scholarly journals Pharmacodynamics of Ceftolozane plus Tazobactam Studied in anIn VitroPharmacokinetic Model of Infection

2015 ◽  
Vol 60 (1) ◽  
pp. 515-521 ◽  
Author(s):  
Alasdair P. MacGowan ◽  
Alan R. Noel ◽  
Sharon G. Tomaselli ◽  
Donna Nicholls ◽  
Karen E. Bowker
Keyword(s):  
High Performance ◽  
Human Pharmacokinetics ◽  
Content Type ◽  
Initial Inoculum ◽  
E Coli ◽  
Wide Range ◽  
Static Effect ◽  
Dose Ranging ◽  
Lactamase Inhibitor

ABSTRACTCeftolozane plus tazobactam is an antipseudomonal cephalosporin combined with tazobactam, an established beta-lactamase inhibitor, and hasin vitropotency against a range of clinically important β-lactamase-producing bacteria, including most extended-spectrum-β-lactamase (ESBL)-positiveEnterobacteriaceae. The pharmacodynamics of β-lactam–β-lactamase inhibitor combinations presents a number of theoretical and practical challenges, including modeling different half-lives of the compounds. In this study, we studied the pharmacodynamics of ceftolozane plus tazobactam againstEscherichia coliandPseudomonas aeruginosausing anin vitropharmacokinetic model of infection. Five strains ofE. coli, including three clinical strains plus two CTX-M-15 (one high and one moderate) producers, and five strains ofP. aeruginosa, including two with OprD overexpression and AmpC β-lactamases, were employed. Ceftolozane MICs (E. coli, 0.12 to 0.25 mg/liter, andP. aeruginosa, 0.38 to 8 mg/liter) were determined in the presence of 4 mg/liter tazobactam. Dose ranging of ceftolozane (percentage of time in which the free-drug concentration exceeds the MIC [fT>MIC], 0 to 100%) plus tazobactam (human pharmacokinetics) was simulated every 8 hours, with half-lives (t1/2) of 2.5 and 1 h, respectively. Ceftolozane and tazobactam concentrations were confirmed by high-performance liquid chromatography (HPLC). The ceftolozane-plus-tazobactamfT>MIC values at 24 h for a static effect and a 1-log and 2-log drop in initial inoculum forE. coliwere 27.8% ± 5.6%, 33.0% ± 5.6%, and 39.6% ± 8.5%, respectively. CTX-M-15 production did not affect the 24-hfT>MIC forE. colistrains. The ceftolozane-plus-tazobactamfT>MIC values for a 24-h static effect and a 1-log and 2-log drop forP. aeruginosawere 24.9% ± 3.0%, 26.6% ± 3.9%, and 31.2% ± 3.6%. Despite a wide range of absolute MICs, the killing remained predictable as long as the MICs were normalized to the correspondingfT>MIC. Emergence of resistance on 4× MIC plates and 8× MIC plates occurred maximally at anfT>MIC of 10 to 30% and increased as time of exposure increased. ThefT>MIC for a static effect for ceftolozane plus tazobactam is less than that observed with other cephalosporins againstE. coliandP. aeruginosaand is more similar to thefT>MIC reported for carbapenems.

scholarly journals 1395. Defining the Magnitude of AUC:MIC Driver for Efficacy of the β-Lactamase Inhibitor VNRX-5133 When Combined with Cefepime Against KPC- and VIM/NDM-Producing Enterobacteriaceae and P. aeruginosa

2018 ◽  
Vol 5 (suppl_1) ◽  
pp. S429-S429 ◽  
Author(s):  
Denis Daigle ◽  
Salvador Vernacchio ◽  
Luigi Xerri ◽  
Daniel Pevear
Keyword(s):  
In Vitro Studies ◽  
Dose Fractionation ◽  
Infection Model ◽  
Type Model ◽  
E Coli ◽  
Dose Ranging ◽  
New Generation ◽  
Lactamase Inhibitor

Abstract Background VNRX-5133 is a cyclic boronate β-lactamase inhibitor (BLI) in clinical development with cefepime for treatment of infections caused by ESBL- and carbapenemase producing Enterobacteriaceae and P. aeruginosa. It is a new generation broad-spectrum BLI with direct inhibitory activity against serine-active site and emerging metallo-β-lactamases (e.g., VIM/NDM). In previous in vivo and in vitro studies, the PK-PD driver of efficacy of VNRX-5133 was defined as AUC:MIC. Described herein are in vitro studies to assess the magnitude of VNRX-5133 exposure (AUC:MIC) required to restore efficacy of cefepime against a broad collection of KPC- and VIM/NDM-producing Enterobacteriaceae (ENT) and P. aeruginosa (PSA) clinical isolates. Methods Dose-fractionation studies, consisting of four VNRX-5133 exposures fractionated into regimens administered every 4, 8, 12 and 24 hours, were performed in an in vitro infection model with simulated 2 g q8h dosing of cefepime against NDM-1 producing E. coli. A Hill-type model described the relationship between change in log10 CFU at 24 hours and VNRX-5133 exposure (AUC:MIC), where cefepime MIC was determined with 4 µg/mL VNRX-5133. To evaluate variability of efficacy enabled by VNRX-5133 between isolates as well as between Serine-BL and Metallo-BL producers, dose-ranging studies were completed for eight isolates (seven ENT and one PSA) producing KPC or VIM/NDM metallo-β-lactamases. Results The PK-PD exposure parameter AUC:MIC accurately described the efficacy of VNRX-5133 in rescuing cefepime activity against KPC and VIM/NDM carbapenemase-producing isolates of ENT and PSA. The AUC:MIC ratios associated with net bacterial stasis, 1-, and 2-log10 reductions in bacterial burden from baseline were 6.1, 18.4 and 45, respectively, for a collection of five VIM/NDM- and three KPC-producing isolates with cefepime MICs ranging from 4–8 µg/mL with no significant differences observed between Ser-BL and MBL producers. Conclusion These data confirm the equivalent in vitro activity of cefepime/VNRX-5133 against organisms producing serine- and metallo-β-lactamases and provides an initial PK-PD target for VNRX-5133 efficacy when used in combination with cefepime for the treatment of ESBL- and carbapenemase-producing ENT and PSA infections. Disclosures D. Daigle, VenatoRx Pharmaceuticals Inc.: Employee and Shareholder, Salary. S. Vernacchio, VenatoRx Pharmaceuticals Inc.: Employee and Shareholder, Salary. L. Xerri, VenatoRx Pharmaceuticals Inc.: Employee and Shareholder, Salary. D. Pevear, VenatoRx Pharmaceuticals Inc.: Employee, Salary.

scholarly journals A Toxic Environment: a Growing Understanding of How Microbial Communities Affect Escherichia coli O157:H7 Shiga Toxin Expression

2020 ◽  
Vol 86 (24) ◽  
Author(s):  
Erin M. Nawrocki ◽  
Hillary M. Mosso ◽  
Edward G. Dudley
Keyword(s):  
Escherichia Coli ◽  
Shiga Toxin ◽  
Microbial Interactions ◽  
Severe Illness ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Lambdoid Prophage

ABSTRACT Enterohemorrhagic Escherichia coli (EHEC) strains, including E. coli O157:H7, cause severe illness in humans due to the production of Shiga toxin (Stx) and other virulence factors. Because Stx is coregulated with lambdoid prophage induction, its expression is especially susceptible to environmental cues. Infections with Stx-producing E. coli can be difficult to model due to the wide range of disease outcomes: some infections are relatively mild, while others have serious complications. Probiotic organisms, members of the gut microbiome, and organic acids can depress Stx production, in many cases by inhibiting the growth of EHEC strains. On the other hand, the factors currently known to amplify Stx act via their effect on the stx-converting phage. Here, we characterize two interactive mechanisms that increase Stx production by O157:H7 strains: first, direct interactions with phage-susceptible E. coli, and second, indirect amplification by secreted factors. Infection of susceptible strains by the stx-converting phage can expand the Stx-producing population in a human or animal host, and phage infection has been shown to modulate virulence in vitro and in vivo. Acellular factors, particularly colicins and microcins, can kill O157:H7 cells but may also trigger Stx expression in the process. Colicins, microcins, and other bacteriocins have diverse cellular targets, and many such molecules remain uncharacterized. The identification of additional Stx-amplifying microbial interactions will improve our understanding of E. coli O157:H7 infections and help elucidate the intricate regulation of pathogenicity in EHEC strains.

scholarly journals Pharmacokinetics/Pharmacodynamics of Vaborbactam, a Novel Beta-Lactamase Inhibitor, in Combination with Meropenem

2018 ◽  
Vol 63 (1) ◽  
Author(s):  
David C. Griffith ◽  
Mojgan Sabet ◽  
Ziad Tarazi ◽  
Olga Lomovskaya ◽  
Michael N. Dudley
Keyword(s):  
Hollow Fiber ◽  
Infection Model ◽  
Beta Lactamase ◽  
Fiber Model ◽  
Content Type ◽  
Development Of Resistance ◽  
Wide Range ◽  
Tract Infections ◽  
Lactamase Inhibitor

ABSTRACT Vaborbactam is a novel beta-lactamase inhibitor with activity against important beta-lactamases, in particular, serine carbapenemases, and is currently approved in combination with meropenem as Vabomere for the treatment of complicated urinary tract infections, including pyelonephritis. This combination is highly active against Gram-negative pathogens, especially Klebsiella pneumoniae carbapenemase (KPC)-producing carbapenem-resistant Enterobacteriaceae. The objective of these studies was to evaluate vaborbactam pharmacokinetics (PK) and pharmacodynamics (PD) relationships for efficacy in a neutropenic mouse thigh infection model, as well as in an in vitro hollow-fiber infection model, in combination with a fixed exposure of meropenem using KPC-containing strains of Enterobacteriaceae. For both models, the meropenem dosage regimen was designed to simulate a 2-g dose administered every eight hours (q8h) by 3-h infusion. Vaborbactam dosage regimens were designed to produce a wide range of 24-h areas under the concentration-time curves (AUCs) in the thigh infection model. However, for the hollow-fiber model, the AUCs were limited to values of 192, 320, or 550 mg · h/liter. In both the animal and in vitro models, the PK-PD parameter that best described the antibacterial activity of vaborbactam, when administered in combination with meropenem at exposures equivalent to 2 g dosed q8h by 3-h infusion in humans, was the 24-h free vaborbactam AUC/meropenem-vaborbactam (with vaborbactam at 8 mg/liter) MIC ratio. The magnitude of this ratio for bacteriostasis was 9 to 12 and the magnitude to observe a 1-log kill was 18 to 38. In addition, a magnitude greater than 24 suppressed the development of resistance in the in vitro hollow-fiber model.

scholarly journals Pharmacokinetics-Pharmacodynamics of a Novel β-Lactamase Inhibitor, CB-618, in Combination with Meropenem in anIn VitroInfection Model

2016 ◽  
Vol 60 (7) ◽  
pp. 3891-3896 ◽  
Author(s):  
Brian D. VanScoy ◽  
Michael Trang ◽  
Jennifer McCauley ◽  
Haley Conde ◽  
Sujata M. Bhavnani ◽  
...  
Keyword(s):  
Antimicrobial Agents ◽  
Dose Fractionation ◽  
Infection Model ◽  
Time Curve ◽  
Content Type ◽  
Concentration Time ◽  
Wide Range ◽  
Dosing Regimens ◽  
Lactamase Inhibitor

ABSTRACTThe usefulness of β-lactam antimicrobial agents is threatened as never before by β-lactamase-producing bacteria. For this reason, there has been renewed interest in the development of broad-spectrum β-lactamase inhibitors. Herein we describe the results of dose fractionation and dose-ranging studies carried out using a one-compartmentin vitroinfection model to determine the exposure measure for CB-618, a novel β-lactamase inhibitor, most predictive of the efficacy when given in combination with meropenem. The challenge panel includedEnterobacteriaceaeclinical isolates, which collectively produced a wide range of β-lactamase enzymes (KPC-2, KPC-3, FOX-5, OXA-48, SHV-11, SHV-27, and TEM-1). Human concentration-time profiles were simulated for each drug, and samples were collected for drug concentration and bacterial density determinations. Using data from dose fractionation studies and a challengeKlebsiella pneumoniaeisolate (CB-618-potentiated meropenem MIC = 1 mg/liter), relationships between change from baseline in log10CFU/ml at 24 h and each of CB-618 area under the concentration-time curve over 24 h (AUC0–24), maximum concentration (Cmax), and percentage of the dosing interval that CB-618 concentrations remained above a given threshold were evaluated in combination with meropenem at 2 g every 8 h (q8h). The exposure measures most closely associated with CB-618 efficacy in combination with meropenem were the CB-618 AUC0–24(r2= 0.835) andCmax(r2= 0.826). Using the CB-618 AUC0–24indexed to the CB-618-potentiated meropenem MIC value, the relationship between change from baseline in log10CFU/ml at 24 h and CB-618 AUC0–24/MIC ratio in combination with meropenem was evaluated using the pooled data from five challenge isolates; the CB-618 AUC0–24/MIC ratio associated with net bacterial stasis and the 1- and 2-log10CFU/ml reductions from baseline at 24 h were 27.3, 86.1, and 444.8, respectively. These data provide a pharmacokinetics-pharmacodynamics (PK-PD) basis for evaluating potential CB-618 dosing regimens in combination with meropenem in future studies.

scholarly journals Development of Broth Microdilution MIC and Disk Diffusion Antimicrobial Susceptibility Test Quality Control Ranges for the Combination of Cefepime and the Novel β-Lactamase Inhibitor Enmetazobactam

2019 ◽  
Vol 57 (8) ◽  
Author(s):  
Adam Belley ◽  
Michael D. Huband ◽  
Kelley A. Fedler ◽  
Amy A. Watters ◽  
Robert K. Flamm ◽  
...  
Keyword(s):  
Quality Control ◽  
Performance Criteria ◽  
Disk Diffusion ◽  
Broth Microdilution ◽  
Content Type ◽  
E Coli ◽  
Wide Range ◽  
Tract Infections ◽  
Reference Strains ◽  
Lactamase Inhibitor

ABSTRACTThird-generation cephalosporin resistance amongEnterobacteriaceae, mediated by the spread of extended-spectrum β-lactamases (ESBLs), is a very serious medical concern with limited therapeutic options. Enmetazobactam (formerly AAI101) is a novel penicillanic sulfone β-lactamase inhibitor active against a wide range of ESBLs. The combination of enmetazobactam and cefepime has entered phase 3 development in patients with complicated urinary tract infections. Using the Clinical and Laboratory Standards Institute (CLSI) M23 tier 2 study design, broth microdilution MIC and disk diffusion quality control (QC) ranges were determined for cefepime-enmetazobactam. Enmetazobactam was tested at a fixed concentration of 8 μg/ml in the MIC assay, and a cefepime-enmetazobactam disk mass of 30/20 μg was used in the disk diffusion assay.Escherichia coliATCC 25922,E. coliATCC 35218,E. coliNCTC 13353,Klebsiella pneumoniaeATCC 700603, andPseudomonas aeruginosaATCC 27853 were chosen as reference strains. The CTX-M-15-producingE. coliNCTC 13353 isolate is recommended for routine testing to control for inhibition of ESBL activity by enmetazobactam. Broth microdilution MIC QC ranges spanned 3 to 4 doubling dilutions and contained 99.6% to 100.0% of obtained MIC values for the five reference strains. Disk diffusion yielded inhibition zone diameter QC ranges that spanned 7 mm and encompassed 97.1% to 100.0% of the obtained values. Quality control ranges were approved by the CLSI in 2017 (broth microdilution MIC) and 2019 (disk diffusion). The established QC ranges will ensure that appropriate assay performance criteria are attained using CLSI reference methodology when determining the susceptibility of clinical isolates to cefepime-enmetazobactam.

scholarly journals Bacterial Microcompartment-Mediated Ethanolamine Metabolism inEscherichia coliUrinary Tract Infection

2019 ◽  
Vol 87 (8) ◽  
Author(s):  
Katherine Dadswell ◽  
Sinead Creagh ◽  
Edward McCullagh ◽  
Mingzhi Liang ◽  
Ian R. Brown ◽  
...  
Keyword(s):  
Urinary Tract ◽  
Carbon Source ◽  
High Performance ◽  
Urine Samples ◽  
Breakdown Product ◽  
Content Type ◽  
E Coli ◽  
Tract Infections

ABSTRACTUrinary tract infections (UTIs) are common and in general are caused by intestinal uropathogenicEscherichia coli(UPEC) ascending via the urethra. Microcompartment-mediated catabolism of ethanolamine, a host cell breakdown product, fuels the competitive overgrowth of intestinalE. coli, both pathogenic enterohemorrhagicE. coliand commensal strains. During a UTI, urease-negativeE. colibacteria thrive, despite the comparative nutrient limitation in urine. The role of ethanolamine as a potential nutrient source during UTIs is understudied. We evaluated the role of the metabolism of ethanolamine as a potential nitrogen and carbon source for UPEC in the urinary tract. We analyzed infected urine samples by culture, high-performance liquid chromatography, reverse transcription-quantitative PCR, and genomic sequencing. The ethanolamine concentration in urine was comparable to the concentration of the most abundant reported urinary amino acid,d-serine. Transcription of theeutoperon was detected in the majority of urine samples containingE. coliscreened. All sequenced UPEC strains had conservedeutoperons, while metabolic genotypes previously associated with UTI (dsdCXA,metE) were mainly limited to phylogroup B2.In vitroethanolamine was found to be utilized as a sole source of nitrogen by UPEC strains. The metabolism of ethanolamine in artificial urine medium (AUM) induced metabolosome formation and provided a growth advantage at the physiological levels found in urine. Interestingly,eutE(which encodes acetaldehyde dehydrogenase) was required for UPEC strains to utilize ethanolamine to gain a growth advantage in AUM, suggesting that ethanolamine is also utilized as a carbon source. These data suggest that urinary ethanolamine is a significant additional carbon and nitrogen source for infectingE. colistrains.

scholarly journals Novel Bacterial Topoisomerase Inhibitors with Potent Broad-Spectrum Activity against Drug-Resistant Bacteria

2017 ◽  
Vol 61 (5) ◽  
Author(s):  
Cédric Charrier ◽  
Anne-Marie Salisbury ◽  
Victoria J. Savage ◽  
Thomas Duffy ◽  
Emmanuel Moyo ◽  
...  
Keyword(s):  
Broad Spectrum ◽  
Safety Data ◽  
Dna Gyrase ◽  
Cross Resistance ◽  
Resistant Bacteria ◽  
Topoisomerase Iv ◽  
Content Type ◽  
E Coli ◽  
Wide Range

ABSTRACT The novel bacterial topoisomerase inhibitor class is an investigational type of antibacterial inhibitor of DNA gyrase and topoisomerase IV that does not have cross-resistance with the quinolones. Here, we report the evaluation of the in vitro properties of a new series of this type of small molecule. Exemplar compounds selectively and potently inhibited the catalytic activities of Escherichia coli DNA gyrase and topoisomerase IV but did not block the DNA breakage-reunion step. Compounds showed broad-spectrum inhibitory activity against a wide range of Gram-positive and Gram-negative pathogens, including biodefence microorganisms and Mycobacterium tuberculosis. No cross-resistance with fluoroquinolone-resistant Staphylococcus aureus and E. coli isolates was observed. Measured MIC90 values were 4 and 8 μg/ml against a panel of contemporary multidrug-resistant isolates of Acinetobacter baumannii and E. coli, respectively. In addition, representative compounds exhibited greater antibacterial potency than the quinolones against obligate anaerobic species. Spontaneous mutation rates were low, with frequencies of resistance typically <10−8 against E. coli and A. baumannii at concentrations equivalent to 4-fold the MIC. Compound-resistant E. coli mutants that were isolated following serial passage were characterized by whole-genome sequencing and carried a single Arg38Leu amino acid substitution in the GyrA subunit of DNA gyrase. Preliminary in vitro safety data indicate that the series shows a promising therapeutic index and potential for low human ether-a-go-go-related gene (hERG) inhibition (50% inhibitory concentration [IC50], >100 μM). In summary, the compounds' distinct mechanism of action relative to the fluoroquinolones, whole-cell potency, low potential for resistance development, and favorable in vitro safety profile warrant their continued investigation as potential broad-spectrum antibacterial agents.

Phytochemical, Analytical and Medicinal Studies of Holoptelea integrifolia Roxb. Planch - A Review

2019 ◽  
Vol 5 (4) ◽  
pp. 270-277 ◽  
Author(s):  
Vijay Kumar ◽  
Simranjeet Singh ◽  
Ragini Bhadouria ◽  
Ravindra Singh ◽  
Om Prakash
Keyword(s):  
Liquid Chromatography ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Performance ◽  
Biologically Active ◽  
Toxicological Studies ◽  
Wide Range ◽  
Layer Chromatography

Holoptelea integrifolia Roxb. Planch (HI) has been used to treat various ailments including obesity, osteoarthritis, arthritis, inflammation, anemia, diabetes etc. To review the major phytochemicals and medicinal properties of HI, exhaustive bibliographic research was designed by means of various scientific search engines and databases. Only 12 phytochemicals have been reported including biologically active compounds like betulin, betulinic acid, epifriedlin, octacosanol, Friedlin, Holoptelin-A and Holoptelin-B. Analytical methods including the Thin Layer Chromatography (TLC), High-Performance Thin Layer Chromatography (HPTLC), High-Performance Liquid Chromatography (HPLC) and Liquid Chromatography With Mass Spectral (LC-MS) analysis have been used to analyze the HI. From medicinal potency point of view, these phytochemicals have a wide range of pharmacological activities such as antioxidant, antibacterial, anti-inflammatory, and anti-tumor. In the current review, it has been noticed that the mechanism of action of HI with biomolecules has not been fully explored. Pharmacology and toxicological studies are very few. This seems a huge literature gap to be fulfilled through the detailed in-vivo and in-vitro studies.

scholarly journals Periplasmic synthesis and purification of the human prolactin antagonist Δ1-11-G129R-hPRL

AMB Express ◽  
2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Miriam F. Suzuki ◽  
Larissa A. Almeida ◽  
Stephanie A. Pomin ◽  
Felipe D. Silva ◽  
Renan P. Freire ◽  
...  
Keyword(s):  
Size Exclusion Chromatography ◽  
High Performance ◽  
Signal Sequence ◽  
Osmotic Shock ◽  
Size Exclusion ◽  
Specific Expression ◽  
E Coli ◽  
Human Prolactin ◽  
Exclusion Chromatography

AbstractThe human prolactin antagonist Δ1-11-G129R-hPRL is a 21.9 kDa recombinant protein with 188 amino acids that downregulates the proliferation of a variety of cells expressing prolactin receptors. Periplasmic expression of recombinant proteins in E. coli has been considered an option for obtaining a soluble and correctly folded protein, as an alternative to cytoplasmic production. The aim of this work was, therefore, to synthesize for the first time, the Δ1-11-G129R-hPRL antagonist, testing different activation temperatures and purifying it by classical chromatographic techniques. E. coli BL21(DE3) strain was transformed with a plasmid based on the pET25b( +) vector, DsbA signal sequence and the antagonist cDNA sequence. Different doses of IPTG were added, activating under different temperatures, and extracting the periplasmic fluid via osmotic shock. The best conditions were achieved by activating at 35 °C for 5 h using 0.4 mM IPTG, which gave a specific expression of 0.157 ± 0.015 μg/mL/A600 at a final optical density of 3.43 ± 0.13 A600. Purification was carried out by nickel-affinity chromatography followed by size-exclusion chromatography, quantification being performed via high-performance size-exclusion chromatography (HPSEC). The prolactin antagonist was characterized by SDS-PAGE, Western blotting, reversed-phase high-performance liquid chromatography (RP-HPLC) and MALDI-TOF–MS. The final product presented > 95% purity and its antagonistic effects were evaluated in vitro in view of potential clinical applications, including inhibition of the proliferation of cancer cells overexpressing the prolactin receptor and specific antidiabetic properties, taking also advantage of the fact that this antagonist was obtained in a soluble and correctly folded form and without an initial methionine.

scholarly journals Enhancing Terpene Yield from Sugars via Novel Routes to 1-Deoxy-d-Xylulose 5-Phosphate

2014 ◽  
Vol 81 (1) ◽  
pp. 130-138 ◽  
Author(s):  
James Kirby ◽  
Minobu Nishimoto ◽  
Ruthie W. N. Chow ◽  
Edward E. K. Baidoo ◽  
George Wang ◽  
...  
Keyword(s):  
Bacterial Species ◽  
Xylose Reductase ◽  
Pentose Phosphate ◽  
Terpene Biosynthesis ◽  
Content Type ◽  
E Coli ◽  
Pathway Expression ◽  
Terpene Synthesis ◽  
Selection For

ABSTRACTTerpene synthesis in the majority of bacterial species, together with plant plastids, takes place via the 1-deoxy-d-xylulose 5-phosphate (DXP) pathway. The first step of this pathway involves the condensation of pyruvate and glyceraldehyde 3-phosphate by DXP synthase (Dxs), with one-sixth of the carbon lost as CO2. A hypothetical novel route from a pentose phosphate to DXP (nDXP) could enable a more direct pathway from C5sugars to terpenes and also circumvent regulatory mechanisms that control Dxs, but there is no enzyme known that can convert a sugar into its 1-deoxy equivalent. Employing a selection for complementation of adxsdeletion inEscherichia coligrown on xylose as the sole carbon source, we uncovered two candidate nDXP genes. Complementation was achieved either via overexpression of the wild-typeE. coliyajOgene, annotated as a putative xylose reductase, or via various mutations in the nativeribBgene.In vitroanalysis performed with purified YajO and mutant RibB proteins revealed that DXP was synthesized in both cases from ribulose 5-phosphate (Ru5P). We demonstrate the utility of these genes for microbial terpene biosynthesis by engineering the DXP pathway inE. colifor production of the sesquiterpene bisabolene, a candidate biodiesel. To further improve flux into the pathway from Ru5P, nDXP enzymes were expressed as fusions to DXP reductase (Dxr), the second enzyme in the DXP pathway. Expression of a Dxr-RibB(G108S) fusion improved bisabolene titers more than 4-fold and alleviated accumulation of intracellular DXP.

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