Metabolic engineering of Escherichia coli to produce (2S, 3R, 4S)-4-hydroxyisoleucine

The stereo-specific l-isoleucine-4-hydroxylase (l-isoleucine dioxygenase (IDO)) was cloned and expressed in an Escherichia coli 2Δ strain lacking the activities of α-ketoglutarate dehydrogenase (EC 1.2.4.2), isocitrate liase (EC 4.1.3.1), and isocitrate dehydrogenase kinase/phosphatase (EC 2.7.11.5). The 2Δ strain could not grow in a minimal-salt/glucose/glycerol medium due to the blockage of TCA during succinate synthesis. The IDO activity in the 2Δ strain was able to “shunt” destroyed TCA, thereby coupling l-isoleucine hydroxylation and cell growth. Using this strain, we performed the direct biotransformation of l-isoleucine into 4-HIL with an 82% yield.     

Synthesis of 5-Chloro-l-(2, 3-Orisopropylidene-4-keto Rhamnopyranosyl)-uracil

Abstract

Syntheses of 5-chloro-l-C2, 3, 4-tri-0-acetyl-αa-L-rhamnopyrano-syl)-uracil (1), 5-chloro-l-(a-L-rhamnopyranosyl)-uracil (2), 5-chloro-l (2, 3-Oisopropylidene-α-L-rhamnopyranosyl)-uracil (3), and 5-chloro-l-(2, 3-O-isopropylidene-4-Tceto-rhamnopyranosyl)-uracil (4 are reported. Oxidation of 3 to 4 was effected using pyridinium chlorochromate.     

Short and efficient synthesis of (2S,3R,4R,5R) and (2S,3R,4R,5S)-tetrahydroxyazepanes via the Henry reaction

The Henry reaction with the easily available alpha-d-xylo-pentodialdose afforded a diastereomeric mixture of nitroaldoses with the alpha-d-gluco- and beta-l-ido-configuration, respectively, in good yield. When n-BuLi was used as the base, the reaction afforded the alpha-d-gluco-nitroaldose as the only product. The reduction of the nitro group in the alpha-d-gluco- and beta-l-ido-nitroaldoses, removal of the protecting groups and intramolecular reductive cyclo-amination afforded the corresponding (2S,3R,4R,5R) and (2S,3R,4R,5S) tetrahydroxyazepanes.     

3-(4-Methyl-3-pentenyl)-2(5H)-furanone, alpha,alpha-acariolide and 4-(4-methyl-3-pentenyl)-2(5H)-furanone, alpha,beta-acariolide: new monoterpene lactones from the astigmatid mites, Schwiebea araujoae and Rhizoglyphus sp. (Astigmata: Acaridae)

A new monoterpene lactone from the acarid mite, Schwiebea araujoae, was elucidated without its isolation by GC/FT-IR and GC/MS analyses to be 3-(4-methyl-3-pentenyl)-2(5H)-furanone (1) and tentatively named as alpha,alpha-acariolide. The structure of 1 was identified by its synthesis from alpha-bromo-gamma-butyrolactone via 4 reaction steps. The synthesized compound gave the same GC/MS and GC/FT-IR spectra as those of the natural product. The other monoterpene lactone was likewise elucidated from the unidentified Rhizoglyphus mite to be 4-(4-methyl-3-pentenyl)-2(5H)-furanone (2) and named as alpha,beta-acariolide; it was also identified by its synthesis in 5 reaction steps from the same butyrolactone as the starting material. GC/MS and GC/FT-IR spectra of the preparation were identical to those of the natural product.     

Metabolic uncoupling of Shewanella oneidensis MR-1, under the influence of excess substrate and 3, 3', 4', 5-tetrachlorosalicylanilide (TCS)

The dissociation between catabolism and anabolism is generally termed as metabolic uncoupling. Experimentally, metabolic uncoupling is characterized by a reduction in the observed biomass yield. This condition can be brought about by: (a) excess-substrate (as measured by S(0)/X(0)), and (b) addition of chemical uncouplers such as 3, 3', 4', 5-Tetrachlorosalicylanilide (TCS). An empirical model is proposed to quantify the uncoupling effects of both excess-substrate and uncoupler addition on the microbial cultures. Metabolic uncoupling of Shewanella oneidensis MR-1, under the influence of excess pyruvate and TCS, has been modeled using the proposed expression. The degree of uncoupling was measured as a fractional reduction in theoretical maximum observed yield. Excess-substrate was observed to successively reduce biomass yield as substrate concentration was increased. In the presence of TCS, conflicting trends were obtained for number yield and protein yield. This could, in part, be attributed to the observed increase in cellular protein content upon addition of TCS. Excess-substrate conditions dominated uncoupling, as compared to uncoupler addition. However, these two approaches were found to have additive effects and could, in conjunction, be employed to control biomass growth during microbial processes such as subsurface bioremediation and activated sludge treatment.     

(2R,3S,4S)-2-(N,N-二乙氨基)甲酰氧基-3-苯基-4-苯甲酰基-N-甲基-Y-内酰胺的合成及工艺研究

目的:通过对黄皮酰胺全合成中间体(2R,3s,4S)-2-羟基-3-苯基-4-苯甲酰基-N-甲基-Y-内酰胺(化合物A)2位羟基的酯化,提高脂水分配系数(1gP),考察对谷丙转氨酶活性的影响。方法:以化合物A为原料,通过酰化反应合成(2R,3S,4S)-2-(N,N-二乙氨基)甲酰氧基-3-苯基-4-苯甲酰基-N-甲基-Y-内酰胺(化合物B),重点考察了摩尔比、反应温度、反应时间等条件对反应的影响。化合物B结构已经元素分析、红外光谱、质谱及核磁共振氢谱确证。结果:化合物A和酰化剂以摩尔比2:3,在160℃下反应1h,目标化合物B,收率78．42%。结论:本合成路线及具体反应方法,具有试剂廉价易得、反应条件温和、后处理简便等优点,是一种较为实用的合成方法。     

Preparation,crystal structure and thermal behaviour of bischromatodihydroxobis-(pyridine)tricopper(II) [Cu3(CrO4)2(OH)2(C5H5N)2]

The title compound was prepared by slow crystallization from a hot aqueous solution of copper(II)- dichromate and pyridine. The structure determination was performed at room temperature on a single crystal in the triclinic space group P$\text{1}$, a = 5.378(1), b = 5.619(1), c = 13.569(2) Å, α = 93.32(1), β = 100.25(1), γ=98.45(1)°. Using 2026 reflections with F_o² > (F_o²) obtained on a CAD-4 single crystal diffractometer the structure was solved by conventional Patterson and Fourier methods and full matrix least-squares refinement to R = 0.047. The structure consists of complex chains built up from two different (4 + 2) distorted copper(II) octahedra sharing common edges. These chains are linked via OCrO bonds thus forming a two-dimensional infinite network. The pyridine rings extending into the space between these layers are disordered due to rotation around the CuN bond. In the course of the refinement two favoured positions with occupation probabilities 50:50 percent were found. During thermal decomposition the compound loses pyridine and water followed by a release of oxygen to yield poly- crystalline CuCr₂O₄ and CuO. An intermediate phase with empirical formula Cu₃O(CrO₄)₂ was detected by X-ray powder diffraction and its unit cell parameters were determined.     

(2')3',5'-Bisphosphate nucleotidase

(2')3',5'-Bisphosphate nucleotidase has been prepared in electrophoretically homogeneous form from guinea pig liver. The enzyme catalyzes the hydrolysis of the 2'- or 3'-phosphate from the appropriate nucleoside 2',5'- and 3',5'-bisphosphates and is active with 3'-phosphoadenosine 5'-phosphosulfate and with coenzyme A but not with ATP. The 40,000-dalton protein is a monomer that requires Mg2+ for activity.     

The formation mechanism by yeast of 4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone in Miso

The mechanism of the formation of 4-hydroxy-2(or 5)-ethyl-5-(or 2)-methyl-3(2H)-furanone (HEMF) with yeast under caltivation in a medium containing amino-carbonyl reactants of ribose and glycine was investigated using stable isotopes of the corresponding compounds. It was confirmed that the skeleton of the five-membered ring and the methyl group of the side chain of HEMF was formed from ribose, and that the ethyl group was derived from the glucose metabolite by yeast. The formation of HEMF was confirmed when acetaldehyde as the glucose metabolite and a cell-free extract from yeast were added to the medium containing amino-carbonyl reactants. These results suggest that the role of yeast in HEMF formation is not only to provide the glucose metabolite, but also in combining the amino-carbonyl reactants with the glucose metabolite.     

Specific phosphorylase from Euglena gracilis splits diadenosine 5',5''-P(1),P(4)-tetraphosphate (Ap(4)A) and diadenosine 5',5''-P(1),P(3)-triphosphate (Ap(3)A)

1. Phosphorolytic cleavage of Ap(4),A was demonstrated in cell-free extracts from two protozoan organisms, Euglena gracilis and Acanthamoeba castellanii. 2. A specific dinucleoside oligophosphate (DNOP) alpha, beta-phosphorylase which degrades substrates with formation of corresponding nucleoside 5'-diphosphate (NDP) as one of the reaction products was purified 625-fold from Euglena gracilis cells. 3. In addition to Ap(4)A, the phosphorylase degrades AP(3)A, Ap(5)A, Gp(4)G and one of phosphonate analogs, ApppCH(2)pA. The K(m) values for Ap(4), A and Ap(3) A are 27 and 25 micron, and relative velocities 100 and 14, respectively. The K(m) for phosphate is 0.5 mM. 4. Some anions (arsenate, chromate, molybdate and vanadate) can substitute for phosphate in the catalyzed reactions and in their presence the DNOPs yield corresponding nucleoside 5'-monophosphate as one of the reactions' product. The enzyme supports also an anion-dependent dephosphorylation of NDPs. 5. Molecular weight of the native Euglena phosphorylase is 30,000. Optimum pH for its activity is at 8.0 Divalent metal cations are essential for the phosphorolysis of DNOPs but are not for the NDP dephosphorylation mentioned.     

Synthesis and antiviral activity assay of novel (E)-3',5'-diamino-5-(2-bromovinyl)-2',3',5'-trideoxyuridine

(E)-3',5'-diamino-5-(2-bromovinyl)-2',3',5'-trideoxyuridine (5), the diamino analogue of BVDU (1), was synthesized from BVDU. In contrast with BVDU, compound 5 did not show activity against herpes simplex virus or varicella-zoster virus.     

(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone reduces corrosion from Desulfotomaculum orientis

(5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone (furanone) from the red marine alga Delisea pulchra was found previously to inhibit the growth, swarming and biofilm formation of Gram-positive bacteria (Ren et al., 2002, Lett Appl Microbiol 34: 293-299). In the present study, the Gram-positive sulphate-reducing bacterium (SRB), Desulfotomaculum orientis, was used to study the inhibition of mild steel corrosion due to the addition of furanone. The weight loss from batch coupon experiments incubated with 40 microg x ml(-1) furanone was reduced fivefold compared with samples that lacked furanone. Analysis of the metal surface with environmental scanning electron microscopy further confirmed the protection afforded by the addition of furanone. In agreement with the corrosion inhibition, most probable number (MPN) analysis showed that 20 and 40 microg x ml(-1) furanone inhibited 58% and 96% of the D. orientis growth respectively. Hence, furanone has the potential to inhibit microbial-induced corrosion related to Gram-positive bacteria.     

Comparative evaluation of 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium,monosodium salt (WST-8) rapid colorimetric assays for antimicrobial susceptibility testing of staphylococci and ESBL-producing clinical isolates

A new generation of water soluble tetrazolium salts have recently become available and in this study we compared a colorimetric assay developed using one of these salts, 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2, 4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-8), with a previously developed 2,3-bis [2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) colorimetric assay to determine which agent is most suitable for use as a colorimetric indicator in susceptibility testing. The MICs of 6 antibiotics were determined for 33 staphylococci using both colorimetric assays and compared with those obtained using the British Society for Antimicrobial Chemotherapy reference broth microdilution method. Absolute categorical agreement between the reference and test methods ranged from 79% (cefuroxime) to 100% (vancomycin) for both assays. No minor or major errors occurred using either assay with very major errors ranging from zero (vancomycin) to seven (cefuroxime). Analysis of the distribution of differences in the log₂ dilution MIC results revealed overall agreement, within the accuracy limits of the standard test (± 1 log₂ dilution), using the XTT and WST-8 assays of 98% and 88%, respectively. Further studies on 31 ESBL-producing isolates were performed using the XTT method with absolute categorical agreement ranging from 87% (nitrofurantoin) to 100% (ofloxacin and meropenem). No errors were noted for either ofloxacin or meropenem with overall agreement of 91%. The data suggests that XTT is more reliable and accurate than WST-8 for use in a rapid antimicrobial susceptibility test.