Terminal heterocyst differentiation in the Anabaena patA mutant as a result of post-transcriptional modifications and molecular leakage.

The Anabaena genus is a model organism of filamentous cyanobacteria whose vegetative cells can differentiate under nitrogen-limited conditions into a type of cell called heterocyst. These heterocysts lose the possibility to divide and are necessary for the colony because they can fix and share environmental nitrogen. In order to distribute the nitrogen efficiently, heterocysts are arranged to form a quasi-regular pattern whose features are maintained as the filament grows. Recent efforts have allowed advances in the understanding of the interactions and genetic mechanisms underlying this dynamic pattern. However, the main role of the patA and hetF genes are yet to be clarified; in particular, the patA mutant forms heterocysts almost exclusively in the terminal cells of the filament. In this work, we investigate the function of these genes and provide a theoretical model that explains how they interact within the broader genetic network, reproducing their knock-out phenotypes in several genetic backgrounds, including a nearly uniform concentration of HetR along the filament for the patA mutant. Our results suggest a role of hetF and patA in a post-transcriptional modification of HetR which is essential for its regulatory function. In addition, the existence of molecular leakage out of the filament in its boundary cells is enough to explain the preferential appearance of terminal heterocysts, without any need for a distinct regulatory pathway.

High throughput 13C-metabolic flux analysis of 3-hydroxypropionic acid producing Pichia pastoris reveals limited availability of acetyl-CoA and ATP due to tight control of the glycolytic flux.

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.

Engineering the synthetic ß-alanine pathway in Komagataella phaffii for conversion of methanol into 3-hydroxypropionic acid.

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 ß-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 PpCß21 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 (PpCß21) 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 PpCß21. 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 ß-alanine pathway.

A long-lasting porcine model of ARDS caused by pneumonia and ventilator-induced lung injury.

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 < 150 mmHg. Four animals (pneumonia-without-VILI group) were protectively ventilated 3 h before inoculum and thereafter. Gas exchange, respiratory mechanics, hemodynamics, microbiological studies and inflammatory markers were analyzed during the 96-h experiment. During necropsy, lobar samples were also analyzed. RESULTS: All animals from pneumonia-with-VILI group reached Berlin criteria for ARDS diagnosis until the end of experiment. The mean duration under ARDS diagnosis was 46.8 ± 7.7 h; the lowest PaO2/FiO2 was 83 ± 5.45 mmHg. The group of pigs that were not subjected to VILI did not meet ARDS criteria, even when presenting with bilateral pneumonia. Animals developing ARDS presented hemodynamic instability as well as severe hypercapnia despite high-minute ventilation. Unlike the pneumonia-without-VILI group, the ARDS animals presented lower static compliance (p = 0.011) and increased pulmonary permeability (p = 0.013). The highest burden of P. aeruginosa was found at pneumonia diagnosis in all animals, as well as a high inflammatory response shown by a release of interleukin (IL)-6 and IL-8. At histological examination, only animals comprising the pneumonia-with-VILI group presented signs consistent with diffuse alveolar damage. CONCLUSIONS: In conclusion, we established an accurate pulmonary sepsis-induced ARDS model.

A coarse-grained approach to model the dynamics of the actomyosin cortex.

BACKGROUND: The dynamics of the actomyosin machinery is at the core of many important biological processes. Several relevant cellular responses such as the rhythmic compression of the cell cortex are governed, at a mesoscopic level, by the nonlinear interaction between actin monomers, actin crosslinkers, and myosin motors. Coarse-grained models are an optimal tool to study actomyosin systems, since they can include processes that occur at long time and space scales, while maintaining the most relevant features of the molecular interactions. RESULTS: Here, we present a coarse-grained model of a two-dimensional actomyosin cortex, adjacent to a three-dimensional cytoplasm. Our simplified model incorporates only well-characterized interactions between actin monomers, actin crosslinkers and myosin, and it is able to reproduce many of the most important aspects of actin filament and actomyosin network formation, such as dynamics of polymerization and depolymerization, treadmilling, network formation, and the autonomous oscillatory dynamics of actomyosin. CONCLUSIONS: We believe that the present model can be used to study the in vivo response of actomyosin networks to changes in key parameters of the system, such as alterations in the attachment of actin filaments to the cell cortex.

Process intensification at the expression system level for the production of 1-phosphate aldolase in antibiotic-free E. coli fed-batch cultures.

To successfully design expression systems for industrial biotechnology and biopharmaceutical applications; plasmid stability, efficient synthesis of the desired product and the use of selection markers acceptable to regulatory bodies are of utmost importance. In this work we demonstrate the application of a set of IPTG-inducible protein expression systems -- harboring different features namely, antibiotic vs auxotrophy marker; two-plasmids vs single plasmid expression system; expression levels of the repressor protein (LacI) and the auxotrophic marker (glyA) -- in high-cell density cultures to evaluate their suitability in bioprocess conditions that resemble industrial settings. Results revealed that the first generation of engineered strain showed a 50% reduction in the production of the model recombinant protein fuculose-1-phosphate aldolase (FucA) compared to the reference system from QIAGEN. The over-transcription of glyA was found to be a major factor responsible for the metabolic burden. The second- and third-generation of expression systems presented an increase in FucA production and advantageous features. In particular, the third-generation expression system is antibiotic-free, autotrophy-selection based and single-plasmid and, is capable to produce FucA at similar levels compared to the original commercial expression system. These new tools open new avenues for high-yield and robust expression of recombinant proteins in E. coli.

Redox Engineering by Ectopic Overexpression of NADH Kinase in Recombinant Pichia pastoris (Komagataella phaffii): Impact on Cell Physiology and Recombinant Production of Secreted Proteins.

High-level expression and secretion of heterologous proteins in yeast cause an increased energy demand, which may result in altered metabolic flux distributions. Moreover, recombinant protein overproduction often results in endoplasmic reticulum (ER) stress and oxidative stress, causing deviations from the optimal NAD(P)H regeneration balance. In this context, overexpression of genes encoding enzymes catalyzing endogenous NADPH-producing reactions, such as the oxidative branch of the pentose phosphate pathway, has been previously shown to improve protein production in Pichia pastoris (syn. Komagataella spp.). In this study, we evaluate the overexpression of the Saccharomyces cerevisiaePOS5-encoded NADH kinase in a recombinant P. pastoris strain as an alternative approach to overcome such redox constraints. Specifically, POS5 was cooverexpressed in a strain secreting an antibody fragment, either by directing Pos5 to the cytosol or to the mitochondria. The physiology of the resulting strains was evaluated in continuous cultivations with glycerol or glucose as the sole carbon source, as well as under hypoxia (on glucose). Cytosolic targeting of Pos5 NADH kinase resulted in lower biomass-substrate yields but allowed for a 2-fold increase in product specific productivity. In contrast, Pos5 NADH kinase targeting to the mitochondria did not affect growth physiology and recombinant protein production significantly. Growth physiological parameters were in silico evaluated using the recent upgraded version (v3.0) of the P. pastoris consensus genome-scale metabolic model iMT1026, providing insights on the impact of POS5 overexpression on metabolic flux distributions.IMPORTANCE Recombinant protein overproduction often results in oxidative stress, causing deviations from the optimal redox cofactor regeneration balance. This becomes one of the limiting factors in obtaining high levels of heterologous protein production. Overexpression of redox-affecting enzymes has been explored in other organisms, such as Saccharomyces cerevisiae, as a means to fine tune the cofactor regeneration balance in order to obtain higher protein titers. In the present work, this strategy is explored in P. pastoris In particular, one NADH kinase enzyme from S. cerevisiae (Pos5) is used, either in the cytosol or in mitochondria of P. pastoris, and its impact on the production of a model protein (antibody fragment) is evaluated. A significant improvement in the production of the model protein is observed when the kinase is directed to the cytosol. These results are significant in the field of heterologous protein production in general and in particular in the development of improved metabolic engineering strategies for P. pastoris.

Carbohydrate Hydrolytic Potential and Redundancy of an Anaerobic Digestion Microbiome Exposed to Acidosis, as Uncovered by Metagenomics.

Increased hydrolysis of easily digestible biomass may lead to acidosis of anaerobic reactors and decreased methane production. Previously, it was shown that the structure of microbial communities changed during acidosis; however, once the conditions are back to optimal, biogas (initially CO2) production quickly restarts. This suggests the retention of the community functional redundancy during the process failure. In this study, with the use of metagenomics and downstream bioinformatics analyses, we characterize the carbohydrate hydrolytic potential of the microbial community, with a special focus on acidosis. To that purpose, carbohydrate-active enzymes were identified, and to further link the community hydrolytic potential with key microbes, bacterial genomes were reconstructed. In addition, we characterized biochemically the specificity and activity of selected enzymes, thus verifying the accuracy of the in silico predictions. The results confirm the retention of the community hydrolytic potential during acidosis and indicate Bacteroidetes to be largely involved in biomass degradation. Bacteroidetes showed higher diversity and genomic content of carbohydrate hydrolytic enzymes that might favor the dominance of this phylum over other bacteria in some anaerobic reactors. The combination of bioinformatic analyses and activity tests enabled us to propose a model of acetylated glucomannan degradation by BacteroidetesIMPORTANCE The enzymatic hydrolysis of lignocellulosic biomass is mainly driven by the action of carbohydrate-active enzymes. By characterizing the gene profiles at the different stages of the anaerobic digestion experiment, we showed that the microbiome retains its hydrolytic functional redundancy even during severe acidosis, despite significant changes in taxonomic composition. By analyzing reconstructed bacterial genomes, we demonstrate that Bacteroidetes hydrolytic gene diversity likely favors the abundance of this phylum in some anaerobic digestion systems. Further, we observe genetic redundancy within the Bacteroidetes group, which accounts for the preserved hydrolytic potential during acidosis. This work also uncovers new polysaccharide utilization loci involved in the deconstruction of various biomasses and proposes the model of acetylated glucomannan degradation by Bacteroidetes Acetylated glucomannan-enriched biomass is a common substrate for many industries, including pulp and paper production. Using naturally evolved cocktails of enzymes for biomass pretreatment could be an interesting alternative to the commonly used chemical pretreatments.

Deregulation of methanol metabolism reverts transcriptional limitations of recombinant Pichia pastoris (Komagataella spp) with multiple expression cassettes under control of the AOX1 promoter.

The methanol-regulated alcohol oxidase promoter (PAOX1 ) of Pichia pastoris (syn. Komagataella spp. ) is one of the strongest promoters for heterologous gene expression. Although increasing the gene dosage is a common strategy to improve recombinant protein productivities, P. pastoris strains harboring more than two copies of a Rhizopus oryzae lipase gene (ROL) have previously shown a decrease in cell growth, lipase production, and substrate consumption, as well as a significant transcriptional downregulation of methanol metabolism. This pointed to a potential titration effect of key transcriptional factors methanol expression regulator 1 (Mxr1) and methanol-induced transcription factor (Mit1) regulating methanol metabolism caused by the insertion of multiple expression vectors. To prove this hypothesis, a set of strains carrying one and four copies of ROL (1C and 4C, respectively) were engineered to coexpress one or two copies of MXR1*, coding for an Mxr1 variant insensitive to repression by 14-3-3 regulatory proteins, or one copy of MIT1. Small-scale cultures revealed that growth, Rol productivity, and methanol consumption were improved in the 4C-MXR1* and 4C-MIT1, strains growing on methanol as a sole carbon source, whereas only a slight increase in productivity was observed for re-engineered 1C strains. We further verified the improved performance of these strains in glycerol-/methanol-limited chemostat cultures.

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.

Metabolic flux analysis and the NAD(P)H/NAD(P)+ ratios in chemostat cultures of Azotobacter vinelandii.

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.

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.

Droplet digital PCR-aided screening and characterization of Pichia pastoris multiple gene copy strains.

Pichia (syn. Komagataella) pastoris is a widely used yeast platform for heterologous protein production. Expression cassettes are usually stably integrated into the genome of this host via homologous recombination. Although increasing gene dosage is a powerful strategy to improve recombinant protein production, an excess in the number of gene copies often leads to decreased product yields and increased metabolic burden, particularly for secreted proteins. We have constructed a series of strains harboring different copy numbers of a Rhizopus oryzae lipase gene (ROL), aiming to find the optimum gene dosage for secreted Rol production. In order to accurately determine ROL gene dosage, we implemented a novel protocol based on droplet digital PCR (ddPCR), and cross validated it with conventional real-time PCR. Gene copy number determination based on ddPCR allowed for an accurate ranking of transformants according to their ROL gene dosage. Results indicated that ddPCR was particularly superior at lower gene dosages (one to five copies) over quantitative real-time PCR (qPCR). This facilitated the determination of the optimal ROL gene dosage as low as two copies. The ranking of ROL gene dosage versus Rol yield was consistent at both small scale and bioreactor chemostat cultures, thereby easing clone characterization in terms of gene dosage dependent physiological effects, which could be discriminated even among strains differing by only one ROL copy. A selected two-copy strain showed twofold increase in Rol specific production in a chemostat culture over the single copy strain. Conversely, strains harboring more than two copies of the ROL gene showed decreased product and biomass yields, as well as altered substrate consumption specific rates, compared to the reference (one-copy) strain. Biotechnol. Bioeng. 2016;113: 1542-1551. © 2015 Wiley Periodicals, Inc.

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.

Use of chemostat cultures mimicking different phases of wine fermentations as a tool for quantitative physiological analysis.

BACKGROUND: Saccharomyces cerevisiae is the most relevant yeast species conducting the alcoholic fermentation that takes place during winemaking. Although the physiology of this model organism has been extensively studied, systematic quantitative physiology studies of this yeast under winemaking conditions are still scarce, thus limiting the understanding of fermentative metabolism of wine yeast strains and the systematic description, modelling and prediction of fermentation processes. In this study, we implemented and validated the use of chemostat cultures as a tool to simulate different stages of a standard wine fermentation, thereby allowing to implement metabolic flux analyses describing the sequence of metabolic states of S. cerevisae along the wine fermentation. RESULTS: Chemostat cultures mimicking the different stages of standard wine fermentations of S. cerevisiae EC1118 were performed using a synthetic must and strict anaerobic conditions. The simulated stages corresponded to the onset of the exponential growth phase, late exponential growth phase and cells just entering stationary phase, at dilution rates of 0.27, 0.04, 0.007 h-1, respectively. Notably, measured substrate uptake and product formation rates at each steady state condition were generally within the range of corresponding conversion rates estimated during the different batch fermentation stages.Moreover, chemostat data were further used for metabolic flux analysis, where biomass composition data for each condition was considered in the stoichiometric model. Metabolic flux distributions were coherent with previous analyses based on batch cultivations data and the pseudo-steady state assumption. CONCLUSIONS: Steady state conditions obtained in chemostat cultures reflect the environmental conditions and physiological states of S. cerevisiae corresponding to the different growth stages of a typical batch wine fermentation, thereby showing the potential of this experimental approach to systematically study the effect of environmental relevant factors such as temperature, sugar concentration, C/N ratio or (micro) oxygenation on the fermentative metabolism of wine yeast strains.

Relationship between Hydration Status and Muscle Catabolism in the Aged Population: A Cross-Sectional Study.

BACKGROUND: The physiopathology of sarcopenia is still not completely understood. AIM: To assess the relationship between dehydration and skeletal muscle catabolism, muscle mass, and sarcopenia in an aged population. METHODS: Observational cross-sectional study of community-dwelling subjects aged 70 years and older. Dehydration was assessed by plasma osmolarity; bioimpedance analysis (BIA) was used to assess body composition and water content; sarcopenia was established according to the EWGSOP-2 criteria; and 3-methyl-histidine (3MH) was used as an indicator of muscle catabolism. RESULTS: 190 participants were recruited (77.4 years; 51.6% women). In total, 22.6% and 20.5% presented plasma osmolarity of 295-300 mOsm/L and >300 mOsm/L, respectively. Age was correlated with plasma osmolarity (rs = 0.439; p < 0.001). Plasma osmolarity was correlated with 3MH (rs = 0.360; p < 0.001) and showed an effect on 3MH levels, with an adjusted (by age, sex, and number of medications) beta of 0.283 (p < 0.001). BIA water content indicators showed no correlation with 3MH. Lower in sarcopenic compared to non-sarcopenic subjects were the intracellular water percentage (60.3 vs. 61.2%; p = 0.004) and intracellular water/free-fat mass ratio (44.3 vs. 45.0; p = 0.004). CONCLUSIONS: Dehydration is a highly prevalent clinical condition in aged populations, increases with age, and is associated with muscle catabolism but not sarcopenia.

Novel CaLB-like Lipase Found Using ProspectBIO, a Software for Genome-Based Bioprospection.

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.

Metabolic flux profiling of recombinant protein secreting Pichia pastoris growing on glucose:methanol mixtures.

BACKGROUND: The methylotrophic yeast Pichia pastoris has emerged as one of the most promising yeast hosts for the production of heterologous proteins. Mixed feeds of methanol and a multicarbon source instead of methanol as sole carbon source have been shown to improve product productivities and alleviate metabolic burden derived from protein production. Nevertheless, systematic quantitative studies on the relationships between the central metabolism and recombinant protein production in P. pastoris are still rather limited, particularly when growing this yeast on mixed carbon sources, thus hampering future metabolic network engineering strategies for improved protein production. RESULTS: The metabolic flux distribution in the central metabolism of P. pastoris growing on a mixed feed of glucose and methanol was analyzed by Metabolic Flux Analysis (MFA) using 13C-NMR-derived constraints. For this purpose, we defined new flux ratios for methanol assimilation pathways in P. pastoris cells growing on glucose:methanol mixtures. By using this experimental approach, the metabolic burden caused by the overexpression and secretion of a Rhizopus oryzae lipase (Rol) in P. pastoris was further analyzed. This protein has been previously shown to trigger the unfolded protein response in P. pastoris. A series of 13C-tracer experiments were performed on aerobic chemostat cultivations with a control and two different Rol producing strains growing at a dilution rate of 0.09 h(-1) using a glucose:methanol 80:20 (w/w) mix as carbon source.The MFA performed in this study reveals a significant redistribution of carbon fluxes in the central carbon metabolism when comparing the two recombinant strains vs the control strain, reflected in increased glycolytic, TCA cycle and NADH regeneration fluxes, as well as higher methanol dissimilation rates. CONCLUSIONS: Overall, a further 13C-based MFA development to characterise the central metabolism of methylotrophic yeasts when growing on mixed methanol:multicarbon sources has been implemented, thus providing a new tool for the investigation of the relationships between central metabolism and protein production. Specifically, the study points at a limited but significant impact of the conformational stress associated to secretion of recombinant proteins on the central metabolism, occurring even at modest production levels.

Quantitative metabolomics analysis of amino acid metabolism in recombinant Pichia pastoris under different oxygen availability conditions.

BACKGROUND: Environmental and intrinsic stress factors can result in the global alteration of yeast physiology, as evidenced by several transcriptional studies. Hypoxia has been shown to have a beneficial effect on the expression of recombinant proteins in Pichia pastoris growing on glucose. Furthermore, transcriptional profiling analyses revealed that oxygen availability was strongly affecting ergosterol biosynthesis, central carbon metabolism and stress responses, in particular the unfolded protein response. To contribute to the better understanding of the effect and interplay of oxygen availability and foreign protein secretion on central metabolism, a first quantitative metabolomic analysis of free amino acids pools in a recombinant P. pastoris strain growing under different oxygen availability conditions has been performed. RESULTS: The values obtained indicate significant variations in the intracellular amino acid pools due to different oxygen availability conditions, showing an overall increase of their size under oxygen limitation. Notably, even while foreign protein productivities were relatively low (about 40-80 µg Fab/g(DCW)·h), recombinant protein production was found to have a limited but significant impact on the intracellular amino acid pools, which were generally decreased in the producing strain compared with the reference strain. However, observed changes in individual amino acids pools were not correlated with their corresponding relative abundance in the recombinant protein sequence, but to the overall cell protein amino acid compositional variations. CONCLUSIONS: Overall, the results obtained, combined with previous transcriptomic and proteomic analyses provide a systematic metabolic fingerprint of the oxygen availability impact on recombinant protein production in P. pastoris.

Corrigendum: Combining metabolic engineering and multiplexed screening methods for 3-hydroxypropionic acid production in Pichia pastoris.

[This corrects the article DOI: 10.3389/fbioe.2022.942304.].