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Botryococcus braunii has long been known as a prodigious producer of liquid hydrocarbon oils that can be converted into combustion engine fuels. This draft genome for the B race of B. braunii will allow researchers to unravel important hydrocarbon biosynthetic pathways and identify possible regulatory networks controlling this unusual metabolism.

In 2010, when the National Alliance for Advanced Biofuels and Bioproducts (NAABB) consortium began, little was known about the molecular basis of algal biomass or oil production. Very few algal genome sequences were available and efforts to identify the best-producing wild species through bioprospecting approaches had largely stalled after the U.S. Department of Energy's Aquatic Species Program. This lack of knowledge included how reduced carbon was partitioned into storage products like triglycerides or starch and the role played by metabolite remodeling in the accumulation of energy-dense storage products. Furthermore, genetic transformation and metabolic engineering approaches to improve algal biomass and oil yields were in their infancy. Genome sequencing and transcriptional profiling were becoming less expensive, however; and the tools to annotate gene expression profiles under various growth and engineered conditions were just starting to be developed for algae. It was in this context that an integrated algal biology program was introduced in the NAABB to address the greatest constraints limiting algal biomass yield. This review describes the NAABB algal biology program, including hypotheses, research objectives, and strategies to move algal biology research into the twenty-first century and to realize the greatest potential of algae biomass systems to produce biofuels.

Recent observations have shed light on some of the endogenous iron-acquisition mechanisms of members of the Bacillus cereus sensu lato group. In particular, pathogens in the B. cereus group use siderophores with both unique chemical structures and biological roles. This review will focus on recent discoveries in siderophore biosynthesis and biology in this group, which contains numerous human pathogens, most notably the causative agent of anthrax, Bacillus anthracis. DOI 10.1099/mic.0.039404-0

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.

Fosmidomycin is a time-dependent nanomolar inhibitor of methylerythritol phosphate (MEP) synthase, which is the enzyme that catalyzes the first committed step in the MEP pathway to isoprenoids. Importantly, fosmidomycin is one of only a few MEP pathway-specific inhibitors that exhibits antimicrobial activity. Most inhibitors identified to date only exhibit activity against isolated pathway enzymes. The MEP pathway is the sole route to isoprenoids in many bacteria, yet has no human homologs. The development of inhibitors of this pathway holds promise as novel antimicrobial agents. Similarly, analyses of the bacterial response toward MEP pathway inhibitors provides valuable information toward the understanding of how emergent resistance may ultimately develop to this class of antibiotics. We have examined the transcriptional response of Salmonella enterica serovar typhimurium LT2 to sub-inhibitory concentrations of fosmidomycin via cDNA microarray and RT-PCR. Within the regulated genes identified by microarray were a number of genes encoding enzymes associated with the mediation of reactive oxygen species (ROS). Regulation of a panel of genes implicated in the response of cells to oxidative stress (including genes for catalases, superoxide dismutases, and alkylhydrogen peroxide reductases) was investigated and mild upregulation in some members was observed as a function of fosmidomycin exposure over time. The extent of regulation of these genes was similar to that observed for comparable exposures to kanamycin, but differed significantly from tetracycline. Furthermore, S. typhimurium exposed to sub-inhibitory concentrations of fosmidomycin displayed an increased sensitivity to exogenous H2O2 relative to either untreated controls or kanamycin-treated cells. Our results suggest that endogenous oxidative stress is one consequence of exposures to fosmidomycin, likely through the temporal depletion of intracellular isoprenoids themselves, rather than other mechanisms that have been proposed to facilitate ROS accumulation in bacteria (e.g. cell death processes or the ability of the antibiotic to redox cycle). [ABSTRACT FROM AUTHOR]

Fosmidomycin is a time-dependent nanomolar inhibitor of methylerythritol phosphate (MEP) synthase, which is the enzyme that catalyzes the first committed step in the MEP pathway to isoprenoids. Importantly, fosmidomycin is one of only a few MEP pathway-specific inhibitors that exhibits antimicrobial activity. Most inhibitors identified to date only exhibit activity against isolated pathway enzymes. The MEP pathway is the sole route to isoprenoids in many bacteria, yet has no human homologs. The development of inhibitors of this pathway holds promise as novel antimicrobial agents. Similarly, analyses of the bacterial response toward MEP pathway inhibitors provides valuable information toward the understanding of how emergent resistance may ultimately develop to this class of antibiotics. We have examined the transcriptional response of Salmonella enterica serovar typhimurium LT2 to sub-inhibitory concentrations of fosmidomycin via cDNA microarray and RT-PCR. Within the regulated genes identified by microarray were a number of genes encoding enzymes associated with the mediation of reactive oxygen species (ROS). Regulation of a panel of genes implicated in the response of cells to oxidative stress (including genes for catalases, superoxide dismutases, and alkylhydrogen peroxide reductases) was investigated and mild upregulation in some members was observed as a function of fosmidomycin exposure over time. The extent of regulation of these genes was similar to that observed for comparable exposures to kanamycin, but differed significantly from tetracycline. Furthermore, S. typhimurium exposed to sub-inhibitory concentrations of fosmidomycin displayed an increased sensitivity to exogenous H2O2 relative to either untreated controls or kanamycin-treated cells. Our results suggest that endogenous oxidative stress is one consequence of exposures to fosmidomycin, likely through the temporal depletion of intracellular isoprenoids themselves, rather than other mechanisms that have been proposed to facilitate ROS accumulation in bacteria (e.g. cell death processes or the ability of the antibiotic to redox cycle). [ABSTRACT FROM AUTHOR]

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.

Isoprenoids constitute the largest class of natural products with greater than 55,000 identified members. They play essential roles in maintaining proper cellular function leading to maintenance of human health, plant defense mechanisms against predators, and are often exploited for their beneficial properties in the pharmaceutical and nutraceutical industries. Most impressively, all known isoprenoids are derived from one of two C 5 -precursors, isopentenyl diphosphate (IPP) or dimethylallyl diphosphate (DMAPP). In order to study the enzyme transformations leading to the extensive structural diversity found within this class of compounds there must be access to the substrates. Sometimes, intermediates within a biological pathway can be isolated and used directly to study enzyme/pathway function. However, the primary route to most of the isoprenoid intermediates is through chemical catalysis. As such, this review provides the first exhaustive examination of synthetic routes to isoprenoid and isoprenoid precursors with particular emphasis on the syntheses of intermediates found as part of the 2 C -methylerythritol 4-phosphate (MEP) pathway. In addition, representative syntheses are presented for the monoterpenes (C 10 ), sesquiterpenes (C 15 ), diterpenes (C 20 ), triterpenes (C 30 ) and tetraterpenes (C 40 ). Finally, in some instances, the synthetic routes to substrate analogs found both within the MEP pathway and downstream isoprenoids are examined.