Parathion Hydrolase Gene from Pseudomonas diminuta MG: Subcloning, Complete Nucleotide Sequence and Expression of the Mature Portion of the Enzyme in Escherichia coli

Serdar, Cüneyt M.; Murdock, Douglas C.; Rohde, Michael F.

doi:10.1038/nbt1189-1151

Cited by 58 publications

(64 citation statements)

References 14 publications

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“…It should be noted that the P. diminuta opd sequence in the DNA sequence databases (accession number M20392) has been reported to be incorrect (17). The corrected sequence, determined by Serdar et al (26), is identical to that for the Flavobacterium pPDL2 sequence, but it does not appear to have been lodged in the databases.…”

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confidence: 99%

Transposon-Like Organization of the Plasmid-Borne Organophosphate Degradation ( opd ) Gene Cluster Found in Flavobacterium sp

Siddavattam

Syed

Manavathi

et al. 2003

Appl Environ Microbiol

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Several bacterial strains that can use organophosphate pesticides as a source of carbon have been isolated from soil samples collected from diverse geographical regions. All these organisms synthesize an enzyme called parathion hydrolase, and in each case the enzyme is encoded by a gene (opd) located on a large indigenous plasmid. These plasmids show considerable genetic diversity, but the region containing the opd gene is highly conserved. Two opd plasmids, pPDL2 from Flavobacterium sp. and pCMS1 from Pseudomonas diminuta, are well characterized, and in each of them a region of about 5.1 kb containing the opd gene shows an identical restriction pattern. We now report the complete sequence of the conserved region of plasmid pPDL2. The opd gene is flanked upstream by an insertion sequence, ISFlsp1, that is a member of the IS21 family, and downstream by a Tn3-like element encoding a transposase and a resolvase. Adjacent to opd but transcribed in the opposite direction is an open reading frame (orf243) with the potential to encode an aromatic hydrolase somewhat similar to Pseudomonas putida TodF. We have shown that orf243 encodes a polypeptide of 27 kDa, which plays a role in the degradation of p-nitrophenol and is likely to act in concert with opd in the degradation of parathion. The linkage of opd and orf243, the organization of the genes flanking opd, and the wide geographical distribution of these genes suggest that this DNA sequence may constitute a complex catabolic transposon.

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confidence: 99%

Transposon-Like Organization of the Plasmid-Borne Organophosphate Degradation ( opd ) Gene Cluster Found in Flavobacterium sp

Siddavattam

Syed

Manavathi

et al. 2003

Appl Environ Microbiol

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“…Although only a single chromosomal copy of the OPH gene was present, it represents the highest OPH titer reported to date (33). In a way very similar to the concept of man-hours, where the product of two variables determines overall productivity, R. eutropha fermentations allow more cells to produce protein at a lower specific rate while achieving higher overall productivities.…”

Section: Discussionmentioning

confidence: 88%

“…Although high OPHspecific activities have been obtained in E. coli in shake flask experiments, successful scale-up has failed due to inclusion body formation (33). In order to demonstrate that high levels of the enzyme can be produced in a simple and efficient fermentation process, we conducted a fermentation to produce approximately 100 g of biomass/liter and over 1 g of OPH/liter.…”

Section: Resultsmentioning

confidence: 99%

A Novel Method for Producing Transgenic Enzymes and Peptides

Gerngross¹

2004

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Public Reporting burden for this collection of information is estimated to average 1 hour per response, including the time for reviewing instructions, searching existing data sources, gathering and maintaining the data needed, and completing and reviewing the collection of information. Send comment regarding this burden estimates or any other aspect of this collection of information, including suggestions for reducing this burden, to Washington Headquarters Services, Directorate for information Operations and Reports, 1215 The views, opinions and/or findings contained in this report are those of the author(s) and should not be construed as an official Department of the Army position, policy or decision, unless so designated by other documentation. We have successfully developed a novel a protein expression system based on the bacterium Ralstonia eutropha. This system has been developed in our laboratory to overcome some of the shortcomings associated W\\.h recombinant protein expression in other bacteria (e.g. poor fermentation performance, inclusion body formation and proteolysis). High level expression of organophosphohydrolase (OPH), an enzyme prone to inclusion body formation in Eschehchia colt has been demonstrated. A proteomics approach identified the promoter of the phaP gene as a strong, inducible promoter. By creating a translational fusion between the phaP promoter and oph gene and introducing this fusion into the chromosome, OPH titers of greater than 1 g/L were achieved in high cell density fermentations. Multiple copies of the Pphap'.'-oph translational fusion were introduced into the chromosome to further increase expression. A proportional increase in OPH titer was found with increasing copy number. An OPH titer of approximately 4.3 g/L was measured in high cell density fermentation using a strain containing three copies of Pphap'-'oph translational fusion This represents the highest titer reported to date for OPH (approximately 30 times greater than previously reported expression levels). Results have reported in two publications (Srinivasan et al. 2003; Srinivasanetal., 2002 REPORT DOCUMENTATION PAGE (SF298) (Continuation Sheet)Scientific progress and accomplishments:Introduction:With advances in protein engineering, there has been a surge in the number of newly discovered proteins and protein function as well as the ability to engineer the properties of such proteins. Enhancement of protein production is a primary goal of recombinant microbial process development. This can be achieved by increasing the amount of recombinant protein per cell (specific productivity) and/or increasing the cell concentration per unit time (cell productivity). In order to improve volumetric productivity in a cost effective manner, recombinant proteins are often produced in high cell density fermentations. High cell density fermentation has many advantages over normal fermentations in that they have a higher final product concentration, reduced waste water and higher overall productivity and hence lower setup and...

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“…Organophosphorus hydrolase (OPH), isolated from both Flavobacterium sp. ATCC 27551 (Mulbry & Karns, 1989) and Pseudomonas diminuta MG (Serdar et al, 1989), is capable of hydrolyzing a wide range of oxon and thion OPs. However, OPH has already been shown to lack any hydrolytic activity toward numerous dimethyl OPs (Horne et al, 2002).…”

Section: Wwwintechopencommentioning

confidence: 99%