A secretion biosensor for monitoring Sec-dependent protein export in Corynebacterium glutamicum.

BACKGROUND: In recent years, the industrial workhorse Corynebacterium glutamicum has gained increasing interest as a host organism for the secretory production of heterologous proteins. Generally, the yield of a target protein in the culture supernatant depends on a multitude of interdependent biological and bioprocess parameters which have to be optimized. So far, the monitoring of such optimization processes depends on the availability of a direct assay for the respective target protein that can be handled also in high throughput approaches. Since simple assays, such as standard enzymatic activity assays, are not always at hand, the availability of a general protein secretion biosensor is highly desirable. RESULTS: High level secretion of proteins via the Sec protein export pathway leads to secretion stress, a phenomenon that is thought to be caused by the accumulation of incompletely or misfolded proteins at the membrane-cell envelope interface. We have analyzed the transcriptional responses of C. glutamicum to the secretory production of two different heterologous proteins and found that, in both cases, the expression of the gene encoding a homologue of the extracytosolic HtrA protease was highly upregulated. Based on this finding, a C. glutamicum Sec secretion biosensor strain was constructed in which the htrA gene on the chromosome was replaced by the eyfp gene. The fluorescence of the resulting reporter strain responded to the secretion of different heterologous proteins (cutinase from Fusarium solani pisi and alkaline phosphatase PhoA from Escherichia coli) in a dose-dependent manner. In addition, three differently efficient signal peptides for the secretory production of the cutinase could be differentiated by the biosensor signal. Furthermore, we have shown that an efficient signal peptide can be separated from a poor signal peptide by using the biosensor signal of the respective cells in fluorescence activated cell sorting experiments. CONCLUSIONS: We have succeeded in the construction of a C. glutamicum biosensor strain that allows for the monitoring of Sec-dependent secretion of heterologous proteins in a dose-dependent manner, independent of a direct assay for the desired target protein.

Optical response of the Cu2 S2 diamond core in Cu2II(NGuaS)2 Cl2.

Density functional theory (DFT) and time-dependent DFT calculations are presented for the dicopper thiolate complex Cu2 (NGuaS)2 Cl2 [NGuaS=2-(1,1,3,3-tetramethylguanidino) benzenethiolate] with a special focus on the bonding mechanism of the Cu2 S2 Cl2 core and the spectroscopic response. This complex is relevant for the understanding of dicopper redox centers, for example, the CuA center. Its UV/Vis absorption is theoretically studied and found to be similar to other structural CuA models. The spectrum can be roughly divided in the known regions of metal d-d absorptions and metal to ligand charge transfer regions. Nevertheless the chloride ions play an important role as electron donors, with the thiolate groups as electron acceptors. The bonding mechanism is dissected by means of charge decomposition analysis which reveals the large covalency of the Cu2 S2 diamond core mediated between Cu dz2 and S-S π and π* orbitals forming Cu-S σ bonds. Measured resonant Raman spectra are shown for 360- and 720-nm excitation wavelength and interpreted using the calculated vibrational eigenmodes and frequencies. The calculations help to rationalize the varying resonant behavior at different optical excitations. Especially the phenylene rings are only resonant for 720 nm. © 2016 Wiley Periodicals, Inc.

Catching an entatic state--a pair of copper complexes.

The structures of two types of guanidine-quinoline copper complexes have been investigated by single-crystal X-ray crystallography, K-edge X-ray absorption spectroscopy (XAS), resonance Raman and UV/Vis spectroscopy, cyclic voltammetry, and density functional theory (DFT). Independent of the oxidation state, the two structures, which are virtually identical for solids and complexes in solution, resemble each other strongly and are connected by a reversible electron transfer at 0.33 V. By resonant excitation of the two entatic copper complexes, the transition state of the electron transfer is accessible through vibrational modes, which are coupled to metal-ligand charge transfer (MLCT) and ligand-metal charge transfer (LMCT) states.

Taking control over control: use of product sensing in single cells to remove flux control at key enzymes in biosynthesis pathways.

Enzymes initiating the biosynthesis of cellular building blocks are frequently inhibited by the end-product of the respective pathway. Here we present an approach to rapidly generate sets of enzymes overriding this control. It is based on the in vivo detection of the desired end-product in single cells using a genetically encoded sensor. The sensor transmits intracellular product concentrations into a graded optical output, thus enabling ultrahigh-throughput screens by FACS. We randomly mutagenized plasmid-encoded ArgB of Corynebacterium glutamicum and screened the library in a strain carrying the sensor pSenLys-Spc, which detects l-lysine, l-arginine and l-histidine. Six of the resulting N-acetyl-l-glutamate kinase proteins were further developed and characterized and found to be at least 20-fold less sensitive toward l-arginine inhibition than the wild-type enzyme. Overexpression of the mutein ArgB-K47H-V65A in C. glutamicumΔargR led to the accumulation of 34 mM l-arginine in the culture medium. We also screened mutant libraries of lysC-encoded aspartate kinase and hisG-encoded ATP phosphoribosyltransferase. We isolated 11 LysC muteins, enabling up to 45 mM l-lysine accumulation, and 13 HisG muteins, enabling up to 17 mM l-histidine accumulation. These results demonstrate that in vivo screening of enzyme libraries by using metabolite sensors is extremely well suited to identify high-performance muteins required for overproduction.

SoxR as a single-cell biosensor for NADPH-consuming enzymes in Escherichia coli.

An ultra-high-throughput screening system for NADPH-dependent enzymes, such as stereospecific alcohol dehydrogenases, was established. It is based on the [2Fe-2S] cluster-containing transcriptional regulator SoxR of Escherichia coli that activates expression of soxS in the oxidized but not in the reduced state of the cluster. As SoxR is kept in its reduced state by NADPH-dependent reductases, an increased NADPH demand of the cell counteracts SoxR reduction and increases soxS expression. We have taken advantage of these properties by placing the eyfp gene under the control of the soxS promoter and analyzed the response of E. coli cells expressing an NADPH-dependent alcohol dehydrogenase from Lactobacillus brevis (LbAdh), which reduces methyl acetoacetate to (R)-methyl 3-hydroxybutyrate. Under suitable conditions, the specific fluorescence of the cells correlated with the substrate concentration added and with LbAdh enzyme activity, supporting the NADPH responsiveness of the sensor. These properties enabled sorting of single cells harboring wild-type LbAdh from those with lowered or without LbAdh activity by fluorescence-activated cell sorting (FACS). In a proof-of-principle application, the system was used successfully to screen a mutant LbAdh library for variants showing improved activity with the substrate 4-methyl-2-pentanone.

Recombineering in Corynebacterium glutamicum combined with optical nanosensors: a general strategy for fast producer strain generation.

Recombineering in bacteria is a powerful technique for genome reconstruction, but until now, it was not generally applicable for development of small-molecule producers because of the inconspicuous phenotype of most compounds of biotechnological relevance. Here, we establish recombineering for Corynebacterium glutamicum using RecT of prophage Rac and combine this with our recently developed nanosensor technology, which enables the detection and isolation of productive mutants at the single-cell level via fluorescence-activated cell sorting (FACS). We call this new technology RecFACS, which we use for genomic site-directed saturation mutagenesis without relying on pre-constructed libraries to directly isolate L-lysine-producing cells. A mixture of 19 different oligonucleotides was used targeting codon 81 in murE of the wild-type, at a locus where one single mutation is known to cause L-lysine production. Using RecFACS, productive mutants were screened and isolated. Sequencing revealed 12 different amino acid exchanges in the targeted murE codon, which caused different L-lysine production titers. Apart from introducing a rapid genome construction technology for C. glutamicum, the present work demonstrates that RecFACS is suitable to simply create producers as well as genetic diversity in one single step, thus establishing a new general concept in synthetic biology.

Effects of omega-3 fatty acids on postprandial triglycerides and monocyte activation.

OBJECTIVE: Epidemiologic studies suggest that elevated postprandial triglycerides (ppTG) are associated with future cardiovascular events. Monocyte activation plays an important role in vascular diseases. Omega-3 fatty acids (n3-FA) lower fasting TG levels. The effects of n3-FA on ppTG and the role of ppTG for monocyte activation are insufficiently understood. METHODS AND RESULTS: 23 healthy volunteers and 30 non-diabetic patients with documented coronary artery disease were subjected to an oral TG tolerance test (OTTT) consisting of 80 g cream fat or to water as control (H(2)O). Patients were treated with 4 g n3-FA/day or placebo for 3 weeks in a randomized, placebo-controlled, double-blind, crossover study. Relative postprandial TG increase reached its maximum 4 h after fat intake (185.1 ± 10.9% of baseline). n3-FA reduced fasting TG from 137.1 ± 12.9 to 112.2 ± 8.6 mg/dl (p < 0.05), and maximum ppTG concentrations from 243.6 ± 24.6 to 205.8 ± 17.1 mg/dl (p < 0.05), while relative TG increase (192.8 ± 12.7%) was comparable to placebo. Relative monocytopenia and neutrophilia were detected following fat intake, which was unaffected by n3-FA and also detectable in the H(2)O group. Serum chemotactic cytokine (MCP1 and fractalkine) concentrations and monocyte migration were not affected by fat intake or n3-FA. Monocyte activation markers CD11b and CD14, monocyte subpopulations CD16(+)CD14(high) and CD16(+)CD14(low), sICAM serum levels and markers of oxidative stress remained unchanged by fat intake or n3-FA. CONCLUSION: The postprandial TG increase does not stimulate monocytes beyond their circadian activation patterns. n3-FA reduce fasting TG and the postprandial TG increase. n3-FA may therefore allow to prospectively study whether selected patients benefit from TG-lowering independent of LDL- and HDL-cholesterol.

A high-throughput approach to identify genomic variants of bacterial metabolite producers at the single-cell level.

We present a novel method for visualizing intracellular metabolite concentrations within single cells of Escherichia coli and Corynebacterium glutamicum that expedites the screening process of producers. It is based on transcription factors and we used it to isolate new L-lysine producing mutants of C. glutamicum from a large library of mutagenized cells using fluorescence-activated cell sorting (FACS). This high-throughput method fills the gap between existing high-throughput methods for mutant generation and genome analysis. The technology has diverse applications in the analysis of producer populations and screening of mutant libraries that carry mutations in plasmids or genomes.

UV-light-induced conversion and aggregation of prion proteins.

Increasing evidence suggests a central role for oxidative stress in the pathology of prion diseases, a group of fatal neurodegenerative disorders associated with structural conversion of the prion protein (PrP). Because UV-light-induced protein damage is mediated by direct photo-oxidation and radical reactions, we investigated the structural consequences of UVB radiation on recombinant murine and human prion proteins at pH 7.4 and pH 5.0. As revealed by circular dichroism and dynamic light scattering measurements, the observed PrP aggregation follows two independent pathways: (i) complete unfolding of the protein structure associated with rapid precipitation or (ii) specific structural conversion into distinct soluble beta-oligomers. The choice of pathway was directly attributed to the chromophoric properties of the PrP species and the susceptibility to oxidation. Regarding size, the oligomers characterized in this study share a high degree of identity with oligomeric species formed after structural destabilization induced by other triggers, which significantly strengthens the theory that partly unfolded intermediates represent initial precursor molecules directing the pathway of PrP aggregation. Moreover, we identified the first suitable photo-trigger capable of inducing refolding of PrP, which has an important biotechnological impact in terms of analyzing the conversion process on small time scales.

First experimental evidence for the preferential stabilization of the natural D- over the nonnatural L-configuration in nucleic acids.

The homochirality of biomolecules is a prerequisite for the origin and evolution of terrestrial life. The unique selection of D-monosaccharides, in particular, D-ribose in RNA and D-deoxyribose in DNA, leads to the construction of proteins by L-amino acids. This points to the exclusive role of stereoselectivity in the most important physiological processes. So far, there is no experimental confirmation for the theoretical calculations of the energy differences between enantiomers used for the explanation of the stereoselection of biomolecules. Therefore, the question of why nature prefers one configuration over the other still lacks a definitive answer. Here, we present the first experimental evidence that the D-enantiomer of RNA has a different electronic structure compared to the corresponding L-enantiomer. When varying the incident photon energy of the ultraviolet Raman probe across 5 eV, D- and L-isomers of the RNA duplex with the sequence [r(CUGGGCGG).r(CCGCCUGG)] show differences in the intensity of the vibrational modes with energies of 124.0 meV to 210.8 meV. The intensity difference of these vibrational modes can be traced back to energy differences in the electronic levels of D- and L-RNA leading to the preferential stabilization of the naturally occurring D-configuration of RNA over the L-configuration.

Molecular interaction of droperidol with human ether-a-go-go-related gene channels: prolongation of action potential duration without inducing early afterdepolarization.

BACKGROUND: The cardiac safety of droperidol given at antiemetic doses is a matter of debate. Although droperidol potently inhibits human ether-a-go-go-related gene (HERG) channels, the molecular mode of this interaction is unknown. The role of amino acid residues typically mediating high-affinity block of HERG channels is unclear. It is furthermore unresolved whether droperidol at antiemetic concentrations induces action potential prolongation and arrhythmogenic early afterdepolarizations in cardiac myocytes. METHODS: Molecular mechanisms of HERG current inhibition by droperidol were established using two-electrode voltage clamp recordings of Xenopus laevis oocytes expressing wild-type and mutant channels. The mutants T623A, S624A, V625A, Y652A, and F656A were generated by site-directed mutagenesis. The effect of droperidol on action potentials was investigated in cardiac myocytes isolated from guinea pig hearts using the patch clamp technique. RESULTS: Droperidol inhibited currents through HERG wild-type channels with a concentration of half-maximal inhibition of 0.6-0.9 microM. Droperidol shifted the channel activation and the steady state inactivation toward negative potentials while channel deactivation was not affected. Current inhibition increased with membrane potential and with increasing duration of current activation. Inhibition of HERG channels was similarly reduced by all mutations. Droperidol at concentrations between 5 and 100 nM prolonged whereas concentrations greater than 300 nm shortened action potentials. Early afterdepolarizations were not observed. CONCLUSIONS: Droperidol is a high-affinity blocker of HERG channels. Amino acid residues typically involved in high-affinity block mediate droperidol effects. Patch clamp results and computational modeling allow the hypothesis that interaction with calcium currents may explain why droperidol at antiemetic concentrations prolongs the action potential without inducing early afterdepolarizations.

Long QT 1 mutation KCNQ1A344V increases local anesthetic sensitivity of the slowly activating delayed rectifier potassium current.

BACKGROUND: Anesthesia in patients with long QT syndrome (LQTS) is a matter of concern. Congenital LQTS is most frequently caused by mutations in KCNQ1 (Kv7.1), whereas drug-induced LQTS is a consequence of HERG (human ether-a-go-go-related gene) channel inhibition. The aim of this study was to investigate whether the LQT1 mutation A344V in the S6 region of KCNQ1, at a position corresponding to the local anesthetic binding site in HERG, may render drug insensitive KCNQ1 channels into a toxicologically relevant target of these pharmacologic agents. This may suggest that LQTS constitutes not only a nonspecific but also a specific pharmacogenetic risk factor for anesthesia. METHODS: The authors examined electrophysiologic and pharmacologic properties of wild-type and mutant KCNQ1 channels. The effects of bupivacaine, ropivacaine, and mepivacaine were investigated using two-electrode voltage clamp and whole cell patch clamp recordings. RESULTS: The mutation A344V induced voltage-dependent inactivation in homomeric KCNQ1 channels and shifted the voltage dependence of KCNQ1/KCNE1 channel activation by +30 mV. The mutation furthermore increased the sensitivity of KCNQ1/KCNE1 channels for bupivacaine 22-fold (KCNQ1wt/KCNE1: IC50 = 2,431 +/- 582 microM, n = 20; KCNQ1A344V/KCNE1: IC50 = 110 +/- 9 microM, n = 24). Pharmacologic effects of the mutant channels were dominant when mutant and wild-type channels were coexpressed. Simulation of cardiac action potentials with the Luo-Rudy model yielded a prolongation of the cardiac action potential duration and induction of early afterdepolarizations by the mutation A344V that were aggravated by local anesthetic intoxication. CONCLUSIONS: The results indicate that certain forms of the LQTS may constitute a specific pharmacogenetic risk factor for regional anesthesia.