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Genome-scale metabolic models (GEMs) are mathematical representations of metabolism that allow for in silico simulation of metabolic phenotypes and capabilities. A prerequisite for these predictions is an accurate representation of the biomolecular composition of the cell necessary for replication and growth, implemented in GEMs as the so-called biomass objective function (BOF). The BOF contains the metabolic precursors required for synthesis of the cellular macro- and micromolecular constituents (e.g. protein, RNA, DNA), and its composition is highly dependent on the particular organism, strain, and growth condition. Despite its critical role, the BOF is rarely constructed using specific measurements of the modeled organism, drawing the validity of this approach into question. Thus, there is a need to establish robust and reliable protocols for experimental condition-specific biomass determination. Here, we address this challenge by presenting a general pipeline for biomass quantification, evaluating its performance on Escherichia coli K-12 MG1655 sampled during balanced exponential growth under controlled conditions in a batch-fermentor set-up. We significantly improve both the coverage and molecular resolution compared to previously published workflows, quantifying 91.6% of the biomass. Our measurements display great correspondence with previously reported measurements, and we were also able to detect subtle characteristics specific to the particular E. coli strain. Using the modified E. coli GEM i ML1515a, we compare the feasible flux ranges of our experimentally determined BOF with the original BOF, finding that the changes in BOF coefficients considerably affect the attainable fluxes at the genome-scale.

Background The yeast Komagataella phaffii (formerly known as Pichia pastoris ) has been widely used for functional expression of recombinant proteins, including plant and animal food proteins. CRISPR/Cas9 genome editing systems can be used for insertion of heterologous genes without the use of selection markers. The study aimed to create a convenient markerless knock-in method for integrating expression cassettes into the chromosome of K. phaffii using CRISPR/Cas9 technology. The approach was based on the hierarchical, modular, Golden Gate assembly employing the Golden Pi CS toolkit. Furthermore, the aim was to evaluate the system’s efficiency and suitability for producing secreted recombinant food proteins. Results Three Cas9/sgRNA plasmids were constructed, along with corresponding donor helper plasmids containing homology regions for chromosomal integration via homology-directed repair. The integration efficiency of an enhanced green fluorescent protein (eGFP) expression cassette was assessed at three genomic loci ( 04576 , PFK1 , and ROX1 ). The 04576 locus showed the highest integration efficiency, while ROX1 had the highest transformation efficiency. Whole genome sequencing revealed variable copy numbers of eGFP expression cassettes among clones, corresponding with increasing levels of fluorescence. Furthermore, the system’s applicability for producing recombinant food proteins was validated by successfully expressing and secreting chicken ovalbumin. This constitutes the first report of CRISPR/Cas9 applied to produce recombinant chicken ovalbumin. Conclusions The adapted Golden Pi CS toolkit combined with CRISPR/Cas9 technology enabled efficient and precise genome integration in K. phaffii . This approach holds promise for expanding the production of high-value recombinant proteins. Future research should focus on optimizing integration sites and improving cloning procedures to enhance the system’s efficiency and versatility.

Precise and accurate quantification is a prerequisite for interpretation of targeted metabolomics data, but this task is challenged by the inherent instability of the analytes. The sampling, quenching, extraction, and sample purification conditions required to recover and stabilize metabolites in representative extracts have also been proven highly dependent on species-specific properties. For Escherichia coli, unspecific leakage has been demonstrated for conventional microbial metabolomics sampling protocols. We herein present a fast filtration-based sampling protocol for this widely applied model organism, focusing on pitfalls such as inefficient filtration, selective loss of biomass, matrix contamination, and membrane permeabilization and leakage. We evaluate the effect of and need for removal of extracellular components and demonstrate how residual salts can challenge analytical accuracy of hyphenated mass spectrometric analyses, even when sophisticated correction strategies are applied. Laborious extraction procedures are bypassed by direct extraction in cold acetonitrile:water:methanol (3:5:2, v/v%), ensuring compatibility with sample concentration and thus, any downstream analysis. By applying this protocol, we achieve and demonstrate high precision and low metabolite turnover, and, followingly, minimal perturbation of the inherent metabolic state. This allows us to herein report absolute intracellular concentrations in E. coli and explore its central carbon metabolome at several commonly applied cultivation conditions.

Background: Nicotinamide adenine dinucleotide (NAD+), its precursors, and its derivatives (collectively NADome) play a crucial role in cellular processes and maintain redox homeostasis. Understanding the dynamics of these metabolic pools and redox reactions can provide valuable insights into metabolic functions, especially cellular regulation and stress response mechanisms. The accurate quantification of these metabolites is challenging due to the interconversion between the redox forms. Methods: Our laboratory previously developed a zwitterionic hydrophilic interaction liquid chromatography (zic-HILIC)–tandem mass spectrometry method for the quantification of five essential pyridine nucleotides, including NAD+ derivatives and it’s reduced forms, with 13C isotope dilution and matrix-matched calibration. In this study, we have improved the performance of the chromatographic method and expanded its scope to twelve analytes for a comprehensive view of NAD+ biosynthesis and utilization. The analytical method was validated and applied to investigate Escherichia coli BL21 under varying oxygen supplies including aerobic, microaerobic, and anaerobic conditions. Conclusions: The intracellular absolute metabolite concentrations ranged over four orders of magnitude with NAD+ as the highest abundant, while its precursors were much less abundant. The composition of the NADome at oxygen-limited conditions aligned more with that in the anaerobic conditions rather than in the aerobic phase. Overall, the NADome was quite homeostatic and E. coli rapidly, but in a minor way, adapted the metabolic activity to the challenging shift in the growth conditions and achieved redox balance. Our findings demonstrate that the zic-HILIC-MS/MS method is sensitive, accurate, robust, and high-throughput, providing valuable insights into NAD+ metabolism and the potential significance of these metabolites in various biological contexts.

The effectiveness of rabbit-sperm cryopreservation is still below average compared to other domestic species. After the sperm cryopreservation process, post-thawing parameters like motility and membrane integrity are significantly compromised. The use of new extender constituents is an approach that can be used to improve the effectiveness of cryopreservation. Accordingly, we used honey (1.25, 2.5, 5, and 10%), coenzyme Q10 (100 and 200 μM), and β-carotene/α-tocopherol (500 μM/620 μM and 250 μM/310 μM) as candidate components for rabbit-sperm extenders during cryopreservation. Ejaculates from commercial adult rabbit bucks (n = 5) were cryopreserved using conventional freezing. Several post-thawing sperm parameters were assessed, including total motility, membrane integrity, viability, nuclear membrane integrity, acrosome reaction, and mitochondrial membrane potential and activation. Additionally, we performed hormonal analyses of the seminal plasma. Moreover, we analyzed the post-thawing levels of a molecular marker of sperm quality, proAKAP4, which was used in rabbits for the first time. Our findings showed that the 2.5% honey supplementation increased the post-thawing sperm motility (13.75 ± 3.75%) compared to the greater concentrations employed. However, the post-thawing motility was negatively affected by the coenzyme Q10 (0%, in both groups) but was not affected by the β-carotene/α-tocopherol supplementation (22 ± 18.15%, and 11.67 ± 10.17%). In conclusion, the cryopreservation protocols of this study did not help to maintain the sperm parameters after thawing. Further studies are required to identify novel protocols to mitigate the damage caused to rabbit sperm during cryopreservation.

Blood serum (BS) and seminal plasma (SP) share a plethora of compounds that might present an individual and/or temporal concentration variation. We aimed to determine whether BS and SP concentrations of albumin, calcium, citrate, creatinine, fructose, glucose, lactate, total protein, urea, zinc, cortisol, anti‐Müllerian hormone (AMH) and testosterone are related to weekly collections in New Zealand White (NZW) adult rabbit bucks. During a 12‐week study, blood samples were obtained at the beginning and the end of the study period, and semen samples were taken twice a week from four NZW adult rabbit bucks, starting at 6–7 months of age. After semen collection, the sperm motility was subjectively assessed, and SP was obtained by centrifugation. BS and SP were evaluated for the above‐mentioned metabolites using a Biosystems BA400 automated analyser with commercial‐specific kits or enzyme immunoassay (EIA) kits, assessing the effects of the male and time of collection. In addition, a correlation analysis aimed at disclosing associations between parameters in BS and SP was performed. Male effect was not significant for BS, but it was significant for SP albumin, citrate, fructose, glucose, lactate and total protein. In addition, all the correlations in BS were positive, whereas they were more balanced in SP, being close to half of the correlations. In conclusion, variations of some metabolites (albumin, citrate, fructose, glucose, lactate and total protein) appear to be potential biomarkers for rabbit SP, although further studies should test their usefulness for sperm fertility assessment. New Zealand White adult rabbit bucks blood serum (BS) and seminal plasma (SP) share a plethora of compounds that might present an individual and/or temporal concentration variation (12‐week study). Our results showed variations of some metabolites (albumin, citrate, fructose, glucose, lactate and total protein) that appear to be potential biomarkers for rabbit SP. However, further studies should test their usefulness for sperm fertility assessment.

El retablo mayor de la localidad guipuzcoana de Lazkao fue realizado por Juan de Ursularre en 1683, siguiendo los parámetros de la retablística barroca. Queriendo engrandecer su aspecto, fue paulatinamente transformado durante el siglo XVIII. Por ello se concibió un nuevo cascarón con pinturas enviadas desde Madrid y más tarde se adecuó a los nuevos gustos neoclásicos mediante una radical reforma realizada en 1791, que consistió en eliminar todo atisbo de decoración barroca.The main altarpiece of Lazkao, Gipuzkoa, was done by Juan de Ursularre in 1683, following the Baroque guidelines. It was transformed during the 18th century to enlarge its appearance. Due to this, it was conceived anew with paintings sent from Madrid. Later, in 1791, the altarpiece was neoclassized with a radical transformation that consisted of the removal of the whole Baroque decoration.

Plant biomass is a promising substrate for biorefinery, as well as a source of bioactive compounds, platform chemicals, and precursors with multiple industrial applications. These applications depend on the hydrolysis of its recalcitrant structure. However, the effective biological degradation of plant cell walls requires several enzymatic groups acting synergistically, and novel enzymes are needed in order to achieve profitable industrial hydrolysis processes. In the present work, a feruloyl esterase (FAE) activity screening of Penicillium spp. strains revealed a promising candidate (Penicillium rubens Wisconsin 54–1255; previously Penicillium chrysogenum), where two FAE-ORFs were identified and subsequently overexpressed. Enzyme extracts were analyzed, confirming the presence of FAE activity in the respective gene products (PrFaeA and PrFaeB). PrFaeB-enriched enzyme extracts were used to determine the FAE activity optima (pH 5.0 and 50–55 °C) and perform proteome analysis by means of MALDI-TOF/TOF mass spectrometry. The studies were completed with the determination of other lignocellulolytic activities, an untargeted metabolite analysis, and upscaled FAE production in stirred tank reactors. The findings described in this work present P. rubens as a promising lignocellulolytic enzyme producer.Key points• Two Penicillium rubens ORFs were first confirmed to have feruloyl esterase activity.• Overexpression of the ORFs produced a novel P. rubens strain with improved activity.• The first in-depth proteomic study of a P. rubens lignocellulolytic extract is shown.

Fourier transform mid-infrared (FT-MIR) spectroscopy has been extensively used as a potent, fast and non-destructive procedure for analyzing cell wall architectures, with the capacity to provide abundant information about their polymers, functional groups, and in muro entanglement. In conjunction with multivariate analyses, this method has proved to be a valuable tool for tracking alterations in cell walls. The present review examines recent progress in the use of FT-MIR spectroscopy to monitor cell wall changes occurring in muro as a result of various factors, such as growth and development processes, genetic modifications, exposition or habituation to cellulose biosynthesis inhibitors and responses to other abiotic or biotic stresses, as well as its biotechnological applications.