Genetic engineering approach to toxic waste management: case study for organophosphate waste treatment.

Currently, there has been limited use of genetic engineering for waste treatment. In this work, we are developing a procedure for the in situ treatment of toxic organophosphate wastes using the enzyme parathion hydrolase. Since this strategy is based on the use of an enzyme and not viable microorganisms, recombinant DNA technology could be used without the problems associated with releasing genetically altered microorganisms into the environment. The gene coding for parathion hydrolase was cloned into a Streptomyces lividans, and this transformed bacterium was observed to express and excrete this enzyme. Subsequently, fermentation conditions were developed to enhance enzyme production, and this fermentation was scaled-up to the pilot scale. The cell-free culture fluid (i.e., a nonpurified enzyme solution) was observed to be capable of effectively hydrolyzing organophosphate compounds under laboratory and simulated in situ conditions.

Regulation of tyrosine biosynthesis by phenylalanine in anthramycin-producing Streptomyces refuineus.

The regulation of tyrosine production in the anthramycin-producing organism Streptomyces refuineus var. thermotolerans has been studied with wild-type and tyrosine auxotrophic organisms. Growth of the auxotroph on minimal medium plus phenylalanine suggested that phenylalanine may increase the supply of tyrosine. In incubation with whole cells, tyrosine levels increased in response to added phenylalanine. However, no radiolabeled tyrosine was detected after incubation with 14C-phenylalanine. Thus, no phenylalanine hydroxylase is present. Phenylalanine was found to feedback inhibit prephenate dehydratase, resulting in an increase in NAD-dependent prephenate dehydrogenase activity, thus channeling prephenic acid toward tyrosine.

Demonstration of a metabolic grid at an early step in the streptonigrin biosynthetic pathway in Streptomyces flocculus.

The enzyme activities which catalyze the conversion of tryptophan to beta-methyltryptophan by two different routes have been demonstrated in cell-free extracts of streptonigrin-producing Streptomyces flocculus. The first route involves direct methylation of tryptophan by a C-methyltransferase. The second involves transamination of tryptophan to indolepyruvate, methylation of indolepyruvate to beta-methylindolepyruvate, followed by a reverse transamination reaction to yield beta-methyltryptophan. The direct methylation route was confirmed by the fact that the methyltransferase activity is still present after the transaminase has been inactivated by hydroxylamine treatment. The L-tryptophan C-methyltransferase has been purified 30-fold by ammonium sulfate precipitation and a Sephadex G-150 column. The indolepyruvate C-methyltransferase activity copurified with the tryptophan C-methyltransferase activity, but the transaminase did not. These results show that a metabolic grid exists for the first antibiotic-committed step of the streptonigrin biosynthetic pathway.

Purification and characterization of S-adenosylhomocysteine deaminase from streptonigrin-producing Streptomyces flocculus.

An S-adenosylhomocysteine deaminase has been isolated and purified from streptonigrin-producing Streptomyces flocculus ATCC 13257. Deamination represents the major metabolic route of S-adenosylhomocysteine in this organism. The protein was found to be monomeric with a molecular weight of 56,100 +/- 1,600. The activity was optimal at pH 7.0 and 37 degrees C, and the deaminase was inactivated by p-chloromercuribenzoate but not by metal chelators. The Km for S-adenosylhomocysteine is 2.5 mM, and the Ki for inhibition by deoxycoformycin is 1.6 nM.

A tryptophan C-methyltransferase involved in streptonigrin biosynthesis in Streptomyces flocculus.

A C-methyltransferase that catalyses the transfer of a methyl group from S-adenosylmethionine to C-3 of tryptophan, resulting in beta-methyltryptophan, has been identified in cell-free extracts of streptonigrin-producing Streptomyces flocculus. The absolute configuration of the product was shown to be (2S,3R)-beta-methyltryptophan by high-pressure liquid chromatography and reactivity with D- and L-amino acid oxidases. In shake culture, maximum specific activity occurs after S. flocculus enters stationary phase, but before significant streptonigrin accumulates.

Radiometric prescreen for antitumor activity with Saccharomyces cerevisiae mutant strain.

After modification, a technique for radiometrically measuring bacterial growth has been applied to a mutant strain of Saccharomyces cerevisiae. The assay is based on inhibition of 14CO2 release from [14C]glucose, which provides an extremely sensitive measure of cellular respiratory activity and growth. The criterion for antitumor activity is the differential inhibition of wild-type and mutant (distorted cell membrane) strains of the yeast. The system was optimized for medium, time of incubation, temperature, and size of inoculum. Known antitumor agents, including bleomycin, actinomycin D, adriamycin, and ellipticine were tested in the system, and differential inhibition was observed. Vincristine showed no inhibitory effects at the concentrations tried. The sensitivity for 20% inhibition ranged from 0.8 micrograms of adriamycin per ml to 0.14 mg of ellipticine per ml. Antifungal agents such as amphotericin B exhibited no differential inhibition. Antibacterial agents were inactive. This method may provide a rapid, sensitive, in vitro quantitative assay for antitumor agents which could be applied to a variety of assay needs and which can be run with facilities and equipment available in most laboratories.

S-adenosylhomocysteine metabolism in Streptomyces flocculus.

S-Adenosylhomocysteine metabolism was studied in cell extracts of streptonigrin-producing Streptomyces flocculus. The major route of metabolism was found to be deamination to form S-inosylhomocysteine. The metabolite was purified by high-performance liquid chromatography and identified by its UV and nuclear magnetic resonance spectra and by its chemical degradation to hypoxanthine.

Ammonium effects on streptonigrin biosynthesis by Streptomyces flocculus.

A defined medium containing glucose and ammonium as the sole carbon and nitrogen sources was developed to support growth and streptonigrin production. In this defined medium, increased initial levels of ammonium resulted in increased growth suggesting that nitrogen is the growth limiting nutrient. In some cases, increased initial ammonium levels resulted in decreased specific streptonigrin productivity, suggesting that nitrogen regulatory mechanisms may adversely affect streptonigrin biosynthesis. This suggestion that nitrogen regulation adversely affects antibiotic biosynthesis is further supported by results from two studies in which the ammonium supply to the cells was controlled. In the first study, streptonigrin productivity and final titer were enhanced by the addition of an ammonium trapping agent. In the second experiment, when ammonium chloride was fed slowly throughout the course of cultivation, the production phase was lengthened and the maximum antibiotic concentration was enhanced compared to the batch controls containing either the same initial or the same total ammonium chloride levels. Although our results indicate streptonigrin production may be subject to nitrogen regulatory mechanisms, the effect of nitrogen on streptonigrin production cannot be strictly correlated to the extracellular ammonium concentration. In fact, we observed that when ammonium was depleted from the medium, streptonigrin production ceased.

Purification and characterization of a secreted recombinant phosphotriesterase (parathion hydrolase) from Streptomyces lividans.

A heterologous phosphotriesterase (parathion hydrolase), previously cloned from a Flavobacterium species into Streptomyces lividans, was secreted at high levels and purified to homogeneity. N-terminal analysis revealed that it had been processed in the same manner as the native membrane-bound Flavobacterium hydrolase. The enzyme consisted of a single polypeptide with an apparent molecular weight of 35,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Substrate specificity studies showed Kms of 68 microM for parathion, 46 microM for O-ethyl O-p-nitrophenyl phenylphosphonothioate, 599 microM for methyl parathion, and 357 microM for p-nitrophenyl ethyl(phenyl)phosphinate. Temperature and pH optima were 45 degrees C and 9.0, respectively. The purified enzyme was inhibited by 1 mM dithiothreitol and 1 mM CuSO4. After chelation and inactivation by o-phenanthroline, however, activity could be partially restored by 1 mM CuCl or 1 mM CuSO4. The results showed that the purified recombinant parathion hydrolase has the same characteristics as the native Flavobacterium hydrolase. This system provides a source of milligram quantities of parathion hydrolase for future structural and mechanism studies and has the potential to be used in toxic waste treatment strategies.

Native and heterologous protein secretion by Streptomyces lividans.

The secretion of the heterologous parathion phosphotriesterase in S. lividans using the Streptomyces beta-galactosidase signal sequence was further characterised using a pulse/chase system. Unsecreted cell-associated protein in both the precursor and signal-cleaved forms was observed when the protein was expressed from both low- and high-copy vectors. Fractionation of the cells followed by immunoprecipitation with phosphotriesterase antibody suggests that the precursor is membrane-bound while the signal cleaved form is present in the soluble fraction. Preliminary data on the processing of alpha-amylase, a native streptomyces protein, showed much more rapid processing and secretion, but nevertheless still revealed cell-associated, signal-cleaved protein.

Isolation and characterization of tryptophan transaminase and indolepyruvate C-methyltransferase. Enzymes involved in indolmycin biosynthesis in Streptomyces griseus.

Two enzymes, tryptophan transaminase and indolepyruvate C-methyltransferase, which are active in the initial steps of the biosynthetic pathway of the antibiotic indolmycin, have been detected and partially purified from cell-free extracts of Streptomyces griseus. The transaminase has been purified 3-fold by ammonium sulfate fractionation. At this stage of purification, it catalyzes the alpha-ketoglutarate and pyridoxal phosphate-dependent transamination of L-tryptophan, 3-methyltryptophan, L-pphenylalanine, and L-tyrosine. The C-methyltransferase catalyzes the transfer of a methyl group from S-adenosylmethionine to position 3 of the aliphatic side chain of indolepyruvate. No cofactors are required. The C-methyltransferase has been purified 110-fold by ammonium sulfate fractionation, Sephadex G-150 gel filtration, DEAE-Sephadex column chromotography, and Bio-Gel A-5m gel filtration. The enzyme has a broad pH optimum of 7.5 to 8.5. A molecular weight of 55,000 +/- 5,000 has been determined by Sephadex G-200 gel filtration with reference proteins and a molecular weight of 58,000 +/- 8,000 has been determined by sucrose density gradient centrifugation. The enzyme is relatively stable at temperatures of 0-5 degrees but is destroyed by freezing or by heating. The C-methyltransferase is inhibited strongly by the thiol reagents p-chloromercuribenzoate and N-ethylmaleimide. The Zn2+ and Fe2+ chelators 1,10-phenanthroline and 2,2'-bipyridine also inhibit the enzyme activity but EDTA does not. Michaelis-Menten constants have been determined for the 110-fold purified enzyme as 1.2 X 10(-5) M for S-adenosylmethionine and 4.8 X 10(-6) M for indolepyruvate. The enzyme activity in the crude extract is inhibited competitively by indolmycin (Ki equals 2.3 mM) and L-tryptophan (Ki equals 0.17 mM), but these effects are not observed after the enzyme has been passed through the Sephades G-150 column during purification. The crude extract is capable of methylating phenylpyruvate and p-hydroxyphenylpyruvate but this capability is lost upon purification of the indolepyruvate C-methyltransferase activity. No methylation of L-tryptophan occurs under the conditions used.

The effect of signal sequences on the efficiency of secretion of a heterologous phosphotriesterase by Streptomyces lividans.

A heterologous phosphotriesterase (parathion hydrolase) containing the native Flavobacterium species signal sequence was previously shown to be secreted by Streptomyces lividans. Western blot analysis of the recombinant phosphotriesterase produced by S. lividans demonstrated only the mature form extracellularly but both processed and unprocessed forms in cell-associated samples. To investigate the efficiency of secretion in Streptomyces, a construction was made that substituted a native Streptomyces beta-galactosidase signal sequence for the Flavobacterium signal sequence. This resulted in a higher proportion of hydrolase in the extracellular fluid and a lower proportion of parathion hydrolase remaining cell-associated. These results suggest that use of a native Streptomyces signal sequence may result in more efficient secretion of heterologous proteins.

Purification and characterization of a novel enoyl coenzyme A reductase from Streptomyces collinus.

A novel NADPH-dependent enoyl reductase, catalyzing the conversion of 1-cyclohexenylcarbonyl coenzyme A (1-cyclohexenylcarbonyl-CoA) to cyclohexylcarbonyl-CoA, was purified to homogeneity from Streptomyces collinus. This enzyme, a dimer with subunits of identical M(r) (36,000), exhibits a Km of 1.5 +/- 0.3 microM for NADPH and 25 +/- 3 microM for 1-cyclohexenylcarbonyl-CoA. It has a pH optimum of 7.5, is most active at 30 degrees C, and is inhibited by both divalent cations and thiol reagents. Two internal peptide sequences were obtained. Ansatrienin A (an antibiotic produced by S. collinus) contains a cyclohexanecarboxylic acid moiety, and it is suggested that the 1-cyclohexenylcarbonyl-CoA reductase described herein catalyzes the final reductive step in the conversion of shikimic acid into this moiety.

Genetic and biochemical evidence for the lack of significant hydrolysis of soman by a Flavobacterium parathion hydrolase.

Pure recombinant Flavobacterium parathion hydrolase (an organophosphorus acid anhydrase) from Streptomyces lividans was found to hydrolyze the toxic nerve agent soman at only 0.1% of the rate observed with parathion as substrate. Studies with wild-type and recombinant strains of S. lividans support the lack of significant soman breakdown by the hydrolase and also indicate the presence in S. lividans of other significant hydrolytic enzymatic activity towards soman.

Purification of crotonyl-CoA reductase from Streptomyces collinus and cloning, sequencing and expression of the corresponding gene in Escherichia coli.

A crotonyl-CoA reductase (EC 1.3.1.38, acyl-CoA:NADP+ trans-2-oxidoreductase) catalyzing the conversion of crotonyl-CoA to butyryl-CoA has been purified and characterized from Streptomyces collinus. This enzyme, a dimer with subunits of identical mass (48 kDa), exhibits a Km = 18 microM for crotonyl-CoA and 15 microM for NADPH. The enzyme was unable to catalyze the reduction of any other enoyl-CoA thioesters or to utilize NADH as an electron donor. A highly effective inhibition by straight-chain fatty acids (Ki = 9.5 microM for palmitoyl-CoA) compared with branched-chain fatty acids (Ki > 400 microM for isopalmitoyl-CoA) was observed. All of these properties are consistent with a proposed role of the enzyme in providing butyryl-CoA as a starter unit for straight-chain fatty acid biosynthesis. The crotonyl-CoA reductase gene was cloned in Escherichia coli. This gene, with a proposed designation of ccr, is encoded in a 1344-bp open reading frame which predicts a primary translation product of 448 amino acids with a calculated molecular mass of 49.4 kDa. Several dispersed regions of highly significant sequence similarity were noted between the deduced amino acid sequence and various alcohol dehydrogenases and fatty acid synthases, including one region that contains a putative NADPH binding site. The ccr gene product was expressed in E. coli and the induced crotonyl-CoA reductase was purified tenfold and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate/polyacrylamide to the native protein.

Relationship of inappropriate drug prescribing to increased length of hospital stay.

The relationship of inappropriate drug prescribing to increased length of hospital stay was studied. The medical records of 77 cases of pyelonephritis were reviewed retrospectively. Appropriateness of antimicrobial drug therapy was judged by three types of explicit screening criteria: drug-specific, patient-specific, and match of drug to infecting organism. Patients whose therapy passed all the criteria were hospitalized, on the average, two days less than those whose therapy failed one or more of the criteria. This was a significant difference (p less than 0.05). Age, seriousness of the pyelonephritis, or method of payment appeared to have no significant moderating effect on this result. However, the increased length of stay may not have been associated with only inappropriate prescribing, because the inappropriately prescribing physicians kept their patients hospitalized longer beyond the point of symptom remission than did the appropriately prescribing physicians. The study suggest that successful interventions for improving drug therapy could result in large cost savings.