High-throughput metabolic engineering: advances in small-molecule screening and selection.

Metabolic engineering for the overproduction of high-value small molecules is dependent upon techniques in directed evolution to improve production titers. The majority of small molecules targeted for overproduction are inconspicuous and cannot be readily obtained by screening. We provide a review on the development of high-throughput colorimetric, fluorescent, and growth-coupled screening techniques, enabling inconspicuous small-molecule detection. We first outline constraints on throughput imposed during the standard directed evolution workflow (library construction, transformation, and screening) and establish a screening and selection ladder on the basis of small-molecule assay throughput and sensitivity. An in-depth analysis of demonstrated screening and selection approaches for small-molecule detection is provided. Particular focus is placed on in vivo biosensor-based detection methods that reduce or eliminate in vitro assay manipulations and increase throughput. We conclude by providing our prospectus for the future, focusing on transcription factor-based detection systems as a natural microbial mode of small-molecule detection.

Synthesis of three advanced biofuels from ionic liquid-pretreated switchgrass using engineered Escherichia coli.

One approach to reducing the costs of advanced biofuel production from cellulosic biomass is to engineer a single microorganism to both digest plant biomass and produce hydrocarbons that have the properties of petrochemical fuels. Such an organism would require pathways for hydrocarbon production and the capacity to secrete sufficient enzymes to efficiently hydrolyze cellulose and hemicellulose. To demonstrate how one might engineer and coordinate all of the necessary components for a biomass-degrading, hydrocarbon-producing microorganism, we engineered a microorganism naïve to both processes, Escherichia coli, to grow using both the cellulose and hemicellulose fractions of several types of plant biomass pretreated with ionic liquids. Our engineered strains express cellulase, xylanase, beta-glucosidase, and xylobiosidase enzymes under control of native E. coli promoters selected to optimize growth on model cellulosic and hemicellulosic substrates. Furthermore, our strains grow using either the cellulose or hemicellulose components of ionic liquid-pretreated biomass or on both components when combined as a coculture. Both cellulolytic and hemicellulolytic strains were further engineered with three biofuel synthesis pathways to demonstrate the production of fuel substitutes or precursors suitable for gasoline, diesel, and jet engines directly from ionic liquid-treated switchgrass without externally supplied hydrolase enzymes. This demonstration represents a major advance toward realizing a consolidated bioprocess. With improvements in both biofuel synthesis pathways and biomass digestion capabilities, our approach could provide an economical route to production of advanced biofuels.

A 1-day imported fire ant rush immunotherapy schedule with and without premedication.

BACKGROUND: Rush immunotherapy (RIT) schedules can expedite protection in individuals sensitive to imported fire ant (IFA) stings. OBJECTIVE: To evaluate the safety and efficacy of 1-day RIT with IFA whole body extract (WBE) and determine the benefit of premedication with antihistamines and prednisone. METHODS: Patients with systemic reactions to IFAs and evidence of specific IgE by skin test or serologic test started a 1-day RIT protocol without premedication. The 1-day RIT protocol consisted of a total of 10 injections every 30 to 60 minutes to achieve a 0.3-mL 1:100 (wt/vol) dose. A higher systemic reaction rate (SRR) prompted protocol revision to include a 3-day course of oral 20 mg of prednisone twice daily, 150 mg of ranitidine, and 10 mg of loratadine started 2 days before the 1-day RIT. Patients returned on days 8 and 15 to receive a 0.5 mL 1:100 (wt/vol) maintenance injection. The effectiveness of the RIT was evaluated with a sting challenge on approximately day 22. RESULTS: Eighty of the 96 patients enrolled initiated the 1-day RIT. The first nonpremedicated group exhibited a SRR of 24.3% (9 of 37 patients), whereas the revised premedicated group had a SRR of 9.5% (4 of 42 patients; P = .07). The most severe reaction during RIT included dizziness, angioedema, and urticaria. Sting challenges on 53 patients resulted in 1 mild rhinitis reaction (efficacy, 98.1%). CONCLUSION: One-day RIT with IFA WBE for IFA hypersensitivity is efficacious. Although there was a trend with premedications to reduce SRRs during the RIT, safety data with premedication require confirmation in a larger trial.

Manipulation of the carbon storage regulator system for metabolite remodeling and biofuel production in Escherichia coli.

BACKGROUND: Microbial engineering strategies that elicit global metabolic perturbations have the capacity to increase organism robustness for targeted metabolite production. In particular, perturbations to regulators of cellular systems that impact glycolysis and amino acid production while simultaneously decreasing fermentation by-products such as acetate and CO(2) make ideal targets. Intriguingly, perturbation of the Carbon Storage Regulator (Csr) system has been previously implicated in large changes in central carbon metabolism in E. coli. Therefore, we hypothesized that perturbation of the Csr system through the CsrA-CsrB ribonucleoprotein complex might increase production of biofuels and their intermediates from heterologous pathways. RESULTS: We engaged the CsrA-CsrB ribonucleoprotein complex of E. coli via overexpression of CsrB. CsrB is a 350-nucleotide non-coding RNA that antagonizes CsrA, an RNA-binding protein that regulates translation of specific mRNA targets. By using shotgun proteomics and targeted metabolomics we established that elevation of CsrB levels leads to alterations in metabolite and protein levels in glycolysis, the TCA cycle and amino acid levels. Consequently, we show that such changes can be suitably applied to improve the production of desired compounds through the native fatty acid and heterologous n-butanol and isoprenoid pathways by up to two-fold. We also observed concomitant decreases in undesirable fermentation by-products such as acetate and CO(2). CONCLUSIONS: We have demonstrated that simple engineering of the RNA-based Csr global regulatory system constitutes a novel approach to obtaining pathway-independent improvements within engineered hosts. Additionally, since Csr is conserved across most prokaryotic species, this approach may also be amenable to a wide variety of production hosts.

Activation of the dopamine 1 and dopamine 5 receptors increase skeletal muscle mass and force production under non-atrophying and atrophying conditions.

BACKGROUND: Control of skeletal muscle mass and force production is a complex physiological process involving numerous regulatory systems. Agents that increase skeletal muscle cAMP levels have been shown to modulate skeletal muscle mass and force production. The dopamine 1 receptor and its closely related homolog, the dopamine 5 receptor, are G-protein coupled receptors that are expressed in skeletal muscle and increase cAMP levels when activated. Thus we hypothesize that activation of the dopamine 1 and/or 5 receptor will increase skeletal muscle cAMP levels thereby modulating skeletal muscle mass and force production. METHODS: We treated isolated mouse tibialis anterior (TA) and medial gastrocnemius (MG) muscles in tissue bath with the selective dopamine 1 receptor and dopamine 5 receptor agonist SKF 81297 to determine if activation of skeletal muscle dopamine 1 and dopamine 5 receptors will increase cAMP. We dosed wild-type mice, dopamine 1 receptor knockout mice and dopamine 5 receptor knockout mice undergoing casting-induced disuse atrophy with SKF 81297 to determine if activation of the dopamine 1 and dopamine 5 receptors results in hypertrophy of non-atrophying skeletal muscle and preservation of atrophying skeletal muscle mass and force production. RESULTS: In tissue bath, isolated mouse TA and MG muscles responded to SKF 81297 treatment with increased cAMP levels. Treating wild-type mice with SKF 81297 reduced casting-induced TA and MG muscle mass loss in addition to increasing the mass of non-atrophying TA and MG muscles. In dopamine 1 receptor knockout mice, extensor digitorum longus (EDL) and soleus muscle mass and force was not preserved during casting with SKF 81297 treatment, in contrast to significant preservation of casted wild-type mouse EDL and soleus mass and EDL force with SKF 81297 treatment. Dosing dopamine 5 receptor knockout mice with SKF 81297 did not significantly preserve EDL and soleus muscle mass and force although wild-type mouse EDL mass and force was significantly preserved SKF 81297 treatment. CONCLUSIONS: These data demonstrate for the first time that treatment with a dopamine 1/5 receptor agonist results in (1) significant preservation of EDL, TA, MG and soleus muscle mass and EDL muscle force production during periods of atrophy and (2) hypertrophy of TA and MG muscle. These effects appear to be mainly mediated by both the dopamine 1 and dopamine 5 receptors.

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.

Comparison of serum-specific IgE (ImmunoCAP) and skin-prick test results for 53 inhalant allergens in patients with chronic rhinitis.

Prior studies comparing skin testing to serum-specific IgE testing for inhalant allergy focused on older technologies or small numbers of allergens. The purpose of this study was to compare ImmunoCAP (CAP) testing to skin prick testing (ST) for 53 inhalant allergens. Subjects > or =18 years old with chronic rhinitis and who had at least 1 positive ST to a 53 inhalant allergen panel underwent testing to an analogous CAP panel. ST was performed with the Quintip device. Using ST as a clinical gold standard, the sensitivity, specificity, positive, and negative predictive values (PPV, NPV) were calculated for CAP for each allergen. Percent agreement between testing methods was also evaluated, and the results were analyzed in association with the subjects' total IgE levels. Two-hundred fifty patients (96 male, 154 female, mean 37.1 years) were enrolled. Mean number of positive ST and CAP results were similar. The ST was more often positive for 69.8% of allergens, and 64% of patients had more positive ST than CAP. Overall, the specificity and NPV (generally 80-90%) of CAP were higher than the sensitivity and PPV. The overall agreement between tests was 80.6%, with 11.7% ST+CAP- results and 7.7% CAP+ST- results. In patients with a total IgE level > or = 200 IU/L, the percentage of positive CAP results for 52/53 allergens was significantly higher with more CAP+ST- results. The performance characteristics of CAP compared to ST vary among 53 inhalant allergens. CAP should be considered complementary, not equivalent, to ST. Total IgE levels should be obtained with serum-specific IgE testing.

Characterization of fibroblast morphology on bioactive surfaces using vertical scanning interferometry.

Tissue donor scarcity is a major hindrance to articular cartilage tissue engineering. Previous research shows that dermal fibroblasts express chondrocytic markers after seeding on aggrecan-coated surfaces. Since cell roundness appears to correlate with chondrocytic behavior of dermal fibroblasts, this study quantified roundness by measuring cell height and surface area-volume ratio. In addition to aggrecan as a surface coating, collagen type II and decorin, two other major extracellular matrix components of articular cartilage, were examined. Aggrecan, collagen type II, and decorin were coated onto a glass substrate using three application techniques: static drying, airbrush, and painting. Vertical scanning interferometry (VSI) is a novel technique that allows for the expedient morphological determination of single cells. Interferometry was used for the characterization of protein-coated surfaces in addition to characterizing the morphology of single dermal fibroblasts after 24 h of seeding. Fibroblast height was found to vary from 1.0 to 4.0 microm and protein coating, application technique, and seeding position were significant factors (p < 0.002). The largest cell heights were observed on aggrecan and collagen type II coated surfaces using the air brush and static applications. Additionally, variations were observed for surface area-volume ratio, ranging from 1.75 to 11.94 microm(-1) with decorin resulting in the lowest ratio, followed by collagen type II and aggrecan. This study identifies optimal coating conditions for stimulating morphology in dermal fibroblasts that is characteristic of the chondrocytic phenotype. These conditions can be employed to attempt articular cartilage regeneration and bypass difficulties due to a paucity of donor tissue.

Transcription factor-based screens and synthetic selections for microbial small-molecule biosynthesis.

Continued advances in metabolic engineering are increasing the number of small molecules being targeted for microbial production. Pathway yields and productivities, however, are often suboptimal, and strain improvement remains a persistent challenge given that the majority of small molecules are difficult to screen for and their biosynthesis does not improve host fitness. In this work, we have developed a generalized approach to screen or select for improved small-molecule biosynthesis using transcription factor-based biosensors. Using a tetracycline resistance gene 3' of a small-molecule inducible promoter, host antibiotic resistance, and hence growth rate, was coupled to either small-molecule concentration in the growth medium or a small-molecule production phenotype. Biosensors were constructed for two important chemical classes, dicarboxylic acids and alcohols, using transcription factor-promoter pairs derived from Pseudomonas putida, Thauera butanivorans, or E. coli. Transcription factors were selected for specific activation by either succinate, adipate, or 1-butanol, and we demonstrate product-dependent growth in E. coli using all three compounds. The 1-butanol biosensor was applied in a proof-of-principle liquid culture screen to optimize 1-butanol biosynthesis in engineered E. coli, identifying a pathway variant yielding a 35% increase in 1-butanol specific productivity through optimization of enzyme expression levels. Lastly, to demonstrate the capacity to select for enzymatic activity, the 1-butanol biosensor was applied as synthetic selection, coupling in vivo 1-butanol biosynthesis to E. coli fitness, and an 120-fold enrichment for a 1-butanol production phenotype was observed following a single round of positive selection.

Reasons for outpatient consultation in allergy/immunology.

There is little data in the literature regarding outpatient consultation in allergy/immunology (A/I). The purpose of this study was to determine the relative frequency of different reasons for A/I outpatient consultation to help guide graduate medical education (GME) and assist with A/I practice management. We retrospectively reviewed the electronic medical records of all outpatient A/I consultations from January 1, 2006 to December 31, 2006. The study was performed at our tertiary care referral center which is a GME training site. There were 1412 A/I consults requested during the 1-year period. The consults per month ranged from a low of 69 to a high of 157. The referrals consisted of 35% pediatric and 65% adult patients. There were 52.8% female and 47.2% male patients. We received 74.3% of referrals from primary care, 19.8% from specialty care, and 5.9% from the emergency department. The most common reasons for consultation included 808 (57.2%) patients for chronic rhinitis, 288 (20.4%) for asthma, 196 (13.9%) for food allergy, 89 (6.3%) for venom allergy, 68 (4.8%) for atopic dermatitis, 66 (4.7%) for drug allergy, 62 (4.4%) for chronic urticaria, 45 (3.2%) for acute urticaria, 34 (2.4%) for immunodeficiency, 31 (2.2%) for anaphylaxis, and 162 (11.5%) for other reasons. More than one reason was given for 27.1% of consults, and there was an average of 1.3 reasons for consultation per patient. Although the allergist/immunologist is consulted for a variety of reasons, the top three reasons make up a majority of outpatient consults, and consults are often requested to address more than one diagnosis.

A novel semi-biosynthetic route for artemisinin production using engineered substrate-promiscuous P450(BM3).

Production of fine chemicals from heterologous pathways in microbial hosts is frequently hindered by insufficient knowledge of the native metabolic pathway and its cognate enzymes; often the pathway is unresolved, and the enzymes lack detailed characterization. An alternative paradigm to using native pathways is de novo pathway design using well-characterized, substrate-promiscuous enzymes. We demonstrate this concept using P450(BM3) from Bacillus megaterium. Using a computer model, we illustrate how key P450(BM3) active site mutations enable binding of the non-native substrate amorphadiene. Incorporating these mutations into P450(BM3) enabled the selective oxidation of amorphadiene artemisinic-11S,12-epoxide, at titers of 250 mg L(-1) in E. coli. We also demonstrate high-yielding, selective transformations to dihydroartemisinic acid, the immediate precursor to the high-value antimalarial drug artemisinin.

Management of stinging insect hypersensitivity: a 5-year retrospective medical record review.

BACKGROUND: The Joint Task Force on Practice Parameters for Allergy and Immunology recommends that patients with a history of a systemic reaction to an insect sting be educated on ways to avoid insect stings, carry injectable epinephrine for emergency self-treatment, undergo specific IgE testing for stinging insect sensitivity, and be considered for immunotherapy. OBJECTIVE: To review frontline providers' documented care and recommendations for imported fire ant and flying insect hypersensitivity reactions. METHODS: A retrospective medical record review was performed of emergency department and primary care clinic visits between November 1, 1999, and November 30, 2004. Using International Classification of Diseases, Ninth Revision, codes, medical records were selected for review to identify patients with potential insect hypersensitivity. RESULTS: A total of 769 medical records from patients who experienced an insect sting were reviewed. Of 120 patients with a systemic reaction, 66 (55.0%) received a prescription for injectable epinephrine, and 14 (11.7%) were given information regarding avoidance of the offending insect. Forty-seven patients with systemic reactions (39.2%) were referred to an allergist. Of 28 patients who kept their appointments and underwent skin testing, 3 had negative results and 25 (89%) had positive results and were advised to start immunotherapy. CONCLUSION: Adherence to the stinging insect hypersensitivity practice parameter recommendations is poor. Many patients who have experienced a systemic reaction after an insect sting and have sought medical care are not afforded an opportunity for potentially lifesaving therapy.