Scalable protein production by Komagataella phaffii enabled by ARS plasmids and carbon source-based selection.

BACKGROUND: Most recombinant Komagataella phaffii (Pichia pastoris) strains for protein production are generated by genomic integration of expression cassettes. The clonal variability in gene copy numbers, integration loci and consequently product titers limit the aptitude for high throughput applications in drug discovery, enzyme engineering or most comparative analyses of genetic elements such as promoters or secretion signals. Circular episomal plasmids with an autonomously replicating sequence (ARS), an alternative which would alleviate some of these limitations, are inherently unstable in K. phaffii. Permanent selection pressure, mostly enabled by antibiotic resistance or auxotrophy markers, is crucial for plasmid maintenance and hardly scalable for production. The establishment and use of extrachromosomal ARS plasmids with key genes of the glycerol metabolism (glycerol kinase 1, GUT1, and triosephosphate isomerase 1, TPI1) as selection markers was investigated to obtain a system with high transformation rates that can be directly used for scalable production processes in lab scale bioreactors. RESULTS: In micro-scale deep-well plate experiments, ARS plasmids employing the Ashbya gossypii TEF1 (transcription elongation factor 1) promoter to regulate transcription of the marker gene were found to deliver high transformation efficiencies and the best performances with the reporter protein (CalB, lipase B of Candida antarctica) for both, the GUT1- and TPI1-based, marker systems. The GUT1 marker-bearing strain surpassed the reference strain with integrated expression cassette by 46% upon re-evaluation in shake flask cultures regarding CalB production, while the TPI1 system was slightly less productive compared to the control. In 5 L bioreactor methanol-free fed-batch cultivations, the episomal production system employing the GUT1 marker led to 100% increased CalB activity in the culture supernatant compared to integration construct. CONCLUSIONS: For the first time, a scalable and methanol-independent expression system for recombinant protein production for K. phaffii using episomal expression vectors was demonstrated. Expression of the GUT1 selection marker gene of the new ARS plasmids was refined by employing the TEF1 promoter of A. gossypii. Additionally, the antibiotic-free marker toolbox for K. phaffii was expanded by the TPI1 marker system, which proved to be similarly suited for the use in episomal plasmids as well as integrative expression constructs for the purpose of recombinant protein production.

The influence of symptom severity of palliative care patients on their family caregivers.

BACKGROUND: This study anlyzed whether family caregivers of patients with advanced cancer suffer impaired sleep quality, increased strain, reduced quality of life or increased care burden due to the presence and heightened intensity of symptoms in the person being cared for. METHOD: A total of 41 patient-caregiver dyads (41 caregivers and 41 patients with advanced cancer) were recruited at six primary care centres in this cross-sectional study. Data were obtained over a seven-month period. Caregiver's quality of sleep (Pittsburgh Sleep Quality Index), caregiver's quality of life (Quality of Life Family Version), caregiver strain (Caregiver Strain Index), patients' symptoms and their intensity (Edmonton Symptom Assessment System), and sociodemographic, clinical and care-related data variables were assessed. The associations were determined using non-parametric Spearman correlation. RESULTS: Total Edmonton Symptom Assessment System was significantly related to overall score of the Pittsburgh Sleep Quality Index (r = 0.365, p = 0.028), the Caregiver Strain Index (r = 0.45, p = 0.005) and total Quality of Life Family Version (r = 0.432, p = 0.009), but not to the duration of daily care (r = -0.152, p = 0.377). CONCLUSIONS: Family caregivers for patients with advanced cancer suffer negative consequences from the presence and intensity of these patients' symptoms. Therefore, optimising the control of symptoms would benefit not only the patients but also their caregivers. Thus, interventions should be designed to improve the outcomes of patient-caregiver dyads in such cases.

Smoking in hospitalized patients. A great opportunity. / Tabaquismo en pacientes hospitalizados. Una gran oportunidad.

The objective of this study is to describe the characteristics of smokers admitted to different medical and surgical services in a university hospital and the perception of patients regarding the need for a specialized intervention. The sample comprises a total of 307 patients (mean age of 59.4 years), being 40% (n = 123) non-smokers, 42.7% (n = 131) ex-smokers, and 17.3% (n = 53) smokers. The average consumption of smokers was 22.2 cigarettes / day and the severity of nicotine dependence evaluated with the Fagerström test exceeded 5 points in more than half of the sample. On the other hand, 77.7% had made at least one previous attempt to quit tobacco use. Almost the entire sample (89.9%) of smokers and ex-smokers considered it necessary to develop tobacco treatment programs during hospitalization. Finally, the importance of the hospital context is argued as an opportunity to address the cessation of smoking. The data obtained in this study will allow focusing more appropriately on the management of these patients and optimizing resources.

El objetivo de este estudio es conocer las características de los fumadores ingresados en diferentes servicios médicos y quirúrgicos en un hospital universitario y la percepción de los pacientes respecto a la necesidad de una intervención especializada. La muestra comprende un total de 307 pacientes (edad media de 59,4 años), siendo un 40% (n = 123) no fumadores, 42,7% (n = 131) exfumadores, y un 17,3% (n = 53) fumadores. El consumo medio de los fumadores era de 22,2 cigarrillos/día y la gravedad de la dependencia a la nicotina evaluado con el test de Fagerström sobrepasaba los 5 puntos en más de la mitad de la muestra. Por otra parte, el 77,7% había realizado al menos un intento previo de abandono del consumo de tabaco. Casi la totalidad de la muestra (89,9%) de los fumadores y ex fumadores consideraba necesario desarrollar programas de tratamiento del tabaquismo en la hospitalización. Finalmente se argumenta la importancia del contexto hospitalario como oportunidad para abordar la cesación del hábito tabáquico. Los datos obtenidos en el presente estudio permitirán enfocar más adecuadamente el manejo de estos pacientes y optimizar los recursos.

Scalable production and application of Pichia pastoris whole cell catalysts expressing human cytochrome P450 2C9.

BACKGROUND: Currently, the numerous and versatile applications in pharmaceutical and chemical industry make the recombinant production of cytochrome P450 enzymes (CYPs) of great biotechnological interest. Accelerating the drug development process by simple, quick and scalable access of human drug metabolites is key for efficient and targeted drug development in response to new and sometimes unexpected medical challenges and needs. However, due its biochemical complexity, scalable human CYP (hCYP) production and their application in preparative biotransformations was still in its infancy. RESULTS: A scalable bioprocess for fine-tuned co-expression of hCYP2C9 and its essential complementary human cytochrome P450 reductase (hCPR) in the yeast Pichia pastoris (Komagataella phaffii) is presented. High-throughput screening (HTS) of a transformant library employing a set of diverse bidirectional expression systems with different regulation patterns and a fluorimetric assay was used in order to fine-tune hCYP2C9 and hCPR co-expression, and to identify best expressing clonal variants. The bioprocess development for scalable and reliable whole cell biocatalyst production in bioreactors was carried out based on rational optimization criteria. Among the different alternatives studied, a glycerol carbon-limiting strategy at high µ showed highest production rates, while methanol co-addition together with a decrease of µ provided the best results in terms of product to biomass yield and whole cell activity. By implementing the mentioned strategies, up to threefold increases in terms of production rates and/or yield could be achieved in comparison with initial tests. Finally, the performance of the whole cell catalysts was demonstrated successfully in biotransformation using ibuprofen as substrate, demonstrating the expected high selectivity of the human enzyme catalyst for 3'hydroxyibuprofen. CONCLUSIONS: For the first time a scalable bioprocess for the production of hCYP2C9 whole cell catalysts was successfully designed and implemented in bioreactor cultures, and as well, further tested in a preparative-scale biotransformation of interest. The catalyst engineering procedure demonstrated the efficiency of the employment of a set of differently regulated bidirectional promoters to identify transformants with most effective membrane-bound hCYP/hCPR co-expression ratios and implies to become a model case for the generation of other P. pastoris based catalysts relying on co-expressed enzymes such as other P450 catalysts or enzymes relying on co-expressed enzymes for co-factor regeneration.

Bioprocess performance analysis of novel methanol-independent promoters for recombinant protein production with Pichia pastoris.

BACKGROUND: Pichia pastoris is a powerful and broadly used host for recombinant protein production (RPP), where past bioprocess performance has often been directed with the methanol regulated AOX1 promoter (PAOX1), and the constitutive GAP promoter (PGAP). Since promoters play a crucial role in an expression system and the bioprocess efficiency, innovative alternatives are constantly developed and implemented. Here, a thorough comparative kinetic characterization of two expression systems based on the commercial PDF and UPP promoters (PPDF, PUPP) was first conducted in chemostat cultures. Most promising conditions were subsequently tested in fed-batch cultivations. These new alternatives were compared with the classical strong promoter PGAP, using the Candida antarctica lipase B (CalB) as model protein for expression system performance. RESULTS: Both the PPDF and PUPP-based expression systems outperformed similar PGAP-based expression in chemostat cultivations, reaching ninefold higher specific production rates (qp). CALB transcription levels were drastically higher when employing the novel expression systems. This higher expression was also correlated with a marked upregulation of unfolded protein response (UPR) related genes, likely from an increased protein burden in the endoplasmic reticulum (ER). Based on the chemostat results obtained, best culture strategies for both PPDF and PUPP expression systems were also successfully implemented in 15 L fed-batch cultivations where qp and product to biomass yield (YP/X*) values were similar than those obtained in chemostat cultivations. CONCLUSIONS: As an outcome of the macrokinetic characterization presented, the novel PPDF and PUPP were observed to offer much higher efficiency for CalB production than the widely used PGAP-based methanol-free alternative. Thus, both systems arise as highly productive alternatives for P. pastoris-based RPP bioprocesses. Furthermore, the different expression regulation patterns observed indicate the level of gene expression can be adjusted, or tuned, which is interesting when using Pichia pastoris as a cell factory for different products of interest.

Impact of digital transformation on the automotive industry.

Digital technologies are transforming the automotive industry and disrupting traditional business models. New business opportunities related to Industry 4.0 are emerging, so companies must adapt to the new environment. The study presents an application of fuzzy-set qualitative comparative analysis (fsQCA) to analyze the future impact of digital transformation on business performance models and the different actors' satisfaction. A wide range of aspects and actors derived from the digital transformation process in the automotive industry are considered. The study covers connected and autonomous driving, mobility as a service, digital information sources in car purchasing, big data, etc. The disruptive effect of the gradual introduction of electric vehicles into the market is also considered, which is boosted by environmental policies on climate change and directives for the potential use of renewable energy sources to power electric vehicles. On the other hand, the study analyses the impacts of digital transformation on the automotive industry from the point of view of different actors, ranging from automobile manufacturers, service providers, public transportation providers, and consumers to governments. The methodology has been successfully applied to a complex case study-based empirical analysis. It presents a novel application of fsQCA to digital transformation in the automotive industry in Spain. The conclusions show that it is necessary to invest in adequate measures for adaptation to digital transformation, and manufacturers will end up having greater profits, productivity, and competitiveness. From the point of view of consumers, there will be access to more and better services and greater satisfaction with the required services.

Complementary music therapy for cancer patients in at-home palliative care and their caregivers: protocol for a multicentre randomised controlled trial.

BACKGROUND: Patients with advanced cancer, receiving at-home palliative care, are subject to numerous symptoms that are changeable and often require attention, a stressful situation that also impacts on the family caregiver. It has been suggested that music therapy may benefit both the patient and the caregiver. We propose a study to analyse the efficacy and cost utility of a music intervention programme, applied as complementary therapy, for cancer patients in palliative care and for their at-home caregivers, compared to usual treatment. METHOD: A randomised, double-blind, multicentre clinical trial will be performed in cancer patients in at-home palliative care and their family caregivers. The study population will include two samples of 40 patients and two samples of 41 caregivers. Participants will be randomly assigned either to the intervention group or to the control group. The intervention group will receive a seven-day programme including music sessions, while the control group will receive seven sessions of (spoken word) therapeutic education. In this study, the primary outcome measure is the assessment of patients' symptoms, according to the Edmonton Symptom Assessment System, and of the overload experienced by family caregivers, measured by the Caregiver Strain Index. The secondary outcomes considered will be the participants' health-related quality of life, their satisfaction with the intervention, and an economic valuation. DISCUSSION: This study is expected to enhance our understanding of the efficacy and cost-utility of music therapy for cancer patients in palliative care and for their family caregivers. The results of this project are expected to be applicable and transferrable to usual clinical practice for patients in home palliative care and for their caregivers. The approach described can be incorporated as an additional therapeutic resource within comprehensive palliative care. To our knowledge, no previous high quality studies, based on a double-blind clinical trial, have been undertaken to evaluate the cost-effectiveness of music therapy. The cost-effectiveness of the project will provide information to support decision making, thereby improving the management of health resources and their use within the health system. TRIAL REGISTRATION: The COMTHECARE study is registered at Clinical Trials.gov, NCT04052074. Registered 9 August, 2019.

Detection and Mitigation of DoS and DDoS Attacks in IoT-Based Stateful SDN : An Experimental Approach.

The expected advent of the Internet of Things (IoT) has triggered a large demand of embedded devices, which envisions the autonomous interaction of sensors and actuators while offering all sort of smart services. However, these IoT devices are limited in computation, storage, and network capacity, which makes them easy to hack and compromise. To achieve secure development of IoT, it is necessary to engineer scalable security solutions optimized for the IoT ecosystem. To this end, Software Defined Networking (SDN) is a promising paradigm that serves as a pillar in the fifth generation of mobile systems (5G) that could help to detect and mitigate Denial of Service (DoS) and Distributed DoS (DDoS) threats. In this work, we propose to experimentally evaluate an entropy-based solution to detect and mitigate DoS and DDoS attacks in IoT scenarios using a stateful SDN data plane. The obtained results demonstrate for the first time the effectiveness of this technique targeting real IoT data traffic.

Intervention to improve quality of sleep of palliative patient carers in the community: protocol for a multicentre randomised controlled trial.

BACKGROUND: Sleep disorders are commonly experienced by community caregivers for persons with cancer, with at least 72% reporting moderate to severe disorders. A consequence of this condition, which is associated with the presence of overload in the caregiver, is the increased risk of clinical depression. The aim of this study is to evaluate the effects of music on the sleep quality achieved by informal caregivers for cancer patients receiving home palliative care. In addition, we will assess the influence of specific variables that could modify these effects, analyse the correlates related to nocturnal wakefulness and consider the diurnal consequences according to the sleep characteristics identified. METHODS: This single-blind, multicentre, randomised clinical trial will focus on informal providers of care for cancer patients. Two samples of 40 caregivers will be recruited. The first, intervention, group will receive seven music-based sessions. The control group will be masked with seven sessions of therapeutic education (reinforcing previous sessions). Outcomes will be evaluated using the Pittsburgh Sleep Quality Index, a triaxial accelerometer, EuroQol-5D-5L, the Caregiver Strain Index, the Epworth Sleepiness Scale and the Client Satisfaction Questionnaire. The caregivers' satisfaction with the intervention performed will also be examined. DISCUSSION: This study is expected to extend our understanding of the efficacy of music therapy in enhancing the sleep quality of caregivers for patients receiving home palliative care. To our knowledge, no reliable scientific investigations of this subject have previously been undertaken. Music is believed to benefit certain aspects of sleep, but this has yet to be proven and, according to a Cochrane review, high-quality research in this field is necessary. One of the main strengths of our study, which heightens the quality of the randomised clinical trial design, is the objective assessment of physical activity by accelerometry and the use of both objective and subjective measures of sleep in caregivers. Music therapy for the caregivers addressed in this study is complementary, readily applicable, provokes no harmful side effects and may produce significant benefits. TRIAL REGISTRATION: The IMECA study is registered at Clinical Trials.gov, ClinicalTrials.gov Identifier: NCT04491110 . Registered 29 July, 2020.

Circumscribed storiform collagenoma, an unusual tumor.

Circumscribed storiform collagenoma is a rare benign tumor. It appears as an isolated skin lesion or as part of the clinical spectrum of Cowden syndrome. The pathogenesis is still controversial. Although its clinical expression is heterogeneous, it has a characteristic histological pattern. We describe a case of a solitary circumscribed storiform collagenoma not associated with Cowden syndrome.

Specific growth rate governs AOX1 gene expression, affecting the production kinetics of Pichia pastoris (Komagataella phaffii) PAOX1-driven recombinant producer strains with different target gene dosage.

BACKGROUND: The PAOX1-based expression system is the most widely used for producing recombinant proteins in the methylotrophic yeast Pichia pastoris (Komagataella phaffii). Despite relevant recent advances in regulation of the methanol utilization (MUT) pathway have been made, the role of specific growth rate (µ) in AOX1 regulation remains unknown, and therefore, its impact on protein production kinetics is still unclear. RESULTS: The influence of heterologous gene dosage, and both, operational mode and strategy, on culture physiological state was studied by cultivating the two PAOX1-driven Candida rugosa lipase 1 (Crl1) producer clones. Specifically, a clone integrating a single expression cassette of CRL1 was compared with one containing three cassettes over broad dilution rate and µ ranges in both chemostat and fed-batch cultivations. Chemostat cultivations allowed to establish the impact of µ on the MUT-related MIT1 pool which leads to a bell-shaped relationship between µ and PAOX1-driven gene expression, influencing directly Crl1 production kinetics. Also, chemostat and fed-batch cultivations exposed the favorable effects of increasing the CRL1 gene dosage (up to 2.4 fold in qp) on Crl1 production with no significant detrimental effects on physiological capabilities. CONCLUSIONS: PAOX1-driven gene expression and Crl1 production kinetics in P. pastoris were successfully correlated with µ. In fact, µ governs MUT-related MIT1 amount that triggers PAOX1-driven gene expression-heterologous genes included-, thus directly influencing the production kinetics of recombinant protein.

Functional expression of aryl-alcohol oxidase in Saccharomyces cerevisiae and Pichia pastoris by directed evolution.

Aryl-alcohol oxidase (AAO) plays a fundamental role in the fungal ligninolytic secretome, acting as a supplier of H2 O2 . Despite its highly selective mechanism of action, the presence of this flavooxidase in different biotechnological settings has hitherto been hampered by the lack of appropriate heterologous expression systems. We recently described the functional expression of the AAO from Pleurotus eryngii in Saccharomyces cerevisiae by fusing a chimeric signal peptide (preαproK) and applying structure-guided evolution. Here, we have obtained an AAO secretion variant that is readily expressed in S. cerevisiae and overproduced in Pichia pastoris. First, the functional expression of AAO in S. cerevisiae was enhanced through the in vivo shuffling of a panel of secretion variants, followed by the focused evolution of the preαproK peptide. The outcome of this evolutionary campaign-an expression variant that accumulated 4 mutations in the chimeric signal peptide, plus two mutations in the mature protein- showed 350-fold improved secretion (4.5 mg/L) and was stable. This secretion mutant was cloned into P. pastoris and fermented in a fed-batch bioreactor to enhance production to 25 mg/L. While both recombinant AAO from S. cerevisiae and P. pastoris were subjected to the same N-terminal processing and had a similar pH activity profile, they differed in their kinetic parameters and thermostability. The strong glycosylation observed in the evolved AAO from S. cerevisiae underpinned this effect, since when the mutant was produced in the glycosylation-deficient S. cerevisiae strain Δkre2, its kinetic parameters and thermostability were comparable to its poorly glycosylated P. pastoris recombinant counterpart.

An improved secretion signal enhances the secretion of model proteins from Pichia pastoris.

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.

The effect of hypoxia on the lipidome of recombinant Pichia pastoris.

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.

Clustering and Beamforming for Efficient Communication in Wireless Sensor Networks.

Energy efficiency is a critical issue for wireless sensor networks (WSNs) as sensor nodes have limited power availability. In order to address this issue, this paper tries to maximize the power efficiency in WSNs by means of the evaluation of WSN node networks and their performance when both clustering and antenna beamforming techniques are applied. In this work, four different scenarios are defined, each one considering different numbers of sensors: 50, 20, 10, five, and two nodes per scenario, and each scenario is randomly generated thirty times in order to statistically validate the results. For each experiment, two different target directions for transmission are taken into consideration in the optimization process (φ = 0° and θ = 45°; φ = 45°, and θ = 45°). Each scenario is evaluated for two different types of antennas, an ideal isotropic antenna and a conventional dipole one. In this set of experiments two types of WSN are evaluated: in the first one, all of the sensors have the same amount of power for communications purposes; in the second one, each sensor has a different amount of power for its communications purposes. The analyzed cases in this document are focused on 2D surface and 3D space for the node location. To the authors' knowledge, this is the first time that beamforming and clustering are simultaneously applied to increase the network lifetime in WSNs.

A macrokinetic model-based comparative meta-analysis of recombinant protein production by Pichia pastoris under AOX1 promoter.

An unstructured macrokinetic model for heterologous production of Rhizopus oryzae lipase (ROL) by a Pichia pastoris PAOX1 based system was developed. Mean specific rates and state variables for various fed-batch cultures, under methanol limited and non-limited conditions were used for modeling. The most representative kinetic functions (viz., Monod, Haldane, Pirt and Luedeking-Piret) were used for the specific rates of cell growth (µ), substrate consumption (qS ) and product formation (qP ). The performance of two different process models was assessed via simulation tests, using an overall validation procedure. The best model describes the cellular growth by a non-monotonic substrate function, substrate uptake via Pirt's equation and product formation through a Luedeking-Piret equation. A comparative meta-analysis of heterologous protein production of various target proteins by Pichia pastoris under AOX1 promoter was conducted and a general strategy for improving protein production from process kinetics was developed as a key to bioprocess optimization.

Monitoring lipase/esterase activity by stopped flow in a sequential injection analysis system using p-nitrophenyl butyrate.

Lipases and esterases are biocatalysts used at the laboratory and industrial level. To obtain the maximum yield in a bioprocess, it is important to measure key variables, such as enzymatic activity. The conventional method for monitoring hydrolytic activity is to take out a sample from the bioreactor to be analyzed off-line at the laboratory. The disadvantage of this approach is the long time required to recover the information from the process, hindering the possibility to develop control systems. New strategies to monitor lipase/esterase activity are necessary. In this context and in the first approach, we proposed a lab-made sequential injection analysis system to analyze off-line samples from shake flasks. Lipase/esterase activity was determined using p-nitrophenyl butyrate as the substrate. The sequential injection analysis allowed us to measure the hydrolytic activity from a sample without dilution in a linear range from 0.05-1.60 U/mL, with the capability to reach sample dilutions up to 1000 times, a sampling frequency of five samples/h, with a kinetic reaction of 5 min and a relative standard deviation of 8.75%. The results are promising to monitor lipase/esterase activity in real time, in which optimization and control strategies can be designed.

Comprehensive clone screening and evaluation of fed-batch strategies in a microbioreactor and lab scale stirred tank bioreactor system: application on Pichia pastoris producing Rhizopus oryzae lipase.

BACKGROUND: In Pichia pastoris bioprocess engineering, classic approaches for clone selection and bioprocess optimization at small/micro scale using the promoter of the alcohol oxidase 1 gene (PAOX1), induced by methanol, present low reproducibility leading to high time and resource consumption. RESULTS: An automated microfermentation platform (RoboLector) was successfully tested to overcome the chronic problems of clone selection and optimization of fed-batch strategies. Different clones from Mut+P. pastoris phenotype strains expressing heterologous Rhizopus oryzae lipase (ROL), including a subset also overexpressing the transcription factor HAC1, were tested to select the most promising clones.The RoboLector showed high performance for the selection and optimization of cultivation media with minimal cost and time. Syn6 medium was better than conventional YNB medium in terms of production of heterologous protein.The RoboLector microbioreactor was also tested for different fed-batch strategies with three clones producing different lipase levels. Two mixed substrates fed-batch strategies were evaluated. The first strategy was the enzymatic release of glucose from a soluble glucose polymer by a glucosidase, and methanol addition every 24 hours. The second strategy used glycerol as co-substrate jointly with methanol at two different feeding rates. The implementation of these simple fed-batch strategies increased the levels of lipolytic activity 80-fold compared to classical batch strategies used in clone selection. Thus, these strategies minimize the risk of errors in the clone selection and increase the detection level of the desired product.Finally, the performance of two fed-batch strategies was compared for lipase production between the RoboLector microbioreactor and 5 liter stirred tank bioreactor for three selected clones. In both scales, the same clone ranking was achieved. CONCLUSION: The RoboLector showed excellent performance in clone selection of P. pastoris Mut+ phenotype. The use of fed-batch strategies using mixed substrate feeds resulted in increased biomass and lipolytic activity. The automated processing of fed-batch strategies by the RoboLector considerably facilitates the operation of fermentation processes, while reducing error-prone clone selection by increasing product titers.The scale-up from microbioreactor to lab scale stirred tank bioreactor showed an excellent correlation, validating the use of microbioreactor as a powerful tool for evaluating fed-batch operational strategies.

Synergic kinetic and physiological control to improve the efficiency of Komagataella phaffii recombinant protein production bioprocesses.

The yeast Komagataella phaffii (Pichia pastoris) is currently considered a versatile and highly efficient host for recombinant protein production (RPP). Interestingly, the regulated application of specific stress factors as part of bioprocess engineering strategies has proven potential for increasing the production of recombinant products. This study aims to evaluate the impact of controlled oxygen-limiting conditions on the performance of K. phaffii bioprocesses for RPP in combination with the specific growth rate (µ) in fed-batch cultivations. In this work, Candida rugosa lipase 1 (Crl1) production, regulated by the constitutive GAP promoter, growing at different nominal µ (0.030, 0.065, 0.100 and 0.120 h-1 ) under both normoxic and hypoxic conditions in carbon-limiting fed-batch cultures is analysed. Hypoxic fermentations were controlled at a target respiratory quotient (RQ) of 1.4, with excellent performance, using an innovative automated control based on the stirring rate as the manipulated variable developed during this study. The results conclude that oxygen limitation positively affects bioprocess efficiency under all growing conditions compared. The shift from respiratory to respiro-fermentative metabolism increases bioprocess productivity by up to twofold for the specific growth rates evaluated. Moreover, the specific product generation rate (qp ) increases linearly with µ, regardless of oxygen availability. Furthermore, this hypoxic boosting effect was also observed in the production of Candida antarctica lipase B (CalB) and pro-Rhizopus oryzae lipase (proRol), thus proving the synergic effect of kinetic and physiological stress control. Finally, the Crl1 production scale-up was conducted successfully, confirming the strategy's scalability and the robustness of the results obtained at the bench-scale level.

Effect of serum from patients with ST-segment elevation myocardial infarction on endothelial cells.

INTRODUCTION AND OBJECTIVES: Clinical and experimental studies have shown that, in patients with reperfused ST-segment elevation myocardial infarction (STEMI), abnormalities in the endothelial monolayer are initiated during ischemia but rapidly intensify upon restoration of blood perfusion to the ischemic area. We aimed to evaluate the effect of serum isolated after revascularization from STEMI patients on the degree of endothelial permeability in vitro, by promoting endothelial cell apoptosis and necrosis in vitro. We also investigated the association between the percentage of serum-induced endothelial cell apoptosis or necrosis in vitro and the extent of cardiovascular magnetic resonance (CMR)-derived parameters of reperfusion injury (edema, hemorrhage, and microvascular obstruction). METHODS: Human coronary artery endothelial cells were incubated with serum isolated 24hours after revascularization from 43 STEMI patients who underwent CMR and 14 control participants. We assessed the effect of STEMI serum on activation of apoptosis and necrosis, as well as on the permeability and structure of the endothelial monolayer. RESULTS: Serum from STEMI patients increased apoptosis (P <.01) and necrosis (P <.05) in human coronary artery endothelial cells and caused increased permeability of the endothelial monolayer in vitro (P <.01), due to enlarged intercellular spaces (P <.05 vs control in all cases). Higher serum-induced necrosis was associated with greater endothelial permeability in vitro (P <.05) and with more extensive CMR-derived indices of reperfusion injury and infarct size. CONCLUSIONS: Postreperfusion serum activates necrosis and apoptosis in endothelial cells and increases the degree of endothelial permeability in vitro. The more potent the necrosis-triggering effect of serum, the more deleterious the consequences in terms of the resulting cardiac structure.