Conversion of 5′-inosinic acid to inosine by Streptomyces aureus

The production of inosine by microbial conversion of 5′-inosinic acid (IMP) was investigated. Among the various strains of Streptomyces and Bacillus tested, Streptomyces aureus NCIB 9803 was selected for the microbial conversion process due to its high IMP-degrading activity. A maximum conversion yield of 0.43 (86% of the theoretical value) was obtained when IMP was added to the culture medium at 24 h. Kinetic studies with [8-¹⁴C] IMP showed that the difference from the theoretical values mainly attributable to the uptake of inosine by S. aureus.     

Inhibitors of Adenosine Deaminase: Synthesis of Coformycin and 3′-Deoxycoformycin

Abstract

Glycosylation of the heterocycle, 6,7-dihydro-imidazo [4,5-d] [1,3] diazepin-8(3H)-one, with suitably protected sugars under the influence of Lewis acid catalysts gave the β-D-ribo- and 3′-deoxy-β-D-erythropento-furanosyl nucleosides. Deprotection and reduction of the keto nucleosides with sodium borohydride gave the (8R)- and (8S)-3-β-D-glycofuranosyl-3,6,7,8-tetrahydroimidazo [4,5-d]-[1,3] diazepin-8-ols, the (8R)-isomers of which are potent inhibitors of adenosine deaminase.     

Enzymatic synthesis of 2′-ara and 2′-deoxy analogues of c-di-GMP

The substrate specificity of recombinant full-length diguanylate cyclase (DGC) of Thermotoga maritima with mutant allosteric site was investigated. It has been originally shown that the enzyme could use GTP closest analogues – 2′-deoxyguanosine-5′-triphosphate (dGTP) and 9-β-D-arabinofuranosyl-guanine-5′-triphosphate (araGTP) as the substrates. The first demonstrations of an enzymatic synthesis of bis-(3′-5′)-cyclic dimeric deoxyguanosine monophosphate (c-di-dGMP) and the previously unknown bis-(3′-5′)-cyclic dimeric araguanosine monophosphate (c-di-araGMP) using DGC of T. maritima in the form of inclusion bodies have been provided.     

2′-N-Acetylation of Butirosin A by Mutants of Streptomyces fradiae

To reveal the mechanism of reducing sugar-induced polymerization of proteins, monomeric lysozymes were isolated at various stages of storage from whole lysozyme (WL) being kept with glucose at 75% relative humidity and 50°C for up to 30 days, and their chemical properties were investigated and compared with the corresponding WL. The impairment of Lys, Arg, and Trp residues was observed in all the isolated monomeric lysozymes (IM) as well as in the WL.

When the IM were stored for another 10 days without glucose, they polymerized and had an additional impairment of Arg and Lys but not Trp residues. All IM exhibited almost the same polymerization rate, but the sum of additional losses of Lys and Arg residues varied. The IM was also found to cross-link untreated lysozymes even in the absence of glucose.

On basis of the results obtained hitherto, it is suggested that the glucose-induced polymerization of lysozymes proceeds through the following paths. At the first step, some bifunctional agents (BF), probably α-dicarbonyl compounds, generated from the reaction between ?-amino groups of lysine residues and glucose, attach to Arg, Lys, and Trp residues through one of their two functional sites. At the second step, some of those proteins with BF attached polymerize by binding of the other unoccupied functional site with the remaining Lys and Arg (not Trp) residues of the other protein molecules. The other of the proteins with BF attached polymerize through the combination between the other unoccupied functional sites themselves with no loss of amino acid residues.     

Enzymatic synthesis of 2′-deoxyadenosine

Summary The title compound was prepared by a two step enzymatic procedure consisting of DNA hydrolysis to the mixture of 2-deoxynucleosides followed by a transdeoxyribosilation of exogenous adenine.     

Conversion of Orotic Acid to Uridine-5′-monophosphate by Enzymes of Micrococcus glutamicus

An enzyme system which could convert orotic acid to uridine-5′-monophosphate (5′-UMP) was found in cell-free extract of a threonine-requiring auxotroph of Micrococcus glutamicus (Syn. Corynebacterium glutamicum) 534 Co-147. This reaction required 5-phosphoribosylpyrophosphate (PRPP) and magnesium ion as essential components. The product of the enzyme reaction was separated by ion exchange resin chromatography and identified to be uridine-5′-monopbosphate. From the stoichiometric studies and other characteristics, it became evident that this enzyme reaction proceeded according to the following equation and was assumed to be catalyzed by orotidine-5′-monophosphate pyrophosphorylase and orotidine-5′-monophosphate decarboxylase. $\text{Orotic}\text{ acid + PRPP}\underset{{\text{Mg}}^{++}}{\to }{5}^{\prime }?\text{UMP}+\text{PPi}+{\text{CO}}_2$     

Synthesis and Photochemical Conversion of Oligonucleotides Containing 2-Chloro-2′-Deoxyadenosine

Abstract

The rate and velocity of the photoconversion of 2-chloro-2′-deoxyadenosine into 2′-deoxyisoguanosine within oligonucleotides was found to be sequence-specific and depends on the nearest neighbor.     

Enzymatic modification of β-lactoglobulin with N-fatty-acyl-dipeptide by transglutaminase from Streptomyces mobaraense

-Lactoglobulin was enzymatically acylated with N-lauroyl-l-glutaminyl-glycine and N-lauroyl-l-glutamyl-l-lysine using transglutaminase from Streptomyces mobaraense. The modification of the protein with N-fatty-acyl-dipeptide was confirmed by SDS-PAGE, gel chromatography, HPLC, amino acid analysis, and TOF-MS. The degrees of the protein modification with N-lauroyl-l-glutaminyl-glycine and N-lauroyl-l-glutamyl-l-lysine were estimated to be 2–4 and 1.5 residues per molecule, respectively.     

Tubercidin: Its Conversion to 5′-Amino-5′-deoxytubercidin

A method for the determination of allethrin and other pesticides in mosquito coils was developed by the combination of shaking extraction and gas chromatography (GC). Allethrin and other pesticides were adequately extracted by shaking for only half an hour with a mixture of toluene and 99% formic acid (5:1). This shaking extraction method was more effective for shortening the extraction time compared with the Soxhlet extraction method, and accurate determination was achieved without the interference from inert materials in the mosquito coils. The recovery of allethrin in various contents from the coils was 96.6 to 97.1%, with a 1.2 to 1.5% coefficient of variation. Furthermore, the recoveries of other pesticides and synergists from the coils were 94 to 102% by this shaking extraction method.     

Effect of Ferrous Ion on ��-Poly-l-Lysine Biosynthesis by Streptomyces diastatochromogenes CGMCC3145

ε-Poly-L-lysine (ε-PL) is known as an amino acid homopolymer occurring in nature. It is mainly produced by bacteria belonging to the family of Streptomycetaceae. In this study, the effect of ferrous ion on the metabolic pathway such as key enzyme activities and substrate consumptions relating to ε-PL production were investigated. When 0.1 mM Fe(2+) was supplemented into the culture medium of Streptomyces diastatochromogenes CGMCC3145 for 72 h of cultivation, the yield of ε-PL and biomass were enhanced by 29.2 and 41.9%, respectively, compared with those of the control. Synchronously, consumptions of glucose, ammoniac nitrogen, and phosphorus were increased by 50, 67.6, and 35.7%, respectively. The enzyme activity measurements showed that the specific activities of proteinase, phosphatase, glutamate dehydrogenase, and aspartate aminotransferase activities were improved for 2.32, 3.68, 1.72, and 1.50 folds, respectively, however, aspartokinase and ε-PL synthetase were hardly affected by ferrous ion. These results manifested that Fe(2+) can improve the yield of ε-PL by promoting aspartate production, and the aspartate was further catalyzed to produce much more L-lysine, precursor for ε-PL biosynthesis.     

Corrections to: Enhanced lincomycin production by co‑overexpression of metK1 and metK2 in Streptomyces lincolnensis

Journal of Industrial Microbiology & Biotechnology - In the online published article, row value “pIB139-metK1-metK2” in table 1 has been processed incorrectly. The correct table is...     

Conversion of 1′-deoxy-2H-ABA to2H-ABA in cell-free extracts fromCercospora rosicola

Abscisic acid (ABA) biosynthesis in the fungusCercospora rosicola has been studied previously by feeding potential intermediates to growing cultures and examining the products. The present work describes the isolation and partial characterization of an enzyme system which catalyzes the last step in this pathway, the conversion of 1′-deoxy-²H-ABA to²H-ABA. Enzyme extracts were prepared from cold-pressed mycelia of actively growingC. rosicola in tricine buffer containing sucrose and β-mercaptoethanol. Low-speed supernatants and resuspended microsomal preparations were active in converting 1′-deoxy-²H-ABA to²H-ABA. Optimum conditions for the reactions were established at 10 μM substrate, 300 μl enzyme extract in a total volume of 1 ml. Reaction products were chromatographed by reverse-phase high-performance liquid chromatography (HPLC). The presumptive ABA fractions were collected and the²H-ABA was measured by combined gas chromatography-mass spectrometry (GC-MS) with an²H-ABA standard. Most of the enzyme activity was found in the microsomal fraction. Typical reaction rates were on the order of 1.5 pmol²H-ABA formed/min/mg protein. The reaction required NADPH and was enhanced by FAD. Triarimol, a substituted pyrimidine, which inhibits ABA synthesis inC. rosicola, had no effect on the conversion of 1′-deoxy-²H-ABA to²H-ABA.     

Biosynthesis of the angiogenesis inhibitor borrelidin by Streptomyces parvulus Tü4055: insights into nitrile formation

The 18-membered polyketide macrolide borrelidin exhibits a number of important biological activities, including potent angiogenesis inhibition. This has prompted two recent total syntheses as well as the cloning of the biosynthetic gene cluster from Streptomyces parvulus Tü4055. Borrelidin possesses some unusual structural characteristics, including a cyclopentane carboxylic acid moiety at C17 and a nitrile moiety at C12 of the macrocyclic ring. Nitrile groups are relatively rare in nature, and little is known of their biosynthesis during secondary metabolism. The nitrile group of borrelidin is shown here to arise from the methyl group of a methylmalonyl-CoA extender unit incorporated during polyketide chain extension. Insertional inactivation of two genes in the borrelidin gene cluster, borI (coding for a cytochrome P450 monooxygenase) and borJ (coding for an aminotransferase), generated borrelidin non-producing mutants. These mutants accumulated different compounds lacking the C12 nitrile moiety, with the product of the borI-minus mutant (12-desnitrile-12-methyl-borrelidin) possessing a methyl group and that of the borJ-minus mutant (12-desnitrile-12-carboxyl-borrelidin) a carboxyl group at C12. The former but not the latter was converted into borrelidin when biotransformed by an S. parvulus mutant that is deficient in the biosynthesis of the borrelidin starter unit. This suggests that 12-desnitrile-12-methyl-borrelidin is a competent biosynthetic intermediate, whereas the carboxylated derivative is a shunt metabolite. Bioconversion of 12-desnitrile-12-methyl-borrelidin into borrelidin was also achieved in a heterologous system co-expressing borI and borJ in Streptomyces albus J1074. This bioconversion was more efficient when borK, which is believed to encode a dehydrogenase, was simultaneously expressed with borI and borJ. On the basis of these findings, a pathway is proposed for the formation of the nitrile moiety during borrelidin biosynthesis.     

Enzymatic Synthesis of 2′,5′-Dideoxv Purine Nucleosides and Related Compounds

Abstract

A wide range of 2′,5′-dideoxy-nucleosides, including 6- substituted purine, pyrazolo[3,4-d]pyrimidine and 1-deazapurine derivatives, has been enzymatically prepared using purine nucleoside phosphorylase. Specificity towards cleavage by bacterial versus mammalian purine nucleoside phosphorylase was evaluated.     

Functionaleation of Nucleoside Analogs: Regiospecific Conversion of 4″-Hydroxyl Groups to Thioesters and Deoxygenation of 3′-Hydroxyl Groups

Abstract

Nucleoside analogs 3a-e were conveniently functionalized by regiospecifically converting 4″-hydroxyl groups to thioesters 4a-e (>90% yield) and reducing 3-hydroxyl groups to give building blocks 2a-e in 85–90% yields.     

Effect of the Bacterial Hemoglobin vhbGene on the Efficiency of Intergeneric Conjugation Escherichia coli–Streptomyces and Biosynthesis of Antibiotics in Streptomyces

The bacterial hemoglobin vhbgene was cloned from sliding bacterium Vitreoscillasp. as an element of the system ensuring survival of this microorganism in an environment that contains insufficient amount of oxygen. The vhbgene was transferred fromEscherichia colito some Streptomycesstrains, producers of antibiotics, by the method of intergeneric conjugation using conjugative–integrative plasmid vectors pIH1 and pCH2. The stability of plasmid DNA inheritance was analyzed in the genomes of exconjugants. A positive effect of the vhbgene on processes of conjugation and antibiotic production in a number of examined strains was shown.     

Inhibition of Rac and Rac-linked functions by 8-oxo-2′-deoxyguanosine in murine macrophages

Rac is a protein involved in the various functions of macrophages (Mφ), including the production of reactive oxygen species (ROS), phagocytosis, chemotaxis and the secretion of cytokines (such as γ-INF). This study tested the effects of nucleosides containing 8-oxoguanine(8-hydroxyguanine) such as 8-oxo-2′-guanosine (8-oxoG) or 8-oxo-2′-deoxyguanosine (8-oxodG), on Rac and the above-listed Rac-associated functions of Mφ using mouse peritoneal Mφ (MpMφ). It is reported that 8-oxodG was able to effectively inhibit Rac and the Rac-associated functions of MpMφ. Compared to 8-oxodG, 8-oxoG showed negligible effects. Furthermore, normal nucleosides such as deoxyguanosine (dG), guanosine (G) and adenosine (A) did not exert any effects. These results suggest that 8-oxodG could be used as a potential tool to modulate the functions of Mφ that are intimately related to various pathological processes.     

Integrating Separation and Conversion—Conversion of Biorefinery Process Streams to Biobased Chemicals and Fuels

The concept of the integrated biorefinery is critical to developing a robust biorefining industry in the USA. Within this model, the biorefinery will produce fuel as a high-volume output addressing domestic energy needs and biobased chemical products (high-value organics) as an output providing necessary economic support for fuel production. This paper will overview recent developments within two aspects of the integrated biorefinery—the fractionation of biomass into individual process streams and the subsequent conversion of lignin into chemical products. Solvent-based separation of switchgrass, poplar, and mixed feedstocks is being developed as a biorefinery “front end” and will be described as a function of fractionation conditions. Control over the properties and structure of the individual biomass components (carbohydrates and lignin) can be observed by adjusting the fractionation process. Subsequent conversion of the lignin isolated from this fractionation leads to low molecular weight aromatics from selective chemical oxidation. Together, processes such as these provide examples of foundational technology that will contribute to a robust domestic biorefining industry.     

β-Lactamase genes of Streptomyces badius,Streptomyces cacaoi and Streptomyces fradiae: Cloning and expression in Streptomyces lividans

Genes encoding extracellular β-lactamases (EC 3.5.2.6) of Gram-positive Streptomyces badius, Streptomyces cacaoi and Streptomyces fradiae have been cloned into Streptomyces lividans. The β-lactamase gene of S. badius was initially isolated on a 7 kb BamHI fragment and further located on a 1300 bp DNA segment. An 11 kb BamHI fragment was isolated encompassing the S. cacaoi β-lactamase gene, which was subcloned to a 1250 bp DNA fragment. The β-lactamase gene of S. fradiae was cloned on an 8 kb BamHI fragment and mapped to a 4 kb DNA segment. Each of the three BamHI fragments encompassing the β-lactamase genes hybridized to a BamHI fragment of the corresponding size in chromosomal DNA from the respective strain used for cloning. The activities of the three β-lactamases were predominantly found to be extracellular in the S. lividans recombinants. The S. badius and S. cacaoi β-lactamases exhibited a 10–100-times lower activity in S. lividans, whereas the S. fradiae β-lactamase showed an approximately 10-fold higher activity in the cloned state, compared with the activities found in the original strains.