Microbiological toxicity tests using standardized ISO/OECD methods-current state and outlook.

Microbial toxicity tests play an important role in various scientific and technical fields including the risk assessment of chemical compounds in the environment. There is a large battery of normalized tests available that have been standardized by ISO (International Organization for Standardization) and OECD (Organization for Economic Co-operation and Development) and which are worldwide accepted and applied. The focus of this review is to provide information on microbial toxicity tests, which are used to elucidate effects in other laboratory tests such as biodegradation tests, and for the prediction of effects in natural and technical aqueous compartments in the environment. The various standardized tests as well as not normalized methods are described and their advantages and disadvantages are discussed. In addition, the sensitivity and usefulness of such tests including a short comparison with other ecotoxicological tests is presented. Moreover, the far-reaching influence of microbial toxicity tests on biodegradation tests is also demonstrated. A new concept of the physiological potential of an inoculum (PPI) consisting of microbial toxicity tests whose results are expressed as a chemical resistance potential (CRP) and the biodegradation adaptation potential (BAP) of an inoculum is described that may be helpful to characterize inocula used for biodegradation tests. KEY POINTS: • Microbial toxicity tests standardized by ISO and OECD have large differences in sensitivity and applicability. • Standardized microbial toxicity tests in combination with biodegradability tests open a new way to characterize inocula for biodegradation tests. • Standardized microbial toxicity tests together with ecotoxicity tests can form a very effective toolbox for the characterization of toxic effects of chemicals.

Psychiatric care for people with Prader-Willi syndrome-characteristics, needs and barriers.

BACKGROUND: Prader-Willi syndrome (PWS) is commonly associated with intellectual disability, but also with a specific behavioural phenotype and a high predisposition to psychiatric comorbidity. This study examines the psychiatric care situation of people with PWS. METHOD: A structured online questionnaire was administered to carers of people with PWS living in Germany, asking about demographic, diagnostic and treatment parameters as well as personal experiences. RESULTS: Of 77 people with PWS, 44.2% had at least one psychiatric comorbid diagnosis. The main reasons for seeking psychiatric care were emotional outbursts and aggressive behaviour. 34.9% reported that they were currently seeking psychiatric care without success. However, 32.5% of PWS had been treated with psychotropic medication, mainly antipsychotics. CONCLUSIONS: Psychiatric comorbidity appears to be undertreated in PWS, especially in the ambulatory setting. Uncertainty among mental health care providers may also lead to frequent off-label use of psychotropic medications.

Psychometric properties of the German Penn Alcohol Craving Scale.

Craving for alcohol is an important diagnostic criterion in alcohol use disorder (AUD) and an established predictor of future relapse. The 5-item Penn Alcohol Craving Scale (PACS) is one of the most widely used questionnaires to quantify craving and has been translated into different languages. It is assumed that the PACS constitutes one factor, although theoretical considerations suggest an additional second factor. We conducted stability and factor analyses (principal component and confirmatory factor analyses) of the German PACS (PACS-G) in samples of patients with AUD from the following three German study sites: Erlangen, N = 188 (mean age: 47.1 years, 43.5% female); Mannheim, N = 440 (45.5 years, 28.6% female); Hannover, N = 107 (48.1 years, 48.6% female). In our samples, the 2-factor solution of the PACS-G version is more stable than the internationally assumed 1-factor solution. The resulting two PACS-G subscores 'difficulty to resist' (items 4 and 5) and 'thoughts about alcohol' (items 1, 2, and 3) have an internal consistency (Cronbach's alpha) of 0.80 ≤ α ≤ 0.90, m = 0.86 and 0.86 ≤ α ≤ 0.91, m = 0.89 with an overlap of R2 = 62%. We found good convergent validity assessed via the Craving Automatized Scale-Alcohol and the Obsessive-Compulsive Drinking Scale, but also correlations with depression and anxiety assessed via the Beck's Depression and Anxiety Inventories. This study is the first to provide evidence for a 2-factor solution ('difficulty to resist' and 'thoughts about alcohol') underlying the PACS-G version.

N-Acetylcysteine provides limited efficacy as treatment option for skin picking in Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a rare neurodevelopmental disorder based on a loss of paternally expressed genes in chromosome region 15q11-13. In addition to typical characteristics such as hyperphagia, PWS is evidenced by a certain behavioral phenotype. Common indicators are repetitive behaviors, temper tantrums, and self-injurious behaviors such as skin- and/or rectal picking. N-Acetylcysteine (NAC) was previously described as a promising therapeutic option for skin picking in PWS. In this case series, we retrospectively investigated the effect of pharmacotherapy with NAC in 14 individuals with PWS suffering from skin- and/or rectal picking. Treatment success was determined using the Clinical Global Impression-Improvement scale (CGI-I). The Clinical Global Impression-Efficacy index (CGI-EI) was used to put treatment success and side effects into perspective. Six of fourteen patients, all of which were female, showed improvement in symptoms (dosage 1800-2400 mg/day), whereas six patients did not show any change during treatment. Moreover, two male patients treated for solitary rectal picking showed new onset of skin picking. Across all cases, a CGI-I of 3 (corresponding to minimal improvement) was seen after 3 months of treatment, with a CGI-EI of 1.6 (corresponding to moderate efficacy). NAC remains a reasonable therapeutic option in certain cases of skin picking in PWS but provides only limited efficacy compared to previous studies on the topic. There was a higher rate of adverse drug reactions than previously reported. The results particularly suggest caution in future treatment in individuals with solitary rectal picking and reduced efficacy when coadministered with neuroleptics.

Electroconvulsive therapy and adiposity-related parameters in treatment-resistant depressed patients.

Obesity is often accompanied by major depressive disorder (MDD), and vice versa. Latest research findings suggest the body mass index (BMI) to play a role in antidepressant treatment response in general. Our study aims to examine whether adiposity-related parameters such as BMI, glucose homeostasis, or serum lipids are associated with remission to electroconvulsive therapy (ECT). A pilot study (PS, n = 9) and a glucose study (GS, n = 29) were conducted. Blood was withdrawn directly before and 15 min (GS) as well as 1 h (PS) after the first ECT and directly before the last one (usually an ECT series comprised up to twelve sessions). BMI was associated with remission in the PS (remitters: M = 28, SD = 2.5; non-remitters: M = 22, SD = 2.08; t(7) = 3.325, p < 0.001, d = 0.24) but not in the GS or when pooled together. Glucose and insulin levels increased significantly after a single ECT session (GS: glucose: F (2,25.66) = 39.04, p < 0.001; insulin: PS: F (2,83) = 25.8, p < 0.001; GS: F (2,25.87) = 3.97, p < 0.05) but no chronic effect was detectable. Serum lipids were neither significantly altered after a single ECT session nor during a whole course of ECT. There was no difference between remitters and non-remitters in insulin, glucose, or serum lipid levels. Our study is lacking the differentiation between abdominal and peripheral fat distribution, and the sample size is small. Unexpectedly, BMI, glucose homeostasis, and lipid serum levels did not differ in patients remitting during ECT. In contrast to recently published studies, we cannot confirm the hypothesis that BMI may have an impact on ECT response.

Sertraline as a treatment option for temper outbursts in Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a rare neurodevelopmental disorder caused by lack of the paternal copy of maternally imprinted, paternally expressed genes at the chromosome 15q11-13 region. In most cases, it is caused by a paternal deletion or a maternal disomy of chromosome 15. Behavioral problems with temper outbursts are common and often combined with physical aggressiveness and self-injury. They are the most frequent cause for a reduced quality of life in adulthood and represent a serious challenge for the individual and those surrounding the individual in everyday life. Until now, no promising pharmaceutical treatment option has been established, and only a few case reports on treatment with selective serotonin reuptake inhibitors (SSRIs) have been reported. In this case series, we investigated the effect of the SSRI sertraline in 14 individuals with PWS frequently showing severe temper outbursts with aggressiveness and self-injuries. After 6 months of treatment with sertraline, 13 of 14 patients (92.6%) either no longer displayed temper outbursts or showed a significant decrease in frequency and severity of temper outbursts. In one case, treatment was stopped due to severe sleep abnormalities. We conclude that sertraline is a promising and safe treatment option for severe temper outbursts in patients with PWS.

Psychotropic drug-induced hyponatremia: results from a drug surveillance program-an update.

Hyponatremia (HN) is the most common electrolyte imbalance (defined as a serum sodium concentration Na(S) of  < 130 mmol/l) and often induced by drugs including psychotropic drugs. AMSP (Arzneimittelsicherheit in der Psychiatrie) is a multicenter drug surveillance program that assesses severe or unusual adverse drug reactions (ADRs) occurring during treatment with psychotropic drugs. This study presents data from 462,661 psychiatric inpatients treated in participating hospitals between 1993 and 2016 and serves as an update of a previous contribution by Letmaier et al. (JAMA 15(6):739-748, 2012). A total of 210 cases of HN were observed affecting 0.05% of patients. 57.1% of cases presented symptomatically; 19.0% presented with severe symptoms (e.g., seizures, vomiting). HN occurred after a median of 7 days following the first dose or dose increase. Incidence of HN was highest among the two antiepileptic drugs oxcarbazepine (1.661% of patients treated) and carbamazepine (0.169%), followed by selective serotonin-norepinephrine reuptake inhibitors (SSNRIs, 0.088%) and selective serotonin reuptake inhibitors (0.071%). Antipsychotic drugs, tricyclic antidepressants, and mirtazapine exhibited a significantly lower incidence of HN. The risk of HN was 16-42 times higher among patients concomitantly treated with other potentially HN-inducing drugs such as diuretic drugs, angiotensin-converting-enzyme inhibitors, angiotensin II receptor blockers, and proton pump inhibitors. Female SSNRI-users aged  ≥ 65 years concomitantly using other HN-inducing drugs were the population subgroup with the highest risk of developing HN. The identification of high-risk drug combinations and vulnerable patient subgroups represents a significant step in the improvement of drug safety and facilitates the implementation of precautionary measures.

Quantification of outer membrane vesicles: a potential tool to compare response in Pseudomonas putida KT2440 to stress caused by alkanols.

The bacterial release of outer membrane vesicles (OMVs) is an important physiological mechanism of Gram-negative bacteria playing numerous key roles. One function of the release of OMVs is related to an increase in surface hydrophobicity. This phenomenon initiates biofilm formation, making bacteria more tolerant to environmental stressors. Recently, it was qualitatively shown for Pseudomonas putida that vesicle formation plays a crucial role in multiple stress responses. Yet, no quantification of OMVs for certain stress scenarios has been conducted. In this study, it is shown that the quantification of OMVs can serve as a simple and feasible tool, which allows a comparison of vesicle yields for different experimental setups, cell densities, and environmental stressors. Moreover, the obtained results provide insight to the underlying mechanism of vesicle formation as it was observed that n-alkanols, with a chain length of C7 and longer, caused a distinct and steep increase in vesiculation (12-19-fold), compared to shorter chain n-alkanols (2-4-fold increase).

Immediate response mechanisms of Gram-negative solvent-tolerant bacteria to cope with environmental stress: cis-trans isomerization of unsaturated fatty acids and outer membrane vesicle secretion.

Bacteria have evolved an array of adaptive mechanisms enabling them to survive and grow in the presence of different environmental stresses. These mechanisms include either modifications of the membrane or changes in the overall energy status, cell morphology, and cell surface properties. Long-term adaptations are dependent on transcriptional regulation, the induction of anabolic pathways, and cell growth. However, to survive sudden environmental changes, bacterial short-term responses are essential to keep the cells alive after the occurrence of an environmental stress factor such as heat shock or the presence of toxic organic solvents. Thus far, two main short-term responses are known. On the one hand, a fast isomerization of cis into trans unsaturated fatty leads to a quick rigidification of the cell membrane, a mechanism known in some genera of Gram-negative bacteria. On the other hand, a fast, effective, and ubiquitously present countermeasure is the release of outer membrane vesicles (OMVs) from the cell surface leading to a rapid increase in cell surface hydrophobicity and finally to the formation of cell aggregates and biofilms. These immediate response mechanisms just allow the bacteria to stay physiologically active and to employ long-term responses to assure viability upon changing environmental conditions. Here, we provide insight into the two aforementioned rapid adaptive mechanisms affecting ultimately the cell envelope of Gram-negative bacteria.

Unraveling the specific regulation of the central pathway for anaerobic degradation of 3-methylbenzoate.

The mbd cluster encodes the anaerobic degradation of 3-methylbenzoate in the ß-proteobacterium Azoarcus sp. CIB. The specific transcriptional regulation circuit that controls the expression of the mbd genes was investigated. The PO, PB 1, and P3 R promoters responsible for the expression of the mbd genes, their cognate MbdR transcriptional repressor, as well as the MbdR operator regions (ATACN10GTAT) have been characterized. The three-dimensional structure of MbdR has been solved revealing a conformation similar to that of other TetR family transcriptional regulators. The first intermediate of the catabolic pathway, i.e. 3-methylbenzoyl-CoA, was shown to act as the inducer molecule. An additional MbdR-dependent promoter, PA, which contributes to the expression of the CoA ligase that activates 3-methylbenzoate to 3-methylbenzoyl-CoA, was shown to be necessary for an efficient induction of the mbd genes. Our results suggest that the mbd cluster recruited a regulatory system based on the MbdR regulator and its target promoters to evolve a distinct central catabolic pathway that is only expressed for the anaerobic degradation of aromatic compounds that generate 3-methylbenzoyl-CoA as the central metabolite. All these results highlight the importance of the regulatory systems in the evolution and adaptation of bacteria to the anaerobic degradation of aromatic compounds.

Identification and characterization of 2-naphthoyl-coenzyme A reductase, the prototype of a novel class of dearomatizing reductases.

The enzymatic dearomatization of aromatic ring systems by reduction represents a highly challenging redox reaction in biology and plays a key role in the degradation of aromatic compounds under anoxic conditions. In anaerobic bacteria, most monocyclic aromatic growth substrates are converted to benzoyl-coenzyme A (CoA), which is then dearomatized to a conjugated dienoyl-CoA by ATP-dependent or -independent benzoyl-CoA reductases. It was unresolved whether or not related enzymes are involved in the anaerobic degradation of environmentally relevant polycyclic aromatic hydrocarbons (PAHs). In this work, a previously unknown dearomatizing 2-naphthoyl-CoA reductase was purified from extracts of the naphthalene-degrading, sulphidogenic enrichment culture N47. The oxygen-tolerant enzyme dearomatized the non-activated ring of 2-naphthoyl-CoA by a four-electron reduction to 5,6,7,8-tetrahydro-2-naphthoyl-CoA. The dimeric 150 kDa enzyme complex was composed of a 72 kDa subunit showing sequence similarity to members of the flavin-containing 'old yellow enzyme' family. NCR contained FAD, FMN, and an iron-sulphur cluster as cofactors. Extracts of Escherichia coli expressing the encoding gene catalysed 2-naphthoyl-CoA reduction. The identified NCR is a prototypical enzyme of a previously unknown class of dearomatizing arylcarboxyl-CoA reductases that are involved in anaerobic PAH degradation; it fundamentally differs from known benzoyl-CoA reductases.

Structured neurological soft signs examination reveals motor coordination deficits in adults diagnosed with high-functioning autism.

Neurological soft signs (NSS), discrete deficits in motor coordination and sensory integration, have shown promise as markers in autism diagnosis. While motor impairments, partly associated with core behavioral features, are frequently found in children with autism, there is limited evidence in adults. In this study, NSS were assessed in adults undergoing initial diagnosis of high-functioning autism (HFA), a subgroup difficult to diagnose due to social adaptation and psychiatric comorbidity. Adults with HFA (n = 34) and 1:1 sex-, age-, and intelligence-matched neurotypical controls were administered a structured NSS examination including motor, sensory, and visuospatial tasks. We showed that adults with HFA have significantly increased motor coordination deficits compared with controls. Using hierarchical cluster analysis within the HFA group, we also identified a subgroup that was particularly highly affected by NSS. This subgroup differed from the less affected by intelligence level, but not severity of autism behavioral features nor global psychological distress. It remains questionable whether motor impairment represents a genuinely autistic trait or is more a consequence of factors such as intelligence. Nevertheless, we conclude that examining NSS in terms of motor coordination may help diagnose adults with HFA and identify HFA individuals who might benefit from motor skills interventions.

Exploring engineered vesiculation by Pseudomonas putida KT2440 for natural product biosynthesis.

Pseudomonas species have become promising cell factories for the production of natural products due to their inherent robustness. Although these bacteria have naturally evolved strategies to cope with different kinds of stress, many biotechnological applications benefit from engineering of optimised chassis strains with specially adapted tolerance traits. Here, we explored the formation of outer membrane vesicles (OMV) of Pseudomonas putida KT2440. We found OMV production to correlate with the recombinant production of a natural compound with versatile beneficial properties, the tripyrrole prodigiosin. Further, several P. putida genes were identified, whose up- or down-regulated expression allowed controlling OMV formation. Finally, genetically triggering vesiculation in production strains of the different alkaloids prodigiosin, violacein, and phenazine-1-carboxylic acid, as well as the carotenoid zeaxanthin, resulted in up to three-fold increased product yields. Consequently, our findings suggest that the construction of robust strains by genetic manipulation of OMV formation might be developed into a useful tool which may contribute to improving limited biotechnological applications.

Unraveling the effects of acrylonitrile butadiene styrene (ABS) microplastic ageing on the sorption and toxicity of ionic liquids with 2,4-D and glyphosate herbicides.

Microplastics represent a novel category of environmental pollutants, and understanding their interactions with typical xenobiotics is crucial. In this study, we investigated the impact of ionic liquids (ILs) containing herbicidal anions, namely glyphosate [Glyph] and 2,4-dichlorophenoxyacetate [2,4-D], and the surfactant cation - dodecyltrimethylammonium [C12TMA] on acrylonitrile butadiene styrene (ABS) microplastics. The aim of the study was to assess the sorption capacity of microplastics that were present in both untreated and aged form using standard and modified Fenton methods. In addition, impact on toxicity and stress adaptation of the model soil bacterium Pseudomonas putida KT2440 was measured. Upon ageing, ABS microplastics underwent a fivefold increase in BET surface area and total pore volume (from 0.001 to 0.004 cm3/g) which lead to a dramatic increase in adsorption of the cations on ABS microplastics from 40 to 45% for virgin ABS to 75-80% for aged ABS. Toxicity was mainly attributed to hydrophobic cations in ILs (EC50 ∼ 60-65 mg/dm3), which was also mitigated by sorption on ABS. Furthermore, both cations and anions behaved similarly across different ILs, corresponding chlorides, and substrates used in the ILs synthesis. These findings highlight microplastics potential as hazardous sorbents, contributing to the accumulation of xenobiotics in the environment.

Characterization of the mbd cluster encoding the anaerobic 3-methylbenzoyl-CoA central pathway.

The mbd cluster encoding genes of the 3-methylbenzoyl-CoA pathway involved in the anaerobic catabolism of 3-methylbenzoate and m-xylene was characterized for the first time in the denitrifying ß-Proteobacterium Azoarcus sp. CIB. The mbdA gene product was identified as a 3-methylbenzoate-CoA ligase required for 3-methylbenzoate activation; its substrate spectrum was unique in activating all three methylbenzoate isomers. An inducible 3-methylbenzoyl-CoA reductase (mbdONQP gene products), displaying significant amino acid sequence similarities to known class I benzoyl-CoA reductases catalysed the ATP-dependent reduction of 3-methylbenzoyl-CoA to a methyldienoyl-CoA. The mbdW gene encodes a methyldienoyl-CoA hydratase that hydrated the methyldienoyl-CoA to a methyl-6-hydroxymonoenoyl-CoA compound. The mbd cluster also contains the genes predicted to be involved in the subsequent steps of the 3-methylbenzoyl-CoA pathway as well as the electron donor system for the reductase activity. Whereas the catabolic mbd genes are organized in two divergent inducible operons, the putative mbdR regulatory gene was transcribed separately and showed constitutive expression. The efficient expression of the mbd genes required the oxygen-dependent AcpR activator, and it was subject of carbon catabolite repression by some organic acids and amino acids. Sequence analyses suggest that the mbd gene cluster was recruited by Azoarcus sp. CIB through horizontal gene transfer.

ATP-dependent/-independent enzymatic ring reductions involved in the anaerobic catabolism of naphthalene.

Polycyclic aromatic hydrocarbons are among the most hazardous environmental pollutants. However, in contrast to aerobic degradation, the respective degradation pathways in anaerobes are greatly unknown which has so far prohibited many environmental investigations. In this work, we studied the enzymatic dearomatization reactions involved in the degradation of the PAH model compounds naphthalene and 2-methylnaphthalene in the sulfate-reducing enrichment culture N47. Cell extracts of N47 grown on naphthalene catalysed the sodium dithionite-dependent four-electron reduction of the key intermediate 2-naphthoyl-coenzyme A (NCoA) to 5,6,7,8-tetrahydro-2-naphthoyl-CoA (THNCoA). The NCoA reductase activity was independent of ATP and was, surprisingly, not sensitive to oxygen. In cell extracts in the presence of various electron donors the product THNCoA was further reduced by a two-electron reaction to most likely a conjugated hexahydro-2-naphthoyl-CoA isomer (HHNCoA). The reaction assigned to THNCoA reductase strictly depended on ATP and was oxygen-sensitive with a half-life time between 30 s and 1 min when exposed to air. The rate was highest with NADH as electron donor. The results indicate that two novel and completely different dearomatizing ring reductases are involved in anaerobic naphthalene degradation. While the THNCoA reducing activity shows some properties of ATP-dependent class I benzoyl-CoA reductases, NCoA reduction appears to be catalysed by a previously unknown class of dearomatizing aryl-carboxyl-CoA reductases.

Epigenetic down regulation of nerve growth factor during alcohol withdrawal.

We investigated the Cytosin-phosphatidyl-Guanin (CpG) island promoter methylation (mean and methylation of individual CpG-sites) of the nerve growth factor (NGF) gene in the blood of alcohol-dependent patients (57 male patients) during withdrawal (days 1, 7 and 14). Methylation and NGF serum levels did not change significantly from days 1-7. From days 7-14, mean methylation increased (F = 30.55, P < 0.001), whereas the NGF serum levels decreased significantly (days 7-14: F = 17.95, P < 0.001). The NGF serum levels were significantly associated with the mean methylation of the investigated CpG-sites (F = 1.55, P < 0.001). These results imply an epigenetic regulation of the NGF gene during alcohol withdrawal.

Hypomethylation of the dopamine transporter (DAT) gene promoter is associated with hyperphagia-related behavior in Prader-Willi syndrome: A case-control study.

Prader-Willi syndrome (PWS), a neurodevelopmental disorder based on the loss of paternally derived but maternally imprinted genes on chromosome 15q11-13, is typically associated with hyperphagia-related behavior leading to massive obesity. Recently, there has been increasing evidence for dysregulated expression patterns of genes outside the PWS locus that influence the behavioral phenotype and for alterations in the dopaminergic system associated with weight regulation in PWS. In this study, we investigated the epigenetic regulation of the promoter regions of the dopamine transporter (DAT) and dopamine receptor D2 (DRD2) genes and their association with hyperphagia-related behavior in PWS. Methylation of the DAT and DRD2 promoter regions was examined by DNA bisulfite sequencing in 32 individuals with PWS and compared with a control group matched for sex, age, and body mass index (BMI). Hyperphagia-related behavior was assessed using the Hyperphagia Questionnaire for Clinical Trials (HQ-CT). Analysis by linear mixed models revealed a significant effect of factor group on mean DAT promoter methylation rate with decreased mean methylation in PWS (7.3 ± 0.4%) compared to controls (18.8 ± 0.6%), p < 0.001. In the PWS group, we further identified effects of HQ-CT score and BMI on DAT promoter methylation. Although also statistically significantly different (8.4 ± 0.2 in PWS, 10.5 ± 0.3 in controls, p < 0.001), DRD2 promoter methylation visually appeared to be evenly distributed between groups, raising concerns regarding a biological effect. Here, we provide evidence for altered epigenetic regulation of the DAT gene in PWS, which is associated with PWS-typical hyperphagia-related behaviors.

Assessment of New and Genome-Reduced Pseudomonas Strains Regarding Their Robustness as Chassis in Biotechnological Applications.

Organic olvent-tolerant strains of the Gram-negative bacterial genus Pseudomonas are discussed as potential biocatalysts for the biotechnological production of various chemicals. However, many current strains with the highest tolerance are belonging to the species P. putida and are classified as biosafety level 2 strains, which makes them uninteresting for the biotechnological industry. Therefore, it is necessary to identify other biosafety level 1 Pseudomonas strains with high tolerance towards solvents and other forms of stress, which are suitable for establishing production platforms of biotechnological processes. In order to exploit the native potential of Pseudomonas as a microbial cell factory, the biosafety level 1 strain P. taiwanensis VLB120 and its genome-reduced chassis (GRC) variants as well as the plastic-degrading strain P. capeferrum TDA1 were assessed regarding their tolerance towards different n-alkanols (1-butanol, 1-hexanol, 1-octanol, 1-decanol). Toxicity of the solvents was investigated by their effects on bacterial growth rates given as the EC50 concentrations. Hereby, both toxicities as well as the adaptive responses of P. taiwanensis GRC3 and P. capeferrum TDA1 showed EC50 values up to two-fold higher than those previously detected for P. putida DOT-T1E (biosafety level 2), one of the best described solvent-tolerant bacteria. Furthermore, in two-phase solvent systems, all the evaluated strains were adapted to 1-decanol as a second organic phase (i.e., OD560 was at least 0.5 after 24 h of incubation with 1% (v/v) 1-decanol), which shows the potential use of these strains as platforms for the bio-production of a wide variety of chemicals at industrial level.

Production of (hydroxy)benzoate-derived polyketides by engineered Pseudomonas with in situ extraction.

Polyketides from (hydroxy)benzoates are an interesting group of plant polyphenolic compounds, whose biotechnological production is so far underrepresented due to their challenging heterologous biosynthesis. Efficient heterologous production of 2,4,6-tri- and 2,3',4,6-tetrahydroxybenzophenone, 3,5-dihydroxybiphenyl, and 4-hydroxycoumarin by whole-cell biocatalysis in combination with in situ product extraction with an organic solvent was demonstrated. Production was highly dependent on the used CoA ligase and polyketide synthase type III. Therefore, different combinations of polyketide synthases and benzoate-CoA ligases were evaluated for their biosynthesis performance in the solvent-tolerant Pseudomonas taiwanensis VLB120. A solvent screening yielded 2-undecanone as biocompatible, extraction-efficient solvent with good phase separation. In aqueous-organic two-phase cultivations, this solvent extraction circumvents product instability in the aqueous cultivation medium, and it increases yields by reducing inhibitory effects. Complete de novo synthesis from glucose of all (hydroxy)benzoate-derived polyketides was achieved in two-phase cultivations with metabolically engineered strains. Additionally, mutasynthesis was applied to obtain fluorinated benzophenone derivatives.