Neuroreceptor Inhibition by Clozapine Triggers Mitohormesis and Metabolic Reprogramming in Human Blood Cells.

The antipsychotic drug clozapine demonstrates superior efficacy in treatment-resistant schizophrenia, but its intracellular mode of action is not completely understood. Here, we analysed the effects of clozapine (2.5-20 µM) on metabolic fluxes, cell respiration, and intracellular ATP in human HL60 cells. Some results were confirmed in leukocytes of clozapine-treated patients. Neuroreceptor inhibition under clozapine reduced Akt activation with decreased glucose uptake, thereby inducing ER stress and the unfolded protein response (UPR). Metabolic profiling by liquid-chromatography/mass-spectrometry revealed downregulation of glycolysis and the pentose phosphate pathway, thereby saving glucose to keep the electron transport chain working. Mitochondrial respiration was dampened by upregulation of the F0F1-ATPase inhibitory factor 1 (IF1) leading to 30-40% lower oxygen consumption in HL60 cells. Blocking IF1 expression by cotreatment with epigallocatechin-3-gallate (EGCG) increased apoptosis of HL60 cells. Upregulation of the mitochondrial citrate carrier shifted excess citrate to the cytosol for use in lipogenesis and for storage as triacylglycerol in lipid droplets (LDs). Accordingly, clozapine-treated HL60 cells and leukocytes from clozapine-treated patients contain more LDs than untreated cells. Since mitochondrial disturbances are described in the pathophysiology of schizophrenia, clozapine-induced mitohormesis is an excellent way to escape energy deficits and improve cell survival.

Itaconate Production from Crude Substrates with U. maydis: Scale-up of an Industrially Relevant Bioprocess.

BACKGROUND: Industrial by-products accrue in most agricultural or food-related production processes, but additional value chains have already been established for many of them. Crude glycerol has a 60% lower market value than commercial glucose, as large quantities are produced in the biodiesel industry, but its valorisation is still underutilized. Due to its high carbon content and the natural ability of many microorganisms to metabolise it, microbial upcycling is a suitable option for this waste product. RESULTS: In this work, the use of crude glycerol for the production of the value-added compound itaconate is demonstrated using the smut fungus Ustilago maydis. Starting with a highly engineered strain, itaconate production from an industrial glycerol waste stream was quickly established on a small scale, and the resulting yields were already competitive with processes using commercial sugars. Adaptive laboratory evolution resulted in an evolved strain with a 72% increased growth rate on glycerol. In the subsequent development and optimisation of a fed-batch process on a 1.5-2 L scale, the use of molasses, a side stream of sugar beet processing, eliminated the need for other expensive media components such as nitrogen or vitamins for biomass growth. The optimised process was scaled up to 150 L, achieving an overall titre of 72 g L- 1, a yield of 0.34 g g- 1, and a productivity of 0.54 g L- 1 h- 1. CONCLUSIONS: Pilot-scale itaconate production from the complementary waste streams molasses and glycerol has been successfully established. In addition to achieving competitive performance indicators, the proposed dual feedstock strategy offers lower process costs and carbon footprint for the production of bio-based itaconate.

Pseudomonas putida KT2440 endures temporary oxygen limitations.

The obligate aerobic nature of Pseudomonas putida, one of the most prominent whole-cell biocatalysts emerging for industrial bioprocesses, questions its ability to be cultivated in large-scale bioreactors, which exhibit zones of low dissolved oxygen tension. P. putida KT2440 was repeatedly subjected to temporary oxygen limitations in scale-down approaches to assess the effect on growth and an exemplary production of rhamnolipids. At those conditions, the growth and production of P. putida KT2440 were decelerated compared to well-aerated reference cultivations, but remarkably, final biomass and rhamnolipid titers were similar. The robust growth behavior was confirmed across different cultivation systems, media compositions, and laboratories, even when P. putida KT2440 was repeatedly exposed to dual carbon and oxygen starvation. Quantification of the nucleotides ATP, ADP, and AMP revealed a decrease of intracellular ATP concentrations with increasing duration of oxygen starvation, which can, however, be restored when re-supplied with oxygen. Only small changes in the proteome were detected when cells encountered oscillations in dissolved oxygen tensions. Concluding, P. putida KT2440 appears to be able to cope with repeated oxygen limitations as they occur in large-scale bioreactors, affirming its outstanding suitability as a whole-cell biocatalyst for industrial-scale bioprocesses.

Parallelized disruption of prokaryotic and eukaryotic cells via miniaturized and automated bead mill.

The application of integrated microbioreactor systems is rapidly becoming of more interest to accelerate strain characterization and bioprocess development. However, available high-throughput screening capabilities are often limited to target extracellular compounds only. Consequently, there is a great demand for automated technologies allowing for miniaturized and parallel cell disruption providing access to intracellular measurements. In this study, a fully automated bead mill workflow was developed and validated for four different industrial platform organisms: Escherichia coli, Corynebacterium glutamicum, Saccharomyces cerevisiae, and Aspergillus niger. The workflow enables up to 48 parallel cell disruptions in microtiter plates and is applicable at-line to running lab-scale cultivations. The resulting cell extracts form the basis for quantitative omics studies where no rapid metabolic quenching is required (e.g., genomics and proteomics).

Comprehensive Analysis of C. glutamicum Anaplerotic Deletion Mutants Under Defined d-Glucose Conditions.

Wild-type C. glutamicum ATCC 13032 is known to possess two enzymes with anaplerotic (C4-directed) carboxylation activity, namely phosphoenolpyruvate carboxylase (PEPCx) and pyruvate carboxylase (PCx). On the other hand, C3-directed decarboxylation can be catalyzed by the three enzymes phosphoenolpyruvate carboxykinase (PEPCk), oxaloacetate decarboxylase (ODx), and malic enzyme (ME). The resulting high metabolic flexibility at the anaplerotic node compromises the unambigous determination of its carbon and energy flux in C. glutamicum wild type. To circumvent this problem we performed a comprehensive analysis of selected single or double deletion mutants in the anaplerosis of wild-type C. glutamicum under defined d-glucose conditions. By applying well-controlled lab-scale bioreactor experiments in combination with untargeted proteomics, quantitative metabolomics and whole-genome sequencing hitherto unknown, and sometimes counter-intuitive, genotype-phenotype relationships in these mutants could be unraveled. In comparison to the wild type the four mutants C. glutamiucm Δpyc, C. glutamiucm Δpyc Δodx, C. glutamiucm Δppc Δpyc, and C. glutamiucm Δpck showed lowered specific growth rates and d-glucose uptake rates, underlining the importance of PCx and PEPCk activity for a balanced carbon and energy flux at the anaplerotic node. Most interestingly, the strain C. glutamiucm Δppc Δpyc could be evolved to grow on d-glucose as the only source of carbon and energy, whereas this combination was previously considered lethal. The prevented anaplerotic carboxylation activity of PEPCx and PCx was found in the evolved strain to be compensated by an up-regulation of the glyoxylate shunt, potentially in combination with the 2-methylcitrate cycle.

A closer look at Aspergillus: online monitoring via scattered light enables reproducible phenotyping.

BACKGROUND: Filamentously growing microorganisms offer unique advantages for biotechnological processes, such as extraordinary secretion capacities, going along with multiple obstacles due to their complex morphology. However, limited experimental throughput in bioprocess development still hampers taking advantage of their full potential. Miniaturization and automation are powerful tools to accelerate bioprocess development, but so far the application of such technologies has mainly been focused on non-filamentous systems. During cultivation, filamentous fungi can undergo remarkable morphological changes, creating challenging cultivation conditions. Depending on the process and product, only one specific state of morphology may be advantageous to achieve e.g. optimal productivity or yield. Different approaches to control morphology have been investigated, such as microparticle enhanced cultivation. However, the addition of solid microparticles impedes the optical measurements typically used by microbioreactor systems and thus alternatives are needed. RESULTS: Aspergillus giganteus IfGB 0902 was used as a model system to develop a time-efficient and robust workflow allowing microscale cultivation with increased throughput. The effect of microtiter plate geometry, shaking frequency and medium additives (talc and calcium chloride) on homogeneity of culture morphology as well as reproducibility were analyzed via online biomass measurement, microscopic imaging and cell dry weight. While addition of talc severely affected online measurements, 2% (w v-1) calcium chloride was successfully applied to obtain a highly reproducible growth behavior with homogenous morphology. Furthermore, the influence of small amounts of complex components was investigated for the applied model strain. By correlation to cell dry weight, it could be shown that optical measurements are a suitable signal for biomass concentration. However, each correlation is only applicable for a specific set of cultivation parameters. These optimized conditions were used in micro as well as lab-scale bioreactor cultivation in order to verify the reproducibility and scalability of the setup. CONCLUSION: A robust workflow for A. giganteus was developed, allowing for reproducible microscale cultivation with online monitoring, where calcium chloride is an useful alternative to microparticle enhanced cultivation in order to control the morphology. Independent of the cultivation volume, comparable phenotypes were observed in microtiter plates and in lab-scale bioreactor.

Identification of modifications procuring growth on xylose in recombinant Saccharomyces cerevisiae strains carrying the Weimberg pathway.

The most prevalent xylose-assimilating pathways in recombinant Saccharomyces cerevisiae, i.e. the xylose isomerase (XI) and the xylose reductase/xylitol dehydrogenase (XR/XDH) pathways, channel the carbon flux through the pentose phosphate pathway and further into glycolysis. In contrast, the oxidative and non-phosphorylative bacterial Weimberg pathway channels the xylose carbon through five steps into the metabolic node α-ketoglutarate (αKG) that can be utilized for growth or diverted into production of various metabolites. In the present study, steps preventing the establishment of a functional Weimberg pathway in S. cerevisiae were identified. Using an original design where a S. cerevisiae strain was expressing the essential four genes of the Caulobacter crescentus pathway (xylB, xylD, xylX, xylA) together with a deletion of FRA2 gene to upregulate the iron-sulfur metabolism, it was shown that the C. crescentus αKG semialdehyde dehydrogenase, XylA was not functional in S. cerevisiae. When replaced by the recently described analog from Corynebacterium glutamicum, KsaD, significantly higher in vitro activity was observed but the strain did not grow on xylose. Adaptive laboratory evolution (ALE) on a xylose/glucose medium on this strain led to a loss of XylB, the first step of the Weimberg pathway, suggesting that ALE favored minimizing the inhibiting xylonate accumulation by restricting the upper part of the pathway. Therefore three additional gene copies of the lower Weimberg pathway (XylD, XylX and KsaD) were introduced. The resulting S. cerevisiae strain (ΔΔfra2, xylB, 4x (xylD-xylX-ksaD)) was able to generate biomass from xylose and Weimberg pathway intermediates were detected. To our knowledge this is the first report of a functional complete Weimberg pathway expressed in fungi. When optimized this pathway has the potential to channel xylose towards value-added specialty chemicals such as dicarboxylic acids and diols.

Análise do impacto do uso de organofosforados e carbamatos em trabalhadores rurais de um município da região noroeste do estado do Rio Grande do Sul / Impact analysis of the use of organophosphates and carbamates rural workers of the municipality in a northwest region of Rio Grande do Sul

Anualmente milhões de agricultores são intoxicados no mundo, e destes, mais de 20 mil morrem em consequência da exposição a agrotóxicos. Intoxicações por organofosforados (OF) e carbamatos (CAR) representam as maiores ameaças à saúde dos trabalhadores rurais. Os OF e CAR atuam na inibição da enzima colinesterase, sendo assim a inibição desta mostra-se um excelente indicador da severidade da intoxicação. O objetivo deste estudo foi analisar o impacto do uso de OF e CAR em trabalhadores rurais na cidade de Mato Queimado/RS. Foi realizado um estudo transversal, prospectivo e experimental. Investigaramse 27 trabalhadores rurais expostos. Foram realizadas coletas sanguíneas e dados epidemiográficos nos meses de fevereiro e março de 2014. A atividade da colinesterase foi determinada através do método bioquímico cinético colorimétrico. A faixa etária média dos participantes foi 34,6 anos (± 8,5). A forma de contato mais prevalente foi a aplicação do produto (88,9%). O tempo médio de exposição foi de 10,7 anos. 70,4% relataram usar equipamentos de proteção individual (EPI), sendo mais frequente o uso de máscara (55,5%). A média dos valores de colinesterase para foi de 3244,45 U/I (± 345,8), níveis estes abaixo dos de referência. Através dos resultados obtidos nesta pesquisa torna-se imprescindível a utilização de meios de monitoramento biológico dos trabalhadores rurais na finalidade de prevenção e promoção da saúde.

Annually millions of rural workers are intoxicated in the world, and of these, more than 20,000 die as a result of exposure to pesticides. Intoxication by insecticides organophosphate (OF) and carbamates (CAR) represent the greatest threats to the health of rural workers. OF CAR and act on the inhibition of cholinesterase enzyme, thus inhibition of this proves to be an excellent indicator of the severity of the intoxication. The objective of this study was to analyze the impact of using OF CAR and in rural workers in the city of Mato Queimado/RS. A cross-sectional, prospective and experimental study was conducted. Twenty-three rural workers exposed were investigated. Sample collection and data demographic were conducted in February and March 2014. The cholinesterase activity was determined by biochemical kinetic colorimetric method. The average age of participants was 34.6 years (± 8.5). The most prevalent form of contact is via the application of the product (88.9%). The mean duration of exposure was 10.7 years. Still, 70.4% reported using personal protective equipment (PPE), more frequent use of mask (55.5%). The average values for cholinesterase was 3244.45 U/l (± 345.8) levels below those of the reference. The results obtained in this study are essential to use biological monitoring means of rural workers in purpose of prevention and health promotion.

Elucidating cellular mechanisms of Saccharomyces cerevisiae tolerant to combined lignocellulosic-derived inhibitors using high-throughput phenotyping and multiomics analyses.

A robust cell factory that can tolerate combined inhibitory lignocellulosic compounds is essential for the cost-effective lignocellulose-based production of second-generation bioethanol and other bulk chemicals. Following high-throughput phenotyping of a yeast genomic overexpression library, we identified a Saccharomyces cerevisiae mutant (denoted AFb.01) with improved growth and fermentation performance under combined toxicity of acetic acid and furfural. AFb.01 carries overexpression of TRX1, which encodes for thioredoxin, a cellular redox machinery. Through comparative proteomics and metabolomics, the resulting cell-wide changes in the mutant were elucidated and these primarily target on the maintenance of energy and redox homeostasis and the minimization of stress-induced cell damages. In particular, the upregulation of the stress-response proteins Hsp26p and Fmp16p conferred tolerance of AFb.01 against protein denaturation and DNA damage. Moreover, increased levels of protectant metabolites such as trehalose, fatty acids, GABA and putrescine provided additional defense mechanisms for the mutant against oxidative and redox stresses. Future studies will concentrate on targeted genetic engineering to validate these mechanisms as well as to support the creation of more robust yeast strains, applicable for industrial, cost-competitive biorefinery production.

pH fluctuations imperil the robustness of C. glutamicum to short term oxygen limitation.

The presence of complex gradients for, e.g., nutrients, oxygen or pH in industrial scale fed batch processes are a major challenge for process performance. To consider such impact of scale-up during laboratory scale process development, scale-down bioreactor simulation, i.e. mimicking inhomogeneous conditions, became the method of choice. However, most scale-down studies simulate combined inhomogeneities of more than one parameter, so that the impact of the individual parameters remains unclear. The presented scale down study addresses this challenge by separating the influence of glucose, pH and oxygen fluctuations in terms of their specific impact in a well-established two compartment scale down device. This was carried out for an 1,5-diaminopentane production process using the industrial production host Corynebacterium glutamicum. Strikingly, oxygen depletion alone showed no effect on the process performance while changes of only one pH unit in acidic as well as alkaline direction reduced the biomass and product formation. Even more pronounced phenotypes up to -13% of µ and -39% of YX/S were observed, when an oscillatory acidic pH shift was combined with dissolved oxygen fluctuations. These losses are accompanied by a missing regulation of fermentative pathways. In conclusion, large-scale C. glutamicum processes seem to be most sensitive to pH variation.

Comprehensive and accurate tracking of carbon origin of LC-tandem mass spectrometry collisional fragments for 13C-MFA.

In recent years the benefit of measuring positionally resolved 13C-labeling enrichment from tandem mass spectrometry (MS/MS) collisional fragments for improved precision of 13C-Metabolic Flux Analysis (13C-MFA) has become evident. However, the usage of positional labeling information for 13C-MFA faces two challenges: (1) The mass spectrometric acquisition of a large number of potentially interfering mass transitions may hamper accuracy and sensitivity. (2) The positional identity of carbon atoms of product ions needs to be known. The present contribution addresses the latter challenge by deducing the maximal positional labeling information contained in LC-ESI-MS/MS spectra of product anions of central metabolism as well as product cations of amino acids. For this purpose, we draw on accurate mass spectrometry, selectively labeled standards, and published fragmentation pathways to structurally annotate all dominant mass peaks of a large collection of metabolites, some of which with a complete fragmentation pathway. Compiling all available information, we arrive at the most detailed map of carbon atom fate of LC-ESI-MS/MS collisional fragments yet, comprising 170 intense and structurally annotated product ions with unique carbon origin from 76 precursor ions of 72 metabolites. Our 13C-data proof that heuristic fragmentation rules often fail to yield correct fragment structures and we expose common pitfalls in the structural annotation of product ions. We show that the positionally resolved 13C-label information contained in the product ions that we structurally annotated allows to infer the entire isotopomer distribution of several central metabolism intermediates, which is experimentally demonstrated for malate using quadrupole-time-of-flight MS technology. Finally, the inclusion of the label information from a subset of these fragments improves flux precision in a Corynebacterium glutamicum model of the central carbon metabolism.

The contest for precursors: channelling L-isoleucine synthesis in Corynebacterium glutamicum without byproduct formation.

L-Isoleucine is an essential amino acid, which is required as a pharma product and feed additive. Its synthesis shares initial steps with that of L-lysine and L-threonine, and four enzymes of L-isoleucine synthesis have an enlarged substrate specificity involved also in L-valine and L-leucine synthesis. As a consequence, constructing a strain specifically overproducing L-isoleucine without byproduct formation is a challenge. Here, we analyze for consequences of plasmid-encoded genes in Corynebacterium glutamicum MH20-22B on L-isoleucine formation, but still obtain substantial accumulation of byproducts. In a different approach, we introduce point mutations into the genome of MH20-22B to remove the feedback control of homoserine dehydrogenase, hom, and threonine dehydratase, ilvA, and we assay sets of genomic promoter mutations to increase hom and ilvA expression as well as to reduce dapA expression, the latter gene encoding the dihydrodipicolinate synthase. The promoter mutations are mirrored in the resulting differential protein levels determined by a targeted LC-MS/MS approach for the three key enzymes. The best combination of genomic mutations was found in strain K2P55, where 53 mM L-isoleucine could be obtained. Whereas in fed-batch fermentations with the plasmid-based strain, 94 mM L-isoleucine with L-lysine as byproduct was formed; with the plasmid-less strain K2P55, 109 mM L-isoleucine accumulated with no substantial byproduct formation. The specific molar yield with the latter strain was 0.188 mol L-isoleucine (mol glucose)(-1) which characterizes it as one of the best L-isoleucine producers available and which does not contain plasmids.

Studies on substrate utilisation in L-valine-producing Corynebacterium glutamicum strains deficient in pyruvate dehydrogenase complex.

The pyruvate dehydrogenase complex was deleted to increase precursor availability in Corynebacterium glutamicum strains overproducing L: -valine. The resulting auxotrophy is treated by adding acetate in addition glucose for growth, resulting in the puzzling fact of gluconeogenic growth with strongly reduced glucose uptake in the presence of acetate in the medium. This result was proven by intracellular metabolite analysis and labelling experiments. To increase productivity, the SugR protein involved in negative regulation of the phosphotransferase system, was inactivated, resulting in enhanced consumption of glucose. However, the surplus in substrate uptake was not converted to L-valine; instead, the formation of up to 289 microM xylulose was observed for the first time in C. glutamicum. As an alternative to the genetic engineering solution, a straightforward process engineering approach is proposed. Acetate limitation resulted in a more efficient use of acetate as cosubstrate, shown by an increased biomass yield Y(X/Ac) and improved L-valine formation.

Analysing overexpression of L-valine biosynthesis genes in pyruvate-dehydrogenase-deficient Corynebacterium glutamicum.

L-valine biosynthesis was analysed by comparing different plasmids in pyruvate-dehydrogenase-deficient Corynebacterium glutamicum strains in order to achieve an optimal production strain. The plasmids contained different combinations of the genes ilvBNCDE encoding for the L-valine forming pathway. It was shown that overexpression of the ilvBN genes encoding acetolactate synthase is obligatory for efficient pyruvate conversion and to prevent L-alanine as a by-product. In contrast to earlier studies, overexpression of ilvE encoding transaminase B is favourable in pyruvate-dehydrogenase-negative strains. Its amplification enhanced L-valine formation and avoided extra- and intracellular accumulation of ketoisovalerate.

Influence of L-isoleucine and pantothenate auxotrophy for L-valine formation in Corynebacterium glutamicum revisited by metabolome analyses.

The effect of different amounts of supplemented L-isoleucine and pantothenate has been analysed with the auxotrophic strain Corynebacterium glutamicum DeltailvA DeltapanB, showing that the final biomass concentration of this preliminary L-valine production strain can be controlled by the amount of added L-isoleucine. One gramme cell dry weight is formed from 48 micromol L-isoleucine. Different amounts of available pantothenate affect the intracellular pyruvate concentration. By limiting pantothenate supplementation from 0.8 to 0.1 microM, a 35-fold increase of cytoplasmic pyruvate up to 14.2 mM can be observed, resulting in the increased formation of L-valine, L-alanine and organic acids in the presence of low pantothenate concentrations. These findings can be used to redirect the carbon flux from glycolysis via pyruvate to the TCA cycle towards the desired product L-valine.