phenol tolerance of biofilms utilizing phenol was observed. The effect of humic acids on phenol killing was described and discussed as well. To allow use of recombinant DNA technology for strain improvement, methods of genetic transfer (transformation and conjugation) in R. erythropolis were established.     

Enhanced production of amidase from <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> MTCC 1526 by medium optimisation using a statistical experimental design

In the present work, statistical experimental methodology was used to enhance the production of amidase from Rhodococcus erythropolis MTCC 1526. R. erythropolis MTCC 1526 was selected through screening of seven strains of Rhodococcus species. The Placket–Burman screening experiments suggested that sorbitol as carbon source, yeast extract and meat peptone as nitrogen sources, and acetamide as amidase inducer are the most influential media components. The concentrations of these four media components were optimised using a face-centred design of response surface methodology (RSM). The optimum medium composition for amidase production was found to contain sorbitol (5 g/L), yeast extract (4 g/L), meat peptone (2.5 g/L), and acetamide (12.25 mM). Amidase activities before and after optimisation were 157.85 units/g dry cells and 1,086.57 units/g dry cells, respectively. Thus, use of RSM increased production of amidase from R. erythropolis MTCC 1526 by 6.88-fold.     

The <Emphasis Type="Italic">dsdA</Emphasis> gene from <Emphasis Type="Italic">Escherichia coli</Emphasis> provides a novel selectable marker for plant transformation

Plants are sensitive to D-serine, but functional expression of the dsdA gene, encoding D-serine ammonia lyase, from Escherichia coli can alleviate this toxicity. Plants, in contrast to many other organisms, lack the common pathway for oxidative deamination of D-amino acids. This difference in metabolism has major consequences for plant responses to D-amino acids, since several D-amino acids are toxic to plants even at relatively low concentrations. Therefore, introducing an enzyme specific for a phytotoxic D-amino acid should generate a selectable characteristic that can be screened. Here we present the use of the dsdA gene as a selectable marker for transformation of Arabidopsis. D-serine ammonia lyase catalyses the deamination of D-serine into pyruvate, water and ammonium. dsdA transgenic seedlings can be clearly distinguished from wild type, having an unambiguous phenotype immediately following germination when selected on D-serine containing medium. The dsdA marker allows flexibility in application of the selective agent: it can be applied in sterile plates, in foliar sprays or in liquid culture. Selection with D-serine resistance was compared with selection based on kanamycin resistance, and was found to generate similar transformation frequencies but also to be more unambiguous, more rapid and more versatile with respect to the way the selective agent can be supplied.     

Gene cloning system for sulfonamide-mineralizing <Emphasis Type="Italic">Microbacterium</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> strain BR1

The wide application of sulfonamide (SA) antibiotics in human and veterinary medicine contributes to the accumulation of these antibiotics in the environment and the corresponding onset of antibiotic resistance among bacteria. Microbacterium sp. BR1 is capable of mineralizing sulfamethoxazole and other SAs via a novel mechanism. The genetic basis of SA elimination by BR1 remains unknown. Development of an efficient plasmid transfer protocol for Microbacterium sp. BR1 is highly desirable, as it would open the door to genetic analysis and manipulation of its genome. Here we report that intergeneric Escherichia coli–Microbacterium spp. BR1 conjugation is an efficient way to introduce various plasmids into BR1. The generated transconjugants were stable in the presence of antibiotics and the plasmids showed no signs of rearrangements. Nevertheless, the plasmids were rapidly lost in the absence of selection. We also show that the cumate-inducible beta-glucuronidase reporter gene functions in BR1 and is strictly regulated. Our results set the working ground for further genetic manipulations of BR1, such as the overexpression of sulfonamide degradation genes or the selection of strong microbacterial promoters.     

Enhanced biodesulfurization by expression of dibenzothiophene uptake genes in <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis>

The transfer of dibenzothiophene (DBT) and its derivatives into cells is a critical step for biodesulfurization. The desulfurization reactions of resting cells and cell lysate were studied, which showed that the desulfurization rate of DBT, especially 4, 6-dimethyldibenzothiophene (4, 6-DMDBT) in Rhodococcus erythropolis LSSE8-1 was seriously affected by the transfer into cells. The inhibited effect of NaN₃ on desulfurization reactions was studied, which confirmed that the transfer of DBT into cells was an active transport in R. erythropolis LSSE8-1. The uptake-genes of DBT and its derivatives (HcuABC) of Pseudomonas delafieldii R-8 were introduced into the specific desulfurization bacterium, R. erythropolis LSSE8-1. Compared with the wild type, the strains bearing HcuABC genes showed a higher desulfurization activity. The desulfurization ratio of DBT showed a 19% increase, and 13% increase of 4, 6-DMDBT.     

Random segment deletion based on IS<Emphasis Type="Italic">31831</Emphasis> and Cre/<Emphasis Type="Italic">loxP</Emphasis> excision system in <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

A simple and random genome deletion method combining insertion sequence (IS) element IS31831 and the Cre/loxP excision system generated 42 Corynebacterium glutamicum mutants (0.2–186 kb). A total of 393.6 kb (11.9% of C. glutamicum R genome) coding for 331 genes was confirmed to be nonessential under standard laboratory conditions. The deletion strains, generated using only two vectors, varied not only in their lengths but also the location of the deletion along the C. glutamicum R genome. By comparing and analyzing the generated deletion strains, identification of nonessential genes, the roles of genes of hitherto unknown function, and gene–gene interactions can be easily and efficiently determined. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

Overexpression of a nitrile hydratase from <Emphasis Type="Italic">Klebsiella oxytoca</Emphasis> KCTC 1686 in <Emphasis Type="Italic">Escherichia coli</Emphasis> and its biochemical characterization

The putative Co-type nitrile hydratase (NHaseK, consisting of α- and β-subunits) genes and the putative activator (17K) gene adjacent to the β subunit region were cloned from Klebsiella oxytoca KCTC 1686. 17K is essential for the functional expression of recombinant NHaseK in Escherichia coli; however, the expression level of 17K was very low when the 17K gene and NHaseK structural genes were expressed as a gene cluster in E. coli BL21(DE3). To improve the 17K expression level and NHaseK activity, the expression cassette was redesigned by placing the 17K gene and NHaseK structural genes under the control of different promoters in the pETDuet-1 expression vector, co-expressing the 17K gene with the gene cluster in a double plasmid or a single plasmid with a double promoter, and introducing an efficient Shine- Dalgarno sequence 5' to the17K gene. The specific activity of NHaseK was improved when 17K was co-expressed with the gene cluster, whereas the production of NHaseK protein decreased. The maximum activity was achieved when an efficient Shine-Dalgarno sequence was introduced 5' to the 17K gene: the expression level of 17K was significantly improved and the expression level of NHaseK did not decrease significantly. The maximum activity was about 63,480 ± 1915.6 U/L broth towards 3-Cyanopyridine. Recombinant NHaseK could hydrolyze a wide range of aliphatic, aromatic, and heterocyclic nitriles, and convert racemic nitriles to the corresponding S-amides, with E values ranging from 9 to 17. The enzyme had a temperature optimum of 35°C and exhibited remarkably stability below 35°C.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

<Emphasis Type="Italic">Drosophila melanogaster</Emphasis> gene <Emphasis Type="Italic">Merlin</Emphasis> interacts with the clathrin adaptor protein gene <Emphasis Type="Italic">lap</Emphasis>

The protein Merlin is involved in the regulation of cell proliferation and differentiation in the eyes and wings of Drosophila and is a homolog of the human protein encoded by the Neurofibromatosis 2 (NF2) gene whose mutations cause auricular nerve tumors. Recent studies show that Merlin and Expanded cooperatively regulate the recycling of membrane receptors, such as the epidermal growth factor receptor (EGFR). By performing a search for potential genetic interactions between Merlin (Mer) and the genes important for vesicular trafficking, we found that ectopic expression in the wing pouch of the clathrin adapter protein Lap involved in clathrin-mediated receptor endocytosis resulted in the formation of extra vein materials. On the one hand, coexpression of wild-type Merlin and lap in the wing pouch restored normal venation, while overexpression of a dominant-negative mutant Mer ^DBB together with lap enhanced ectopic vein formation. Using various constructs with Merlin truncated copies, we showed the C-terminal portion of the Merlin protein to be responsible for the Merlin-lap genetic interaction. Furthermore, we showed that the Merlin and Lap proteins colocalized at the cortex of the wing imaginal disc cells.     

Discovery and characterization of a putrescine oxidase from <Emphasis Type="Italic">Rhodococcus erythropolis</Emphasis> NCIMB 11540

A gene encoding a putrescine oxidase (PuO_Rh, EC 1.4.3.10) was identified from the genome of Rhodococcus erythropolis NCIMB 11540. The gene was cloned in the pBAD vector and overexpressed at high levels in Escherichia coli. The purified enzyme was shown to be a soluble dimeric flavoprotein consisting of subunits of 50 kDa and contains non-covalently bound flavin adenine dinucleotide as a cofactor. From all substrates, the highest catalytic efficiency was found with putrescine (K _M = 8.2 μM, k _cat = 26 s⁻¹). PuO_Rh accepts longer polyamines, while short diamines and monoamines strongly inhibit activity. PuO_Rh is a reasonably thermostable enzyme with t _1/2 at 50°C of 2 h. Based on the crystal structure of human monoamine oxidase B, we constructed a model structure of PuO_Rh, which hinted to a crucial role of Glu324 for substrate binding. Mutation of this residue resulted in a drastic drop (five orders of magnitude) in catalytic efficiency. Interestingly, the mutant enzyme showed activity with monoamines, which are not accepted by wt-PuO_Rh. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biotransformation of polychlorinated dibenzo-<Emphasis Type="Italic">p</Emphasis>-dioxin by <Emphasis Type="Italic">Coprinellus</Emphasis> species

A degradation experiment on dibenzo-p-dioxin (DD) and 2,7-dichlorodibenzo-p-dioxin (2,7-DCDD) was carried out using basidiomycetous fungi belonging to the genera Coprinus, Coprinellus, and Coprinopsis. Some species showed a high rate of decrease in DD for the 2-week test period. Among them, Coprinellus disseminatus showed the highest ability to decrease the DD level, close to 100% by the end of 2 weeks. Further examination showed that Coprinellus disseminatus and Coprinellus micaceus, belonging to the genus Coprinellus, were able to metabolize 2,7-DCDD to a monohydroxylated compound, probably mediated by the P450 system. The metabolism of chlorinated DD by fungi capable of living in soil conditions is reported here for the first time.