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The introduction of heterologous pathways into microbial cell compartments offers several potential advantages, including increasing enzyme concentrations and reducing competition with native pathways, making this approach attractive for producing complex metabolites like fatty acids and fatty alcohols. However, measuring subcellular concentrations of these metabolites remains technically challenging. Here, we explored 3-hydroxypropionic acid (3-HP), readily quantifiable and sharing the same precursors—acetyl-CoA, NADPH, and ATP—with the above-mentioned products, as a reporter metabolite for peroxisomal engineering in the yeast Komagataella phaffii. To this end, the malonyl-CoA reductase pathway for 3-HP production was targeted into the peroxisome of K. phaffii using the PTS1-tagging system, and further tested with different carbon sources. Thereafter, we used compartmentalized 3-HP production as a reporter system to showcase the impact of different strategies aimed at enhancing the peroxisomal NADPH pool. Co-overexpression of genes encoding a NADPH-dependent redox shuttle from Saccharomyces cerevisiae (IDP2/IDP3) significantly increased 3-HP yields across all substrates, whereas peroxisomal targeting of the S. cerevisiae NADH kinase Pos5 failed to improve 3-HP production. This study highlights the potential of using peroxisomal 3-HP production as a biosensor for evaluating peroxisomal acetyl-CoA and NAPDH availability by simply quantifying 3-HP, demonstrating its potential for peroxisome-based metabolic engineering in yeast.

Abstract Background Methanol is increasingly gaining attraction as renewable carbon source to produce specialty and commodity chemicals, as it can be generated from renewable sources such as carbon dioxide (CO2). In this context, native methylotrophs such as the yeast Komagataella phaffii (syn Pichia pastoris) are potentially attractive cell factories to produce a wide range of products from this highly reduced substrate. However, studies addressing the potential of this yeast to produce bulk chemicals from methanol are still scarce. 3-Hydroxypropionic acid (3-HP) is a platform chemical which can be converted into acrylic acid and other commodity chemicals and biopolymers. 3-HP can be naturally produced by several bacteria through different metabolic pathways. Results In this study, production of 3-HP via the synthetic β-alanine pathway has been established in K. phaffii for the first time by expressing three heterologous genes, namely panD from Tribolium castaneum, yhxA from Bacillus cereus, and ydfG from Escherichia coli K-12. The expression of these key enzymes allowed a production of 1.0 g l−1 of 3-HP in small-scale cultivations using methanol as substrate. The addition of a second copy of the panD gene and selection of a weak promoter to drive expression of the ydfG gene in the PpCβ21 strain resulted in an additional increase in the final 3-HP titer (1.2 g l−1). The 3-HP-producing strains were further tested in fed-batch cultures. The best strain (PpCβ21) achieved a final 3-HP concentration of 21.4 g l−1 after 39 h of methanol feeding, a product yield of 0.15 g g−1, and a volumetric productivity of 0.48 g l−1 h−1. Further engineering of this strain aiming at increasing NADPH availability led to a 16% increase in the methanol consumption rate and 10% higher specific productivity compared to the reference strain PpCβ21. Conclusions Our results show the potential of K. phaffii as platform cell factory to produce organic acids such as 3-HP from renewable one-carbon feedstocks, achieving the highest volumetric productivities reported so far for a 3-HP production process through the β-alanine pathway.

Introduction: There are several tests that provide information about physical fitness and functionality in older adults. The aims of this study were: (i) to analyze the differences between sex and age in functional, strength and cardiorespiratory tests; and (ii) to study the correlations between functional, strength and cardiorespiratory tests according to sex and age.Methods: A total of 171 older adults (72.09 ± 13.27 kg; 1.59 ± 0.09 m; 72.72 ± 6.05 years) were divided according to sex (men: n = 63; women: n = 108) and age (≥60

Abstract Background Animal models of acute respiratory distress syndrome (ARDS) do not completely resemble human ARDS, struggling translational research. We aimed to characterize a porcine model of ARDS induced by pneumonia—the most common risk factor in humans—and analyze the additional effect of ventilator-induced lung injury (VILI). Methods Bronchoscopy-guided instillation of a multidrug-resistant Pseudomonas aeruginosa strain was performed in ten healthy pigs. In six animals (pneumonia-with-VILI group), pulmonary damage was further increased by VILI applied 3 h before instillation and until ARDS was diagnosed by PaO2/FiO2

Abstract Background Production of 3-hydroxypropionic acid (3-HP) through the malonyl-CoA pathway has yielded promising results in Pichia pastoris (Komagataella phaffii), demonstrating the potential of this cell factory to produce this platform chemical and other acetyl-CoA-derived products using glycerol as a carbon source. However, further metabolic engineering of the original P. pastoris 3-HP-producing strains resulted in unexpected outcomes, e.g., significantly lower product yield and/or growth rate. To gain an understanding on the metabolic constraints underlying these observations, the fluxome (metabolic flux phenotype) of ten 3-HP-producing P. pastoris strains has been characterized using a high throughput 13C-metabolic flux analysis platform. Such platform enabled the operation of an optimised workflow to obtain comprehensive maps of the carbon flux distribution in the central carbon metabolism in a parallel-automated manner, thereby accelerating the time-consuming strain characterization step in the design-build-test-learn cycle for metabolic engineering of P. pastoris. Results We generated detailed maps of the carbon fluxes in the central carbon metabolism of the 3-HP producing strain series, revealing the metabolic consequences of different metabolic engineering strategies aimed at improving NADPH regeneration, enhancing conversion of pyruvate into cytosolic acetyl-CoA, or eliminating by-product (arabitol) formation. Results indicate that the expression of the POS5 NADH kinase leads to a reduction in the fluxes of the pentose phosphate pathway reactions, whereas an increase in the pentose phosphate pathway fluxes was observed when the cytosolic acetyl-CoA synthesis pathway was overexpressed. Results also show that the tight control of the glycolytic flux hampers cell growth due to limited acetyl-CoA biosynthesis. When the cytosolic acetyl-CoA synthesis pathway was overexpressed, the cell growth increased, but the product yield decreased due to higher growth-associated ATP costs. Finally, the six most relevant strains were also cultured at pH 3.5 to assess the effect of a lower pH on their fluxome. Notably, similar metabolic fluxes were observed at pH 3.5 compared to the reference condition at pH 5. Conclusions This study shows that existing fluoxomics workflows for high-throughput analyses of metabolic phenotypes can be adapted to investigate P. pastoris, providing valuable information on the impact of genetic manipulations on the metabolic phenotype of this yeast. Specifically, our results highlight the metabolic robustness of P. pastoris’s central carbon metabolism when genetic modifications are made to increase the availability of NADPH and cytosolic acetyl-CoA. Such knowledge can guide further metabolic engineering of these strains. Moreover, insights into the metabolic adaptation of P. pastoris to an acidic pH have also been obtained, showing the capability of the fluoxomics workflow to assess the metabolic impact of environmental changes.

Recently, the monitoring of air pollution by means of low-cost sensors has become a growing research field due to the study of techniques based on machine learning to improve the sensors’ data quality. For this purpose, sensors undergo a calibration process, where these are placed in-situ nearby a regulatory reference station. The data set explained in this paper contains data from two self-built low-cost air pollution nodes deployed for four months, from January 16, 2021 to May 15, 2021, at an official air quality reference station in Barcelona, Spain. The goal of the deployment was to have five electrochemical sensors at a high sampling rate of 0.5 Hz; two NO2 sensors, two O3 sensors, and one NO sensor. It should be noted that the reference stations publish air pollution data every hour, thus at a rate of 2.7×10−4 Hz. In addition, the nodes have also captured temperature and relative humidity data, which are typically used as correctors in the calibration of low-cost sensors. The availability of the sensors’ time series at this high resolution is important in order to be able to carry out analysis from the signal processing perspective, allowing the study of sensor sampling strategies, sensor signal filtering, and the calibration of low-cost sensors among others.

Summary The use of the methylotrophic yeast Pichia pastoris (Komagataella phaffi) to produce heterologous proteins has been largely reported. However, investigations addressing the potential of this yeast to produce bulk chemicals are still scarce. In this study, we have studied the use of P. pastoris as a cell factory to produce the commodity chemical 3‐hydroxypropionic acid (3‐HP) from glycerol. 3‐HP is a chemical platform which can be converted into acrylic acid and to other alternatives to petroleum‐based products. To this end, the mcr gene from Chloroflexus aurantiacus was introduced into P. pastoris. This single modification allowed the production of 3‐HP from glycerol through the malonyl‐CoA pathway. Further enzyme and metabolic engineering modifications aimed at increasing cofactor and metabolic precursors availability allowed a 14‐fold increase in the production of 3‐HP compared to the initial strain. The best strain (PpHP6) was tested in a fed‐batch culture, achieving a final concentration of 3‐HP of 24.75 g l−1, a product yield of 0.13 g g−1 and a volumetric productivity of 0.54 g l−1 h−1, which, to our knowledge, is the highest volumetric productivity reported in yeast. These results benchmark P. pastoris as a promising platform to produce bulk chemicals for the revalorization of crude glycerol and, in particular, to produce 3‐HP.

Production of 3-hydroxypropionic acid (3-HP) in Pichia pastoris (syn. Komagataella phaffii) via the malonyl-CoA pathway has been recently demonstrated using glycerol as a carbon source, but the reported metrics were not commercially relevant. The flux through the heterologous pathway from malonyl-CoA to 3-HP was hypothesized as the main bottleneck. In the present study, different metabolic engineering approaches have been combined to improve the productivity of the original 3-HP producing strains. To do so, an additional copy of the gene encoding for the potential rate-limiting step of the pathway, i.e., the C-terminal domain of the malonyl-CoA reductase, was introduced. In addition, a variant of the endogenous acetyl-CoA carboxylase (ACC1S1132A) was overexpressed with the aim to increase the delivery of malonyl-CoA. Furthermore, the genes encoding for the pyruvate decarboxylase, aldehyde dehydrogenase and acetyl-CoA synthase, respectively, were overexpressed to enhance conversion of pyruvate into cytosolic acetyl-CoA, and the main gene responsible for the production of the by-product D-arabitol was deleted. Three different screening conditions were used to classify the performance of the different strains: 24-deep-well plates batch cultures, small-scale cultures in falcon tubes using FeedBeads® (i.e., slow release of glycerol over time), and mini bioreactor batch cultures. The best two strains from the FeedBeads® screening, PpHP8 and PpHP18, were tested in bioreactor fed-batch cultures using a pre-fixed exponentially increasing feeding rate. The strain PpHP18 produced up to 37.05 g L−1 of 3-HP at 0.712 g L−1 h−1 with a final product yield on glycerol of 0.194 Cmol−1 in fed-batch cultures. Remarkably, PpHP18 did not rank among the 2-top producer strains in small scale batch cultivations in deep-well plates and mini bioreactors, highlighting the importance of multiplexed screening conditions for adequate assessment of metabolic engineering strategies. These results represent a 50% increase in the product yield and final concentration, as well as over 30% increase in volumetric productivity compared to the previously obtained metrics for P. pastoris. Overall, the combination of glycerol as carbon source and a metabolically engineered P. pastoris strain resulted in the highest 3-HP concentration and productivity reported so far in yeast.

Enzymes have been highly demanded in diverse applications such as in the food, pharmaceutical, and industrial fuel sectors. Thus, in silico bioprospecting emerges as an efficient strategy for discovering new enzyme candidates. A new program called ProspectBIO was developed for this purpose as it can find non-annotated sequences by searching for homologs of a model enzyme directly in genomes. Here we describe the ProspectBIO software methodology and the experimental validation by prospecting for novel lipases by sequence homology to Candida antarctica lipase B (CaLB) and conserved motifs. As expected, we observed that the new bioprospecting software could find more sequences (1672) than a conventional similarity-based search in a protein database (733). Additionally, the absence of patent protection was introduced as a criterion resulting in the final selection of a putative lipase-encoding gene from Ustilago hordei (UhL). Expression of UhL in Pichia pastoris resulted in the production of an enzyme with activity towards a tributyrin substrate. The recombinant enzyme activity levels were 4-fold improved when lowering the temperature and increasing methanol concentrations during the induction phase in shake-flask cultures. Protein sequence alignment and structural modeling showed that the recombinant enzyme has high similarity and capability of adjustment to the structure of CaLB. However, amino acid substitutions identified in the active pocket entrance may be responsible for the differences in the substrate specificities of the two enzymes. Thus, the ProspectBIO software allowed the finding of a new promising lipase for biotechnological application without the need for laborious and expensive conventional bioprospecting experimental steps.

The simultaneous measurement of soil water content and water table levels is of great agronomic and hydrological interest. Not only does soil moisture represent the water available for plant growth but also water table levels can affect crop productivity. Furthermore, monitoring soil saturation and water table levels is essential for an early warning of extreme rainfall situations. However, the measurement of these parameters employing commercial instruments has certain disadvantages, with a high cost of purchase and maintenance. In addition, the handling of commercial devices makes it difficult to adapt them to the specific requirements of farmers or decision-makers. Open-source IoT hardware platforms are emerging as an attractive alternative to developing flexible and low-cost devices. This paper describes the design of a datalogger device based on open-source hardware platforms to register water table levels and soil moisture data for agronomic applications. The paper begins by describing energy-saving and wireless transmission techniques. Then, it summarizes the linear calibration of the phreatimeter sensor obtained with laboratory and field data. Finally, it shows how non-linear machine-learning techniques improve predictions over classical tools for the moisture sensor (SKU: SEN0193).

The H2020 CAPTOR project deployed three testbeds in Spain, Italy and Austria with low-cost sensors for the measurement of tropospheric ozone (O3). The aim of the H2020 CAPTOR project was to raise public awareness in a project focused on citizen science. Each testbed was supported by an NGO in charge of deciding how to raise citizen awareness according to the needs of each country. The data presented in this document correspond to the raw data captured by the sensor nodes in the Spanish testbed using SGX Sensortech MICS 2614 metal-oxide sensors. The Spanish testbed consisted of the deployment of twenty-five nodes. Each sensor node included four SGX Sensortech MICS 2614 ozone sensors, one temperature sensor and one relative humidity sensor. Each node underwent a calibration process by co-locating the node at an EU reference air quality monitoring station, followed by a deployment in a sub-urban or rural area in Catalonia, Spain. All nodes spent two to three weeks co-located at a reference station in Barcelona, Spain (urban area), followed by two to three weeks co-located at three sub-urban reference stations near the final deployment site. The nodes were then deployed in volunteers' homes for about two months and, finally, the nodes were co-located again at the sub-urban reference stations for two weeks for final calibration and assessment of potential drifts. All data presented in this paper are raw data taken by the sensors that can be used for scientific purposes such as calibration studies using machine learning algorithms, or once the concentration values of the nodes are obtained, they can be used to create tropospheric ozone pollution maps with heterogeneous data sources (reference stations and low-cost sensors).

The use of low-cost sensors in conjunction with high-precision instrumentation for air pollution monitoring has shown promising results in recent years. One of the main challenges for these sensors has been the quality of their data, which is why the main efforts have focused on calibrating the sensors using machine learning techniques to improve the data quality. However, there is one aspect that has been overlooked, that is, these sensors are mounted on nodes that may have energy consumption restrictions if they are battery-powered. In this paper, we show the usual sensor data gathering process and we study the existing trade-offs between the sampling of such sensors, the quality of the sensor calibration, and the power consumption involved. To this end, we conduct experiments on prototype nodes measuring tropospheric ozone, nitrogen dioxide, and nitrogen monoxide at high frequency. The results show that the sensor sampling strategy directly affects the quality of the air pollution estimation and that each type of sensor may require different sampling strategies. In addition, duty cycles of 0.1 can be achieved when the sensors have response times in the order of two minutes, and duty cycles between 0.01 and 0.02 can be achieved when the sensor response times are negligible, calibrating with hourly reference values and maintaining a quality of calibrated data similar to when the node is connected to an uninterruptible power supply.

Abstract Background Proteins can be secreted from a host organism with the aid of N-terminal secretion signals. The budding yeast Pichia pastoris (Komagataella sp.) is widely employed to secrete proteins of academic and industrial interest. For this yeast, the most commonly used secretion signal is the N-terminal portion of pre-pro-α-factor from Saccharomyces cerevisiae. However, this secretion signal promotes posttranslational translocation into the endoplasmic reticulum (ER), so proteins that can fold in the cytosol may be inefficiently translocated and thus poorly secreted. In addition, if a protein self-associates, the α-factor pro region can potentially cause aggregation, thereby hampering export from the ER. This study addresses both limitations of the pre-pro-α-factor secretion signal. Results We engineered a hybrid secretion signal consisting of the S. cerevisiae Ost1 signal sequence, which promotes cotranslational translocation into the ER, followed by the α-factor pro region. Secretion and intracellular localization were assessed using as a model protein the tetrameric red fluorescent protein E2-Crimson. When paired with the α-factor pro region, the Ost1 signal sequence yielded much more efficient secretion than the α-factor signal sequence. Moreover, an allelic variant of the α-factor pro region reduced aggregation of the E2-Crimson construct in the ER. The resulting improved secretion signal enhanced secretion of E2-Crimson up to 20-fold compared to the levels obtained with the original α-factor secretion signal. Similar findings were obtained with the lipase BTL2, which exhibited 10-fold enhanced secretion with the improved secretion signal. Conclusions The improved secretion signal confers dramatic benefits for the secretion of certain proteins from P. pastoris. These benefits are likely to be most evident for proteins that can fold in the cytosol and for oligomeric proteins.

Abstract Background Azotobacter vinelandii is a bacterium that produces alginate and polyhydroxybutyrate (P3HB); however, the role of NAD(P)H/NAD(P)+ ratios on the metabolic fluxes through biosynthesis pathways of these biopolymers remains unknown. The aim of this study was to evaluate the NAD(P)H/NAD(P) + ratios and the metabolic fluxes involved in alginate and P3HB biosynthesis, under oxygen-limiting and non-limiting oxygen conditions. Results The results reveal that changes in the oxygen availability have an important effect on the metabolic fluxes and intracellular NADPH/NADP+ ratio, showing that at the lowest OTR (2.4 mmol L−1 h−1), the flux through the tricarboxylic acid (TCA) cycle decreased 27.6-fold, but the flux through the P3HB biosynthesis increased 6.6-fold in contrast to the cultures without oxygen limitation (OTR = 14.6 mmol L−1 h−1). This was consistent with the increase in the level of transcription of phbB and the P3HB biosynthesis. In addition, under conditions without oxygen limitation, there was an increase in the carbon uptake rate (twofold), as well as in the flux through the pentose phosphate (PP) pathway (4.8-fold), compared to the condition of 2.4 mmol L−1 h−1. At the highest OTR condition, a decrease in the NADPH/NADP+ ratio of threefold was observed, probably as a response to the high respiration rate induced by the respiratory protection of the nitrogenase under diazotrophic conditions, correlating with a high expression of the uncoupled respiratory chain genes (ndhII and cydA) and induction of the expression of the genes encoding the nitrogenase complex (nifH). Conclusions We have demonstrated that changes in oxygen availability affect the internal redox state of the cell and carbon metabolic fluxes. This also has a strong impact on the TCA cycle and PP pathway as well as on alginate and P3HB biosynthetic fluxes.

Currently, three human papillomavirus (HPV) vaccines are already licensed and all of them are based on virus-like particles (VLPs) of HPV L1 capsid protein but not worldwide accessible. While about 38.0 million people were living with HIV in 2019, only 68% of HIV-infected individuals were accessing antiretroviral therapy as of the end of June 2020 and there is no HIV vaccine yet. Therefore, safe, effective, and affordable vaccines against those two viruses are immediately needed. Both HPV and HIV are sexually transmitted infections and one of the main access routes is the mucosal genital tract. Thus, the development of a combined vaccine that would protect against HPV and HIV infections is a logical effort in the fight against these two major global pathogens. In this study, a recombinant Pichia pastoris producing chimeric HPV-HIV L1P18 protein intracellularly was constructed. After cell disruption, the supernatant was collected, and the VLPs were purified by a combination of ammonium sulfate precipitation, size exclusion chromatography, ultracentrifugation, and ultrafiltration. At the end of purification process, the chimeric VLPs were recovered with 96% purity and 9.23% overall yield, and the morphology of VLPs were confirmed by transmission electron microscopy. This work contributes towards the development of an alternative platform for production of a bivalent vaccine against HPV and HIV in P. pastoris.

Abstract Background Cultivation of recombinant Pichia pastoris (Komagataella sp.) under hypoxic conditions has a strong positive effect on specific productivity when the glycolytic GAP promoter is used for recombinant protein expression, mainly due to upregulation of glycolytic conditions. In addition, transcriptomic analyses of hypoxic P. pastoris pointed out important regulation of lipid metabolism and unfolded protein response (UPR). Notably, UPR that plays a role in the regulation of lipid metabolism, amino acid metabolism and protein secretion, was found to be upregulated under hypoxia. Results To improve our understanding of the interplay between lipid metabolism, UPR and protein secretion, the lipidome of a P. pastoris strain producing an antibody fragment was studied under hypoxic conditions. Furthermore, lipid composition analyses were combined with previously available transcriptomic datasets to further understand the impact of hypoxia on lipid metabolism. Chemostat cultures operated under glucose-limiting conditions under normoxic and hypoxic conditions were analyzed in terms of intra/extracellular product distribution and lipid composition. Integrated analysis of lipidome and transcriptome datasets allowed us to demonstrate an important remodeling of the lipid metabolism under limited oxygen availability. Additionally, cells with reduced amounts of ergosterol through fluconazole treatment were also included in the study to observe the impact on protein secretion and its lipid composition. Conclusions Our results show that cells adjust their membrane composition in response to oxygen limitation mainly by changing their sterol and sphingolipid composition. Although fluconazole treatment results a different lipidome profile than hypoxia, both conditions result in higher recombinant protein secretion levels.

Pichia pastoris is recognized as a biotechnological workhorse for recombinant protein expression. The metabolic performance of this microorganism depends on genetic makeup and culture conditions, amongst which the specific growth rate and oxygenation level are critical. Despite their importance, only their individual effects have been assessed so far, and thus their combined effects and metabolic consequences still remain to be elucidated. In this work, we present a comprehensive framework for revealing high-order (i.e., individual and combined) metabolic effects of the above parameters in glucose-limited continuous cultures of P. pastoris, using thaumatin production as a case study. Specifically, we employed a rational experimental design to calculate statistically significant metabolic effects from multiple chemostat data, which were later contextualized using a refined and highly predictive genome-scale metabolic model of this yeast under the simulated conditions. Our results revealed a negative effect of the oxygenation on the specific product formation rate (thaumatin), and a positive effect on the biomass yield. Notably, we identified a novel positive combined effect of both the specific growth rate and oxygenation level on the specific product formation rate. Finally, model predictions indicated an opposite relationship between the oxygenation level and the growth-associated maintenance energy (GAME) requirement, suggesting a linear GAME decrease of 0.56 mmol ATP/gDCW per each 1% increase in oxygenation level, which translated into a 44% higher metabolic cost under low oxygenation compared to high oxygenation. Overall, this work provides a systematic framework for mapping high-order metabolic effects of different culture parameters on the performance of a microbial cell factory. Particularly in this case, it provided valuable insights about optimal operational conditions for protein production in P. pastoris. Keywords: Pichia pastoris, Experimental design, Metabolic modelling, Dissolved oxygen, Recombinant protein, Thaumatin

Antimicrobial peptides (AMPs) have potent and durable antimicrobial activity to a wide range of fungi and bacteria. The growing problem of drug-resistant pathogenic microorganisms, together with the lack of new effective compounds, has stimulated interest in developing AMPs as anti-infective molecules. PAF102 is an AMP that was rationally designed for improved antifungal properties. This cell penetrating peptide has potent and specific activity against major fungal pathogens. Cecropin A is a natural AMP with strong and fast lytic activity against bacterial and fungal pathogens, including multidrug resistant pathogens. Both peptides, PAF102 and Cecropin A, are alternative antibiotic compounds. However, their exploitation requires fast, cost-efficient production systems. Here, we developed an innovative system to produce AMPs in Pichia pastoris using the oleosin fusion technology. Oleosins are plant-specific proteins with a structural role in lipid droplet formation and stabilization, which are used as carriers for recombinant proteins to lipid droplets in plant-based production systems. This study reports the efficient production of PAF102 in P. pastoris when fused to the rice plant Oleosin 18, whereas no accumulation of Cecropin A was detected. The Ole18-PAF102 fusion protein targets the lipid droplets of the heterologous system where it accumulates to high levels. Interestingly, the production of this fusion protein induces the formation of lipid droplets in yeast cells, which can be additionally enhanced by the coexpression of a diacylglycerol transferase gene that allows a three-fold increase in the production of the fusion protein. Using this high producer strain, PAF102 reaches commercially relevant yields of up to 180 mg/l of yeast culture. Moreover, the accumulation of PAF102 in the yeast lipid droplets facilitates its downstream extraction and recovery by flotation on density gradients, with the recovered PAF102 being biologically active against pathogenic fungi. Our results demonstrate that plant oleosin fusion technology can be transferred to the well-established P. pastoris cell factory to produce the PAF102 antifungal peptide, and potentially other AMPs, for multiple applications in crop protection, food preservation and animal and human therapies.

Pichia pastoris has been recognized as an effective host for recombinant protein production. In this work, we combine metabolomics and instationary 13C metabolic flux analysis (INST 13C-MFA) using GC-MS and LC-MS/MS to evaluate the potential impact of the production of a Rhizopus oryzae lipase (Rol) on P. pastoris central carbon metabolism. Higher oxygen uptake and CO2 production rates and slightly reduced biomass yield suggest an increased energy demand for the producing strain. This observation is further confirmed by 13C-based metabolic flux analysis. In particular, the flux through the methanol oxidation pathway and the TCA cycle was increased in the Rol-producing strain compared to the reference strain. Next to changes in the flux distribution, significant variations in intracellular metabolite concentrations were observed. Most notably, the pools of trehalose, which is related to cellular stress response, and xylose, which is linked to methanol assimilation, were significantly increased in the recombinant strain.

New advances in sensor technologies and communications in wireless sensor networks have favored the introduction of low-cost sensors for monitoring air quality applications. In this article, we present the results of the European project H2020 CAPTOR, where three testbeds with sensors were deployed to capture tropospheric ozone concentrations. One of the biggest challenges was the calibration of the sensors, as the manufacturer provides them without calibrating. Throughout the paper, we show how short-term calibration using multiple linear regression produces good calibrated data, but instead produces biases in the calculated long-term concentrations. To mitigate the bias, we propose a linear correction based on Kriging estimation of the mean and standard deviation of the long-term ozone concentrations, thus correcting the bias presented by the sensors.

MOTIVATION:Genome-scale metabolic models (GEMs) are tools that allow predicting a phenotype from a genotype under certain environmental conditions. GEMs have been developed in the last ten years for a broad range of organisms, and are used for multiple purposes such as discovering new properties of metabolic networks, predicting new targets for metabolic engineering, as well as optimizing the cultivation conditions for biochemicals or recombinant protein production. Pichia pastoris is one of the most widely used organisms for heterologous protein expression. There are different GEMs for this methylotrophic yeast of which the most relevant and complete in the published literature are iPP668, PpaMBEL1254 and iLC915. However, these three models differ regarding certain pathways, terminology for metabolites and reactions and annotations. Moreover, GEMs for some species are typically built based on the reconstructed models of related model organisms. In these cases, some organism-specific pathways could be missing or misrepresented. RESULTS:In order to provide an updated and more comprehensive GEM for P. pastoris, we have reconstructed and validated a consensus model integrating and merging all three existing models. In this step a comprehensive review and integration of the metabolic pathways included in each one of these three versions was performed. In addition, the resulting iMT1026 model includes a new description of some metabolic processes. Particularly new information described in recently published literature is included, mainly related to fatty acid and sphingolipid metabolism, glycosylation and cell energetics. Finally the reconstructed model was tested and validated, by comparing the results of the simulations with available empirical physiological datasets results obtained from a wide range of experimental conditions, such as different carbon sources, distinct oxygen availability conditions, as well as producing of two different recombinant proteins. In these simulations, the iMT1026 model has shown a better performance than the previous existing models.

In this study, we have engineered a new mycobacterial vaccine design by using an antibiotic-free plasmid selection system. We assembled a novel Escherichia coli (E. coli)–mycobacterial shuttle plasmid p2auxo.HIVA, expressing the HIV-1 clade A immunogen HIVA. This shuttle vector employs an antibiotic resistance-free mechanism for plasmid selection and maintenance based on glycine complementation in E. coli and lysine complementation in mycobacteria. This plasmid was first transformed into glycine auxotroph of E. coli strain and subsequently transformed into lysine auxotroph of Mycobacterium bovis BCG strain to generate vaccine BCG.HIVA2auxo. We demonstrated that the episomal plasmid p2auxo.HIVA was stable in vivo over a 7-week period and genetically and phenotypically characterized the BCG.HIVA2auxo vaccine strain. The BCG.HIVA2auxo vaccine in combination with modified vaccinia virus Ankara (MVA). HIVA was safe and induced HIV-1 and Mycobacterium tuberculosis-specific interferon-γ-producing T-cell responses in adult BALB/c mice. Polyfunctional HIV-1-specific CD8+ T cells, which produce interferon-γ and tumor necrosis factor-α and express the degranulation marker CD107a, were induced. Thus, we engineered a novel, safer, good laboratory practice–compatible BCG-vectored vaccine using prototype immunogen HIVA. This antibiotic-free plasmid selection system based on “double” auxotrophic complementation might be a new mycobacterial vaccine platform to develop not only recombinant BCG-based vaccines expressing second generation of HIV-1 immunogens but also other major pediatric pathogens to prime protective response soon after birth.

As a consequence of the increase in global average temperature, grapes with the adequate phenolic and aromatic maturity tend to be overripe by the time of harvest, resulting in increased sugar concentrations and imbalanced C/N ratios in fermenting musts. This fact sets obvious additional hurdles in the challenge of obtaining wines with reduced alcohols levels, a new trend in consumer demands. It would therefore be interesting to understand Saccharomyces cerevisiae physiology during the fermentation of must with these altered characteristics. The present study aims to determine the distribution of metabolic fluxes during the yeast exponential growth phase, when both carbon and nitrogen sources are in excess, using continuous cultures. Two different sugar concentrations were studied under two different winemaking temperature conditions. Although consumption and production rates for key metabolites were severely affected by the different experimental conditions studied, the general distribution of fluxes in central carbon metabolism was basically conserved in all cases. It was also observed that temperature and sugar concentration exerted a higher effect on the pentose phosphate pathway and glycerol formation than on glycolysis and ethanol production. Additionally, nitrogen uptake, both quantitatively and qualitatively, was strongly influenced by environmental conditions. This work provides the most complete stoichiometric model used for Metabolic Flux Analysis of S. cerevisiae in wine fermentations employed so far, including the synthesis and release of relevant aroma compounds and could be used in the design of optimal nitrogen supplementation of wine fermentations.