Enzymatic bioremediation of organophosphorus insecticides by recombinant organophosphorous hydrolase

The gene for organophosphorous hydrolase (OPH) (GenBank accession no. M20392) was chemically synthesised with a codon bias toward E. coli, followed by its cloning and heterologous over-expression in E. coli under induction with 0.1 M isopropyl-d-thiogalactopyranoside (IPTG). The protein was localised in the membrane fraction and no amount of the expressed protein was soluble, thus hindering its purification and further downstream utility. The expressed enzyme was solubilised by mild treatment with ionic detergent [sodium dodecyl sulphate (SDS 1.0% (w/v))]. An innovative step of incubation at 4 °C was used to precipitate SDS, resulting in catalytically active OPH in the supernatant and detergent at the bottom. This solubilised, SDS-free, recombinant OPH was able to detoxify parathion and methylparathion ranging between 10-80% and 3.6-45% in enzyme reaction cycles after immobilization on Ca-alginate and agar-agar, respectively.     

Versatile signal peptide of Flavobacterium‐originated organophosphorus hydrolase for efficient periplasmic translocation of heterologous proteins in Escherichia coli

Organophosphorus hydrolase (OPH) from Flavobacterium species is a membrane‐associated homodimeric metalloenzyme and has its own signal peptide in its N‐terminus. We found that OPH was translocated into the periplasmic space when the original signal peptide‐containing OPH was expressed in recombinant Escherichia coli even though its translocation efficiency was relatively low. To investigate the usability of this OPH signal peptide for periplasmic expression of heterologous proteins in an E. coli system, we employed green fluorescent protein (GFP) as a cytoplasmic folding reporter and alkaline phosphatase (ALP) as a periplasmic folding reporter. We found that the OPH signal peptide was able to use both twin‐arginine translocation (Tat) and general secretory (Sec) machineries by switching translocation pathways according to the nature of target proteins in E. coli. These results might be due to the lack of Sec‐avoidance sequence in the c‐region and a moderate hydrophobicity of the OPH signal peptide. Interestingly, the OPH signal peptide considerably enhanced the translocation efficiencies for both GFP and ALP compared with commonly used TorA and PelB signal peptides that have Tat and Sec pathway dependences, respectively. Therefore, this OPH signal peptide could be successfully used in recombinant E. coli system for efficient periplasmic production of target protein regardless of the subcellular localization where functional folding of the protein occurs. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:848–854, 2016     

Expression of recombinant organophosphorus hydrolase in the original producer of the enzyme,Sphingobium fuliginis ATCC 27551

The plasmid encoding His-tagged organophosphorus hydrolase (OPH) cloned from Sphingobium fuliginis was modified to be transferred back to this bacterium. The replication function of S. amiense plasmid was inserted at downstream of OPH gene, and S. fuliginis was transformed with this plasmid. The transformant produced larger amount of active OPH with His-tag than E. coli.     

Enhanced detoxification of organophosphates using recombinant Escherichia coli with co-expression of organophosphorus hydrolase and bacterial hemoglobin

A significantly improved, recombinant Escherichia coli has been developed to degrade the toxic organophosphorus compound, Paraoxon, to non-toxic materials by co-expression of organophosphorus hydrolase (OPH) under trc promoter and Vitreoscilla hemoglobin (VHb) under O₂dependent nar promoter. VHb-expressing whole cells had significant enhancement of OPH activity (48%, 18.7 vs. 27.8 unit l^–1) and bioconversion efficiency V _max/K _m (44%, 0.14 vs. 0.2 min^–1) compared to VHb-free system.     

A transferable heterogeneous two-hybrid system in <Emphasis Type="Italic">Escherichia coli</Emphasis> based on polyhydroxyalkanoates synthesis regulatory protein PhaR

Background  

Polyhydroxyalkanoate (PHA) synthesis regulatory protein PhaR contains a DNA binding domain (DBD) and a PHA granule binding domain (GBD), it anchors to the promoter region of PHA granule-associated protein (PhaP) to repress phaP expression. However, PhaR will bind to PHB granules and be released from phaP promoter region when PHA granules are formed in vivo, initiating expression of phaP gene. Based on this regulatory mechanism, a bacterial two-hybrid system was developed: PhaR was separated into two parts: DBD was used to fuse with the bait, GBD with the prey, and phaP was replaced by a reporter gene lacZ. However, GBD protein expressed in vivo formed inclusion bodies. Thus, PhaP with strong binding ability to PHB granules was employed to replace GBD.     

A System for Dual Protein Expression in Pichia pastoris and Escherichia coli

We have constructed a novel Pichia pastoris/Escherichia coli dual expression vector for the production of recombinant proteins in both host systems. In this vector, an E. coli T7 promoter region, including the ribosome binding site from the phage T7 major capsid protein for efficient translation is placed downstream from the yeast alcohol oxidase promoter (AOX). For detection and purification of the target protein, the vector contains an amino-terminal oligohistidine domain (His6) followed by the hemaglutinine epitope (HA) adjacent to the cloning sites. A P. pastoris autonomous replicating sequence (PARS) was integrated enabling simple propagation and recovery of plasmids from yeast and bacteria (1). In the present study, the expression of human proteins in P. pastoris and E. coli was compared using this single expression vector. For this purpose we have subcloned a cDNA expression library deriving from human fetal brain (2) into our dual expression T7 vector and investigated 96 randomly picked clones. After sequencing, 29 clones in the correct reading frame have been identified, their plasmids isolated and shuttled from yeast to bacteria. All proteins were expressed soluble in P. pastoris, whereas in E. coli only 31% could be purified under native conditions. Our data indicates that this dual expression vector allows the economic expression and purification of proteins in different hosts without subcloning.     

Expression of four <Emphasis Type="Italic">pha</Emphasis> genes involved in poly-β-hydroxybutyrate production and accumulation in <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> FJ1

A cluster of genes encoding polyhydroxybutyrate (PHB) depolymerase (phaZ), PHB synthase (phaC), phasin (phaP), and the regulator protein (phaR) was previously identified in Rhodobacter sphaeroides FJ1 (R. sphaeroides FJ1). In this study, we investigated the role of the PhaR protein on the expression of the pha genes. Immunoblot analysis revealed that the expressions of phaP, phaZ and phaR genes in wild-type cells of R. sphaeroides FJ1 are repressed during the active growth phase, with the exception of phaC. A phaR deletion mutant of R. sphaeroides FJ1 was constructed, and the basal level of phaP and phaZ expression in this mutant was markedly increased. Electrophoretic mobility shift assays demonstrated that PhaR binds to the promoter region of phaP as well as those of phaR and phaZ. These results suggest that the PhaR protein is a repressor of phaP, phaR, and phaZ genes in R. sphaeroides FJ1.     

Expression and fermentation optimization of oxidized polyvinyl alcohol hydrolase in E. coli

Oxidized polyvinyl alcohol (PVA) hydrolase (OPH) is a key enzyme in the degradation of PVA, suggesting that OPH has a great potential for application in textile desizing processes. In this study, the OPH gene from Sphingopyxis sp. 113P3 was modified, by artificial synthesis, for overexpression in Escherichia coli. The OPH gene, lacking the sequence encoding the original signal peptide, was inserted into pET-20b (+) expression vector, which was then used to transform E. coli BL21 (DE3). OPH expression was detected in culture medium in which the transformed E. coli BL21 (DE3) was grown. Nutritional and environmental conditions were investigated for improved production of OPH protein by the recombinant strain. The highest OPH activity measured was 47.54 U/mL and was reached after 84 h under optimal fermentation conditions; this level is 2.64-fold higher that obtained under sub-optimal conditions. The productivity of recombinant OPH reached 565.95 U/L/h. The effect of glycine on the secretion of recombinant OPH was examined by adding glycine to the culture medium to a final concentration of 200 mM. This concentration of glycine reduced the fermentation time by 24 h and increased the productivity of recombinant OPH to 733.17 U/L/h. Our results suggest that the recombinant strain reported here has great potential for use in industrial applications.     

Gene silencing in Escherichia coli using antisense RNAs expressed from doxycycline‐inducible vectors

Here, we report on the construction of doxycycline (tetracycline analogue)‐inducible vectors that express antisense RNAs in Escherichia coli. Using these vectors, the expression of genes of interest can be silenced conditionally. The expression of antisense RNAs from the vectors was more tightly regulated than the previously constructed isopropyl‐β‐D‐galactopyranoside‐inducible vectors. Furthermore, expression levels of antisense RNAs were enhanced by combining the doxycycline‐inducible promoter with the T7 promoter‐T7 RNA polymerase system; the T7 RNA polymerase gene, under control of the doxycycline‐inducible promoter, was integrated into the lacZ locus of the genome without leaving any antibiotic marker. These vectors are useful for investigating gene functions or altering cell phenotypes for biotechnological and industrial applications.

Significance and Impact of the Study

A gene silencing method using antisense RNAs in Escherichia coli is described, which facilitates the investigation of bacterial gene function. In particular, the method is suitable for comprehensive analyses or phenotypic analyses of genes essential for growth. Here, we describe expansion of vector variations for expressing antisense RNAs, allowing choice of a vector appropriate for the target genes or experimental purpose.     

Gene Cloning,Expression, and Characterization of the <Emphasis Type="Italic">Geobacillus Thermoleovorans</Emphasis> CCR11 Thermoalkaliphilic Lipase

The gene for a Geobacillus thermoleovorans CCR11 thermostable lipase was recovered by PCR and cloned. Four genetic constructions were designed and successfully expressed in E. coli: (i) the lipase structural gene (lipCCR11) in the PinPoint Xa vector; (ii) the lipase structural gene (lipACCR11) in the pET-28a(+) vector; (iii) the lipase structural gene minus the signal peptide (lipMatCCR11) in the pET-3b vector; and (iv) the lipase structural gene plus its own promoter (lipProCCR11) in the pGEM-T cloning vector. The lipase gene sequence analysis showed an open reading frame of 1,212 nucleotides coding for a mature lipase of 382 residues (40 kDa) plus a 22 residues signal peptide. Expression under T7 and T7lac promoter resulted in a 40- and 36-fold increase in lipolytic activity with respect to the original strain lipase. All recombinant lipases showed an optimal activity at pH 9.0, but variations were found in the temperature for maximum activity and the substrate specificity among them and when compared with the parental strain lipase, especially in the recombinant lipases that contained fusion tags. Therefore, it is important to find the appropriate expression system able to attain a high concentration of the recombinant lipase without compromising the proper folding of the protein.     

Polychlorinated biphenyl (PCB) degradation and persistence of a<Emphasis Type="Italic"> gfp</Emphasis>-marked<Emphasis Type="Italic"> Ralstonia eutropha</Emphasis> H850 in PCB-contaminated soil

Ralstonia eutropha H850 was labelled chromosomally with a gfp marker gene encoding for the green fluorescent protein, and designated R. eutropha H850g13. Visual observation of green fluorescent cells under an epifluorescence microscope, and PCR amplification products, confirmed that the bacterium was labelled with gfp. Southern blot hybridization products further confirmed the gfp was chromosomally labelled. Using resting cell assays, it was determined that insertion of the gfp gene decreased the microorganisms' ability to degrade biphenyl compared to the parent strain. However, this marker facilitated the identification and monitoring of R. eutropha H850g13 survival in soil microcosm experiments. Survival and polychlorinated biphenyl degradation by R. eutropha H850g13 was analysed in soil microcosms spiked with 2,2,5,5-tetrachlorobiphenyl (TeCB). R. eutropha H850g13 was detected by viable plate counts and most-probable-number/PCR after 102 days in TeCB-contaminated soil microcosms, and was likely outcompeted by indigenous soil microorganisms in microcosms amended with oil and Daramend (an organic amendment, ). R. eutropha H850g13 did not degrade TeCB in any of the soil microcosms. This research confirmed that gfp was useful as a marker to distinguish R. eutropha H850g13 from indigenous soil microorganisms over a 102 day period and that, under the experimental conditions used, R. eutropha H850g13 did not degrade TeCB.     

Production of 3-hydroxypropionate homopolymer and poly(3-hydroxypropionate-co-4-hydroxybutyrate) copolymer by recombinant Escherichia coli

Conversion of 3-hydroxypropionate (3HP) from 1,3-propanediol (PDO) was improved by expressing dehydratase gene (dhaT) and aldehyde dehydrogenase gene (aldD) of Pseudomonas putida KT2442 under the promoter of phaCAB operon from Ralstonia eutropha H16. Expression of these genes in Aeromonas hydrophila 4AK4 produced up to 21 g/L 3HP in a fermentation process. To synthesize homopolymer poly(3-hydroxypropionate) (P3HP), and copolymer poly(3-hydroxypropionate-co-3-hydroxybutyrate) (P3HP4HB), dhaT and aldD were expressed in E. coli together with the phaC1 gene encoding polyhydroxyalkanoate (PHA) synthase gene of Ralstonia eutropha, and pcs' gene encoding the ACS domain of the tri-functional propionyl-CoA ligase (PCS) of Chloroflexus aurantiacus. Up to 92 wt% P3HP and 42 wt% P3HP4HB were produced by the recombinant Escherichia coli grown on PDO and a mixture of PDO+1,4-butanediol (BD), respectively.     

Improvement in organophosphorus hydrolase activity of cell surface-engineered yeast strain using Flo1p anchor system

Organophosphorus hydrolase (OPH) hydrolyzes organophosphorus esters. We constructed the yeast-displayed OPH using Flo1p anchor system. In this system, the N-terminal region of the protein was fused to Flo1p and the fusion protein was displayed on the cell surface. Hydrolytic reactions with paraoxon were carried out during 24 h of incubation of OPH-displaying cells at 30°C. p-Nitrophenol produced in the reaction mixture was detected by HPLC. The strain with highest activity showed 8-fold greater OPH activity compared with cells engineered using glycosylphosphatidylinositol anchor system, and showed 20-fold greater activity than Escherichia coli using the ice nucleation protein anchor system. These results indicate that Flo1p anchor system is suitable for display of OPH in the cell surface-expression systems.     

High-level Expression of a Synthetic Gene Encoding a Sweet Protein,Monellin, in <Emphasis Type="Italic">Escherichia coli</Emphasis>

The expression of a synthetic gene encoding monellin, a sweet protein, in E. coli under the control of T7 promoter from phage is described. The single-chain monellin gene was designed based on the biased codons of E. coli so as to optimize its expression. Monellin was produced and accounted for 45% of total soluble proteins. It was purified to yield 43 mg protein per g dry cell wt. The purity of the recombinant protein was confirmed by SDS-PAGE. Revisions requested 13 April 2005 and 26 May 2005; Revisions received 19 May 2005 and 30 August 2005     

Development of HIV-1 protease expression methods using the T7 phage promoter system

New and simple human immunodeficiency virus type 1 (HIV-1) protease expression methods in Escherichia coli were developed using the T7 phage promoter system. In order to suppress leaky HIV-1 protease expression under the control of the T7 polymerase, two new methods were tested. One involved the introduction of supplementary T7 promoter regions into host cells [E. coli BL-21(DE3)] containing the HIV-1 protease gene under the control of the T7 promoter. It was expected that the supplementary T7 promoter regions would compete with the HIV-1 protease expression vector for the T7 polymerase binding. The other involved the infection of late-log-phase cultures of E.␣coli JM109 harboring the same HIV-1 protease expression vector with the M13 phage expressing T7 polymerase. Both methods were effective, and transformants with the mature HIV-1 protease expression vector showed ten times higher HIV-1 protease activity than activities obtained with the autoprocessing vector. The expression systems described here are convenient and are also easily applicable for the expression of other proteins toxic for E. coli. Received: 5 September 1996 / Received last revision: 1 November 1996 / Accepted: 15 November 1996     

A novel T7 RNA polymerase dependent expression system for high-level protein production in the phototrophic bacterium Rhodobacter capsulatus

The functional expression of heterologous genes using standard bacterial expression hosts such as Escherichia coli is often limited, e.g. by incorrect folding, assembly or targeting of recombinant proteins. Consequently, alternative bacterial expression systems have to be developed to provide novel strategies for protein synthesis exceeding the repertoire of the standard expression host E. coli.Here, we report on the construction of a novel expression system that combines the high processivity of T7 RNA polymerase with the unique physiological properties of the facultative photosynthetic bacterium Rhodobacter capsulatus. This system basically consists of a recombinant R. capsulatus T7 expression strain (R. capsulatus B10S-T7) harboring the respective polymerase gene under control of a fructose inducible promoter. In addition, a set of different broad-host-range vectors (pRho) was constructed allowing T7 RNA polymerase dependent and independent target gene expression in R. capsulatus and other Gram-negative bacteria. The expression efficiency of the novel system was studied in R. capsulatus and E. coli using the yellow fluorescent protein (YFP) as model protein. Expression levels were comparable in both expression hosts and yielded up to 80 mg/l YFP in phototrophically grown R. capsulatus cultures. This result clearly indicates that the novel R. capsulatus-based expression system is well suited for the high-level expression of soluble proteins.     

Morphology and Antigenic Properties of Recombinant Analogs of the Marburg Virus Nucleoprotein

The full-length gene for Marburg virus (MV) nucleoprotein (NP) was cloned in prokaryotic pQE32 under the control of the T5 promoter and in eukaryotic pTM1 under the control of the T7 RNA polymerase promoter. Recombinant NP was synthesized in Escherichia coliand in human kidney cell line 293 cotransfected with recombinant vaccinia virus vTF7-3 expressing T7 RNA polymerase. On evidence of electron microscopy with immune detection, recombinant NP formed tubules of two types in E. coliand of a single type in cell line 293. ELISA and immunoblotting with polyclonal and monoclonal antibodies revealed common antigenic determinants in recombinant NP and natural MV NP.     

Purification of the Early Sporulation Gene spo0F Product of Bacillus subtilis

The RsaI fragment (750 base pairs) containing the entire early sporulation gene spo0F of Bacillus subtilis was inserted in the downstream region of the P_R promoter of the expression vector, pEBR-151. The recombinant plasmid thus obtained was introduced into Escherichia coli HB101 and the synthesis of the spo0F gene product was induced by a temperature shift up. After induction for 7 hr, a protein of molecular weight 14,000 (14 K protein) was overproduced to about 9% of the total cellular protein. The 14 K protein was purified to 94% purity by four steps of column chromatography. Deletion analysis and the sequence determination of the NH₂-terminal amino acid residues of the purified 14 K protein confirmed that the 14 K protein is the spo0F gene product.     

Construction of PHB and PHBV multiple-gene vectors driven by an oil palm leaf-specific promoter

One of the targets in oil palm genetic engineering programme is the production of polyhydroxybutyrate (PHB) and polyhydroxybutyrate-co-valerate (PHBV) in the oil palm leaf tissues. Production of PHB requires the use of phbA (β-ketothiolase type A), phbB (acetoacetyl-CoA reductase) and phbC (PHB synthase) genes of Ralstonia eutropha, whereas bktB (β-ketothiolase type B), phbB, phbC genes of R. eutropha and tdcB (threonine dehydratase) gene of Escherichia coli were used for PHBV production. Each of these genes was fused with a transit peptide (Tp) of oil palm acyl-carrier-protein (ACP) gene, driven by an oil palm leaf-specific promoter (LSP1) to genetically engineer the PHB/PHBV pathway to the plastids of the leaf tissues. In total, four transformation vectors, designated pLSP15 (PHB) and pLSP20 (PHBV), and pLSP13 (PHB) and pLSP23 (PHBV), were constructed for transformation in Arabidopsis thaliana and oil palm, respectively. The phosphinothricin acetyltransferase gene (bar) driven by CaMV35S promoter in pLSP15 and pLSP20, and ubiquitin promoter in pLSP13 and pLSP23 were used as the plant selectable markers. Matrix attachment region of tobacco (RB7MAR) was also included in the vectors to stabilize the transgene expression and to minimize silencing due to positional effect. Restriction digestion, PCR amplification and/or sequencing were carried out to ensure sequence integrity and orientation.     

An expression vector system providing plasmid stability and conditional suicide of plasmid-containing cells

Summary A cloning vector system was constructed on the basis of the pBR322 derivative pEG1 by introducing the whole parB locus of plasmid R1 cloned behind the promoter of the alkaline phosphatase gene (phoA) of Escherichia coli. The parB locus in combination with the phoA promoter ensures both (i) plasmid stabilization due to the post-segregational killing of plasmid-free cells during growth and (ii) killing of the cells induced by the potential environmental signal phosphate limitation. This vector, therefore, appears to be a model system for increasing the stability of recombinant plasmids and for decreasing the potential risks in the application of recombinant bacteria in industrial fermentations.Correspondence to: T. Schweder