LacdiNAc synthase B4GALNT3 has a unique PA14 domain and suppresses N-glycan capping.

Structural variation of N-glycans is essential for the regulation of glycoprotein functions. GalNAcß1-4GlcNAc (LacdiNAc or LDN), a unique subterminal glycan structure synthesized by B4GALNT3 or B4GALNT4, is involved in the clearance of N-glycoproteins from the blood and maintenance of cell stemness. Such regulation of glycoprotein functions by LDN is largely different from that by the dominant subterminal structure, N-acetyllactosamine (Galß1-4GlcNAc, LacNAc). However, the mechanisms by which B4GALNT activity is regulated and how LDN plays different roles from LacNAc remain unclear. Here, we found that B4GALNT3 and four have unique domain organization containing a noncatalytic PA14 domain, which is a putative glycan-binding module. A mutant lacking this domain dramatically decreases the activity toward various substrates, such as N-glycan, O-GalNAc glycan, and glycoproteins, indicating that this domain is essential for enzyme activity and forms part of the catalytic region. In addition, to clarify the mechanism underlying the functional differences between LDN and LacNAc, we examined the effects of LDN on the maturation of N-glycans, focusing on the related glycosyltransferases upstream and downstream of B4GALNT. We revealed that, unlike LacNAc synthesis, prior formation of bisecting GlcNAc in N-glycan almost completely inhibits LDN synthesis by B4GALNT3. Moreover, the presence of LDN negatively impacted the actions of many glycosyltransferases for terminal modifications, including sialylation, fucosylation, and human natural killer-1 synthesis. These findings demonstrate that LDN has significant impacts on N-glycan maturation in a completely different way from LacNAc, which could contribute to obtaining a comprehensive overview of the system regulating complex N-glycan biosynthesis.

A versatile in situ cofactor enhancing system for meeting cellular demands for engineered metabolic pathways.

Cofactor imbalance obstructs the productivities of metabolically engineered cells. Herein, we employed a minimally perturbing system, xylose reductase and lactose (XR/lactose), to increase the levels of a pool of sugar phosphates which are connected to the biosynthesis of NAD(P)H, FAD, FMN, and ATP in Escherichia coli. The XR/lactose system could increase the amounts of the precursors of these cofactors and was tested with three different metabolically engineered cell systems (fatty alcohol biosynthesis, bioluminescence light generation, and alkane biosynthesis) with different cofactor demands. Productivities of these cells were increased 2-4-fold by the XR/lactose system. Untargeted metabolomic analysis revealed different metabolite patterns among these cells, demonstrating that only metabolites involved in relevant cofactor biosynthesis were altered. The results were also confirmed by transcriptomic analysis. Another sugar reducing system (glucose dehydrogenase) could also be used to increase fatty alcohol production but resulted in less yield enhancement than XR. This work demonstrates that the approach of increasing cellular sugar phosphates can be a generic tool to increase in vivo cofactor generation upon cellular demand for synthetic biology.

Efficacy and Findings of a Blinded Randomized Reintroduction Phase for the Low FODMAP Diet in Irritable Bowel Syndrome.

BACKGROUND & AIMS: The efficacy of a low fermentable oligo-, di-, monosaccharides and polyols (FODMAP) diet in irritable bowel syndrome (IBS) is well established. After the elimination period, a reintroduction phase aims to identify triggers. We studied the impact of a blinded reintroduction using FODMAP powders to objectively identify triggers and evaluated the effect on symptoms, quality of life, and psychosocial comorbidities. METHODS: Responders to a 6-week low FODMAP diet, defined by a drop in IBS symptom severity score (IBS-SSS) compared with baseline, entered a 9-week blinded randomized reintroduction phase with 6 FODMAP powders (fructans, fructose, galacto-oligosaccharides, lactose, mannitol, sorbitol) or control (glucose). A rise in IBS-SSS (≥50 points) defined a FODMAP trigger. Patients completed daily symptom diaries and questionnaires for quality of life and psychosocial comorbidities. RESULTS: In 117 recruited patients with IBS, IBS-SSS improved significantly after the elimination period compared with baseline (150 ± 116 vs 301 ± 97, P < .0001, 80% responders). Symptom recurrence was triggered in 85% of the FODMAP powders, by an average of 2.5 ± 2 FODMAPs/patient. The most prevalent triggers were fructans (56%) and mannitol (54%), followed by galacto-oligosaccharides, lactose, fructose, sorbitol, and glucose (respectively 35%, 28%, 27%, 23%, and 26%) with a significant increase in abdominal pain at day 1 for sorbitol/mannitol, day 2 for fructans/galacto-oligosaccharides, and day 3 for lactose. CONCLUSION: We confirmed the significant benefit of the low FODMAP diet in tertiary-care IBS. A blinded reintroduction revealed a personalized pattern of symptom recurrence, with fructans and mannitol as the most prevalent, and allows the most objective identification of individual FODMAP triggers. Ethical commission University hospital of Leuven reference number: s63629; Clinicaltrials.gov number: NCT04373304.

Hepatoprotective activity of Lactéol® forte and quercetin dihydrate against thioacetamide-induced hepatic cirrhosis in male albino rats.

Liver cirrhosis is a silent disease in humans and is experimentally induced by many drugs and toxins as thioacetamide (TAA) in particular, which is the typical model for experimental induction of hepatic fibrosis. Thus, the objective of the present study was to elucidate the possible protective effects of lactéol® forte (LF) and quercetin dihydrate (QD) against TAA-induced hepatic damage in male albino rats. Induction of hepatotoxicity was performed by TAA injection (200 mg/kg I/P, twice/ week) in rats. LF (1 × 109 CFU/rat 5 times/week) and QD (50 mg/kg 5 times/week) treated groups were administered concurrently with TAA injection (200 mg/kg I/P, twice/ week). The experimental treatments were conducted for 12 weeks. Hepatotoxicity was evaluated biochemically by measuring alanine aminotransferase (ALT), aspartate aminotransferase (AST) and gamma-glutamyl transferase (GGT) in the serum and histopathologically with the scoring of histopathological changes besides histochemical assessment of collagen by Masson's trichrome and immunohistochemical analysis for α-smooth muscle actin (α-SMA), Ki67 and caspase-3 expression in liver sections. Our results indicated that LF and QD attenuated some biochemical changes and histochemical markers in TAA-mediated hepatotoxicity in rats by amelioration of biochemical markers and collagen, α-SMA, Ki67 and caspase3 Immunoexpression. Additionally, LF and QD supplementation downregulated the proliferative, necrotic, fibroblastic changes, eosinophilic intranuclear inclusions, hyaline globules and Mallory-like bodies that were detected histopathologically in the TAA group. In conclusion, LF showed better hepatic protection than QD against TAA-induced hepatotoxicity in rats by inhibiting inflammatory reactions with the improvement of some serum hepatic transaminases, histopathological picture and immunohistochemical markers.

LacdiNAc to LacNAc: remodelling of bovine α-lactalbumin N-glycosylation during the transition from colostrum to mature milk.

α -Lactalbumin, an abundant protein present in the milk of most mammals, is associated with biological, nutritional and technological functionality. Its sequence presents N-glycosylation motifs, the occupancy of which is species-specific, ranging from no to full occupancy. Here, we investigated the N-glycosylation of bovine α-lactalbumin in colostrum and milk sampled from four individual cows, each at 9 time points starting from the day of calving up to 28.0 d post-partum. Using a glycopeptide-centric mass spectrometry-based glycoproteomics approach, we identified N-glycosylation at both Asn residues found in the canonical Asn-Xxx-Ser/Thr motif, i.e. Asn45 and Asn74 of the secreted protein. We found similar glycan profiles in all four cows, with partial site occupancies, averaging at 35% and 4% for Asn45 and Asn74, respectively. No substantial changes in occupancy occurred over lactation at either site. Fucosylation, sialylation, primarily with N-acetylneuraminic acid (Neu5Ac), and a high ratio of N,N'-diacetyllactosamine (LacdiNAc)/N-acetyllactosamine (LacNAc) motifs were characteristic features of the identified N-glycans. While no substantial changes occurred in site occupancy at either site during lactation, the glycoproteoform (i.e. glycosylated form of the protein) profile revealed dynamic changes; the maturation of the α-lactalbumin glycoproteoform repertoire from colostrum to mature milk was marked by substantial increases in neutral glycans and the number of LacNAc motifs per glycan, at the expense of LacdiNAc motifs. While the implications of α-lactalbumin N-glycosylation on functionality are still unclear, we speculate that N-glycosylation at Asn74 results in a structurally and functionally different protein, due to competition with the formation of its two intra-molecular disulphide bridges.

Dietary human milk oligosaccharides reduce allergic airway inflammation by modulating SCFAs level and ILC2 activity.

Group 2 innate lymphoid cells (ILC2s) play a crucial role in the progression of asthma, yet the regulatory mechanisms modulating ILC2 responses in asthma remain underexplored. Human milk oligosaccharides (HMOs), vital non-nutritive components of breast milk, are known to significantly shape immune system development and influence the incidence of allergic diseases. However, their impact on ILC2-driven asthma is not fully understood. Our research reveals that dietary HMOs act as potent inhibitors of ILC2 responses and allergic airway inflammation. Treatment with 2'-fucosyllactose (2'-FL) and 6'-sialyllactose (6'-SL) significantly reduced ILC2-related airway inflammation induced by papain or Alternaria alternata in mice, evidenced by decreased eosinophil (EOS) infiltration and lower IL-5 and IL-13 levels in BALF. Notably, while ILC2 expresses HMO receptors, HMO did not act directly on ILC2 but potentially modulated their activity through alterations in gut microbiota derived SCFAs. HMO treatments alleviated airway inflammation in SCFA-dependent manners, with SCFA depletion or receptor blocking reversing these beneficial effects. This study reveals the potential of dietary HMOs in managing asthma through modulation of ILC2 activity and the gut-lung axis, proposing a new therapeutic avenue that utilises the immunomodulatory capacities of nutritional components to combat respiratory diseases.

Transcriptomic characterization of recombinant Clostridium beijerinckii NCIMB 8052 expressing methylglyoxal synthase and glyoxal reductase from Clostridium pasteurianum ATCC 6013.

Bioconversion of abundant lactose-replete whey permeate to value-added chemicals holds promise for valorization of this expanding food processing waste. Efficient conversion of whey permeate-borne lactose requires adroit microbial engineering to direct carbon to the desired chemical. An engineered strain of Clostridium beijerinckii NCIMB 8052 (C. beijerinckii_mgsA+mgR) that produces 87% more butanol on lactose than the control strain was assessed for global transcriptomic changes. The results revealed broadly contrasting gene expression patterns in C. beijerinckii_mgsA+mgR relative to the control strain. These were characterized by widespread decreases in the abundance of mRNAs of Fe-S proteins in C. beijerinckii_mgsA+mgR, coupled with increased differential expression of lactose uptake and catabolic genes, iron uptake genes, two-component signal transduction and motility genes, and genes involved in the biosynthesis of vitamins B5 and B12, aromatic amino acids (particularly tryptophan), arginine, and pyrimidines. Conversely, the mRNA patterns suggest that the L-aspartate-dependent de novo biosynthesis of NAD as well as biosynthesis of lysine and asparagine and metabolism of glycine and threonine were likely down-regulated. Furthermore, genes involved in cysteine and methionine biosynthesis and metabolism, including cysteine desulfurase-a central player in Fe-S cluster biosynthesis-equally showed reductions in mRNA abundance. Genes involved in biosynthesis of capsular polysaccharides and stress response also showed reduced mRNA abundance in C. beijerinckii_mgsA+mgR. The results suggest that remodeling of cellular and metabolic networks in C. beijerinckii_mgsA+mgR to counter anticipated effects of methylglyoxal production from heterologous expression of methylglyoxal synthase led to enhanced growth and butanol production in C. beijerinckii_mgsA+mgR. IMPORTANCE: Biological production of commodity chemicals from abundant waste streams such as whey permeate represents an opportunity for decarbonizing chemical production. Whey permeate remains a vastly underutilized feedstock for bioproduction purposes. Thus, enhanced understanding of the cellular and metabolic repertoires of lactose-mediated production of chemicals such as butanol promises to identify new targets that can be fine tuned in recombinant and native microbial strains to engender stronger coupling of whey permeate-borne lactose to value-added chemicals. Our results highlight new genetic targets for future engineering of C. beijerinckii for improved butanol production on lactose and ultimately in whey permeate.

A unique metabolic gene cluster regulates lactose and galactose metabolism in the yeast Candida intermedia.

Lactose assimilation is a relatively rare trait in yeasts, and Kluyveromyces yeast species have long served as model organisms for studying lactose metabolism. Meanwhile, the metabolic strategies of most other lactose-assimilating yeasts remain unknown. In this work, we have elucidated the genetic determinants of the superior lactose-growing yeast Candida intermedia. Through genomic and transcriptomic analyses, we identified three interdependent gene clusters responsible for the metabolism of lactose and its hydrolysis product galactose: the conserved LAC cluster (LAC12, LAC4) for lactose uptake and hydrolysis, the conserved GAL cluster (GAL1, GAL7, and GAL10) for galactose catabolism through the Leloir pathway, and a "GALLAC" cluster containing the transcriptional activator gene LAC9, second copies of GAL1 and GAL10, and a XYL1 gene encoding an aldose reductase involved in carbon overflow metabolism. Bioinformatic analysis suggests that the GALLAC cluster is unique to C. intermedia and has evolved through gene duplication and divergence, and deletion mutant phenotyping proved that the cluster is indispensable for C. intermedia's growth on lactose and galactose. We also show that the regulatory network in C. intermedia, governed by Lac9 and Gal1 from the GALLAC cluster, differs significantly from the galactose and lactose regulons in Saccharomyces cerevisiae, Kluyveromyces lactis, and Candida albicans. Moreover, although lactose and galactose metabolism are closely linked in C. intermedia, our results also point to important regulatory differences.IMPORTANCEThis study paves the way to a better understanding of lactose and galactose metabolism in the non-conventional yeast C. intermedia. Notably, the unique GALLAC cluster represents a new, interesting example of metabolic network rewiring and likely helps to explain how C. intermedia has evolved into an efficient lactose-assimilating yeast. With the Leloir pathway of budding yeasts acting like a model system for understanding the function, evolution, and regulation of eukaryotic metabolism, this work provides new evolutionary insights into yeast metabolic pathways and regulatory networks. In extension, the results will facilitate future development and use of C. intermedia as a cell-factory for conversion of lactose-rich whey into value-added products.

Rational Design of Highly Selective Sialyllactose-Imprinted Nanogels.

We describe a facile method to prepare water-compatible molecularly imprinted polymer nanogels (MIP NGs) as synthetic antibodies against target glycans. Three different phenylboronic acid (PBA) derivatives were explored as monomers for the synthesis of MIP NGs targeting either α2,6- or α2,3-sialyllactose, taken as oversimplified models of cancer-related sT and sTn antigens. Starting from commercially available 3-acrylamidophenylboronic acid, also its 2-substituted isomer and the 5-acrylamido-2-hydroxymethyl cyclic PBA monoester derivative were initially evaluated by NMR studies. Then, a small library of MIP NGs imprinted with the α2,6-linked template was synthesized and tested by mobility shift Affinity Capillary Electrophoresis (msACE), to rapidly assess an affinity ranking. Finally, the best monomer 2-acrylamido PBA was selected for the synthesis of polymers targeting both sialyllactoses. The resulting MIP NGs display an affinity constant≈106 M-1 and selectivity towards imprinted glycans. This general procedure could be applied to any non-modified carbohydrate template possessing a reducing end.

Lactobacillus amylovorus Promotes Lactose Utilization in Small Intestine and Enhances Intestinal Barrier Function in Intrauterine Growth Restricted Piglets.

BACKGROUND: Intrauterine growth restriction (IUGR) resulted in high mortality and many physiological defects of piglets, causing huge economic loss in the swine industry. Lactobacillus amylovorus (L. amylovorus) was identified as one of the main differential bacteria between IUGR and normal piglets. However, the effects of L. amylovorus on the growth performance and intestinal health in IUGR piglets remained unclear. OBJECTIVES: This study aimed to investigate the promoting effects of L. amylovorus Mafic1501, a new strain isolated from normal piglets, on the growth performance and intestinal barrier functions in IUGR piglets. METHODS: Newborn mice or piglets were assigned into 3 groups: CON (normal birth weight, control), IUGR (low birth weight), and IUGR+L. amy (low birth weight), administered with sterile saline or L. amylovorus Mafic1501, respectively. Growth performance, lactose content in the digesta, intestinal lactose transporter, and barrier function parameters were profiled. IPEC-J2 cells were cultured to verify the effects of L. amylovorus Mafic1501 on lactose utilization and intestinal barrier functions. RESULTS: L. amylovorus Mafic1501 elevated body weight and average daily gain of IUGR mice and piglets (P < 0.05). The lactose content in the ileum was decreased, whereas gene expression of glucose transporter 2 (GLUT2) was increased by L. amylovorus Mafic1501 in IUGR piglets during suckling period (P < 0.05). Besides, L. amylovorus Mafic1501 promoted intestinal barrier functions by increasing the villus height and relative gene expressions of tight junctions (P < 0.05). L. amylovorus Mafic1501 and its culture supernatant decreased the lactose level in the medium and upregulated gene expressions of transporter GLUT2 and tight junction protein Claudin-1 of IPEC-J2 cells (P < 0.05). CONCLUSION: L. amylovorus Mafic1501 improved the growth performance of IUGR piglets by promoting the lactose utilization in small intestine and enhancing intestinal barrier functions. Our results provided the new evidence of L. amylovorus Mafic1501 for its application in the swine industry.

A FRET based ultrasensitive fluorescent aptasensor for 6'-sialyllactose detection.

As a kind of human milk oligosaccharide, 6'-sialyllactose (6'-SL) plays an important role in promoting infant brain development and improving infant immunity. The content of 6'-SL in infant formula milk powder is thus one of the important nutritional indexes. Since the lacking of efficient and rapid detection methods for 6'-SL, it is of great significance to develop specific recognition elements and establish fast and sensitive detection methods for 6'-SL. Herein, using 6'-SL specific aptamer as the recognition element, catalytic hairpin assembly as the signal amplification technology and quantum dots as the signal label, a fluorescence biosensor based on fluorescence resonance energy transfer (FRET) was constructed for ultra-sensitive detection of 6'-SL. The detection limit of this FRET-based fluorescent biosensor is 0.3 nM, and it has some outstanding characteristics such as high signal-to-noise ratio, low time-consuming, simplicity and high efficiency in the actual sample detection. Therefore, it has broad application prospect in 6'-SL detection.

Development and Validation of Novel Z-360-Based Macromolecules for the Active Targeting of CCK2-R.

The cholecystokinin type 2 receptor (CCK2-R) represents an ideal target for cancer therapy since it is overexpressed in several tumors and is associated with poor prognosis. Nastorazepide (Z-360), a selective CCK2-R antagonist, has been widely investigated as a CCK2-R ligand for targeted therapy; however, its high hydrophobicity may represent a limit to cell selectivity and optimal in vivo biodistribution. Here, we present three new fluorescent Z-360 derivatives (IP-002G-Rho, IP-002L-Rho, and IP-002M-Rho) in which nastorazepide was linked, through spacers bearing different saccharides (glucose (G), lactose (L), and maltotriose (M)), to sulforhodamine B. A fourth compound (IP-002H-Rho) with no pendant sugar was also synthesized as a control. Through two-dimensional (2D) and three-dimensional (3D) in vitro studies, we evaluated the compound association with and selectivity for CCK2-R-overexpressing cells (A431-CCK2-R+) vs CCK2-R-underexpressing cells (A431 WT). 2D in vitro studies highlighted a progressive increase of IP-002x-Rho association with A431-CCK2-R+ cells according to the linker hydrophilicity, that is, maltotriose > lactose > glucose > hydrogen, with IP-002M-Rho showing a 2.4- and a 1.36-fold higher uptake than IP-002G-Rho and IP-002L-Rho, respectively. Unexpectedly, IP-002H-Rho showed a similar cell association to that of IP-002L-Rho but with no difference between the two tested cell lines. On the contrary, association with A431-CCK2-R+ cells as compared to the A431 WT was found to be 1.08-, 1.14-, and 1.37-fold higher for IP-002G-Rho, IP-002L-Rho, and IP-002M-Rho, respectively, proving IP-002M-Rho to be the best-performing compound, as also confirmed by competition studies. Trafficking studies on A431-CCK2-R+ cells incubated with IP-002M-Rho suggested the coexistence of receptor-mediated endocytosis and simple diffusion. On the contrary, a high and selective uptake of IP-002M-Rho by A431-CCK2-R+ cells only was observed on 3D scaffolds embedded with cells, underlining the importance of 3D models in in vitro preliminary evaluation.

Inhaled JAK Inhibitor GDC-0214 Nanoaggregate Powder Exhibits Improved Pharmacokinetic Profile in Rats Compared to the Micronized Form: Benefits of Thin Film Freezing.

Asthma is a common chronic disease affecting the airways in the lungs. The receptors of allergic cytokines, including interleukin (IL)-4, IL-5, and IL-13, trigger the Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway, which involves the pathogenesis of asthma. GDC-0214 is a JAK inhibitor that was developed as a potent and selective target for the treatment of asthma, specifically targeting the lungs. While inhaled GDC-0214 is a promising novel treatment option against asthma, improvement is still needed to achieve increased potency of the powder formulation and a reduced number of capsules containing powder to be inhaled. In this study, high-potency amorphous powder formulations containing GDC-0214 nanoaggregates for dry powder inhalation were developed using particle engineering technology, thin film freezing (TFF). A high dose per capsule was successfully achieved by enhancing the solubility of GDC-0214 and powder conditioning. Lactose and/or leucine as excipients exhibited optimum stability and aerosolization of GDC-0214 nanoaggregates, and aerosolization of the dose was independent of air flow through the device between 2 and 6 kPa pressure drops. In the rat PK study, formulation F20, which contains 80% GDC-0214 and 20% lactose, resulted in the highest AUC0-24h in the lungs with the lowest AUC0-24h in the plasma that corresponds to a 4.8-fold higher ratio of the lung-to-plasma exposures compared to micronized crystalline GDC-0214 powder administered by dry powder inhalation. Therefore, GDC-0214 nanoaggregates produced by TFF provided an improved dry powder for inhalation that can lead to enhanced therapeutic efficacy with a lower risk of systemic toxicity.

Efficient heterologous expression of cellobiose 2-epimerase gene in Escherichia coli under the control of T7 lac promoter without addition of IPTG and lactose.

In this study, the cellobiose 2-epimerase gene csce from Caldicellulosiruptor saccharolyticus was expressed in Escherichia coli using TB medium containing yeast extract Oxoid and tryptone Oxoid. Interesting, it was found that when the concentration of isopropyl-beta-d-thiogalactopyranoside (IPTG) and lactose was 0 (no addition), the activity of cellobiose 2-epimerase reached 5.88 U/mL. It was 3.70-fold higher than the activity observed when 1.0 mM IPTG was added. When using M9 medium without yeast extract Oxoid and tryptone Oxoid, cellobiose 2-epimerase gene could not be expressed without IPTG and lactose. However, cellobiose 2-epimerase gene could be expressed when yeast extract Oxoid or tryptone Oxoid was added, indicating that these supplements contained inducers for gene expression. In the absence of IPTG and lactose, the addition of soy peptone Angel-1 or yeast extract Angel-1 to M9 medium significantly upregulated the expression of cellobiose 2-epimerase gene in E. coli BL21 pET28a-csce, and these inductions led to higher expression levels compared to tryptone Oxoid or yeast extract Oxoid. The relative transcription level of csce was consistent with its expression level in E. coli BL21 pET28a-csce. In the medium TB without IPTG and lactose and containing yeast extract Angel-1 and soy peptone Angel-1, the activity of cellobiose 2-epimerase reached 6.88 U/mL, representing a 2.2-fold increase compared to previously reported maximum activity in E. coli. The significance of this study lies in its implications for efficient heterologous expression of recombinant enzyme proteins in E. coli without the need for IPTG and lactose addition.

Glycan-Driven Formation of Raft-Like Domains with Hierarchical Periodic Nanoarrays on Dendrimersome Synthetic Cells.

The accurate spatial segregation into distinct phases within cell membranes coordinates vital biochemical processes and functionalities in living organisms. One of nature's strategies to localize reactivity is the formation of dynamic raft domains. Most raft models rely on liquid-ordered L0 phases in a liquid-disordered Ld phase lacking correlation and remaining static, often necessitating external agents for phase separation. Here, we introduce a synthetic system of bicomponent glycodendrimersomes coassembled from Janus dendrimers and Janus glycodendrimers (JGDs), where lactose-lactose interactions exclusively drive lateral organization. This mechanism results in modulated phases across two length scales, yielding raft-like microdomains featuring nanoarrays at the nanoscale. By varying the density of lactose and molecular architecture of JGDs, the nanoarray type and size, shape, and spacing of the domains were controlled. Our findings offer insight into the potential primordial origins of rudimentary raft domains and highlight the crucial role of glycans within the glycocalyx.

Enzymatically Oxidized Carbohydrates As Dicarbonyl Biobased Cross-Linkers for Polyamines.

Carbonyl cross-linkers are used to modify textiles and form resins, and are produced annually in megatonne volumes. Due to their toxicity toward the environment and human health, however, less harmful biobased alternatives are needed. This study introduces carbonyl groups to lactose and galactose using galactose oxidase from Fusarium graminearum (FgrGalOx) and pyranose dehydrogenase from Agaricus bisporus (AbPDH1) to produce four cross-linkers. Differential scanning calorimetry was used to compare cross-linker reactivity, most notably resulting in a 34 °C decrease in reaction peak temperature (72 °C) for FgrGalOx-oxidized galactose compared to unmodified galactose. Attenuated total reflectance Fourier-transform infrared (ATR-FTIR) spectroscopy, X-ray photoelectron spectroscopy (XPS), and proton nuclear magnetic resonance (1H NMR) spectroscopy were used to verify imine formation and amine and aldehyde depletion. Cross-linkers were shown to form gels when mixed with polyallylamine, with FgrGalOx-oxidized lactose forming gels more effectively than all other cross-linkers, including glutaraldehyde. Further development of carbohydrate cross-linker technologies could lead to their adoption in various applications, including in adhesives, resins, and textiles.

Enhancement of essential cofactors for in vivo biocatalysis.

A scarcity of cofactors, necessary metabolites or substrates for in vivo enzymatic reactions, is among the major barriers for product synthesis in metabolically engineered cells. This work compares our recently developed cofactor-boosting strategy, which uses xylose reductase (XR) and lactose to increase the intracellular levels of reduced or oxidized nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), adenosine triphosphate (ATP) and acetyl coenzymeA (acetyl-CoA), with other previously reported methods. We demonstrated that the XR/lactose approach enhances levels of sugar alcohols and sugar phosphates, which leads to elevated levels of crucial cofactors required by specific metabolic pathways. The patterns of cofactor enhancement are not uniform and depend upon the specific pathway components that are overexpressed. We term this model the "user-pool" model. Here, we investigated metabolite alteration in the fatty-alcohol-producing system in the presence of XR/lactose within an early time frame (5 min after the bioconversion started). All metabolite data were analyzed using untargeted metabolomics. We found that the XR/lactose system could improve fatty-alcohol production as early as 5 min after the bioconversion started. The enhancement of key cofactors and intermediates, such as hexitol, NAD(P)H, ATP, 3-phosphoglycerate, acetyl-CoA, 6-phosphogluconate (6-PG) and glutathione, was consistent with those previously reported on a longer time scale (after 1 h). However, measurements performed at the early time reported here showed detectable differences in metabolite enhancement patterns, such as those of ATP, NADPH, acetyl-CoA and glutathione. These data could serve as a basis for future analysis of metabolic flux alteration by the XR/lactose system. Comparative analysis of the cofactor enhancement by XR and other methods suggests that XR/lactose can serve as a simple tool to increase levels of various cofactors for microbial cell factories.

All lactose-oxidizing enzymes of Pseudomonas taetrolens, a highly efficient lactobionic acid-producing microorganism, are pyrroloquinoline quinone-dependent enzymes.

In previous and present studies, four enzymes (GCD1, GCD3, GCD4, and MQO1) have been found to act as lactose-oxidizing enzymes of Pseudomonas taetrolens. To investigate whether the four enzymes were the only lactose-oxidizing enzymes of P. taetrolens, we performed the inactivation of gcd1, gcd3, gcd4, and mqo1 genes in P. taetrolens. Compared to the wild-type strain, the lactobionic acid (LBA)-producing ability of P. taetrolens ∆gcd1 ∆gcd3 ∆gcd4 ∆mqo1 was only slightly decreased, implying that P. taetrolens possesses more lactose-oxidizing enzymes. Interestingly, the four lactose-oxidizing enzymes were all pyrroloquinoline quinone (PQQ)-dependent. To identify other unidentified lactose-oxidizing enzymes of P. taetrolens, we prevented the synthesis of PQQ in P. taetrolens by inactivating the genes related to PQQ synthesis such as pqqC, pqqD, and pqqE. Surprisingly, all three knocked-out strains were unable to convert lactose to LBA, indicating that all lactose-oxidizing enzymes in P. taetrolens were inactivated by eliminating PQQ synthesis. In addition, external PQQ supplementation restored the LBA production ability of P. taetrolens ∆pqqC, comparable to the wild-type strain. These results indicate that all lactose-oxidizing enzymes in P. taetrolens are PQQ-dependent.

Optimizing microbioreactor cultivation strategies for Trichoderma reesei: from batch to fed-batch operations.

BACKGROUND: Filamentous fungi have long been recognized for their exceptional enzyme production capabilities. Among these, Trichoderma reesei has emerged as a key producer of various industrially relevant enzymes and is particularly known for the production of cellulases. Despite the availability of advanced gene editing techniques for T. reesei, the cultivation and characterization of resulting strain libraries remain challenging, necessitating well-defined and controlled conditions with higher throughput. Small-scale cultivation devices are popular for screening bacterial strain libraries. However, their current use for filamentous fungi is limited due to their complex morphology. RESULTS: This study addresses this research gap through the development of a batch cultivation protocol using a microbioreactor for cellulase-producing T. reesei strains (wild type, RutC30 and RutC30 TR3158) with offline cellulase activity analysis. Additionally, the feasibility of a microscale fed-batch cultivation workflow is explored, crucial for mimicking industrial cellulase production conditions. A batch cultivation protocol was developed and validated using the BioLector microbioreactor, a Round Well Plate, adapted medium and a shaking frequency of 1000 rpm. A strong correlation between scattered light intensity and cell dry weight underscores the reliability of this method in reflecting fungal biomass formation, even in the context of complex fungal morphology. Building on the batch results, a fed-batch strategy was established for T. reesei RutC30. Starting with a glucose concentration of 2.5 g l - 1 in the batch phase, we introduced a dual-purpose lactose feed to induce cellulase production and prevent carbon catabolite repression. Investigating lactose feeding rates from 0.3 to 0.75 g (l h) - 1 , the lowest rate of 0.3 g (l h) - 1 revealed a threefold increase in cellobiohydrolase and a fivefold increase in ß -glucosidase activity compared to batch processes using the same type and amount of carbon sources. CONCLUSION: We successfully established a robust microbioreactor batch cultivation protocol for T. reesei wild type, RutC30 and RutC30 TR3158, overcoming challenges associated with complex fungal morphologies. The study highlights the effectiveness of microbioreactor workflows in optimizing cellulase production with T. reesei, providing a valuable tool for simultaneous assessment of critical bioprocess parameters and facilitating efficient strain screening. The findings underscore the potential of microscale fed-batch strategies for enhancing enzyme production capabilities, revealing insights for future industrial applications in biotechnology.

Recombinant production of Paenibacillus wynnii ß-galactosidase with Komagataella phaffii.

BACKGROUND: The ß-galactosidase from Paenibacillus wynnii (ß-gal-Pw) is a promising candidate for lactose hydrolysis in milk and dairy products, as it has a higher affinity for the substrate lactose (low KM value) compared to industrially used ß-galactosidases and is not inhibited by the hydrolysis-generated product D-galactose. However, ß-gal-Pw must firstly be produced cost-effectively for any potential industrial application. Accordingly, the yeast Komagataella phaffii was chosen to investigate its feasibility to recombinantly produce ß-gal-Pw since it is approved for the regulated production of food enzymes. The aim of this study was to find the most suitable way to produce the ß-gal-Pw in K. phaffii either extracellularly or intracellularly. RESULTS: Firstly, 11 different signal peptides were tested for extracellular production of ß-gal-Pw by K. phaffii under the control of the constitutive GAP promoter. None of the signal peptides resulted in a secretion of ß-gal-Pw, indicating problems within the secretory pathway of this enzyme. Therefore, intracellular ß-gal-Pw production was investigated using the GAP or methanol-inducible AOX1 promoter. A four-fold higher volumetric ß-galactosidase activity of 7537 ± 66 µkatoNPGal/Lculture was achieved by the K. phaffii clone 27 using the AOX1 promoter in fed-batch bioreactor cultivations, compared to the clone 5 using the GAP promoter. However, a two-fold higher specific productivity of 3.14 ± 0.05 µkatoNPGal/gDCW/h was achieved when using the GAP promoter for ß-gal-Pw production compared to the AOX1 promoter. After partial purification, a ß-gal-Pw enzyme preparation with a total ß-galactosidase activity of 3082 ± 98 µkatoNPGal was obtained from 1 L of recombinant K. phaffii culture (using AOX1 promoter). CONCLUSION: This study showed that the ß-gal-Pw was produced intracellularly by K. phaffii, but the secretion was not achieved with the signal peptides chosen. Nevertheless, a straightforward approach to improve the intracellular ß-gal-Pw production with K. phaffii by using either the GAP or AOX1 promoter in bioreactor cultivations was demonstrated, offering insights into alternative production methods for this enzyme.