Studies on the biosynthesis and heterologous expression of complex secondary metabolites from streptomycetes

The heterologous expression of natural product biosynthetic pathways is of increasing interest in biotechnology and drug discovery. This approach enables the production of complex metabolites in more amenable host organisms and provides the basis for the generation of novel analogs through genetic engineering. In this thesis, we describe a straightforward strategy for the heterologous expression of the highly complex phenalinolactone biosynthetic pathway, which was recently cloned from Streptomyces sp. Tü6071. Using Red/ET recombineering, the phenalinolactone pathway was reconstituted from two cosmids and heterologously expressed in several Streptomyces strains. The established expression system now provides a convenient platform for functional investigations of the biosynthetic genes and the generation of novel analogs, by genetic engineering of the pathway in Escherichia coli. The second part of this thesis describes work on a distinct class of secondary metabolites, the GE81112 tetrapeptide family. We developed a strategy for the cloning and identification of the GE81112 biosynthetic gene cluster, in order to investigate the biosynthetic pathway in detail. Generation of a cosmid library enabled us to identify the corresponding biosynthetic gene cluster on two overlapping cosmids. In parallel, we established methods to manipulate the strain genetically, allowing us to verify the identity of the GE81112 gene cluster by gene inactivation experiments. In addition, we characterized several proteins from the pathway using enzymatic assays in vitro. Taken together, these data have enabled us to propose a preliminary model for GE81112 biosynthesis. The results also open the door to developing new derivatives of these promising compounds by genetic engineering.Die heterologe Expression komplexer Naturstoff-Biosynthesewege spielt eine immer größer werdende Rolle bei der Entdeckung und Modifikation von Wirkstoffen aus der Natur und gewinnt somit zunehmend an Bedeutung in der biotechnologischen Forschung. Diese Methode ermöglicht die Produktion komplexer Metabolite in leicht handhabbaren Wirtsorganismen und bildet so die Grundlage für die Entwicklung neuer Naturstoffderivate durch genetische Manipulation der Biosynthesewege. Die vorliegende Arbeit beschreibt eine innovative Strategie für die heterologe Expression des komplexen Phenalinolacton- Biosynthesewegs aus dem Streptomyceten Tü6071. Mit Hilfe der Red/ET Rekombination wurde dieses Gencluster, das auf 2 Cosmiden vorlag, kloniert und in verschiedenen Streptomyceten-Stämmen heterolog exprimiert. Das somit etablierte Expressionssystem bietet nun die Möglichkeit, durch gezielte genetische Manipulation der Biosynthesewege in Escherichia coli und anschließende heterologe Expression neue, möglicherweise wirksamere Naturstoffe und Naturstoffderivate zu entwickeln. Der zweite Teil dieser Arbeit beschäftigt sich mit Untersuchungen zur Biosynthese einer ungewöhnlichen Klasse von Sekundärstoffen: den GE81112 Tetrapeptiden. Um die zugrundeliegende Biosynthese dieser Naturstoffe nun detailliert zu untersuchen, wurde eine Strategie für die Klonierung und Identifizierung des entsprechenden Genclusters entwickelt, das schließlich auf zwei überlappenden Cosmiden identifiziert wurde. Gleichzeitig konnten Methoden zur genetischen Manipulation des Stammes entwickelt werden, die es ermöglichten das Gencluster durch Geninaktivierungsexperimente zu identifizieren. Mit Hilfe der erhaltenen Ergebnisse konnte schließlich ein erstes Model für die GE81112 Biosynthese erarbeitet werden. Die in dieser Arbeit erhaltenen Einblicke in die Biosynthese können in Zukunft einen wichtigen Beitrag zur Entwicklung neuer GE81112 Derivate leisten

Advances in testing for sample manipulation in clinical and forensic toxicology—part B: hair samples

As a continuation of part A, focusing on advances in testing for sample manipulation of urine samples in clinical and forensic toxicology, part B of the review article relates to hair, another commonly used matrix for abstinence control testing. Similar to urine manipulation, relevant strategies to manipulate a hair test are lowering drug concentrations in hair to undercut the limits of detection/cut-offs, for instance, by forced washout effects or adulteration. However, distinguishing between usual, common cosmetic hair treatment and deliberate manipulation to circumvent a positive drug test is often impossible. Nevertheless, the identification of cosmetic hair treatment is very relevant in the context of hair testing and interpretation of hair analysis results. Newly evaluated techniques or elucidation of specific biomarkers to unravel adulteration or cosmetic treatment often focused on specific structures of the hair matrix with promising strategies recently proposed for daily routine work. Identification of other approaches, e.g., forced hair-washing procedures, still remains a challenge in clinical and forensic toxicology

Single sample preparation for the simultaneous extraction of drugs, pharmaceuticals, cannabinoids and endogenous steroids in hair

A combined sample preparation for the simultaneous extraction of drugs, pharmaceuticals, cannabinoids and endogenous steroids was developed based on three independent fully validated analytical methods. Recently, we published a multi-analyte method for the simultaneous analysis of 116 drugs and pharmaceuticals including different substance groups like opioids, stimulants, benzodiazepines, z-drugs, antidepressants and neuroleptics based on a single sample workup followed by a single analytical measurement with LC-MS/MS. However, in some cases, additional analysis of further substance groups, such as cannabinoids and endogenous steroids, is required, which are analyzed in our laboratory using separate sample preparation and separate analytical methods. The goal of this study was to use the knowledge from the different sample preparations and combine them into a single sample preparation and extraction workflow for the simultaneous extraction of drugs, pharmaceuticals, cannabinoids, and endogenous steroids to be analyzed with the appropriate analytical methods. A partial validation of selected parameters such as selectivity, linearity, limit of quantification (LOQ), accuracy, precision and robustness for the different analytical methods was carried out and revalidated. In addition, comparative measurements of quality controls and authentic pools were performed and statistically evaluated using the unpaired t-test or the non-parametric Mann-Whitney test. The results using the newly established sample preparation and extraction were in good agreement with the original data. In conclusion, the newly established sample preparation is suitable for the combined extraction of drugs, pharmaceuticals, cannabinoids and endogenous steroids, and gives reliable results for quantification of various substances

Simultaneous quantification of steroid hormones and endocannabinoids (ECs) in human hair using an automated supported liquid extraction (SLE) and LC-MS/MS – Insights into EC baseline values and correlation to steroid concentrations

Endogenous steroid hormones and endocannabinoids (ECs) are important regulators in the stress response of the human body. For the measurement of chronic stress, hair analysis has been established as method of choice for long-term and retrospective determination of endogenous stress markers. A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the quantification of five steroid hormones (cortisone, cortisol, androstenedione, testosterone, progesterone) and four endocannabinoids (anandamide, palmitoylethanolamide, 2-arachidonylglycerol, oleoylethanolamide) in hair was developed and validated. The hair samples were extracted with methanol and cleaned up with a fully automated supported liquid extraction (SLE) before analysis. Special attention was paid to the difficulties accompanying the quantification of endogenous analytes in hair. Five different strategies for endogenous compound quantification in hair (surrogate analyte, standard addition, background correction, stripped matrix and solvent calibration) were tested and compared. As a result, the approach of the surrogate analyte was used for the quantification of steroid hormones whereas background correction was used for endocannabinoids. The measurement of 58 samples from healthy young adults allowed insights into endocannabinoid ranges in hair and the correlation to steroid hormones. No significant differences in steroid and EC concentration levels of male and female in hair were found, except for testosterone (p < 0.001) and androstenedione (p < 0.0001). Cortisol to cortisone and testosterone to androstenedione concentrations were significantly and positively correlated. There were significant intercorrelations between endocannabinoids

Associations between hair-derived cannabinoid levels, self-reported use, and cannabis-related problems

Rationale As cannabis potency and cannabis use are increasing in newly legalized markets, it is increasingly important to measure and examine the effects of cannabinoid exposure. Objectives The current study aims to assess how hair-derived cannabinoid concentrations – offering insight into three-month cumulative exposure – are associated with common self-report measures of cannabis use and cannabis use-related problems. Methods 74 near-daily dependent cannabis users self-reported their quantity of cannabis use, cannabis use-related problems, and estimated cannabis potency. Hair samples were provided to quantify Δ9-THC, CBD, and CBN using LC–MS/MS and THC-consumption was verified by analyzing THC-COOH in hair using GC–MS/MS. Results Cannabinoids were detectable in 95.95% of the hair samples from individuals who tested positive on a urine screen for cannabis. Δ9-THC concentrations were positively associated with measures of self-reported potency (relative potency, potency category, and perceived ‘high’), but Δ9-THC, CBD, CBN concentrations and THC/CBD ratio were not associated with self-reported quantity of use. Self-reported potency, but not hair-derived concentrations, were associated with withdrawal and craving. Self-reported quantity of cannabis use, but not cannabinoid concentrations, were associated with cannabis use-related problems. Conclusions The use of hair-derived cannabinoid quantification is supported for detecting cannabis use in near-daily users, but the lack of associations between hair-derived cannabinoid concentrations and self-report measures of use does not support the use of hair analyses alone for quantification of cannabinoid exposure. Further research comparing hair-derived cannabinoid concentrations with other biological matrices (e.g. plasma) and self-report is necessary to further evaluate the validity of hair analyses for this purpose

Analytical description of adolescent binge drinking patients

Background Binge drinking is a widespread health compromising behavior among adolescents and young adults, leading to significant health problems, injuries and mortality. However, data on alcohol consumption is often unreliable, as it is mainly based on self-reporting surveys. In this five-year study (2014–2019) at the University Children’s Hospital Zurich, we analyzed blood samples from adolescent binge drinking patients to investigate blood alcohol concentrations (BACs), co-ingestion of drugs, assess compliance between self-reported and measured substance use, and test for genetic components of innate alcohol tolerance. Furthermore, hair analysis was performed to retrospectively access drug exposure and to evaluate the potential of hair analysis to assess binge drinking. Methods In a prospective, single-center study, patients with alcohol intoxications aged 16 years and younger were included. Blood and hair samples were analyzed by sensitive liquid chromatography – tandem mass spectrometry drug analysis. HTTLPR genotyping was performed with PCR and fragment analysis. Results Among 72 cases, 72 blood and 13 hair samples were analyzed. BACs ranged from 0.08–3.20‰ (mean 1.63‰, median 1.60‰), while a mean concentration of 3.64 pg/mg hair (median 3.0 pg/mg) of the alcohol marker ethyl glucuronide (EtG) was detected in eleven hair samples, providing no evidence of chronic excessive drinking. In 47% of the cases, co-ingested drugs were qualitatively detected next to ethanol, but only 9% of the detected drugs had blood concentrations classified as pharmacologically active. Cannabis consumption (22%) and stimulant intake (16%) were the most frequently observed drugs. Compliance between patients’ statements and measured substances matched well. Although we investigated the genetic contribution to innate alcohol tolerance via the 5-HTTLPR polymorphism, the diverse genetic background of the cohort and small sample size did not allow any conclusions to be drawn. Conclusion Almost half of our binge drinking patients tested positive for other substances, primarily cannabis. We anticipate that our study enhances understanding of consumption behavior of young people and encourage continued efforts to address the harmful effects of binge drinking and co-occurring substance use

Development and validation of a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method including 25 novel synthetic opioids in hair and subsequent analysis of a Swiss opioid consumer cohort

Major public health concern is raised by the evidence that common drugs like heroin are now frequently laced or replaced with highly potent novel synthetic opioids (NSOs). The objective of this study was to explore the prevalence and patterns of NSOs in a cohort of Swiss opioid users by hair analysis. Hair analysis is considered an ideal tool for retrospective consumption monitoring. Hair samples from 439 opioid users in Zurich were analyzed. Study inclusion required a previous positive hair test result for heroin metabolites, oxycodone, fentanyl, methadone, or tramadol. The samples were extracted with a two‐step extraction procedure, followed by a targeted LC–MS/MS (QTRAP® 6500+) analysis in multiple reaction monitoring mode for a total of 25 NSOs. The method underwent full validation and demonstrated good selectivity and sensitivity with limits of detection (LOD) as low as 0.1 pg/mg. The analyzed sample cohort demonstrated a positivity rate for NSOs of 2.5%, including the following NSOs: butyrylfentanyl, acrylfentanyl, furanylfentanyl, methoxyacetylfentanyl, ocfentanil, U‐47700, isobutyrylfentanyl and benzylfentanyl. Furthermore, we were able to identify specific consumption patterns among drug users. The results indicate that hair analysis is a valuable tool for investigating the prevalence of NSOs in drug‐using populations, which seems to be low in the case of Swiss opioid users. Nevertheless, the results highlight the need for sensitive analytical detection methods in forensic toxicology to identify and monitor substance distribution in different populations

Stereoselective Syntheses of Deuterated Pipecolic Acids as Tools to Investigate the Stereoselectivity of the Hydroxylase GetF

Members of the GE81112 family are interesting candidates for the development of antibiotics. The configuration of the OH group on the pipecolic acid moiety plays a pivotal role in antibiotic activity. To investigate the stereoselectivity of the corresponding hydroxylase GetF, involved in the biosynthetic pathway, we synthesized the two deuterium-labeled pipecolic acid diastereomers in a highly stereoselective fashion via chelate-enolate Claisen rearrangement. The stereochemical outcome of the enzymatic hydroxylation step could easily be determined by analysis of mass differences between the products

Stereoselective Syntheses of Deuterated Pipecolic Acids as Tools to Investigate the Stereoselectivity of the Hydroxylase GetF

Members of the GE81112 family are interesting candidates for the development of antibiotics. The configuration of the OH group on the pipecolic acid moiety plays a pivotal role in antibiotic activity. To investigate the stereoselectivity of the corresponding hydroxylase GetF, involved in the biosynthetic pathway, we synthesized the two deuterium-labeled pipecolic acid diastereomers in a highly stereoselective fashion via chelate-enolate Claisen rearrangement. The stereochemical outcome of the enzymatic hydroxylation step could easily be determined by analysis of mass differences between the products

How Do Road Traffic Noise and Residential Greenness Correlate with Noise Annoyance and Long-Term Stress? Protocol and Pilot Study for a Large Field Survey with a Cross-Sectional Design

Urban areas are continuously growing, and densification is a frequent strategy to limit urban expansion. This generally entails a loss of green spaces (GSs) and an increase in noise pollution, which has negative effects on health. Within the research project RESTORE (Restorative potential of green spaces in noise-polluted environments), an extended cross-sectional field study in the city of Zurich, Switzerland, is conducted. The aim is to assess the relationship between noise annoyance and stress (self-perceived and physiological) as well as their association with road traffic noise and GSs. A representative stratified sample of participants from more than 5000 inhabitants will be contacted to complete an online survey. In addition to the self-reported stress identified by the questionnaire, hair cortisol and cortisone probes from a subsample of participants will be obtained to determine physiological stress. Participants are selected according to their dwelling location using a spatial analysis to determine exposure to different road traffic noise levels and access to GSs. Further, characteristics of individuals as well as acoustical and non-acoustical attributes of GSs are accounted for. This paper presents the study protocol and reports the first results of a pilot study to test the feasibility of the protocol