Redesigning enzymes based on adaptive evolution for optimal function in synthetic metabolic pathways

Nature has balanced most metabolic pathways such that no one enzyme in the pathway controls the flux through that pathway. However, unnatural or nonnative, constructed metabolic pathways may have limited product flux due to unfavorable in vivo properties of one or more enzymes in the pathway. One such example is the mevalonate-based isoprenoid biosynthetic pathway that we previously reconstructed in Escherichia coli. We have used a probable mechanism of adaptive evolution to engineer the in vivo properties of two enzymes (3-hydroxy-3-methylglutaryl-CoA reductase [tHMGR] and many terpene synthases) in this pathway and thereby eliminate or minimize the bottleneck created by these inefficient or nonfunctional enzymes. Here, we demonstrate how we significantly improved the productivity (by approximately 1000 fold) of this reconstructed biosynthetic pathway using this strategy. We anticipate that this strategy will find broad applicability in the functional construction (or reconstruction) of biological pathways in heterologous hosts.     

Effect of Processing Conditions on Heat Seal Strength for Peelable Heat Sealing of Multilayered Polyethylene Films with Different Sealant Layers

The effect of heat sealing temperature on peelable heat seal strength (HSS) before and after the sterilization, often used industrially, was investigated for one of either polyethylene (PE) multilayered films with various inner sealant layers consisting of two high density PEs (HDPEs), a linear low density PE (LLDPE) or low density PE (LDPE). To attain similar HSS, heat sealing at a temperature close to the melting peak temperature (Tm-p) was needed only in the case of LDPE. Heat sealing behavior at higher temperatures, to attain higher HSS, was also investigated for HDPE and LDPE. High HSS, as well as suppressed reduction of HSS after the sterilization, was achieved for HDPE at a heat sealing temperature well below its Tm-p. An Increase of HSS with the increase of dwell time was also observed. On the other hand, for LDPE, high HSS and its stability after the sterilization were achieved only when the heat sealing temperature was increased to the value equivalent to its Tm-p. Improvement of the processing conditions for the shortening of the processing period was also investigated. For HDPE, higher sealing bar temperature was needed to gain similar HSS when the dwell time of the heat sealing was shortened from 4 to 2 s, whereas the attained maximum temperature at the interface was found to be similar. The characteristics of the heat sealed portion, judged from the relation between HSSs before and after the sterilization, were not affected by the dwell time of heat sealing, indicating that the shortening of the processing time by appropriate raising of the sealing bar temperature is possible.     

Colchicine Models: Synthesis and Binding to Tubulin of Tertamethoxybiphenyls

Syntheis of tetramethoxybiphenyl 21 was accomplished from 4-phenylcylohexane-1,3-dione 13 by aromatization to biphenyl 19 and reductive removal of the phenolic OH group as phenyltetrazolyl ether. Tertramethoxybiphenyls 34 and 40 were obtained from 4-phenylcyclohexenone 26 via ester 27 . The tetramethoxybiphenyls 21, 34 , and 40 , and analogs 28, 29 , and 31 were evaluated for antitubulin activity and as antimitotic agents with L1210 murine leukemia cells. Compounds 31 and 34 had significant effects on the in-vitro polymerization of tubulin. Compound 31 was the most cytotoxic of the six new biphenyls studied (IC₅₀ for cell growth, 0.6M ) and caused the accumulation of cells in metaphase arrest.     

Rapid decomposition and dissolution of silicate rocks by fusion with lithium tetraborate and lithium sulphate

Fusion with 1.0 g of Li(2)B(4)O(7)Li(2)SO(4) (2:1) mixture in a platinum crucible at 1000 degrees will decompose 0.1 g of silicate rock in less than 10-15 min, and the cooled fusion cake can be completely dissolved by 20 ml of 1.2M hydrochloric acid at 90-100 degrees in approximately 5 min.     

Colchicine models: Synthesis and Antitubulin Activity of 2′-Monosubstituted and 2′, 5-Disubstituted 2,3,4,4′-Tetramethoxy-1,1′-biphenyls. Synthesis of 4,4′, 5′, 6′-tetramethoxy-1,1′-biphenyl-2,3′-dicarboxylic acid

Reductive amination of 2,3,4,4′-tetramethoxtybiphenyl-2-carbaldehyde ( 4 ) with MeNH₂ afforded methylamine 5 (Scheme 1), Hydroxymethylation of amine 8 , prepared similarly from 4 by reductive amination with benzylamine followed by N-methylation, afforded alcohol 12 which was converted the 5-methyl-substituted methylamine 14 by conventional chemical reactions (Scheme 2), Methylamine 14 was also obtained from ester 16 after hydroxymethylation to alcohol 17 and conventional manipulation of alcohol and ester functions (Scheme 2). Both amines 5 and 14 as well as the 2′, 5-dimethyl-substituted biphenyl 26 prepared from the dialdehyde 25 by a Wolff-Kishner reduction, did not show noteworthy activity in the tubulin binding assay or as inhibitors of tubulin polymerization (Table). However, the 2′ethyl-substituted biphebyl 11 prepared from 4 by reaction with MeLi followed by dehyderation and catalytic reduction of styrene 10 (Scheme 1) showed appreciable activity in both assays, coming close to that of known phenyltropolone models. The X-ray analysis of 14 ·HCl and 11 showed significant difference in the orientation of the rings with respect to one another (Fig.).     

Computer-aided measurement of kinetic parameters of electrode reactions of cobalt(III)—ammine complexes at mercury electrodes

A computer-aided technique based on Tast polarography is examined for the determination of kinetic parameters of electrode reactions. It is particularly useful for the investigation of unstable species because of the simple and rapid processing of data. Kinetic parameters of cobalt(III)—ammine complexes at mercury electrodes are given for [Co(NH₃)₆]Cl₃, [Co(H₂O)(NH₃)₅](ClO₄)₃, [Co(NO₃)(NH₃)₅](NO₃)₂ [CoF(NH₃)₅](Cl0₄)₂, [Co(C0₃)(NH₃)₅] NO₃ ,cis-[Co(H₂O)₂(NH₃)₄] (Cl0₄)₃ [Co(CO₃)(NH₃)₄] NO₃ · 0.5H₂0, [Co(ox)(NH₃)₄]Cl · H₂0, and NH₄[Co(ox)₂(NH₃)₂] · H₂O, which are obtained in solutions containing 0.1 M acetate buffer and 0.005% gelatin at 25°C.     

Adsorption Equilibrium Equation of Carboxylic Acids on Anion-Exchange Resins in Water

The adsorption of propionic acid and benzoic acid on anion-exchange resins was analyzed, and an adsorption equilibrium equation of carboxylic acids was proposed. The adsorption of carboxylic acids on the anion-exchange resins was considered to be the sum of the physical adsorption of the molecule and the ion-exchange adsorption of the ion, which were independent of each other. For the physical adsorption of carboxylic acids, it was conformed to the Freundlich equation. For the ion-exchange adsorption of carboxylate ions, the equilibrium equation corresponded well with the experimental results for wide ranges of concentration and pH. The equation contains a selectivity coefficient S(A)(Cl) for the chloride ion versus the carboxylate ion, which was considered essentially a constant. The influent of the bicarbonate ion from carbon dioxide in air could also be expressed by the additional equilibrium equation with the selectivity coefficient S(HCO(3))(Cl) for the chloride ion versus the bicarbonate ion. Consequently, an adsorption equilibrium equation can estimate the equilibrium adsorption amounts. Even the effect of a coexisting bicarbonate ion is inconsequential when the parameters of the Freundlich isotherm equation and the selectivity coefficients of the carboxylate ion and the bicarbonate ion in each resin are determined in advance. Copyright 2001 Academic Press.     

Novel rapid decomposition and dissolution method for silicates using a mixed potassium metaborate/potassium carbonate flux

A novel method for the rapid dissolution of fused silicates in mineral acid is described. Fusion with 2.5 g of a KBO(2)-K(2)CO(3) (3:2, w/w) mixture in a platinum crucible at 1000 degrees C will decompose 0.1 g of silicate samples such as basalt rock, glass sand and powdered glass in 10 min, and the cooled fusion cakes can be completely dissolved by 20 ml of 3 N mineral acids such as hydrochloric, nitric and sulfuric acids in less than 1 min at about 50 degrees C. Fusion with 5.0 g of a KBO(2)-K(2)CO(3) (3:2, w/w) mixture can completely decompose 0.1 g of chrome refractory in 20 min and the cooled melt can be dissolved by 80 ml of 3 N hydrochloric or sulfuric acids in less than 30 s.     

Parity non-conserving effect in the neutron radiative capture reaction

Helicity dependence was observed for the resonance of the neutron radiative capture reaction of¹³⁹La using a polarized neutron beam. The polarized neutron beam was produced by a dynamically polarized proton filter. The neutron polarization was about 70 % throughout the measurement. The preliminary result of the asymmetry in the-ray count was (9.5±1.2) %. Experiments were also carried out for⁹⁸Mo,¹⁰⁸Pd and¹²⁹Xe. Such a large asymmetry as that found in¹³⁹La was not observed in these nuclei.