Microbial transformation of precocene II: oxidative reactions by Streptomyces griseus.

Various species of "Streptomyces," "Aspergillus," "Rhodotorula," "Brevilegnia," "Syncephalastrum," and "Stysanus" were found to transform precocene II to three major metabolites. These major biotransformation products were isolated from a preparative-scale incubation of precocene II with Streptomyces griseus and were conclusively identified as (-)cis- and (+)trans-precocene II-3,4-dihydrodiols and (+)-3-chromenol. 18O2 incorporation studies indicated the involvement of a monooxygenase enzyme system in precocene II transformation by S. griseus. A mechanism is proposed for the formation of (+)-3-chromenol.     

Involvement of an Inducible Cytochrome P-450 in Precocene II Oxidation by Streptomyces Griseus

Streptomyces griseus oxidizes the insecticide precocene II to its cis- and trans-dihydrodiols and 3-chromenol after growth on an enriched medium containing soybean flour. Oxidation of precocene II is dependent on the level of cytochrome P-450 in this organism. Extracts of cells grown on media lacking soybean flour were devoid of cytochrome P-450 and could not oxidize precocene II. In an in vitro reconstituted system containing NADPH, spinach ferredoxin reductase, spinach ferredoxin and ammonium sulfate fractions enriched in cytochrome P-450, precocene II was oxidized to its dihydrodiols. An aerial mycelium-negative variant of S. griseus (AMY mutant), that was unable to elicit cytochrome P-450 when grown on soybean flour-enriched medium, failed to oxidize precocene II.     

Purification and characterization of a soybean flour-induced cytochrome P-450 from Streptomyces griseus.

A soybean flour-induced, soluble cytochrome P-450 (P-450soy) was purified 130-fold to homogeneity from Streptomyces griseus. Native cytochrome P-450soy is a single polypeptide, with a molecular weight of 47,500, in association with one ferriprotoporphyrin IX prosthetic group. Oxidized P-450soy exhibited visible absorption maxima at 394, 514, and 646 nm, characteristic of a high-spin cytochrome P-450. The CO-reduced difference spectrum of P-450soy had a Soret maximum at 448 nm. When reconstituted with spinach ferredoxin and spinach ferredoxin:NADP+ oxidoreductase, purified cytochrome P-450soy catalyzed the NADPH-dependent oxidation of the xenobiotic substrates precocene II and 7-ethoxycoumarin. In vitro proteolysis of cytochrome P-450soy generated a stable and catalytically active cytochrome P-450, designated P-450soy delta.     

Metabolism and hepatotoxicity of the naturally occurring benzo[b]pyran precocene I

The in vitro metabolism of precocene I by liver microsomes from control and treated rats and the effects of precocene I on the function and histology of the rat liver were examined. The major metabolites (80-90% of total metabolites) from all microsomal preparations were the cis and trans 3,4-diols of precocene I produced with a cis/trans isomer ratio of 1:2. These diols appear to arise mainly by spontaneous hydrolysis of precocene I 3,4-oxide. (+)-(3R,4R)-cis- and (-)-(3R,4S)-trans-precocene I 3,4-diols were the predominant enantiomers of the 3,4-diol formed. The enantiomeric excess of these diols (2-50%) is dependent on the microsomal preparation, with microsomes from control rats exhibiting the highest stereoselectivity and microsomes from phenobarbital-treated rats the least. 6-Hydroxyprecocene I was the next major metabolite and was formed to the extent of 5% (control), 10% and 17% (phenobarbital and 3-methylcholanthrene treatment, respectively) of total metabolites. Treatment of rats with a single i.p. dose of precocene I (300 mg/kg) resulted in extensive hepatic damage as evidenced by a marked increase of plasma glutamic pyruvic transaminase levels and histologic observation in liver sections of severe centrolobular necrosis. Although phenobarbital treatment of rats increased the rate of liver microsomal metabolism of precocene I by approximately 50% (nmol products/nmol cytochrome P-450/min) compared to liver microsomes from control rats, hepatic damage caused by precocene I was not significantly affected. Depletion of glutathione levels in the rats with diethyl maleate prior to precocene I treatment dramatically increased the severity of hepatic insult, whereas treatment of the rats with the mixed function oxidase inhibitor piperonyl butoxide prior to treatment with precocene I blocked hepatic damage. Treatment of rats with cysteamine prior to treatment with precocene I protected the animals against the toxic effects. Neither cis nor trans precocene I 3,4-diol nor 3,4-dihydroprecocene I elicited impaired liver function or cellular damage. The above results are consistent with the view that precocene I 3,4-oxide is the metabolite responsible for the hepatotoxic effects observed when precocene I is injected into rats.     

Characterization of a novel sesquiterpene cyclase involved in (+)-caryolan-1-ol biosynthesis in Streptomyces griseus

Most terpenoids have been isolated from plants and fungi and only a few from bacteria. However, an increasing number of genome sequences indicate that bacteria possess a variety of terpenoid cyclase genes. We characterized a sesquiterpene cyclase gene (SGR2079, named gcoA) found in Streptomyces griseus. When expressed in Streptomyces lividans, gcoA directed production of a sesquiterpene, isolated and determined to be (+)-caryolan-1-ol using spectroscopic analyses. (+)-Caryolan-1-ol was also detected in the crude cell lysate of wild-type S. griseus but not in a gcoA knockout mutant, indicating that GcoA is a genuine (+)-caryolan-1-ol synthase. Enzymatic properties were characterized using N-terminally histidine-tagged GcoA, produced in Escherichia coli. As expected, incubation of the recombinant GcoA protein with farnesyl diphosphate yielded (+)-caryolan-1-ol. However, a small amount of another sesquiterpene was also detected. This was identified as the bicyclic sesquiterpene hydrocarbon (+)-β-caryophyllene by comparison with an authentic sample using GC-MS. Incorporation of a deuterium atom into the C-9 methylene of (+)-caryolan-1-ol in an in vitro GcoA reaction in deuterium oxide indicated that (+)-caryolan-1-ol was synthesized by a proton attack on the C-8/C-9 double bond of (+)-β-caryophyllene. Several β-caryophyllene synthases have been identified from plants, but these cannot synthesize caryolan-1-ol. Although caryolan-1-ol has been isolated previously from several plants, the enzyme responsible for its biosynthesis has not been identified previously. GcoA is thus the first known caryolan-1-ol synthase. Isolation of caryolan-1-ol from microorganisms is unprecedented.     

Age-dependent physiological responses of last instarHelicoverpa zea larvae to oral and topical application of precocene II

Newly-ecdysed last instar larvae ofH. zea grouped into 100-, 200-, 300-, or 400-mg categories were fed diet containing precocene II or given precocene II topically on the abdomen. The time for larvae to reach a maximal weight, time to pupation, growth rate, and the amount of precocene II excreted were calculated. Younger larvae of lower weights, which were fed or topically treated with precocene II required more time to reach their maximal weight, had a lower maximal weight, a lower growth rate, and required more time to pupate than control larvae. Older larvae represented by the largest weight category were less sensitive to precocene II, had a shorter delay in reaching maximal weight, and a shorter delay in the time to pupation than control larvae; larvae in the largest weight category that were fed precocene II also had smaller decreases in the growth rate. Growth rate declines for larvae given topical doses of precocene II, however, were largest for the oldest larvae. All larvae given a single topical dose excreted precocene II for several days and were most efficient at eliminating smaller doses; larger, older larvae excreted more precocene II than smaller, younger larvae. Age-dependent responses to precocene II indicate that growth and metabolic processes, as well as xenobiotic metabolism, change in last instar larvae.     

Lack of regio- and stereospecificity in oxidation of (+) camphor by Streptomyces griseus enriched in cytochrome P-450soy

Oxidation of (+) camphor by cytochrome P-450soy-enriched intact cells of Streptomyces griseus resulted in the formation of one major and several minor metabolites. The minor metabolites were identified as 3-endo-hydroxycamphor (2%), 5-endo-hydroxycamphor (7%), 5-exo-hydroxycamphor (9%), 2,5-diketobornane (2%), and camphorquinone (3%). The major metabolite was isolated and conclusively identified as 6-endo-hydroxycamphor (60%). When supplemented with NADPH, spinach ferredoxin:NADP oxidoreductase and spinach ferredoxin, homogeneous preparations of cytochrome P-450soy oxidized camphor to a mixture of 3-endo-, 5-endo-, 5-exo-and 6-endo-hydroxycamphor. The data presented indicates that cytochrome P-450soy resembles its mammalian counterparts in its lack of regio- and stereospecificity in camphor oxidation.     

Streptomycin biosynthesis in Streptomyces griseus I. Characterization of streptomycin-idiotrophic mutants

Abstract A total of 16 idiotrophic mutants unable to produce the aminoglycoside antibiotic streptomycin ( smi ) were isolated from Streptomyces griseus N2-3-11. Cosynthesis of streptomycin, its formation from various precursors and analysis of accumulated intermediates allowed grouping of the mutants in 3 classes, blocked: (I) in the first transamination step of the streptidine pathway; (II) in later steps of the streptidine pathway; or (III) outside streptidine biosynthesis.     

响应面法优化灰色链霉菌产纤维素酶的工艺研究

以灰色链霉菌为原料,在单因素试验的基础上,采用响应面法试验,优化灰色链霉菌产纤维素酶活性的发酵条件。结果表明,单因素试验灰色链霉菌产纤维素酶活性的最适发酵条件:碳源为CMC-Na,氮源为明胶,温度为28℃,pH为7.0,转速为130 r/min。响应面法试验优化灰色链霉菌产纤维素酶活性最佳发酵条件为:温度27.7℃,pH值6.9,转数130.3 r/min,在此优化条件下,灰色链霉菌产纤维素平均酶活性为6.103 U/mL(n=3),与模型的预测值(6.217 U/mL)比较接近,误差为1.83%,证明了该响应面模型具有可靠性。     

Lethal and mutagenic effect of fast neutrons of different energies on the spores of Streptomyces griseus

A study was made of lethal and mutagenic effects of fast neutrons of different energy on spores of prototrophic and auxotrophic strains of Streptomyces griseus. Relative biological effectiveness of fast neutrons is higher than that of gamma-rays and depends on beam energy. Neutrons of 22-50 MeV induce Streptomyces griseus mutations more frequently (by one order of magnitude) than neutrons of 1.4-1.6 MeV do. The obtained mutants can be used in studying Streptomyces griseus genetics.     

Detection and properties of A-factor-binding protein from Streptomyces griseus.

The optically active form of tritium-labeled A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone), a pleiotropic autoregulator responsible for streptomycin production, streptomycin resistance, and sporulation in Streptomyces griseus, was chemically synthesized. By using the radioactive A-factor, a binding protein for A-factor was detected in the cytoplasmic fraction of this organism. The binding protein had an apparent molecular weight of approximately 26,000, as determined by gel filtration. Scatchard analysis suggested that A-factor bound the protein in the molar ratio of 1:1 with a binding constant, Kd, of 0.7 nM. The number of the binding protein was roughly estimated to be 37 per genome. The "inducing material" virginiae butanolide C (VB-C), which has a structure very similar to that of A-factor and is essential for virginiamycin production in Streptomyces virginiae, did not inhibit binding. In addition, no protein capable of specifically binding 3H-labeled VB-C was found in S. griseus. Together with the observation that VB-C had almost no biological activity on the restoration of streptomycin production or sporulation in an A-factor-deficient mutant of S. griseus, these results indicated that the binding protein had a strict ligand specificity. Examination for an A-factor-binding protein in Streptomyces coelicolor A3(2) and Streptomyces lividans showed the absence of any specifically binding protein.     

Nonactin biosynthesis: the potential nonactin biosynthesis gene cluster contains type II polyketide synthase-like genes

Nonactin is the parent compound of a group of highly atypical polyketide metabolites produced by Streptomyces griseus subsp. griseus ETH A7796. In this paper we describe the isolation, sequencing, and analysis of 15? omitted?559 bp of chromosomal DNA, containing the potential nonactin biosynthesis gene cluster, from S. griseus subsp. griseus ETH A7796. Fourteen open reading frames were observed in the DNA sequence. Significantly, type II polyketide synthase (PKS) homologues were discovered in an apparent operon structure, which also contained the nonactate synthase gene (nonS), clustered with the tetranactin resistance gene. The deduced products of two of the genes (nonK and nonJ) are quite unusual ketoacyl synthase (KAS) alpha and KASbeta homologues. We speculate that nonactic acid, the polyketide precursor of nonactin, is synthesized by a type II PKS system.     

Construction and properties of hybrids obtained in interspecific crosses between Streptomyces coelicolor A3(2) and Streptomyces griseus Kr-15.

Recombinants between Streptomyces coelicolor A3(2) and Streptomyces griseus Kr-15 were obtained using methods of hybrid construction. Recombinant Rcg1, obtained from a cross between S. griseus and a S. coelicolor UF (SCPI-) strain, phenotypically resembled S. coelicolor UF strains and in crosses with a S. coelicolor NF donor strin produced recombinatn progeny at a frequency of 100%. Recominant Rcg3, like SCP1-carrying S. coelicolor strains, inhibited SCP1-strains of S. coelicolor and in crosses with a UF recipient strain of S. coelicolor generated recombinants at high frequency. In crosses between S. griseus and Rcgi the frequency of recombinant formation was increased about 100-fold relative to crosses between S. griseus and S. coelicolor. Effective transfer of S. grieseus and Rcg3 chromosomal markers into Rcg1 and S. coelicolor, respectively, indicated that S. griseus had donor properties. Studies of the ability of recombinants to support phage growth indicated that parental chromosomal fragments containing genes involved in control of phage-receptor formation and intracellular growth were present in the hybrids. Grisin-producing recombinants, capable of restricting phages attacking S. coelicolor and S. griseus, were obtained.     

Molecular cloning and nucleotide sequence of Streptomyces griseus trypsin gene.

Streptomyces griseus trypsin (E.C. 3.4.21.4) is one of the major extracellular proteinase, which is secreted by S. griseus. The gene encoding S. griseus trypsin was isolated from a S. griseus genomic library by using a synthetic oligonucleotide probe. Fragments containing the gene for S. griseus trypsin were characterized by hybridization and demonstration of proteolytic activity in S. lividans. Deduced amino acid sequence from the nucleotide sequence suggests that S. griseus trypsin is produced as a precursor, consisting of three portions; an amino-terminal pre sequence (32 amino acid residues), a pro sequence (4 residues), and the mature trypsin. The S. griseus trypsin consists of 223 amino acids with a computed molecular weight of 23,112. The existence of proline at the pro and mature junction suggests that the processing of S. griseus trypsin is non-autocatalytic.     

Cloning of a DNA fragment from Streptomyces griseus which directs streptomycin phosphotransferase activity

DNA from Streptomyces griseus ATCC 12475 was partially digested with Sau3A and fragments were ligated into BglII-cleaved pIJ702. When the ligation mixture was used to transform protoplasts of Streptomyces lividans TK54, two transformants resistant to both thiostrepton and streptomycin were isolated. The hybrid plasmids pBV3 and pBV4 which they contained, carrying inserts of sizes 4.45 and 11.55 kbp respectively, each retransformed S. lividans to streptomycin resistance at high efficiency. Both plasmids hybridized to restriction digests of S. griseus chromosomal DNA in Southern blot experiments. In vitro deletion and sub-cloning experiments showed the sequence conferring streptomycin resistance to lie within a segment of 1.95 kbp. Extracts of TK54(pBV3) and TK54(pBV4) contained a streptomycin phosphotransferase similar to that in extracts of S. griseus. Streptomycin phosphotransferase activity appeared in extracts of S. griseus, TK54(pBV3) and TK54(pBV4) within 2 d of inoculation. When pBV3 and pBV4 were retransformed into S. griseus with selection for thiostrepton resistance, plasmid DNA of sizes corresponding to the incoming plasmids was found in the transformants. In these transformants the phosphotransferase appeared at 1.5 rather than 2 d, and reached a level over twice that of the original S. griseus strain.     

Biosynthesis of N-methyl-L-glucosamine from D-glucose by Streptomyces griseus.

Biosynthesis of N-methyl-L-glucosamine moiety of streptomycin from D-glucose by Streptomyces griseus was studied. A mixture of D-[1-(14) C] glucose and D-[6(-3)H]glucose was given to the culture of S. griseus. The 3H/14C ratio found in N-methyl-L-glucosamine further supports a mechanism that the conversion of D-glucose to L-hexose is carried out without scission of carbon skeleton. When D-[1-14C]glucose and D-[3-3H]glucose were used, the fall of 3H/14C ratio in N-metyl-L-glucosamine showed that the hydrogen atom at C-3 plays a r?le in such a transformation.