A Gene Coding for a Membrane–Bound Hydrolase is Expressed as a Secreted, Soluble Enzyme in Streptomyces Lividans

Steiert, John G.; Pogell, Burton M.; Speedie, Marilyn K.; Laredo, James

doi:10.1038/nbt0189-65

Cited by 47 publications

(34 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…as an RNA polymerase promoter, then it is unclear why they do not function in E. coli. Nevertheless, expression of the opd gene in E. coli is entirely dependent on exogenous promoters (6; W. Mulbry (18). The responsible promoter sequences were localized by a deletion experiment to the region that contains our putative promoter sequences.…”

Section: Resultsmentioning

confidence: 99%

Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene and protein

1989

View full text Add to dashboard Cite

The sequence of a 1,693-base-pair plasmid DNA fragment from Flavobacterium sp. strain ATCC 27551 containing the parathion hydrolase gene (opd) was determined. Within this sequence, there is only one open reading frame large enough to encode the 35,000-dalton membrane-associated hydrolase protein purified from Flavobacterium extracts. Amino-terminal sequence analysis of the purified Flavobacterium hydrolase demonstrated that serine is the amino-terminal residue of the hydrolase protein. The amino-terminal serine corresponds to a TCG codon located 87 base pairs downstream of the presumptive ATG initiation codon in the nucleotide sequence. The amino acid composition of the purified protein agrees well with that predicted from the nucleotide sequence, using serine as the amino-terminal residue. These data suggest that the parathion hydrolase protein is processed at its amino terminus in Flavobacterium sp. Construction in Escherichia coli of a lacZ-opd gene fusion in which the first 33 amino-terminal residues of opd were replaced by the first 5 residues of lacZ resulted in the production of an active hydrolase identical in molecular mass to the hydrolase isolated from Flavobacterium sp. E. coli cells containing the lacZ-opd fusion showed higher levels of hydrolase activity than did cells containing the parent plasmid.The microbial degradation of hazardous waste offers a promising strategy by which such some wastes may be economically and safely detoxified. For selected situations, microbial processes have considerable advantages over other technologies in that microbial processes can yield precise products, function at low concentrations of solute, and require relatively low levels of technology for construction and maintenance. However, there are relatively few instances in which microbial processes are being actively used to control hazardous wastes.Organophosphate compounds such as the insecticide parathion (O,O-diethyl-0-4-nitrophenyl phosphorothioate) are susceptible to microbial hydrolysis by bacterial parathion hydrolases. In fact, such soil hydrolases are thought to play a role in the relatively low persistence of these compounds. Since organophosphates constitute a large fraction of the insecticides used in the industrialized countries, there is a need for economical and reliable methods to detoxify organophosphate wastes (such as residual pesticide concentrates in their original containers, contaminated stock solutions, and dilute pesticide solutions resulting from the washing of spraying equipment). This need has stimulated recent research on parathion hydrolases.Parathion hydrolase activities have been described in a variety of bacterial isolates and are characterized by broad substrate ranges for compounds structurally related to parathion, broad temperature and pH optima, and high stability (1,10,13,14). Of the parathion hydrolases that have been characterized in detail, the enzymes encoded by the related opd genes of Flavobacterium sp. strain ATCC 27551 and Pseudomonas diminuta MG are noteworthy becaus...

show abstract

Section: Resultsmentioning

confidence: 99%

Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene and protein

1989

View full text Add to dashboard Cite

show abstract

“…Moreover, less manipulation and downstream processing is required to obtain pure protein. The amount of OpdA secreted in our system is similar to that in the S. lividans system (23.6 compared to 21.3 mg/l after 72 h) [Rowland et al, 1992;Steiert et al, 1989], but the B. choshinensis expressed protein is purer, suggesting that very little downstream processing would be needed, mainly to remove media constituents. In the S. lividans system, ammonium sulphate precipitation and then ion exchange chromatography were required to obtain pure protein [Rowland et al, 1992].…”

Section: Discussionmentioning

confidence: 53%

“…ATCC27551 and Brevundimonas diminuta MG (90% amino acid identity), which has been expressed as a fusion protein in Escherichia coli [Mulchandani et al, 1999;Richins et al, 1997Richins et al, , 2000Shimazu et al, 2001], albeit in a form still associated with the E. coli cell. OPH has been successfully secreted in the Gram-positive bacterial species Streptomyces lividans using its own signal peptide, but the majority of the expressed protein was still retained in the cytoplasm of the cell [Steiert et al, 1989]. Changing the signal peptide to the S. lividans ß-galactosidase signal peptide (according to our analysis, a TAT type) greatly enhanced the efficiency of secretion to almost 70% [Rowland et al, 1992].…”

Section: Introductionmentioning

confidence: 98%

See 1 more Smart Citation

A <i>Brevibacillus choshinensis</i> System That Secretes Cytoplasmic Proteins

Horne¹,

Williams²,

Sutherland³

et al. 2004

Microb Physiol

View full text Add to dashboard Cite

Brevibacillus choshinensis has previously been shown to be a useful strain for the secretion of heterologous proteins via the Sec secretory pathway. This pathway involves the secretion of proteins prior to folding, whereas the alternative TAT (twin-arginine translocation) pathway enables pre-folded proteins to be secreted. We have modified the signal peptide of the Brevibacillus expression vector pNCMO2 to accommodate a Sec avoidance signal as well as the twin arginines required for secretion via the TAT system. Use of this modified signal peptide with the phosphotriesterase OpdA enabled B. choshinensis transformants to express and secrete the enzyme in an active and substantially pure form. The system was also used successfully to secrete two cytoplasmic proteins, the phosphotriesterase HocA from Pseudomonas monteilii and the phenylcarbamate-degrading enzyme, PCD, from Arthrobacter oxydans. The inhibitors carbonyl cyanide m-chlorophenyl hydrazine and sodium azide were used to confirm that secretion was occurring via the TAT secretion pathway. The modified B. choshinensis system we have developed may have general utility in secreting a wide range of heterologous proteins in active and conveniently processed form.

show abstract

“…The hydrolysis ofparaoxon results in the production of diethyl phosphate and p-nitrophenol by a single stereochemical inversion at the phosphorus center (Lewis et al 1988). The enzyme has been expressed in Streptomyces lividans (Steiert et al 1989), Escherichia coli (Serdar et al 1989;Mulbry and Karns 1989;Dave et al 1993) and insect cells (Dumas et al 1989a;Dave et al 1994). Supak (1990) demonstrated integration and low levels of expression of recombinant DNA plasmids containing the opd gene in the fungus Gliocladium virens.…”

mentioning

confidence: 96%

Expression of organophosphate hydrolase in the filamentous fungus Gliocladium virens

Dave

Lauriano

et al. 1994

Appl Microbiol Biotechnol

View full text Add to dashboard Cite

The broad-spectrum organophosphate hydrolase (OPH; EC 3.1.8.1) encoded by the organophosphate-degrading gene (opd) from Pseudomonas diminuta MG and Flavobacterium sp. ATCC 27551 possesses capabilities of both P-O bond hydrolysis (e.g. paraoxon) and P-F bond hydrolysis [e.g. sarin and diisopropylfluorophosphate (DFP)]. In the present study a 9.4-kb plasmid, pCL1, was used to transform the saprophytic fungus Gliocladium virens. pCL1 was derived from pJS294 by placing the fungal promoter (prom1) from Cochliobolus heterostrophus upstream and the trpC terminator from Aspergillus nidulans down-stream of the opd gene. Southern analysis of restricted genomic DNA from various transformants indicated that integration occurred non-specifically at multiple sites. Western blot analysis of mycelial extracts from transformants confirmed the production of a processed form of the enzyme in the fungus. Maximal levels of OPH activity (rate of p-nitrophenol production from paraoxon) were observed after 168 h of culture and activity levels correlated with biomass production in mature vegetative growth.

show abstract

A Gene Coding for a Membrane–Bound Hydrolase is Expressed as a Secreted, Soluble Enzyme in Streptomyces Lividans

Cited by 47 publications

References 18 publications

Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene and protein

Parathion hydrolase specified by the Flavobacterium opd gene: relationship between the gene and protein

A <i>Brevibacillus choshinensis</i> System That Secretes Cytoplasmic Proteins

Expression of organophosphate hydrolase in the filamentous fungus Gliocladium virens

Contact Info

Product

Resources

About