Longissimus thoracis (LT) muscle samples were taken for metabolite profile analysis using nuclear magnetic resonance. Spectrum was analyzed using chenomx software, and multi- and mega-variate data analyses were performed. The PLS-DA showed clear separation between FH and PL groups and overlap among treatments with different finishing systems but similar for matching ADG (FL and PH) treatments. Using a VIP cut-off of around 1.0, ATP and fumarate were shown to be greater in meat from PL cattle, while succinate, leucine, AMP, glutamate, carnosine, inosine, methionine, G1P, and choline were greater in meat from FH. Comparing FL and PH treatments, glutamine, carnosine, urea, NAD+, malonate, lactate, isoleucine, and alanine were greater in the meat of PH cattle, while G6P and betaine were elevated in that of FL cattle. Relevant pathways were also identified by differences in growth rate (FH versus PL) and finishing system were also noted. Growth rate caused a clear difference in meat metabolism that was highlighted by energy metabolism and associated pathways, while the feeding system tended to alter protein and lipid metabolism.The present study explored changes in the meat metabolome of animals subjected to different finishing systems and growth rates. Thirty-six Angus × Nellore crossbred steers were used in a completely randomized design with four treatments: (1) feedlot system with high average daily gain ; (2) feedlot system with low ADG (FL); (3) pasture system with high ADG (PH); and (4) pasture system with low ADG (PL). After harvest and chilling,  Feedlot finishing systems are typically based on high-energy diets meticulously balanced to provide all essential nutrients necessary to support maximal tissue growth. As a result, this approach to feeding cattle may alter muscle metabolism to support greater average daily gains (ADG) and greater fat deposition, all leading to greater performance in feedlot cattle compared to animals finished on pasture . On the Many studies have reported differences in meat quality characteristics that arise from changes in beef cattle growth rate, especially regarding the vast differences created between grass- and grain-fed production systems. This is not particularly surprising given changes in nutritional status and(or) environmental stimuli alter gene expression and elicit a whole host of changes in tissue metabolic pathways and subsequent metabolite abundances ,6,7,8,9.Metabolomics is a powerful foodomics tool that, in recent years, has allowed for broad exploration of skeletal muscle metabolism in farm animals ,14,15,161D H1-NMR analyses, 37 compounds were identified in the meat extracts, including essential and non-essential amino acids, metabolites belonging to glycolysis, the citric acid cycle, metabolism of purines, fatty acids, nitrogen, and others. The descriptive statistics are given in Based on the The PLS-DA score and loading plot analysis allowed for visualizing the differences in the metabolite profiles among the treatments A,B. A clA SAM plot displayed a linear correlation between the expected and observed values determined for the quantified metabolites  for the The VIP analysis based on the PLS-DA shows the top 15 metabolites that changed in relative amounts between treatments. The y-axis represents the molecules in order of importance for group classification from top to bottom. Using a VIP cut-off of around 1.0, ATP and fumarate were greater in beef from PL cattle, while succinate, leucine, AMP, glutamate, carnosine, inosine, methionine, G1P, and choline were greater in that of FH cattle A. ComparRelevant pathways identified by differences in growth rate (FH versus PL) were correlated with energy and protein metabolism, which included alanine, aspartate, and glutamate metabolism, aminoacyl-tRNA biosynthesis, arginine biosynthesis, butanoate metabolism, histidine metabolism, citrate cycle (TCA cycle), galactose metabolism, D-glutamine, and D-glutamate metabolism and nitrogen metabolism . When paThis study provides a comprehensive analysis of changes in metabolites and metabolic pathways in meat from beef cattle fed one of two feeding regimes at one of two different growth rates. The totality of these results shows the complexity of metabolic regulation possible in producing this highly coveted food source over a broad range of production scenarios and argues the necessity of understanding the molecular mechanisms driving meat quality characteristics in animal production and meat science. The present metabolomic data are supported by meat quality data previously described by Gómez et al. . The paiIt is well-known that growth is a complex process controlled by many genes, environmental factors, and epigenetic and pleiotropic mechanisms . Data prHigher concentrations of free amino acids, such as leucine, glutamate, and methionine in meat from FH animals compared to that of PL cattle are likely related to changes in muscle protein metabolism, where faster-growing livestock, in this case FH cattle, generally produce more glycolytic muscle, while slower growing (PL) animals likely have more oxidative muscle ,2,7. TheNotwithstanding, Imaz et al.  evaluateCholine was higher in meat from FH cattle than PL animals. This metabolite plays an important role in phospholipid synthesis , possiblDifferences in muscle adenonucleotide data reported herein further support our contention that muscle type changes with growth rate, as facilitated by diet. Basal energy (ATP) charge between oxidative and glycolytic muscle differs, as do resting phosphocreatine levels, which is an immediate source of high-energy phosphates during the rapid loss of ATP. Muscle samples collected from these cattle occurred at 24 h postmortem. However, completion of rigor mortis in beef carcasses varies . It coulRegarding the pairwise comparison between FL and PH cattle, similarities in animal performance and carcass traits were reported by Gómez et al. , so compThe overlapping nature of the metabolic profiles between FL and PH treated cattle suggests the similarities in their meat metabolites profiles are related to similar performance parameters, even though some differences in meat quality have been observed between these two groups . HoweverBecause pastures are rich in antioxidant compounds, it is unsurprising that meat from grass-fed animals possesses large amounts of these compounds. Consistent with this theme, meat from grass-fed animals has more glutamine and carnosine, two metabolites that play a versatile role in cell metabolism, including cellular redox reactions and antioxidative capacities . GreaterDifferences in protein metabolism are also suggested by the observation that greater urea and amino acids are detected in the meat of PH cattle. While this suggests greater protein turnover in the muscle of PH cattle, it may also be related to the catabolism of amino acids for a carbon source, hence greater deamination. Animals fed pasture-based diets most likely consume less fermentable carbohydrates compared to FL cattle. In this regard, PH cattle probably produced less propionate than feedlot-managed cattle, which is more glucogenic than acetate. These changes mirror muscle fiber type in pasture-fed that moved to a more oxidative type of metabolism than cattle in feedlot systems . This isFinally, an interesting metabolite differing between the tissue of FL and PH cattle was malonate. Malonate is a key compound in malonyl-CoA synthesis and represents the rate-limiting and committed metabolite in de novo fatty acid synthesis . The lowad libitum while FL animals were fed 70% of ad libitum diets. Animals finished in the pasture system were housed in paddocks consisting of Marandu grass (Brachiaria brizantha cv. Marandu), and the ADG was controlled by stocking density. Moreover, animals were harvested based on a final body weight (530 ± 20 kg BW). Animals reached a final weight of 530 kg after 116, 228, 262 and 292 d of feeding with an average daily gain of 1.50, 0.94, 0.75 and 0.62 kg/ in FH, FL, PH and PL groups, respectively. More details on rearing practices, animals performance, carcass, and meat quality traits are described in Gómez et al. [The present data is part of a larger experiment partially published by Gómez et al. . Thirty-Longissimus thoracis (LT) muscle samples (approximately 10 g) were taken from twenty-four animals, six per treatment, adjacent to the 12th rib and stored at −80 °C for further analysis and metabolite profiles.When each treatment reached the target BW, animals were transported to an abattoir located at the University of Sao Paulo , and harvested according to the Sanitary and Industrial Inspection Regulation for Animal Origin Products of Humanitarian Slaughter Guidelines as required by Brazilian law. All procedures were similar to those used in commercial settings. After 24 h of chilling (0 to 2 °C), carcasses were fabricated, and n = 24, 6 per group) was macerated and homogenized using a blender . Metabolites were extracted with 3.5 mL of cold methanol/chloroform/water solution (2:2:1 v/v) while vortexing for 1 min, as previously described [g at 4 °C to remove precipitated protein and connective tissue. Supernatants were carefully transferred to Eppendorf tubes and freeze-dried . The freeze-dried residue was reconstituted in 600 μL 100 mM phosphate buffer  and 60 μL internal standard solution  as a quantitation standard and chemical shift reference, and 100 mM imidazole as a pH indicator was added. Samples were centrifuged at 10,000× g for 3 min at 4 °C to remove any precipitate. For the analysis, 600 μL of the supernatant was transferred to standard 5 × 178 mm thin-walled NMR tubes .Six samples per group were randomly selected, and approximately 0.5 g of meat frozen  following the methodology described by Cônsolo et al. [o et al. . Briefly1D 1H NMR spectra of LT muscle extracts using the Profiler module of the Chenomx NMR Suite Professional software with an in-built 1D spectral library. Quantitation was based on comparing the area of selected metabolite peaks with the area under the DSS methyl peak, which corresponded to a known concentration of 0.5 mM in all samples. The resulting metabolite concentration tables (37 metabolites × 24 samples) were exported to Excel, where sample identifiers were added.Spectra were processed using the Chenomx NMR Suite Professional 7.7 software : phasing and baseline correction were performed, and the pH was calibrated using the resonances from imidazole. Spectra were referenced to DSS methyl peaks at 0.00 ppm. The same peak was also used as a chemical shape indicator, i.e., as an internal standard for quantitation. Thirty-nine metabolites were quantified in the http://www.metaboanalyst.ca/, accessed on 22 February 2022). Metabolite concentration tables were first uploaded to MetaboAnalyst, and data were log-transformed and Pareto-scaled before analysis. Supervised Partial Least Squares-Discriminant Analysis (PLS-DA) analyzes were performed with cross-validation using the LOOCV method and the performance measure “accuracy”. To investigate the production system and the growth rate effects on meat metabolomics, the following pairwise comparisons using the significance analysis of metabolites (SAM) approach were conducted to elucidate the specific differences between groups: FH versus PL, elucidating the effect of growth rate; and FL versus PH in regards to the feeding system. The more the variable deviates from the ‘‘observed-expected’’ line, the more likely it is to be significant. Colored dots represent features that exceed the specified threshold. The variable importance in the projection (VIP) was used to rank the metabolites based on their importance in discriminating the different groups. Metabolites with the highest VIP values (>1.0) are the most powerful group discriminators. Metabolomic data were analyzed using the open-source MetaboAnalyst 5.0 tool (Bos taurus.Metabolomics data were further processed with bioinformatics by performing the pathway enrichment analyses using metabolite data sets for each group, according to Xia and Wishart . The comThe type of beef cattle production system and growth rate modulate the animal metabolism, changing meat metabolites composition and metabolic pathways, which impacts meat composition and quality development. This study provided great insight into differential meat metabolomics across animals finished on two divergent types of feeding systems and growth rates commonly used globally. Growth rate impacts meat metabolism through changes in energy metabolism and associated pathways, with FH animals presenting greater meat concentration of compounds correlated with energy production, such as succinate, AMP, inosine, G1P, and choline. Thus, the growth rate appears to be the main factor responsible for manipulating energy metabolism in fed cattle. The feeding system may be more responsible for altering protein and lipid metabolisms, with a depletion of protein regarding meeting the energy demand of grass-fed cattle. Despite the robustness of the model and accuracy of the data, further validation is needed using a high number of animals.


Chromohalobacter canadensis is one of the early halophiles to show promising biotechnology potential, which has not been explored to date. Advanced high throughput technologies such as whole‐genome sequencing allow in‐depth insight into the potential of organisms while at the frontiers of systems biology. At the same time, genome‐scale metabolic models (GEMs) enable phenotype predictions through a mechanistic representation of metabolism. Here, we sequence and analyze the genome of C. canadensis 85B, and we use it to reconstruct a GEM. We then analyze the GEM using flux balance analysis and validate it against literature data on C. canadensis. We show that C. canadensis 85B is a metabolically versatile organism with many features for stress and osmotic adaptation. Pathways to produce ectoine and polyhydroxybutyrates were also predicted. The GEM reveals the ability to grow on several carbon sources in a minimal medium and reproduce osmoadaptation phenotypes. Overall, this study reveals insights from the genome of C. canadensis 85B, providing genomic data and a draft GEM that will serve as the first steps towards a better understanding of its metabolism, for novel applications in industrial biotechnology.Salt tolerant organisms are increasingly being used for the industrial production of high‐value biomolecules due to their better adaptability compared to mesophiles.  Chromohalobacter canadensis 85B was sequenced and its genome‐scale metabolic model was reconstructed and validated, enabling a better understanding of its metabolism. We show that C. canadensis is a metabolically versatile organism with many features for stress and osmotic adaptation, including pathways for compatible solute and polyhydroxybutyrate (PHB) synthesis. The model reveals the ability to grow on several carbon sources in minimal medium, and produce PHBs and compatible solutes such as ectoine, 5‐hydroxyectoine, and betaine.The whole genome of  They can also have a tolerance in the same environment to other conditions such as pH and temperature, thus widening the applications of their bioproducts , MgSO4.7H2O (14 g), KCl (2.8 g), NaBr (0.32 g), NaHCO3 (0.008 g), CaCl2.2H2O (2 g), yeast extract (1 g), Peptone (5 g), and glucose (20 g) as carbon source. The culture was incubated for 3 days at 35°C and 150 rpm in 250 mL Erlenmeyer flasks for polymer production  and visualized on a gel documentation system (BIO‐RAD).From the bacterial cultures, 2 mL of bacterial suspension was obtained for genomic DNA extraction. Genomic DNA was extracted using the PureLink Microbiome DNA purification kit (Invitrogen) according to the manufacturer's instructions. Upon extraction of the pure DNA, an electrophoresis gel was prepared to confirm the presence of a single band corresponding to the whole bacterial genome. A 5 µL of the sample was run on 1% agarose gel for 30 min at 100 v. Gels were stained with ethidium bromide (10 mg mL2.3https://www.patricbrc.org/) using the auto assembly strategy with default parameters  and PATRIC   . Predicted complementary DNA sequences were blasted in the NCBI nonredundant database as well as Swiss‐Prot and UniProt,  and an average G + C content of 60.90%. The N50 length, which is defined as the shortest sequence length at 50% of the genome, was 186,789 bp. The L50 count, which is defined as the smallest number of contigs whose length sum produces N50, was 5  of annotated proteins were included in the subsystems analysis according to the RAST pipeline. An overview of the subsystems for this genome as produced by the annotation pipeline is provided in Figure C. salexigens the first seven groups seem to be the most abundant despite the subtle differences in the relative abundance of orthologous genes between both species. This further emphasizes the importance of these systems in this group of microorganisms.An analysis of orthologous genes shows amino acid metabolism and transport and transcription containing the highest number of orthologous genes Figure . When co3.4C. canadensis 85B and nine subsystems representing biosynthesis and degradation pathways. Predictions show genes for the metabolization of various carbohydrate substrates such as sugar alcohols, C‐1 compounds, sugar acids, monosaccharides, polysaccharides, and fermentation. Enzymes able to metabolize the following substrates were predicted: glucose, starch, sucrose, fructose, mannose, xylose, glycerol, and galactose. The presence of many different pathways for carbohydrate metabolism has significant implications for the adaptation of halophiles.There were 177 carbohydrate metabolism genes in C. salexigens, glucose metabolism occurs exclusively through the Entner–Doudoroff pathway while fructose metabolism occurs through the Entner–Doudoroff and Embden–Meyerhof–Parnas pathways. Fructose metabolism seems to give more metabolic flexibility in response to energy and biosynthetic demands. The Entner–Doudoroff pathway, on the other hand, is inefficient for growth when salinity is low, as a result of metabolite overflow. However, in high salinity, there is a high metabolic burden on this pathway due to the use of NADPH and ATP for the synthesis of compatible solutes. This allows the organism to use other pathways to meet other metabolic requirements  ‐3‐Hydroxybutanoyl‐CoA intermediates that link it to the PHB synthesis pathway.The fatty acid composition of salt‐tolerant organisms is influenced by salt concentrations. This is observed through decreased saturation of fatty acids at suboptimal concentrations. Therefore by varying the ratio of saturated to unsaturated fatty acids adaptation to salt stress can be achieved  is the key enzyme in the PHB synthesis pathway, catalyzing the polymerization of hydroxyalkanoate subunits ‐hydroxyalkanoic acid synthase (C. japonicus). Only one hit was obtained each in the Gene3D, InterPro, Pfam, SUPFAM, and TIGRFAMs, all corresponding to PHA synthase class I. The class I subfamily PHA synthases can polymerize hydroxyacyl‐CoAs with three to five carbons in the hydroxyacyl into PHA esters in this case most likely PHB. These can be accumulated up to 90% of the cell's dry weight. The PhaR genes play a posttranscriptional role and help prevent protease degradation or act directly or indirectly to activate PHA synthase . The annotations are as follows: passive transport (SBO:0000658), active transport (SBO:0000657), cotransport:symport (SBO:0000659), cotransport:antiport (SBO:0000660) other transport reactions (SBO:0000655), general metabolic reactions (SBO:0000176), exchange reactions (SBO:0000627), biomass reactions (SBO:0000629), genes (SBO:0000243), and species (SBO:0000247) , and zinc/iron permease which have been reported to be associated with adaptations to high salt environments by sensing salt stress and regulating intracellular ion homeostasis respectively  showed a decrease in PHB production with increasing nitrogen uptake rates, although with a steeper slope after uptake rates of about 39 mmol/gDW/h , Formal analysis , Funding acquisition , Visualization , Writing – review & editing . Belma Nural Yaman: Conceptualization , Funding acquisition , Writing – review & editing . Chaimaa Tarzi: Formal analysis , Visualization , Writing – review & editing . Pınar Aytar Çelik: Conceptualization , Funding acquisition , Supervision , Writing – review & editing . Mehmet Mutlu: Supervision , Writing – review & editing . Claudio Angione: Conceptualization , Funding acquisition , Supervision , Visualization , Writing – review & editing .None declared.None required.


Mus musculus laboratory strains as model, thus neglecting the wide diversity of sperm traits of other species of Mus. Focus on closely related species with distinct evolutionary histories, which may be the result of different selective pressures, could shed light on diversity of metabolic processes. Here, we analyzed variations in sperm bioenergetics associated with capacitation in spermatozoa of the steppe mouse, Mus spicilegus, a species with high sperm performance. Furthermore, we compared sperm metabolic traits of this species with similar traits previously characterized in M. musculus. We found that the metabolism of M. spicilegus sperm responded to capacitation in a manner similar to that of M. musculus sperm. However, M. spicilegus sperm showed distinct metabolic features, including the ability to perform cross-pathway metabolic compensation in response to either respiratory or glycolytic inhibition, thus revealing a delicate fine-tuning of its metabolic capacities.The acquisition of fertilizing ability by mammalian spermatozoa, known as “capacitation,” includes processes that depend on particular metabolic pathways. This has led to the hypothesis that ATP demands might differ between capacitated and non-capacitated cells. Mouse sperm can produce ATP via OXPHOS and aerobic glycolysis, an advantageous characteristic considering that these cells have to function in the complex and variable environment of the female reproductive tract. Nonetheless, despite evidence showing that both metabolic pathways play a role in events associated with mouse sperm capacitation, there is contradictory evidence regarding changes promoted by capacitation in this species. In addition, the vast majority of studies regarding murine sperm metabolism use The online version contains supplementary material available at 10.1007/s00018-022-04652-0. In mammals, the ATP used to maintain flagellar beating and, hence, cell motility is produced through two distinct metabolic pathways, oxidative phosphorylation (OXPHOS) and aerobic glycolysis –3. TheseThe availability of metabolic substrates varies between the different regions of the female reproductive tract and is also subjected to changes along the female reproductive cycle –9. In th2 [Mammalian sperm are unable to fertilize the egg immediately after ejaculation and must acquire fertilizing ability through a series of structural, biochemical, physiological, and behavioral modifications, collectively known as capacitation –16. Sper2 , activat2 –26, and 2 –29. Thes2 , 31 and 2 –34. Furt2 , 36, whi2 . This le2 , 38.Mouse sperm use OXPHOS and glycolysis as energy-producing pathways  and appeThe evidence regarding how capacitation affects changes in mouse sperm bioenergetics is somewhat contradictory. It has been reported that pharmacologically induced capacitation causes an elevation in glucose consumption, which was associated with an increase in the rates of both respiration and glycolysis during capacitation time . HoweverMus musculus domesticus). Nonetheless, there is a wide range of variation in sperm motility and bioenergetic performance between mouse species, which represents an opportunity to dissect metabolic pathways in different physiological scenarios. A study using extracellular flux analyses to compare sperm metabolism between the house mouse and other species of the genus Mus revealed that the ratio of usage of each metabolic pathway varied in a species-specific manner [Mus species has only been compared in one study [M. spretus and M. spicilegus) experienced a more severe decline in sperm ATP concentration, as a consequence of capacitation, than M. musculus.The vast majority of evidence regarding murine sperm metabolism corresponds to studies on strains of the laboratory mouse . Furthermore, we compared these bioenergetic parameters with the results shown for M. musculus in a previous study [M. spicilegus sperm toward a more glycolytic metabolism, similar to what occurs in M. musculus. On the other hand, the sperm of M. spicilegus exhibits particular metabolic features, such as greater metabolic flexibility, which may be regarded as adaptations to more intense levels of performance required to ensure fertilization in competitive situations.In the present study, we assessed the variations promoted by capacitation in the energy-producing metabolism of us study . We concUnless otherwise stated, reagents were acquired from Merck .Mus spicilegus (3–5 months old) were obtained from the Institut des Sciences de l’Évolution-Montpellier, CNRS-Université Montpellier 2, France. The mice were kept at the animal facilities of the Museo Nacional de Ciencias Naturales  in individual cages at 20–24 °C with a 14 h light and 10 h darkness photoperiod. Animals had water and food available ad libitum.Adult male 2/air. The composition of both incubation media was based on a Hepes-buffered modified Tyrode’s medium [−1). The composition of the non-capacitating medium was: 132 mM NaCl, 2.68 mM KCl, 0.49 mM MgCl2.6H2O, 0.36 mM NaH2PO4.2H2O, 5.56 mM glucose, 20 mM Hepes, 1.80 mM CaCl2, 0.02 mM phenol red, 0.09 mM kanamycin, 4 mg ml−1 fatty acid-free BSA, 20 mM Na lactate, and 0.5 mM Na pyruvate. The capacitating medium had an equivalent formulation, but 15 mM NaCl were replaced with 15 mM NaHCO3 to maintain osmolarity.Mice were dissected after sacrifice and both caudae epididymides were extracted. After removing blood vessels, fat and surrounding connective tissue, each cauda epididymis was placed in a 35-mm Petri dish containing 1 ml of one of two variants of culture medium at 37 °C. One cauda epididymis was placed in non-capacitating medium under air, and the other one was placed in capacitating medium under 5% COs medium , that wa6 sperm ml−1 in each suspension after estimating concentration with a modified Neubauer chamber. Sperm suspensions were then incubated for 1 h at 37 °C under the corresponding atmosphere for each medium. This time of incubation was selected since M. spicilegus sperm populations achieve the peak percentage of capacitated cells (~ 70%) at this time [6 sperm ml−1 prior to measurements.Sperm were collected by performing three to five incisions in the distal region of the caudae and allowing the cells to swim out for 5 min . After shis time . Large-bThe animals used in this study were cared and maintained according to the guidelines of European Union Regulation 2010/63 and the Royal Decree for the Protection of Experimental Animals RD53/2013; animal handling was performed with the approval of CSIC's ethics committee and the Comunidad de Madrid (28079-47-A). Animals were sacrificed by cervical dislocation. No other procedures were included in this study.Oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in real-time using an XF24 extracellular flux analyzer . OCR indicates the level of respiratory activity in a population of cells, and ECAR, which is proportional to the rate of lactate excretion, is used as a proxy for the rate of aerobic glycolysis. The method used in this study to assess OCR and ECAR follows the original technique developed by Tourmente et al. , with mi6 sperm) that were previously incubated under non-capacitating conditions, other 10 wells were seeded with sperm suspension incubated under capacitating conditions, and four wells were left without cells to perform background corrections. After 3 min, the plate was centrifuged for 1 min at 1200 × g in each direction to ensure an even distribution of attached cells throughout the bottom of the well. The incubation medium was then discarded from each well and was immediately replaced with 500 µl of assay medium. Since the assessment of ECAR is only possible in a medium with low pH buffering capacity, an unbuffered assay medium was used for the measurements and dilution of the metabolic modulators. In this medium, Hepes and NaHCO3 were replaced with NaCl to preserve osmolality, and pH was adjusted to 7.4 at 37 °C. Finally, the plate with the sperm was placed in the XF24 apparatus, and a 12-min equilibration step was allowed before assessments. The use of laminin as sperm adhesive, instead of the concanavalin A used in the original protocol [Before the experiment, 30 µl of 0.2 mg/ml natural mouse laminin  in PBS were added to each well of a 24-well XF24 plastic microplate and incubated for at least 3 h at room temperature. Before adding cells to the wells, the excess laminin was rinsed with 1 ml of ultrapure water and discarded. After the 1-h incubation in either non-capacitating or capacitating medium, sperm were transferred to the laminin-coated plate. Ten wells were seeded with 100 µl of sperm suspension . During the first four cycles, sperm metabolic rates were measured without any additions. Subsequently, cells in each well were treated with either assay medium (baseline conditions), or one of four metabolically active compounds via automatic injection, and sperm metabolism was recorded for 3 additional cycles. Metabolic modulators were: (a) 5 μM oligomycin A, an inhibitor of the mitochondrial ATP synthase, (b) 1 µM carbonyl cyanide p-trifluoro-methoxyphenylhydrazone (FCCP), an uncoupler of mitochondrial respiration, (c) 50 mM 2-deoxy-d-glucose (2DOG), a glucose analog that competitively inhibits the first step of glycolysis, and (d) 30 mM sodium oxamate, a competitive inhibitor of the enzyme lactate dehydrogenase (LDH). Finally, 1 µM rotenone and 1 µM antimycin A were added to the wells and two final measurements were performed to complete a total experiment duration of 54 min. Oxygen levels were able to fully recover after the marked descent occurring during the measurement , indicating that the sperm concentration used in the experiment was not enough to cause hypoxia. Cells were treated with oligomycin in four separate experiments, and with 2DOG, oxamate and FCCP in three experiments since the large number of wells made possible the application of more than one treatment in each experiment. The first measurement was discarded in all experiments since, according to our previous experience, it tends to show a higher degree of instability.2 min−1 sperm−1, and nano-pH min−1 sperm−1, respectively.After the end of the experiment, the sperm in each well were resuspended by scratching the bottom of the well with a pipette tip under a phase-contrast microscope at 10 × , and 10 µl of sperm suspension were collected to estimate sperm concentration in each well. OCR and ECAR values for each well were normalized by the number of sperm present and reported as amol of OThe assessment of OCR and ECAR on sperm treated with metabolic modulators led to the estimation of metabolic parameters Table  that areIn order to facilitate comparisons with previous studies, we calculated the response of OCR and ECAR to metabolic modulators as a percentage of the averaged basal levels (measures 2–4) for each well. For OCR measurements, the basal and stimulated/inhibited values were first corrected by subtracting the average of measures after the addition of A + R (cycles 8–9).N = 3). A luciferase-based ATP assay kit (Roche ATP Bioluminescence Assay Kit HS II) was used, based on the protocol of Tourmente et al. [g for 2 min. The supernatant was recovered and frozen in liquid N2. The bioluminescent signal was measured in triplicate in 96-well plates using a luminometer . 50 μl of Luciferase reagent was added to 50 μl of sample in each well via automatic injection, and, following a 1 s delay, light emission was measured over a 5-s integration period. The ATP content for each sample was estimated using standard curves constructed for each plate with solutions containing known concentrations of ATP. ATP content per well was normalized by the number of cells represented in 50 μl of lysate and expressed as amol sperm−1.Sperm ATP content was compared between sperm incubated in non-capacitating or capacitating conditions  [M. musculus metabolic data were also collected using extracellular flux analysis (EFA), differences between studies in the measuring equipment (Agilent Seahorse XFp vs XF24) and the state in which the cells were assessed (free-swimming vs attached sperm) preclude direct comparisons between absolute OCR and ECAR values. Thus, variations in the OCR/ECAR ratio as a consequence of sperm capacitation, and the response of OCR and ECAR to metabolic modulators  in non-capacitated sperm, were compared between species. The effect of the metabolic modulators on OCR and ECAR was expressed as percentages relative to their baseline levels (without any metabolic modulator). The effect of oligomycin and 2DOG on OCR was calculated as the average across cycles 5–7 for each well, while the effect of these modulators on ECAR was represented by the highest (oligomycin) and lowest (2DOG) values among cycles 5–7 for each well.The results from the metabolic measurements in the present study were compared with those of a similar study performed recently in the laboratory mouse ( B6D2F1) . Althougα = 0.05. The effects of incubation conditions and metabolic modulators on OCR and ECAR of M. spicilegus sperm, expressed as percentages relative to the baseline level, were assessed via mixed effect models  using incubation condition medium (non-capacitating and capacitating) and treatment  as fixed factors, and experiment as a random factor. The values of the metabolic parameters calculated after EFAs were compared between incubation conditions using MMs with incubation medium as a fixed factor and experiment as a random factor. The inclusion of the experiment as a random factor allowed for the consideration of each well as an individual value in the statistical analyses, thus decreasing the impact of outliers and increasing the statistical powers of the tests by controlling for the between-experiment variability [All analyses were conducted using R version 4.2.1 , with iability .The interspecific comparison of the OCR/ECAR ratio and the response of metabolic rates to metabolic modulators was also carried out by means of MMs. In the first case, OCR/ECAR was used as a dependent variable and species and incubation conditions as fixed factors. In the second case, the changes in OCR and ECAR after treatment with oligomycin, and 2DOG (expressed as the percentage relative to the baseline) were used as dependent variables and species as a fixed factor. In the two cases, the experiment was used as a random factor.10-transformation applied to the response variable, or a generalized linear mixed effect model (GLMM) with gamma distribution and inverse link function. The type of analysis chosen in each case was the one that produced residuals that better fit the model assumptions. Significant differences between levels of fixed factors were analyzed via post-hoc estimated marginal means tests .Each analysis used either a linear mixed effect model (LMM) with logM. spicilegus sperm are presented in Fig. 3− to the acidification is decreased because the OXPHOS-derived CO2 production is lower [M. spicilegus. The sperm ATP content was lower in sperm preincubated in capacitating medium  and an increase in basal ECAR . The elevation in ECAR observed in M. spicilegus was non-significant, and the change in the OCR/ECAR was more pronounced in M. musculus  has a series of features that make it a relevant model to study murine sperm metabolism. M. spicilegus has the highest total sperm number [Mus species examined so far. Its sperm cells also exhibit higher sperm ATP levels [M. spicilegus sperm have higher OXPHOS rates and their motility is more dependent on oxidative metabolism than M. musculus sperm [M. spicilegus has the largest relative testes size of Palearctic Mus species [M. spicilegus belongs to the sister taxon to M. musculus, having diverged from their common ancestor approximately 1.2 million years ago [The steppe mouse , sperm exhibited a marked change in the usage ratio of the energy-producing pathways in favor of glycolytic metabolism, without decreasing ATP levels. These results supported the hypothesis that the function of the metabolic changes associated with sperm capacitation would be to sustain local ATP concentrations in the distal flagellar regions instead of attending to increased global ATP demands [Previous studies concerning the variations imposed by capacitation on mouse sperm have yielded conflicting results despite using similar measuring techniques (EFA). One study  found thP demand , 38, 73.P demand  showed t demands .M. spicilegus toward a less oxidative metabolism, similarly to what Tourmente et al. [M. musculus. These findings are further supported by recent publications showing that, in the sperm of laboratory mice, sperm capacitation occurs concomitantly with hyperpolarization of the mitochondrial membrane [In all, the results of the present study suggest that capacitation induces a shift in the usage ratio of the energy-producing pathways in the sperm of e et al.  found inmembrane , 53, 54,membrane –76, the membrane , 53 woulM. musculus, the metabolic rates of M. spicilegus sperm responded as expected to metabolic modulation, regardless of incubation conditions. Respiratory rate was reduced when sperm were treated with oligomycin or antimycin plus rotenone and elevated when FCCP was added to the medium, while glycolytic rate decreased after the addition of 2DOG or sodium oxamate. Nonetheless, there are several bioenergetic features in which the sperm of these two species differ.Consistently with what was reported in M. spicilegus swim significantly faster and have more prolonged motility [M. musculus. This seems to be a consequence of their ability to consume ATP at faster rates [M. spicilegus sperm have higher OXPHOS rates and are more dependent on respiration for motility maintenance [Under non-capacitating conditions, the sperm of motility , 57 thaner rates  while suntenance , possiblM. spicilegus. Whereas sperm capacitation promoted a substantial decrease in respiratory rate in our experiments, the increase in glycolytic rate under these conditions was less pronounced. Furthermore, although ATP consumption rate did not vary between incubation conditions, exposure to capacitating medium resulted in lower ATP concentrations, probably as a consequence of the concomitant decrease in respiratory rate. Previous evidence is consistent with this conclusion since sperm ATP levels have been shown to decrease throughout capacitation in M. spicilegus [M. spicilegus than in its M. musculus counterpart [M. spicilegus principal piece is 20% shorter than that of M. musculus [Our findings also support the idea of OXPHOS playing a greater role in sperm bioenergetics in icilegus , and theicilegus  are simiicilegus . Furthernterpart . Finallynterpart . Interesmusculus , ATP proM. spicilegus sperm is their ability to respond to inhibition of one metabolic pathway with an increase in the activity of the other. The sperm of M. spicilegus (this study) and M. musculus [M. musculus sperm to levels similar to those reached with oligomycin [M. spicilegus sperm to 2DOG elicits an increase in OCR in both incubation conditions, indicating that these cells are capable of (a) utilizing exogenous respiratory substrates to fuel OXPHOS in absence of endogenous pyruvate, and (b) perform metabolic compensation toward any of the two main energy production pathways. This represents a higher level of metabolic flexibility that would allow M. spicilegus sperm to fully cope with the changing metabolic landscape of the female reproductive tract, which is known to vary between regions [A striking feature of the metabolism of musculus  are ableigomycin , 55, mea regions , 42.M. musculus, M. spicilegus sperm are able to metabolize external respiratory substrates, the inhibition of LDH by oxamate would, at the same time, preclude glycolysis in the principal piece and lactate reoxidation within the mitochondria, effectively starving both metabolic pathways.Remarkably, the above-mentioned compensation ability is not exhibited when glycolysis is inhibited with sodium oxamate, a competitive inhibitor of the enzyme lactate dehydrogenase (LDH). LDH is present in mouse sperm as a sperm-specific isoform (LDHC) that is located both in the cytosol and the mitochondrial matrix , 79. AloM. spicilegus sperm that are similar in direction to those exhibited by M. musculus. Nonetheless, the sperm of M. spicilegus presents unique metabolic characteristics, such as a prevalence of respiratory processes over glycolytic ones, and a greater capacity for metabolic compensation, that make this species a relevant model for the study of murine sperm bioenergetics. Furthermore, these outstanding characteristics might shed light on adaptations to selective pressures leading to increased gamete performance in very competitive scenarios.Altogether, the results of our study revealed that capacitation elicits changes in the usage ratio of OXPHOS and glycolysis in M. spicilegus and M. musculus suggests that significant differences in sperm metabolic features may arise quickly through evolutionary time. Thus, given the highly species-specific nature of sperm metabolism, the translation of conclusions between different model species should be taken with utmost caution.Finally, these results may pave the way for future studies dealing with sperm metabolic variations and capacitation in human sperm, since these phenomena also appear to be related in this species –84. HoweSupplementary file1 (PDF 10060 KB)Below is the link to the electronic supplementary material.


Komagataella phaffii (formerly known as Pichia pastoris) has become an increasingly important microorganism for recombinant protein production. This yeast species has gained high interest in an industrial setting for the production of a wide range of proteins, including enzymes and biopharmaceuticals. During the last decades, relevant bioprocess progress has been achieved in order to increase recombinant protein productivity and to reduce production costs. More recently, the improvement of cell features and performance has also been considered for this aim, and promising strategies with a direct and substantial impact on protein productivity have been reported. In this review, cell engineering approaches including metabolic engineering and energy supply, transcription factor modulation, and manipulation of routes involved in folding and secretion of recombinant protein are discussed. A lack of studies performed at the higher-scale bioreactor involving optimisation of cultivation parameters is also evidenced, which highlights new research aims to be considered. Komagataella phaffii, formerly known as Pichia pastoris [The methylotrophic yeast pastoris , is one pastoris ,3. As a pastoris . Given ipastoris ,5,6,7.K. phaffii stands out among other yeasts and microorganisms due to several beneficial features  [K. phaffii can perform diverse protein processing and post-translational modifications typical of higher eukaryotes, such as glycosylation and disulphide bond formation [a et al. ). In addht (DCW) ,10,11. Ght (DCW) ,12. In aormation ,13, and ormation . Using tK. phaffii for rProt synthesis has been facilitated by strong methanol-inducible promoters from the alcohol oxidase genes , and also from the glyceraldehyde 3-phosphate dehydrogenase (GAP) promoter GAP,P which exhibits strong constitutive expression in the presence of glucose and glycerol [AOX1P to drive transgene expression, this production system presents some drawbacks associated with methanol utilisation. Indeed, methanol is toxic to cells, inducing cell oxidative stress, and its use comes with a subsequent high oxygen demand for catabolism [The success of glycerol . Notwithtabolism . AdditioK. phaffii under various genetic backgrounds (engineered strains) and bioprocess operations [K. phaffii’s promising future as a robust and highly efficient host for the production of a huge variety of recombinant proteins.Important efforts have been carried out in order to improve the understanding of the physiology and cell response of erations ,18,19 wierations ,21, geneerations ,23,24, aerations . HoweverK. phaffii can metabolise methanol as the sole carbon source; however, it can also metabolise other non-frequently used alkylated nitrogen sources, such as methylamine and choline. The main metabolic drawback of methanol catabolism is the production of toxic metabolites such as formaldehyde and hydrogen peroxide [rProt production and secretion generate a high metabolic burden, as protein synthesis leads to increased nutrient and energy demands . In addiperoxide . At highperoxide ,30. Furtperoxide . TherefoAOX1P expression system, co-substrates such as sorbitol can be used with the goal of dedicating methanol mainly as the inducer for the expression system, while sorbitol is used for biomass and energy formation [AOX1P induction level , compared to cultures with 100% methanol supplementation (7.8 ± 0.7 × 103 Miller unit) [In processes based on the ormation ,33,34. Tormation . In thiser unit) . Glyceroer unit) .Metabolic engineering of the host cell has also been considered, aiming at improving rProt productivity. Recent published work on engineering of the catabolic pathway of methanol and alternative carbon sources, along with co-factor engineering, is described in the following sections and summarised in The MUT pathway can be divided into two main stages. First, methanol is oxidised by two alcohol oxidases  into formaldehyde in the peroxisome, before being further metabolised in two distinct metabolic routes. In the assimilatory branch, formaldehyde is converted into dihydroxyacetone (DHA) and GAP by the peroxisomal dihydroxyacetone synthase (Das). This pathway ends in the cytosol with the generation of fructose 1,6-biphosphate. In the dissimilatory branch, formaldehyde is oxidised to carbon dioxide in three steps by the enzymatic route formed by formaldehyde dehydrogenase (Fld), formyl glutathione hydrolase (Fgh), and formate dehydrogenase (Fdh), with the release of NADH , as illuS/P) and rProt productivity. In a MutS strain (strain with disrupted AOX1), the overexpression of DAS resulted in a 2- to 3-fold increase in YS/P from methanol to produce horseradish peroxidase (Hrp) and Candida antarctica lipase B , with reported values of 3.06 U/mmol and 2.05 U/mmol, respectively, in comparison with the reference strains for each case (~1 U/mmol). Meanwhile, the overexpression of FLD with the same reporter proteins exhibited a 2-fold increase in YS/P from methanol  [DAS isoforms (DAS1 and DAS2) has increased green fluorescent protein (GFP) expression in single and double knockout strains. The highest rProt production was observed in the DAS1 deletion strain, which produced 30% more GFP than the control strain. This yield was followed by the DAS2 deletion strain and finally the double mutant DAS1-DAS2, with GFP expression levels of ~22% and ~15% in each case, both higher than the wild-type strain [Engineering of the MUT pathway has led to higher substrate-product conversion yield (YlB/mmol) . On the e strain .AOX1 and AOX2 disruption), which, contrary to its generally recognised inability for methanol metabolization [S strain, due to the promiscuous activity of the alcohol dehydrogenase enzyme Adh2. Based on these results and using metabolic engineering, the overexpression of ADH2 revealed a significant increase in the productivity (qP) of the camelid antibody fragment vHH (237 μg/g∙h) in the Mut-AOX1PvHHpFLD1Adh2 strain compared to the Mut-AOX1PvHH parent strain (88 μg/g∙h), and a slightly higher increase with the strain MutSAOX1PvHH (205 μg/g∙h). It is worth noting that despite the similarity in productivity between the Mut- improved strain and the MutS strain, the latter showed an additional advantage by having lower rates of oxygen uptake and heat production [An alternative methanol metabolism implemented for recombinant protein production has been presented by Zavec et al.  with thelization , showed oduction .AOX1/2P promoters is concerned, few studies have ventured to question whether methanol could be the sole inducer of the system. Tyurin and Kozlov [AOX1/2P system. Thus, to rule out the possibility of intracellular methanol reduction using potassium formate salt as an inducer, FDH was deleted. With this modification, the expression levels of β-galactosidase were increased by 2-fold (~4500 Miller units) compared to the wild-type strain (~2200 Miller units) [As far as the main mechanism for induction of the d Kozlov  discusser units) .FDL and DAS was introduced [Modifying the MUT pathway and the redistribution of metabolic flux for rProt expression in key metabolic steps directed to a target product has also been successful ,38,39,40troduced ,49. ThusAOX1P promoter, while glycerol and glucose do exert repression of this inducible promoter. Nevertheless, glycerol is mainly used in co-feeding strategies with methanol because it generates higher biomass yield, higher growth rate, and improved productivity [K. phaffii as the initial acceptor of the pexophagy process (ATG30) was deleted to favour peroxisome retention in cells under the influence of the addition of carbon sources that trigger this cellular response. This modification improved xylanase production, reporting an activity of ~1140.7 U/mL in cultures supplemented with 2% sorbitol, representing an increase of ~11.4% compared to the control culture containing 0.5% sorbitol [Due to the already described problems with the use of methanol in rProt production, co-feeding strategies of methanol with auxiliary carbon sources have been studied to mitigate this problem. Based on the results of Inan and Meagher , it was uctivity . The useuctivity . On the uctivity ,33,52. Isorbitol . This desorbitol .K. phaffii does not normally metabolise sucrose because of the absence of an invertase enzyme [K. phaffii the Suc2 invertase enzyme from Saccharomyces cerevisiae [AOX1P. When this strain was grown in sucrose-supplemented medium, both the antibody fragment concentration (93.7 mg/L) and the specific yield on biomass (3.96 mg/g DCW) showed comparable values to glycerol-supplemented cultures . Additionally, when evaluating the use of glucose, this condition showed the lowest productivity (63.3 mg/L), even though using this medium generated a higher cell volume compared to sucrose or glycerol  [K. phaffii in sucrose-based cultures may be mainly related to cell number rather than cell concentration, in addition to the repressive effect on the AOX1P promoter from both glycerol and glucose [e enzyme . Howevere enzyme , expressrevisiae  under thlycerol) ,57. This glucose ,57.The study of these auxiliary carbon sources in recent years has mainly focused on managing the conditions and operating parameters in culture systems ,28,35,58S. cerevisiae POS5-encoded NADH kinase, which increased 2-fold the specific productivity of a Fab antibody fragment [The metabolic burden that arises from processing rProt folding, posttranslational modifications, and secretion has shown that the regeneration of the oxidised co-factor significantly affects energy metabolism, becoming a bottleneck ,59,60. Tfragment .ZWF1) and 6-gluconolactonase (SOL3) that catalyse NADPH-generating reactions led to a 3.8-fold increase in the productivity of human superoxide dismutase (hSod) [SOL3 and the gene encoding 6-phosphogluconate dehydrogenase (GND2) resulted in a 2.2-fold increase (~6.5 mg/L) in recombinant human interferon gamma (hIFN-γ) in comparison with the control strain GS115/hIFN-γ (2.5 mg/L) [Constitutive overexpression of the genes encoding the glucose-6-phosphate dehydrogenase (e (hSod) . Besides.5 mg/L) .K. phaffii to incorporate additional metabolic skills in order to metabolise substrates such as xylose, cellobiose, and cellulose [In the context of circular economy, the trend has been the valorisation or increased added value of renewable raw materials, such as agricultural, forestry, food and industry bioproducts and/or waste streams. This goal would require ellulose ,61.XI) from Orpinomyces spp. and an endogenous xylulokinase, allowed xylose conversion resulting in a yield (Y x/s) of 0.378 g/g, nearly 2-fold higher than that of the parental strain GS115, reaching values comparable to those achieved with glucose or glycerol  [The heterologous expression of genes involved in xylose catabolism, namely the xylose isomerase (ctively) . Using tAspergillus niger β-glucosidase (AnBGL1), A. niger endoglucanase (AnEG-A), and a Trichoderma reesei exoglucanase (TrCBH2), allowed the resulting strain to grow on cellobiose and carboxymethyl cellulose [In a different research study, the expression of three heterologous cellulases, namely ellulose .K. phaffii. Indeed, acetate, which can be obtained from syngas by the hydrolysis of cellulosic biomass or by anaerobic fermentation of different agroindustrial wastes [GAPP-driven overexpression of the native gene PAS_Chr3_1091 encoding the putative serine/threonine protein kinase Hrk1, together with the S. cerevisiae gene ScAcs1* encoding acetyl-CoA [Another metabolic engineering strategy in the search for alternative carbon source metabolism consisted of increasing the acetate tolerance in l wastes , is a chetyl-CoA .K. phaffii was engineered for CO metabolization through a reorganisation of the MUT pathway as well as the xylulose monophosphate (XuMP) pathway, in order to generate a Calvin–Benson–Bassham resembling cycle. This modification was obtained by the expression of eight recombinant proteins from five different organisms: from Ogataea polymorpha , Ogataea parapolymorpha , Spinacia oleracea (phosphoribulokinase Prk), Thiobacillus denitrificans , and Escherichia coli (chaperones GroEL and GroES); and the deletion of three native genes  [Recently, 1, DAS2) . This stNumerous investigations have focused on how to modify gene expression patterns and regulatory networks during rProt production ,66,67. TGAPP [For instance, Yap1 has been identified as a transcription factor associated with the control of the oxidative stress response by modulating the expression level of more than 150 genes ,88. OverPGAP .K. phaffii, further evaluating the variation in copy number and promoter used in the overexpression of HAC1 [GS4J-AMYA gene driven by AOX1P and a basal expression of 305 U/mL was used, and upon incorporation of six copies of AOX1P-driven HAC1, amylase activity increased to 2200 U/mL. However, the excessive number of genes under the control of the AOX1 promoter seemed to interfere, limiting the transcription of GS4J-AMYA; therefore, by using the GAPP constitutive promoter to regulate the expression of HAC1, the amylase activity increased to 3700 U/mL. This illustrates the importance of the strategy followed with each auxiliary gene (in this case HAC1), this being as important as the choice of the auxiliary gene itself.Several groups have also investigated transcription factors associated with protein maturation and secretion. The protein Hac1 is a transcription regulator involved in the unfolded protein response (UPR), which increases the synthesis of endoplasmic reticulum resident proteins required for protein folding, as well as components of the secretory pathway ,82. Over of HAC1 . In thisATT1, a homologue of the GAL4 gene in S. cerevisiae, which specifically recognises sequences called galactose upstream activating sequence (GALUAS) and is mainly associated with genes related to sugar metabolism [K. phaffii strains are very valuable tools to produce more complex recombinant glycoproteins [ATT1 increased the rProt yield with a 1.5-fold change in comparison to the control strain, reaching 1.98 g/L of human epidermal growth factor receptor 2 (anti-HER2) mAb. In addition, this increased the cell temperature tolerance by enduring 35 °C for up to 150 h with a low cell lysis rate in a 15 L bioreactor [In some cases, an improvement can be achieved by decreasing or eliminating the expression of an auxiliary gene. One example of this comes from the transcriptional factor associated with galactose metabolism tabolism . In relaproteins . Howeverproteins . Althougproteins ,97. In toreactor .GAPP, where they found 41 putative transcription factor binding sites (TFBS) in K. phaffii. As GAPP is a carbon-source-related promoter [GAPP in fed-batch bioreactor cultivation [K. phaffii Gal4-like transcription factor overexpressing mutants when compared to the wild-type, and it was noticed that the overexpression of the Gal4-like transcription factor resulted in a switch from Crabtree negative to Crabtree positive behaviour [Additionally, Ata et al.  construcpromoter , TFs reltivation . The speehaviour .AOX1P to convert it into a methanol-free expression system [MRX1 and MIT1, activated AOX1P when the glycerol as repressing carbon source was depleted (derepressed expression) [MIG1, MIG2, and NRG1 alongside MIT1 overexpression also resulted in induction of AOX1P [AOX1P, the goal here resides in the development of a methanol-free bioprocess that offers a safer alternative, hugely relevant to industry.A similar approach was carried out for n system . The indression) . In pararession)  reportedof PAOX1 . AlthougAOX1P regulation, on GAPP-driven k-carrageenase production, including seven chaperones  and two transcription factors Yap1 and Rpn4 (proteasome subunit transcription factors) [AOX1P for these and a constitutively expressed promoter such as GAPP for the rProt of interest.Yu et al.  reportedfactors) ,102. OveK. phaffii are unknown [Consequently, the modulation of expression of genes encoding transcriptional regulators offers a variety of alternatives enabling an increment in the rProt production yield. However, the high number of genes affected by the modulated expression of these factors should be considered ,79,103.  unknown .The main hindrances in the production and secretion of proteins are the folding and processing of complex proteins in the ER. When proteins fold inappropriately or exceed the capacity of the ER, unfolded proteins can accumulate and tend to form aggregates. The cellular response generally involves synthesis and induction of folding-assisting proteins such as chaperones or foldases ,104,105.K. phaffii [A. niger, whose sequence was subjected to codon optimisation, evaluating its performance under the regulation of two promoters (AOXP and ADH2P). The highest production level was achieved with the codon-optimised PGL using the pADH2 obtaining a productivity of 42.33 U/mL (4-fold increase) in shake flasks.Regarding codon sequence optimisation, the codon usage bias can be adapted to the host , and thi phaffii . Karaoğl phaffii  describeStreptomyces sp. lipase Mas1 increased 1.7-fold when it was co-expressed with the chaperone Pdi1, compared to the control strain harbouring only MAS1 [The co-expression of chaperones can promote the correct folding of rProts and reduce the intracellular aggregation of proteins . The pronly MAS1 .Another chaperone protein frequently co-expressed with rProt is Kar2, a homologue of the mammalian immunoglobulin-binding protein (BiP) ,111 thatAnother strategy that also improves protein folding and secretion is the expression of transcription factors such as Yap1 and Hac1, as it was presented in the previous section. However, when these are expressed together with other folding facilitator proteins such as Pdi1 and Kar2, although it was expected to result in an increase in the production of the rProt of interest, the work of Sun et al.  and DuanPDI1 or ERO1 remarkably increased the expression level of RABV-G by 9.5-fold and 3.3-fold, respectively, in the high copy strains of RABV-G , and also the potential conversion of organism .AOX1/2P induction system and to the metabolic system in general.Further study of metabolic modifications directed to assimilation routes of carbon and energy sources in response to the culture media will have a repressive or derepressive response to the highly used On the other hand, modifications to increase/improve protein folding and secretion, such as codon usage optimisation, increase in gene copy number, co-expression/modulation of transcription factors, co-expression of chaperones, and modification of culture conditions have allowed the expression of a wide range of proteins with diverse application fields. However, the use of one or many of these modifications does not ensure an increase in rProt expression, especially in the case of proteins that are difficult to express, such as antibodies, membrane proteins, toxins, and proteins with non-standard amino acids, among others ,114,116.K. phaffii used to produce recombinant proteins, it is necessary to have a model that integrates information related to cell behaviour in a way that is capable of developing hypotheses focused on optimisation of production processes [K. phaffii metabolic network to improve recombinant protein production [Furthermore, due to the variety of available strains of rocesses . The genrocesses . This cooduction ,125.K.-phaffii-based bioprocesses [Finally, the latest advances and improvements addressed in this review have been reported mainly at laboratory scale (flask) with only a few examples at bioreactor level or high cell density. This highlights the need to expand the research scope on the scale-up of these processes, in order to determine cultivation parameters suited for higher-scale and for industrially relevant cultivation modalities  and strain performance in controlled conditions. In this regard, engineering tools such as response surface methodology can be used, as they have been successfully applied to improve rocesses ,128,129.


Lactococcus cremoris, we selected two carbon sources  with predicted coutilization constraints. By evolving L. cremoris on either fructose, galactose, or a mix of both sugars, we imposed selection favoring divergent metabolic specializations or coutilization of both resources, respectively. Phenotypic characterization revealed the evolution of either fructose or galactose specialists in the single-sugar treatments. In the mixed-sugar regime, we observed adaptive diversification: both specialists coexisted, and no generalist evolved. Divergence from the ancestral phenotype occurred at key pathway junctions in the central carbon metabolism. Fructose specialists evolved mutations in the fbp and pfk genes that appear to balance anabolic and catabolic carbon fluxes. Galactose specialists evolved increased expression of pgmA  and silencing of ptnABCD (the main glucose transporter) and ldh (regulator/enzyme of downstream carbon metabolism). Overall, our study shows how metabolic network architecture and historical contingency serve to predict targets of selection and inform the functional interpretation of evolved mutations. The elucidation of the relationship between molecular constraints and phenotypic trade-offs contributes to an integrative understanding of evolutionary specialization and diversification.Mitigating trade-offs between different resource-utilization functions is key to an organism’s ecological and evolutionary success. These trade-offs often reflect metabolic constraints with a complex molecular underpinning; therefore, their consequences for evolutionary processes have remained elusive. Here, we investigate how metabolic architecture induces resource-utilization constraints and how these constraints, in turn, elicit evolutionary specialization and diversification. Guided by the metabolic network structure of the bacterium  Diversity in metabolic strategy is a principal factor underlying variation within and between major groups of organisms  . ResourA functional consequence of metabolic constraints is that they can give rise to trade-offs between resource-utilization strategies . By prevMicrobial laboratory evolution experiments, followed by genomic analyses (‘evolve and resequence’), have been widely used to evaluate this theory, and study the evolution of resource specialization and metabolic diversification . A remaiL. cremoris.To explore the potential of this functional network perspective, we evaluate whether knowledge of the metabolic architecture, that is, the way in which metabolic processes are connected through a series of biochemical reactions, can successfully predict how multiple interdependent functions interact agonistically or antagonistically to facilitate or constrain targeted metabolic adaptations. In particular, we investigate the potential to predict target loci for adaptation in an evolutionary diversification experiment, where we selected alternative carbon-utilization strategies in the lactic acid bacterium L. cremoris MG1363 , has been resolved in detail, owing to its importance in milk product fermentation (L. cremoris has been adapted for fast ‘batch mode’ growth on the glucose moiety of lactose (milk sugar)  . Dairy swth rate . Lactocovailable .A). Growth on fructose and galactose, therefore, requires a different organization of catabolic versus anabolic fluxes than in the glucose-adapted ancestor, so that the metabolic flux pattern expressed by the ancestral strain is likely suboptimal for growth on both fructose and galactose  branch off from the core glycolytic pathway A. Growthfig. 2A;  online.L. cremoris  but a low growth rate  when cells are growing on glucose, but generates a major metabolic bottleneck during growth on galactose (when the G1P to G6P reaction functions as part of the catabolic pathway).Previous analyses of glucose-adapted dairy strains support these hypotheses. First, metabolic flux optimization is known to prioritize high catabolic fluxes at the branch points with anabolic pathways, because only a limited carbon flux suffices to supply the anabolic pathways. Catabolic fluxes outweigh anabolic fluxes by a factor of 10–20 in the carbon metabolism of cremoris , and thicremoris . Second,−1; pgmA , 2010, tL. cremoris. Moreover, given that the required modifications of the anabolic versus catabolic flux relationships are opposite between fructose and galactose metabolism, we also expect that the growth rates on these sugars are subject to a strong trade-off relative to each other.Collectively, these differences suggest that galactose metabolism is a relatively more difficult metabolic specialization to evolve for the glucose-adapted ancestral L. cremoris—specifically, the prediction that resource adaptation will target pathway branch points and reactions in glycolysis where the distribution or net direction of metabolic fluxes needs to be adjusted relative to the ancestral (glucose preadapted) state—we imposed selection on carbon utilization in L. cremoris in an evolution experiment (B). To apply a controlled selective pressure and, at the same time, allow frequency-dependent ecological feedback mechanisms to operate, we evolved strains in a customizable chemostat system (B). The mix treatment is predicted to yield either a polymorphism of metabolic specialists , if the trade-off between fructose and galactose metabolism is strong  was generally associated with decreased performance on fructose compared with the ancestor  appears to be a generalist, it evolved only once (in one replicate) and in a treatment where improvement on fructose was not selected for . Moreover, GS*gal did not evolve directly from the ancestral strain, it only evolved secondarily from a monomorphic GS population (T2). Surprisingly, such a generalist strategy did not evolve in the mix treatment where high performance on galactose and fructose was favored by natural selection.Toward the end of the experiment (T3), we observed the emergence of a phenotype that exhibited high growth rate on both galactose and fructose in one replicate of the galactose treatment  online. gal as an indication that trade-offs can change as phenotypes continue to evolve in response to selection for alternative resource utilization. To test this hypothesis, we selected a subset of representative specialist strains from each treatment and evaluated their growth rate and yield on three additional carbon sources . Glucose was the preferred sugar for the ancestral strain, which was preadapted for efficient glucose utilization; the other two sugars were selected because of the large metabolic overlap between the galactose and trehalose pathways, and between the glucose and mannose pathways, respectively. Fructose does not share overlap in peripheral metabolic pathways with other monosaccharides.We interpreted the evolution of GS*A; gal and GSmix phenotypes display a lower growth rate on glucose, mannose and, to a lesser extent, fructose. Conversely, the FSfru and FSmix strains showed similar, or slightly reduced, growth rates on glucose and mannose when compared with the ancestor. An opposite, but overall less consistent pattern was observed for growth on trehalose: relative to the ancestor, GS strains exhibited an increased growth rate on this sugar, whereas FS strains maintained a similar growth rate as the ancestral strain. For growth yield, no consistent pattern was found for GS strains, and FS strains consistently exhibited a high yield for mannose .The combined population and single-strain genetic analyses showed that FSectively A and B.Fbp—In our experiment, mutations in fbp spread in the fructose control treatment at timepoint T1, T2, and T3. Fbp codes for FBPase (fructose bisphosphatase), which catalyzes the conversion of FBP to F6P. Fbp expression was significantly increased 3.62-fold in the fbp intergenic FSfru mutant and 2.35-fold in the FSmix strain without a mutation compared with the ancestral expression levels (D). The expression data suggest that the FSfru and FSmix evolved an increased anabolic flux from FBP to F6P. This result aligns with earlier work where fbp expression was shown to limit both growth rate and yield when L. cremoris is exclusively grown on fructose . Pfk codes for PFK (phosphofructokinase) and is regulated by allosteric inhibition and activation by ATP and ADP, respectively . Based on these results, we compared the pfk expression of evolved FSs growing on fructose. We found that expression of the pfk mutant with a mutation in proximity of ATP allosteric site was equal to that of the ancestral strain (E). However, the pfk mutant showed a decrease in enzymatic activity of 25% compared with the ancestor (F).pfk gene A and B. ectively . This reion site C. Based l strain E. Howeveancestor F.fbp and pfk mutations occur in the same strain, but we also found strains with a mutation in only one of these genes, indicating that these two mutations can be beneficial independently but also in combination. We speculate that the mutations in pfk and fbp in the FSs decrease catabolic and increase anabolic activity, respectively, both resulting in an increased net carbon flux in the anabolic direction.We found that both pgmA, ptnABCD, and ldh(x) (A). Most of the variants resided in intergenic regions of the genes or were copy-number variants (CNVs) (B). Four structural variants were detected in ldh (C) and one in ptnABCD (D).The combined population and single-strain genetic analyses showed that GS feature mutations in the genes d ldh(x) A. Most os (CNVs) B. Four sd in ldh C and one ptnABCD D.PgmA—The gene pgmA codes for α-PGM (α-phosphoglucomutase), which catalyzes the reversible conversion of G1P to G6P. Its activity in L. cremoris is essential for the catabolism of galactose using the Leloir pathway in the G1P to G6P direction  and T3  featured a mutation in the intergenic region upstream of the pgmA gene (B). The mutation was not found in GSmix, which instead evolved a 6-fold duplication of the chromosome region containing the pgmA gene (B). RT-qPCR expression experiments of pgmA revealed that expression increased 2-fold in both strains . The mutation of pgmA in GSmix resulted in a 3-fold expression increase (fig. 6E). Previous experiments have shown that a plasmid-based 3.8-fold overexpression of pgmA in a ΔpgmA strain boosted α-PGM activity, increasing galactose consumption by 19%  is coded by ptnABCD and was mutated in GSgal and GSmix. PTSman is the primary transporter of glucose and mannose metabolism in L. cremoris (ptnAB intracellular components and in the membrane-embedded permease component (ptnC) as a structural mutation in the transmembrane section of the protein (D). Expression analysis of the ptnABCD mutant strains from GSgal  showed a full inhibition (≤99%) of the expression of ptnABCD, indicating that the mutation suppresses the activity of ptnABCD (F). GSmix without a mutation in ptnABCD (derived from a population that did contain ptnABCD mutants) also displayed full inhibition of ptnABCD (F). This indicates that galactose transport in the GS does not occur via the PTSman. The complete silencing of PTSman in both strains suggests that its expression inhibits fast growth on galactose. This result is consistent with a study where mutants of L. cremoris that evolved a reduced expression of PTSman display decreased growth on glucose, but enhanced growth on galactose . Single-strain genetic analysis found ldh mutation only in one strain, the GS*gal. LDH catalyzes the reversible reduction of pyruvate to lactate with concomitant conversion of NADH to NAD+ (A). This reaction serves to balance redox potential via oxidation . All four were frame-shift mutations; one resulted in the gain of a stop codon which occurred in the GS*gal strain, the other three mutations occurred in the proximity of the LDH protein domains that are associated with substrate binding, allosteric activation and NAD+ binding (C). Besides these structural mutations, we also found a large 2- and 3-fold duplicated section that contained ldhX (a lactate dehydrogenase homolog). Metabolic analysis of a ldh mutant showed a strongly reduced production of lactic acid when grown on a variety of carbon sources (G) combined with a high growth yield (B). We conclude that ldh mutations have a deleterious effect on lactic acid fermentation and appear to shift the phenotype toward increased mixed-acid fermentation.in GSmix B. Single to NAD+ A. This rxidation . Four stll GSgal B and C.  binding C. Beside sources G combineth yield B. We conOur experimental results provide a striking example of how evolutionary adaptation and divergence are shaped by constraints connected to the architecture of a metabolic network. By considering the requirement of maintaining adequate balance between anabolic and catabolic fluxes, we were able to predict the evolvability of alternative resource-utilization strategies  for an ancestral strain preadapted for growth on glucose. Here, we treated network topology  as a ‘hard’ constraint that imposed restrictions on the range of feasible phenotypes that could potentially evolve over the course of the experiment. Additionally, we considered the control of metabolic fluxes  as evolvable ‘soft’ constraints that reflected the result of past selection for optimized glucose metabolism and that biased the rate at which alternative specializations could evolve.L. cremoris appears to be able to resolve the soft constraints related to the unique bottlenecks of fructose and galactose metabolism, but at markedly different rates, consistent with the expectation that fructose utilization requires a smaller adjustment of the glucose preadapted ancestral state than galactose utilization. Second, we hypothesize that metabolic network topology acted as a hard constraint preventing efficient simultaneous utilization of fructose and galactose, and the resulting trade-off induced the evolution of a population-level polymorphism in resource specialization in an environment where both sugars were available simultaneously. Taken together, our data exemplify that careful consideration of the constraints and trade-offs that are exposed under a specific environmental-selection regime provides a useful framework to predict, interpret and link phenotypic and genetic evolutionary change. Further validation of the proposed mechanisms can be obtained from in-depth metabolomics measurements to verify that the observed mutations indeed resolve rate-limiting steps in fructose and galactose metabolism.Data from our experiment indicate that both types of constraints shape the outcome of evolution. First, Escherichia coli growing on a mixture of glucose and acetate, has been explained based on the fact that the ability to switch rapidly from glucose to acetate consumption requires an upstream flux from acetate toward central glycolysis, whereas efficient growth on glucose favors a maximum downstream flux capacity to enable rapid excretion of acetate as a metabolic waste product . These mutations were located at positions in the metabolic map that were expected to undergo directional  or quantitative (ptnABCD and ldh) changes in metabolic fluxes during the adaptation from glucose to fructose or galactose utilization (A). The transcriptional and enzymatic changes induced by these key mutations were consistent with the predicted directionality of selection . Finally, it is possible that selection on fructose utilization has been weaker in the mix treatment, especially if the constraints originating at pfk-fbp are exposed only during selection at higher metabolic rates. Such conditions have potentially occurred less frequently in the mix treatment, which had generally lower fructose availability (50% less) and lower dilution rates . Taken together, this pattern could indicate that the FSs from the mix treatment adapted to cometabolize galactose to supply anabolic fluxes. This principle was demonstrated earlier for the cometabolization of glucose, where fbp limitation of fructose-grown L. lactis cells could be resolved by the addition of a small amount of glucose to the medium , whereas in In our sequence analysis, we considered not only single nucleotide polymorphisms (SNPs) and insertions and deletions (indels) but also performed CNV analysis . CNVs comprised most of the observed mutations 89%;  online, analyzed . An addiL. lactis only approaches high growth rates under homolactic fermentation, which is crucially dependent on LDH activity. The GS*gal strain had a loss-of-function ldh mutation (G), but showed the highest growth rate on galactose and, surprisingly, a high increase in growth on fructose together with low growth on glucose and mannose (A). During our experiments, we noticed that overnight cultures of the evolved GS*gal strain produce a strong diacetyl aroma; yet, we did not follow-up on this observation with quantitative analysis to confirm the increased activity of the acetoin branch of the downstream metabolism. Performing such an experiment would be worthwhile to explore potential applications in the dairy industry, where a strain with the unusual combination of a high growth rate on galactose, low growth rate on glucose, and mixed-acid mode of growth could find potential use as a galactose-scavenger  (A). This pattern is consistent with the unique low entry-point of fructose in glycolysis compared with the other sugars (L. lactis can lead to 57–70% decrease in growth rate on glucose (A). The observed pattern of pleiotropy is consistent with the predicted flux constraints based on their pathway topology , we observed that evolutionary specialization generalizes to a broader pattern of resource-utilization trade-offs. In order to explore whether the observed pleiotropic effects are predictable from the metabolic network architecture, we mapped the observed metabolic adaptations of FS and GS onto the hardwired glycolytic backbone  online, ehalose) A. This pr sugars  online.  glucose . The met glucose A. The obtopology  online. topology , so the tabolism  online. tabolism . The obsh PTSman  leads toTaken together, the observed pleiotropic growth patterns suggest that the signatures of architectural trade-offs can be detected in other resources, warranting further investigation. This would allow us to answer how such constraints manifest themselves dependent on the preadapted initial state of the population and how predictably we may observe evolutionary diversification in populations subject to resource-utilization trade-offs.The metabolic adaptations observed in our evolution experiment manifested themselves at multiple levels of organization: as molecular changes in the organization and regulation of metabolic pathways  and 6, aInteresting future work may include reevolving our galactose and fructose optimized specialists on glucose, mannose, and trehalose, and/or evolving the strains on different pairs of sugar combinations. Such experiments will provide additional insight into the generality and nature of the metabolic architectural constraints identified by our study. This would allow us to answer how such constraints manifest themselves dependent on the preadapted initial state of the population and how predictably we may observe evolutionary diversification in populations subject to resource-utilization trade-offs.L. cremoris with its low biofilm forming properties instead of more commonly used model organisms. Lactococcus cremoris MG1363  was grown in 60 ml of chemically defined medium for prolonged cultivation (CDMPC) (2 headspace flow) under continuous dilution and stirring (330 rpm) at 30 °C and maintained at constant pH 6.5. Three treatments were run in parallel, using media supplemented with either 1% wt/v galactose, 0.5% wt/v fructose, or a mix of 0.5% wt/v galactose + 0.25% wt/v fructose as sole carbon source(s). For each of the three treatments, we ran four replicates in parallel  in steady state, we monitored its change throughout the experiment for signs of evolutionary adaptation, and selected three timepoints for subsequent phenotypic analysis accordingly  . Cultureparallel  online. ordingly  online. F/[F + G]) ratio was also computed for the median colony size.After the evolution experiment was completed, a library of evolved strains was created to phenotypically characterize the evolved mutants in each treatment  online. Greiner Bio-one 781906) under semianaerobic conditions  at 30 °C, whereas keeping track of the OD600 in a plate reader (Tecan F200). After background correction, estimates of the instantaneous growth rate were obtained by performing local linear regression analyses on the ln-transformed growth curve data, using a sliding window of five data points (measurements were taken every 10 min) and OD600 > 0.16. We then determined the maximum growth rate from these regression curves and averaged the three-replicate maximum growth-rate values for each genotype on each sugar  after preculturing them overnight in F-CDMPC and G-CDMPC. The –80 °C stocks were diluted 100 times in PBS, 1 µl of this cell suspension was used to inoculate 100 µl of fresh fructose, galactose, and mixed sugar CDMCP (pH = 6.5). Each strain was grown in triplo in 384-well plates (Fmax (maximum growth rate on fructose) on the y-axis and Gmax  on the x-axis. The growth rate coordinates in the Fmax–Gmax space were clustered per timepoint per treatment. To estimate the number of clusters (phenotypic groups), we applied normal (Gaussian) mixture models and a maximum likelihood approach using the R package mclust .We used a model-based clustering method to identify and classify the different phenotypic groups that evolved during the evolution experiment  online. IlluminaHiSeq (GATC) with a mean coverage of 300, read length, and insert size of 150 bases. To characterize genetic variation across all strains, adaptors were removed with cutadapt 1.18 (Lactococcus lactis subsp cremoris v. MG1363) using BWA 0.7.13 (We sequenced 48 strains by sampling three strains from two out of four replicates for each timepoint for each treatment. We set a threshold of only considering mutated loci that exclusively occurred in one phenotype (FS or GS) and which entailed five or more strains or variants. The isolation of chromosomal DNA of the population and single-genotype cultures was performed as described in apt 1.18 , and paiapt 1.18  and FastA 0.7.13 . DuplicaA 0.7.13 . GenotypA 0.7.13 , settingA 0.7.13  online.CNV along the genome was identified by measuring changes in local coverage relative to the flanking genomic regions and/or genome-wide coverage. CNV was inferred using CNVnator 0.3.3 , with a pfk and ldh proteins was performed using Phyre2  genetic variants were annotated relative to the reference genome using SnpEff 4.3 . Annotatg Phyre2 . EzMol w regions . Protternose PTS .600 ≈ 0.45) and the cell pellet was immediately frozen in liquid nitrogen. RNA extraction and cDNA preparation were performed in duplo. cDNA samples were used to run a qRT-pcr with custom primers for the selected genes  and executed as stated by the manufacturer’s protocol.Protein extracts were prepared from midexponentially harvested cells grown on F-CDMPC. Essays were performed with a Megazyme L-lactic acid assay kit and performed as stated by the manufacturer’s protocol.Cultures were grown on CDMPC supplemented by 0.5% sugar  and harvested midexponentially. Essays were performed on supernatant with a Molecular Biology and Evolution online.msac124_Supplementary_DataClick here for additional data file.