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Four deoxyxylulose phosphate (DXP) analogues were synthesized and evaluated as substrates/inhibitors for methylerythritol phosphate (MEO) synthase. In analogues CF(sub 3)-DXP, CF(sub 2)-DXP, and CF-DXP, the three methyl hydrogens at C1 of DXP were sequentially replaced by fluorine, while in Et-DP, the methyl group in DXP was replaced by an ethyl moiety.

Research has been conducted on the 2-C-methyl-D-erythritol-4-phosphate synthase which catalyzes the reduction of 1-D-deoxyxylulose-5-phosphate to methylerythriol-4-phosphate. The purification of the Escherichia coli recombinant 2-C-methyl-D-erythritol-4-phosphate synthase has been carried out and the results are reported.

The research report on the siderophore petrobactin produced by the causative agent of anthrax (Bacillus anthracis) and number of other pathogenic species such as Bacillus thuringiensis 97-27 and B. cereus G9241 is reviewed. The gene encoding AsbF from Bacillus thuringiensis 97-27, which is overexpressed in an Escherichia coli host, has rapidly and efficiently transformed (-)-3-dehydroshikimate (DHS) into 3,4-dihydroxybenzoate.

Several isotopic feeding experiments are conducted to explain the mechanisms involved in the biosynthesis of the 3,4-dihydroxybenzoate moieties of the siderophore petrobactin by Bacillus anthracis. Phosphophenol pyruvate and erythrose-4-phosphate are shown to act as the main precursors for the construction of carbon backbone of the complex.

A study on mechanism of the reaction catalyzed by 2-C-methyl-D-erthritol 4-phosphate (MEP) synthesis from Escherichia coli is studied. The isotope effect with 1-flouro-deoxy-D-xylulose 5-phospheric acid(CF-DXP) and 4S-[(super 2 H)]NADPH but not DXP indicates that the rearrangement step, that precedes hydride transfer, is rate-limiting for DXP but becomes partially rat-limiting for CF-DXP.

Research has been conducted on the isoprenoid compounds. The syntheses of (E)-1-hydroxy-2-methyl-2-buten-4-yl diphosphate and (E)-[4- (super)2 H](E)-4-hydroxydimethylallyl diphosphate have been carried out from (E)-4-chloro-2-methyl-2-butenal and the details are reported.

Thermostabilization of an enzyme with complete retention of catalytic efficiency was demonstrated on recombinant 3-dehydroshikimate dehydratase (DHSase or wtAsbF) from Bacillus thuringiensis serovar konkukian 97-27 (hereafter, B. thuringiensis 97-27). The wtAsbF is relatively unstable at 37 °C, in vitro (t 1/2 = 15 min), in the absence of divalent metal. We adopted a structure-based design to identify stabilizing mutations and created a combinatorial library based upon predicted mutations at specific locations on the enzyme surface. A diversified asbF library (∼2000 variants) was expressed in E. coli harboring a green fluorescent protein (GFP) reporter system linked to the product of wtAsbF activity (3,4-dihydroxybenzoate, DHB). Mutations detrimental to DHSase function were rapidly eliminated using a high throughput fluorescence activated cell sorting (FACS) approach. After a single sorting round and heat screen at 50 °C, a triple AsbF mutant (Mut1), T61N, H135Y, and H257P, was isolated and characterized. The half-life of Mut1 at 37 °C was >10-fold higher than the wtAsbF (t 37 = 15 min), in the absence of divalent metal. We adopted a structure-based design to identify stabilizing mutations and created a combinatorial library based upon predicted mutations at specific locations on the enzyme surface. A diversified asbF library (∼2000 variants) was expressed in E. coli harboring a green fluorescent protein (GFP) reporter system linked to the product of wtAsbF activity (3,4-dihydroxybenzoate, DHB). Mutations detrimental to DHSase function were rapidly eliminated using a high throughput fluorescence activated cell sorting (FACS) approach. After a single sorting round and heat screen at 50 °C, a triple AsbF mutant (Mut1), T61N, H135Y, and H257P, was isolated and characterized. The half-life of Mut1 at 37 °C was >10-fold higher than the wtAsbF (t 1/2 37 = 169 min). Further, the second-order rate constants for both wtAsbF and Mut1 were approximately equal (9.9 × 10 5 M -1 s -1 , 7.8 × 10 5 M -1 s -1 , respectively), thus demonstrating protein thermostability did not come at the expense of enzyme thermophilicity. In addition, in vivo overexpression of Mut1 in E. coli resulted in a ∼60-fold increase in functional enzyme when compared to the wild-type enzyme under the identical expression conditions. Finally, overexpression of the thermostable AsbF resulted in an approximate 80-120% increase in DHB accumulation in the media relative to the wild-type enzyme.

Until recently, engineering strategies for altering gene expression have focused on transcription control using strong inducible promoters or one of several methods to knock down wasteful genes. Recently, synthetic riboregulators have been developed for translational regulation of gene expression. Here, we report a new modular synthetic riboregulator class that has the potential to finely tune protein expression and independently control the concentration of each enzyme in an engineered metabolic pathway. This development is important because the most straightforward approach to altering the flux through a particular metabolic step is to increase or decrease the concentration of the enzyme. Our design includes a cis-repressor at the 5' end of the mRNA that forms a stem-loop helix, occluding the ribosomal binding sequence and blocking translation. A trans-expressed activating-RNA frees the ribosomal-binding sequence, which turns on translation. The overall architecture of the riboregulators is designed using Watson-Crick base-pairing stability. We describe here a cis-repressor that can completely shut off translation of antibiotic-resistance reporters and a trans-activator that restores translation. We have established that it is possible to use these riboregulators to achieve translational control of gene expression over a wide dynamic range. We have also found that a targeting sequence can be modified to develop riboregulators that can, in principle, independently regulate translation of many genes. In a selection experiment, we demonstrated that by subtly altering the sequence of the trans-activator it is possible to alter the ratio of the repressed and activated states and to achieve intermediate translational control.