Modification of the endoplasmic reticulum morphology enables improved recombinant antibody expression in Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae is a versatile cell factory used for manufacturing of a wide range of products, among them recombinant proteins. Protein folding is one of the rate-limiting processes and this shortcoming is often overcome by the expression of folding catalysts and chaperones in the endoplasmic reticulum (ER). In this work, we aimed to establish the impact of ER structure on cellular productivity. The reticulon proteins Rtn1p and Rtn2p, and Yop1p are membrane curvature inducing proteins that define the morphology of the ER and depletion of these proteins creates yeast cells with a higher ER sheet-to-tubule ratio. We created yeast strains with different combinations of deletions of Rtn1p, Rtn2p, and Yop1p coding genes in cells with a normal or expanded ER lumen. We identified strains that reached up to 2.2-fold higher antibody titres compared to the control strain. The expanded ER membrane reached by deletion of the lipid biosynthesis repressor OPI1 was essential for the increased productivity. The improved specific productivity was accompanied by an up to 2-fold enlarged ER surface area and a 1.5-fold increased cross-sectional cell area. Furthermore, the strains with enlarged ER displayed an attenuated unfolded protein response. These results underline the impact that ER structures have on productivity and support the notion that reprogramming subcellular structures belongs into the toolbox of synthetic biology.

Immunotherapy utilization in stage IIIA melanoma: less may be more.

Background: Immunotherapy agents are approved for adjuvant treatment of stage III melanoma; however, evidence for survival benefit in early stage III disease is lacking. Current guidelines for adjuvant immunotherapy utilization in stage IIIA rely on clinician judgment, creating an opportunity for significant variation in prescribing patterns. This study aimed to characterize current immunotherapy practice variations and to compare patient outcomes for different prescribing practices in stage IIIA melanoma. Study design: Patients with melanoma diagnosed from 2015-2019 that met American Joint Committee on Cancer 8th edition criteria for stage IIIA and underwent resection were identified in the National Cancer Database. Multiple imputation by chained equations replaced missing values. Factors associated with receipt of adjuvant immunotherapy were identified. Multivariable Cox proportional hazards regression compared overall survival across groups. Results: Of 4,432 patients included in the study, 34% received adjuvant immunotherapy. Patients had lower risk-adjusted odds of receiving immunotherapy if they were treated at an academic center (OR=0.48, 95%CI=0.33-0.72, p<0.001 vs. community facility) or at a high-volume center (OR=0.69, 0.56-0.84, p<0.001 vs. low-volume). Immunotherapy receipt was not associated with risk-adjusted survival (p=0.095). Moreover, patients treated at high-volume centers experienced longer overall risk-adjusted survival than those treated at low-volume centers (HR=0.52, 0.29-0.93, p=0.030). Risk-adjusted survival trended toward being longer at academic centers than at community centers, but the difference was not statistically significant. Conclusion: Academic and high-volume centers utilize significantly less adjuvant immunotherapy in stage IIIA melanoma than community and low-volume centers without compromise in overall survival. These findings suggest that this population may benefit from more judicious immunotherapy utilization.

Immunotherapy Initiation at the End of Life in Patients With Metastatic Cancer in the US.

Importance: While immunotherapy is being used in an expanding range of clinical scenarios, the incidence of immunotherapy initiation at the end of life (EOL) is unknown. Objective: To describe patient characteristics, practice patterns, and risk factors concerning EOL-initiated (EOL-I) immunotherapy over time. Design, Setting, and Participants: Retrospective cohort study using a US national clinical database of patients with metastatic melanoma, non-small cell lung cancer (NSCLC), or kidney cell carcinoma (KCC) diagnosed after US Food and Drug Administration approval of immune checkpoint inhibitors for the treatment of each disease through December 2019. Mean follow-up was 13.7 months. Data analysis was performed from December 2022 to May 2023. Exposures: Age, sex, race and ethnicity, insurance, location, facility type, hospital volume, Charlson-Deyo Comorbidity Index, and location of metastases. Main Outcomes and Measures: Main outcomes were EOL-I immunotherapy, defined as immunotherapy initiated within 1 month of death, and characteristics of the cohort receiving EOL-I immunotherapy and factors associated with its use. Results: Overall, data for 242 371 patients were analyzed. The study included 20 415 patients with stage IV melanoma, 197 331 patients with stage IV NSCLC, and 24 625 patients with stage IV KCC. Mean (SD) age was 67.9 (11.4) years, 42.5% were older than 70 years, 56.0% were male, and 29.3% received immunotherapy. The percentage of patients who received EOL-I immunotherapy increased over time for all cancers. More than 1 in 14 immunotherapy treatments in 2019 were initiated within 1 month of death. Risk-adjusted patients with 3 or more organs involved in metastatic disease were 3.8-fold more likely (95% CI, 3.1-4.7; P < .001) to die within 1 month of immunotherapy initiation than those with lymph node involvement only. Treatment at an academic or high-volume center rather than a nonacademic or very low-volume center was associated with a 31% (odds ratio, 0.69; 95% CI, 0.65-0.74; P < .001) and 30% (odds ratio, 0.70; 95% CI, 0.65-0.76; P < .001) decrease in odds of death within a month of initiating immunotherapy, respectively. Conclusions and Relevance: Findings of this cohort study show that the initiation of immunotherapy at the EOL is increasing over time. Patients with higher metastatic burden and who were treated at nonacademic or low-volume facilities had higher odds of receiving EOL-I immunotherapy. Tracking EOL-I immunotherapy can offer insights into national prescribing patterns and serve as a harbinger for shifts in the clinical approach to patients with advanced cancer.

Causal identification of single-cell experimental perturbation effects with CINEMA-OT.

Recent advancements in single-cell technologies allow characterization of experimental perturbations at single-cell resolution. While methods have been developed to analyze such experiments, the application of a strict causal framework has not yet been explored for the inference of treatment effects at the single-cell level. Here we present a causal-inference-based approach to single-cell perturbation analysis, termed CINEMA-OT (causal independent effect module attribution + optimal transport). CINEMA-OT separates confounding sources of variation from perturbation effects to obtain an optimal transport matching that reflects counterfactual cell pairs. These cell pairs represent causal perturbation responses permitting a number of novel analyses, such as individual treatment-effect analysis, response clustering, attribution analysis, and synergy analysis. We benchmark CINEMA-OT on an array of treatment-effect estimation tasks for several simulated and real datasets and show that it outperforms other single-cell perturbation analysis methods. Finally, we perform CINEMA-OT analysis of two newly generated datasets: (1) rhinovirus and cigarette-smoke-exposed airway organoids, and (2) combinatorial cytokine stimulation of immune cells. In these experiments, CINEMA-OT reveals potential mechanisms by which cigarette-smoke exposure dulls the airway antiviral response, as well as the logic that governs chemokine secretion and peripheral immune cell recruitment.

Surgical treatment of stage IV gastroenteropancreatic neuroendocrine carcinoma: Experience and outcomes in the United States.

BACKGROUND AND OBJECTIVES: Surgery for metastatic gastroenteropancreatic neuroendocrine carcinoma (GEP-NEC) has not been well-studied. This retrospective cohort study describes patients in the United States with stage IV GEP-NEC and their survival outcomes segregated by surgery. METHODS: Patients diagnosed with stage IV GEP-NEC from 2004 to 2017 in the National Cancer Database were categorized into three groups: no surgery, primary site or metastatic site ("single-site") surgery, and primary site and metastatic site ("multisite") surgery. Factors associated with surgical treatment were identified, and risk-adjusted overall survival of each group was compared. RESULTS: Of 4171 patients included, 958 (23.0%) underwent single-site surgery and 374 (9.0%) underwent multisite surgery. The strongest predictor of surgery was primary tumor type. Compared with no surgery, the risk-adjusted mortality reduction associated with single-site surgery ranged from 63% for small bowel (HR = 0.37, 0.23-0.58, p < 0.001) NEC to 30% for colon and appendix NEC (HR = 0.70, 0.61-0.80, p < 0.001), while the mortality reduction associated with multisite surgery ranged from 77% for pancreas NEC (HR = 0.23, 0.17-0.33, p < 0.001) to 48% for colon and appendix NEC (HR = 0.52, 0.44-0.63, p < 0.001). CONCLUSIONS: We observed an association between extent of surgical intervention and overall survival for patients with stage IV GEP-NEC. Surgical resection should be further investigated as a treatment option for highly-selected patients with this aggressive disease.

Hepatopancreatobiliary malignancies: time to treatment matters.

Background: Initiation of oncologic care is often delayed, yet little is known about delays in hepatopancreatobiliary (HPB) cancers or their impact. This retrospective cohort study describes trends in time to treatment initiation (TTI), assesses the association between TTI and survival, and identifies predictors of TTI in HPB cancers. Methods: The National Cancer Database was queried for patients with cancers of the pancreas, liver, and bile ducts between 2004 and 2017. Kaplan-Meier survival analysis and Cox regression were used to investigate the association between TTI and overall survival for each cancer type and stage. Multivariable regression identified factors associated with longer TTI. Results: Of 318,931 patients with HPB cancers, median TTI was 31 days. Longer TTI was associated with increased mortality in patients with stages I-III extrahepatic bile duct (EHBD) cancer and stages I-II pancreatic adenocarcinoma. Patients treated within 3-30, 31-60, and 61-90 days had median survivals of 51.5, 34.9, and 25.4 months (log-rank P<0.001), respectively, for stage I EHBD cancer, and 18.8, 16.6, and 15.2 months for stage I pancreatic cancer, respectively (P<0.001). Factors associated with increased TTI included stage I disease (+13.7 days vs. stage IV, P<0.001), treatment with radiation only (ß=+13.9 days, P<0.001), Black race (+4.6 days, P<0.001) and Hispanic ethnicity (+4.3 days, P<0.001). Conclusions: Some HPB cancer patients with longer time to definitive care experienced higher mortality than patients treated expeditiously, particularly in non-metastatic EHBD cancer. Black and Hispanic patients are at risk for delayed treatment. Further research into these associations is needed.

The Impact of Glycoengineering on the Endoplasmic Reticulum Quality Control System in Yeasts.

Yeasts are widely used and established production hosts for biopharmaceuticals. Despite of tremendous advances on creating human-type N-glycosylation, N-glycosylated biopharmaceuticals manufactured with yeasts are missing on the market. The N-linked glycans fulfill several purposes. They are essential for the properties of the final protein product for example modulating half-lives or interactions with cellular components. Still, while the protein is being formed in the endoplasmic reticulum, specific glycan intermediates play crucial roles in the folding of or disposal of proteins which failed to fold. Despite of this intricate interplay between glycan intermediates and the cellular machinery, many of the glycoengineering approaches are based on modifications of the N-glycan processing steps in the endoplasmic reticulum (ER). These N-glycans deviate from the canonical structures required for interactions with the lectins of the ER quality control system. In this review we provide a concise overview on the N-glycan biosynthesis, glycan-dependent protein folding and quality control systems and the wide array glycoengineering approaches. Furthermore, we discuss how the current glycoengineering approaches partially or fully by-pass glycan-dependent protein folding mechanisms or create structures that mimic the glycan epitope required for ER associated protein degradation.

Similarity Index for the Fat Fraction between Breast Milk and Infant Formulas.

The similarity of the fat fraction in infant formulas rich in either bovine milk fat (MF) or vegetable oil (VO) to breast milk was evaluated by analyzing their lipid composition. Milk fat-rich formulas were highly similar (average similarity index 0.68) to breast milk compared to the VO-rich formulas (average similarity index 0.56). The highest difference in the indices was found in the contents of cholesterol (0.66 vs 0.28 in MF- and VO-rich formulas, respectively, on average) and polar lipids (0.84 vs 0.53), the positional distribution of fatty acids in the sn-2 position of triacylglycerols (0.53 vs 0.28), and fatty acid composition (0.72 vs 0.54). The VO-based formulas were superior in similarity in n - 6 PUFA. Thus, the addition of bovine MF fractions is an effective way to increase the similarity between the lipid composition of infant formulas and human milk.

Cumulus cell acetyl-CoA metabolism from acetate is associated with maternal age but only partially with oocyte maturity.

Cumulus cell (CC) clumps that associate with oocytes provide the oocytes with growth and signaling factors. Thus, the metabolism of the CCs may influence oocyte function, and CC metabolism may be predictive of oocyte competence for in vitro fertilization. CCs are thought to be highly glycolytic, but data on the use of other potential carbon substrates are lacking in humans. This prospective and blinded cohort study was designed to examine the substrate utilization of CCs by age and oocyte competence. Individual sets of CC clumps from participants were removed after oocyte retrieval procedure then, incubated with stable isotope labeled substrates, and analyzed using liquid chromatography-high resolution mass spectrometry (LC-HRMS) for isotopologue enrichment of major metabolic intermediates, including acetyl-CoA. The acyl-chain of acetyl-CoA contains 2 carbons that can be derived from 13C-labeled substrates resulting in an M + 2 isotopologue that contains 2 13C atoms. Comparing the fate of three major carbon sources, mean enrichment of M + 2 acetyl-CoA (mean, standard deviation) was for glucose (3.6, 7.7), for glutamine (9.4, 6.2), and for acetate (20.7, 13.9). Due to this unexpected high and variable labeling from acetate, we then examined acetyl-CoA mean % enrichment from acetate in 278 CCs from 21 women ≤34 (49.06, 12.73) decreased with age compared to 124 CCs from 10 women >34 (43.48, 16.20) (p = 0.0004, t-test). The CCs associated with the immature prophase I oocytes had significantly lower enrichment in M + 2 acetyl CoA compared to the CCs associated with the metaphase I and metaphase II oocytes (difference: -6.02, CI: -1.74,-13.79, p = 0.013). Acetate metabolism in individual CC clumps was positively correlated with oocyte maturity and decreased with maternal age. These findings indicate that CC metabolism of non-glucose substrates should be investigated relative to oocyte function and age-related fertility.Abbreviations: CCs: cumulus cells; COC: cumulus-oocyte complex; LC-MS: liquid chromatography-mass spectrometry; acetyl-CoA: acetyl-Coenzyme A; CoA: Coenzyme A.

Production of galactosylated complex-type N-glycans in glycoengineered Saccharomyces cerevisiae.

N-glycosylation is an important posttranslational modification affecting the properties and quality of therapeutic proteins. Glycoengineering in yeast aims to produce proteins carrying human-compatible glycosylation, enabling the production of therapeutic proteins in yeasts. In this work, we demonstrate further development and characterization of a glycoengineering strategy in a Saccharomyces cerevisiae Δalg3 Δalg11 strain where a truncated Man3GlcNAc2 glycan precursor is formed due to a disrupted lipid-linked oligosaccharide synthesis pathway. We produced galactosylated complex-type and hybrid-like N-glycans by expressing a human galactosyltransferase fusion protein both with and without a UDP-glucose 4-epimerase domain from Schizosaccharomyces pombe. Our results showed that the presence of the UDP-glucose 4-epimerase domain was beneficial for the production of digalactosylated complex-type glycans also when extracellular galactose was supplied, suggesting that the positive impact of the UDP-glucose 4-epimerase domain on the galactosylation process can be linked to other processes than its catalytic activity. Moreover, optimization of the expression of human GlcNAc transferases I and II and supplementation of glucosamine in the growth medium increased the formation of galactosylated complex-type glycans. Additionally, we provide further characterization of the interfering mannosylation taking place in the glycoengineered yeast strain. KEY POINTS: • Glycoengineered Saccharomyces cerevisiae can form galactosylated N-glycans. • Genetic constructs impact the activities of the expressed glycosyltransferases. • Growth medium supplementation increases formation of target N-glycan structure.

Early Postoperative Basal Insulin Therapy versus Standard of Care for the Prevention of Diabetes Mellitus after Kidney Transplantation: A Multicenter Randomized Trial.

BACKGROUND: Post-transplantation diabetes mellitus (PTDM) might be preventable. METHODS: This open-label, multicenter randomized trial compared 133 kidney transplant recipients given intermediate-acting insulin isophane for postoperative afternoon glucose ≥140 mg/dl with 130 patients given short-acting insulin for fasting glucose ≥200 mg/dl (control). The primary end point was PTDM (antidiabetic treatment or oral glucose tolerance test-derived 2 hour glucose ≥200 mg/dl) at month 12 post-transplant. RESULTS: In the intention-to-treat population, PTDM rates at 12 months were 12.2% and 14.7% in treatment versus control groups, respectively (odds ratio [OR], 0.82; 95% confidence interval [95% CI], 0.39 to 1.76) and 13.4% versus 17.4%, respectively, at 24 months (OR, 0.71; 95% CI, 0.34 to 1.49). In the per-protocol population, treatment resulted in reduced odds for PTDM at 12 months (OR, 0.40; 95% CI, 0.16 to 1.01) and 24 months (OR, 0.54; 95% CI, 0.24 to 1.20). After adjustment for polycystic kidney disease, per-protocol ORs for PTDM (treatment versus controls) were 0.21 (95% CI, 0.07 to 0.62) at 12 months and 0.35 (95% CI, 0.14 to 0.87) at 24 months. Significantly more hypoglycemic events (mostly asymptomatic or mildly symptomatic) occurred in the treatment group versus the control group. Within the treatment group, nonadherence to the insulin initiation protocol was associated with significantly higher odds for PTDM at months 12 and 24. CONCLUSIONS: At low overt PTDM incidence, the primary end point in the intention-to-treat population did not differ significantly between treatment and control groups. In the per-protocol analysis, early basal insulin therapy resulted in significantly higher hypoglycemia rates but reduced odds for overt PTDM-a significant reduction after adjustment for baseline differences-suggesting the intervention merits further study.Clinical Trial registration number: NCT03507829.

Production of Glucose 6-Phosphate From a Cellulosic Feedstock in a One Pot Multi-Enzyme Synthesis.

Glucose 6-phosphate is the phosphorylated form of glucose and is used as a reagent in enzymatic assays. Current production occurs via a multi-step chemical synthesis. In this study we established a fully enzymatic route for the synthesis of glucose 6-phosphate from cellulose. As the enzymatic phosphorylation requires ATP as phosphoryl donor, the use of a cofactor regeneration system is required. We evaluated Escherichia coli glucokinase and Saccharomyces cerevisiae hexokinase (HK) for the phosphorylation reaction and Pseudomonas aeruginosa polyphosphate kinase 2 (PPK2) for ATP regeneration. All three enzymes were characterized in terms of temperature and pH optimum and the effects of substrates and products concentrations on enzymatic activities. After optimization of the conditions, we achieved a 85% conversion of glucose into glucose 6-phosphate using the HK/PPK2 activities within a 24 h reaction resulting in 12.56 g/l of glucose 6-phosphate. Finally, we demonstrated the glucose 6-phosphate formation from microcrystalline cellulose in a one-pot reaction comprising Aspergillus niger cellulase for glucose release and HK/PPK2 activities. We achieved a 77% conversion of released glucose into glucose 6-phosphate, however at the expense of a lower glucose 6-phosphate yield of 1.17 g/l. Overall, our study shows an alternative approach for synthesis of glucose 6-phosphate that can be used to valorize biomass derived cellulose.

Meconium androgens are correlated with ASD-related phenotypic traits in early childhood in a familial enriched risk cohort.

BACKGROUND: Prenatal exposure to increased androgens has been suggested as a risk factor for autism spectrum disorder (ASD). This hypothesis has been examined by measurement of steroids in amniotic fluid, cord blood, saliva, and blood with mixed results. METHODS: To provide an orthogonal measure of fetal exposure, this study used meconium, the first stool of a newborn, to measure prenatal androgen exposure from infants in the Early Autism Risk Longitudinal Investigation (EARLI). EARLI is a familial-enriched risk cohort that enrolled pregnant mothers who already had a child with an ASD diagnosis. In the younger child, we investigated the association between meconium unconjugated (u) and total (t) concentrations of major androgens testosterone (T), dehydroepiandrosterone (DHEA), and androstenedione (A4), and ASD-related traits at 12 and 36 months of age. Traits were measured at 12 months with Autism Observation Scale for Infants (AOSI) and at 36 months with total score on the Social Responsiveness Scale (SRS). One hundred and seventy children had meconium and AOSI, 140 had meconium and SRS, and 137 had meconium and both AOSI and SRS. RESULTS: Separate robust linear regressions between each of the log-transformed androgens and log-transformed SRS scores revealed three-way interaction between sex of the child, sex of the proband, and testosterone concentration. In the adjusted analyses, t-T, u-A4, and u-DHEA (P ≤ 0.01) were positively associated with AOSI scores, while u-T (P = 0.004) and u-DHEA (P = 0.007) were positively associated with SRS total score among females with female probands (n = 10). Additionally, higher concentrations of u-T (P = 0.01) and t-T (P = 0.01) predicted higher SRS total score in males with male probands (n = 63). Limitations Since we explored three-way interactions, this resulted in a limited sample size for some analyses. This study was from an enriched-risk cohort which may limit generalizability, and this study used ASD-assessment scales as outcomes instead of diagnostic categories. Additionally, the novel use of meconium in this study limits the ability to compare the results in this cohort to others due to the paucity of research on meconium. CONCLUSIONS: This study supports the utility of meconium for studies of endogenous fetal metabolism and suggests the sex of older siblings with autism should be considered as a biological variable in relevant studies.

Mining Data From Plasma Cell Differentiation Identified Novel Genes for Engineering of a Yeast Antibody Factory.

Saccharomyces cerevisiae is a common platform for production of therapeutic proteins, but it is not intrinsically suited for the manufacturing of antibodies. Antibodies are naturally produced by plasma cells (PCs) and studies conducted on PC differentiation provide a comprehensive blueprint for the cellular transformations needed to create an antibody factory. In this study we mined transcriptomics data from PC differentiation to improve antibody secretion by S. cerevisiae. Through data exploration, we identified several new target genes. We tested the effects of 14 genetic modifications belonging to different cellular processes on protein production. Four of the tested genes resulted in improved antibody expression. The ER stress sensor IRE1 increased the final titer by 1.8-fold and smaller effects were observed with PSA1, GOT1, and HUT1 increasing antibody titers by 1. 6-, 1. 4-, and 1.4-fold. When testing combinations of these genes, the highest increases were observed when co-expressing IRE1 with PSA1, or IRE1 with PSA1 and HUT1, resulting in 3.8- and 3.1-fold higher antibody titers. In contrast, strains expressing IRE1 alone or in combination with the other genes produced similar or lower levels of recombinantly expressed endogenous yeast acid phosphatase compared to the controls. Using a genetic UPR responsive GFP reporter construct, we show that IRE1 acts through constitutive activation of the unfolded protein response. Moreover, the positive effect of IRE1 expression was transferable to other antibody molecules. We demonstrate how data exploration from an evolutionary distant, but highly specialized cell type can pinpoint new genetic targets and provide a novel concept for rationalized cell engineering.

Investigating the role of ERAD on antibody processing in glycoengineered Saccharomyces cerevisiae.

N-glycosylation plays an important role in the endoplasmic reticulum quality control (ERQC). N-glycan biosynthesis pathways have been engineered in yeasts and fungi to enable the production of therapeutic glycoproteins with human-compatible N-glycosylation, and some glycoengineering approaches alter the synthesis of the lipid-linked oligosaccharide (LLO). Because the effects of LLO engineering on ERQC are currently unknown, we characterized intracellular processing of IgG in glycoengineered Δalg3 Δalg11 Saccharomyces cerevisiae strain and analyzed how altered LLO structures affect endoplasmic reticulum-associated degradation (ERAD). Intracellular IgG light and heavy chain molecules expressed in Δalg3 Δalg11 strain are ERAD substrates and targeted to ERAD independently of Yos9p and Htm1p, whereas in the presence of ALG3 ERAD targeting is dependent on Yos9p but does not require Htm1p. Blocking of ERAD accumulated ER and post-Golgi forms of IgG and increased glycosylation of matα secretion signal but did not improve IgG secretion. Our results show ERAD targeting of a heterologous glycoprotein in yeast, and suggest that proteins in the ER can be targeted to ERAD via other mechanisms than the Htm1p-Yos9p-dependent route when the LLO biosynthesis is altered.

Production and characterization of Aspergillus niger GH29 family α-fucosidase and production of a novel non-reducing 1-fucosyllactose.

Fucosylated oligosaccharides are interesting molecules due to their bioactive properties. In particular, their application as active ingredient in milk powders is attractive for dairy industries. The objective of this study was to characterize the glycosyl hydrolase family 29 α-fucosidase produced by Aspergillus niger and test its ability to transfucosylate lactose with a view towards potential industrial applications such as the valorization of the lactose side stream produced by dairy industry. In order to reduce costs and toxicity the use of free fucose instead of environmentally questionable fucose derivatives was studied. In contrast to earlier studies, a recombinantly produced A. niger α-fucosidase was utilized. Using pNP-fucose as substrate, the optimal pH for hydrolytic activity was determined to be 3.8. The optimal temperature for a 30-min reaction was 60 °C, and considering temperature stability, the optimal temperature for a 24-h reaction was defined as 45 °C For the same hydrolysis reaction, the kinetic values were calculated to be 0.385 mM for the KM and 2.8 mmol/(mg*h) for the Vmax. Transfucosylation of lactose occurred at high substrate concentrations when reaction time was elongated to several days. The structure of the product trisaccharide was defined as 1-fucosyllactose, where fucose is α-linked to the anomeric carbon of the ß-glucose moiety of lactose. Furthermore, the enzyme was able to hydrolyze its own transfucosylation product and 2'-fucosyllactose but only poorly 3-fucosyllactose. As a conclusion, α-fucosidase from A. niger can transfucosylate lactose using free fucose as substrate producing a novel non-reducing 1-fucosyllactose.

Tailoring N-Glycan Biosynthesis for Production of Therapeutic Proteins in Saccharomyces cerevisiae.

The ability to control and adjust the N-glycosylation pathway of Saccharomyces cerevisiae is a key step toward production of therapeutic glycoproteins such as antibodies or erythropoietin. The focus of this chapter is to describe the road from yeast-type N-glycosylation to human-type complex N-glycosylation. The chapter describes the cell engineering and provides the detailed analytical procedures required to perform glycan analysis using MALDI-TOF mass spectrometry.

Enzymatic synthesis of fucose-containing galacto-oligosaccharides using ß-galactosidase and identification of novel disaccharide structures.

Fucosylated oligosaccharides have an important role in maintaining a healthy immune system and homeostatic gut microflora. This study employed a commercial ß-galactosidase in the production of fucose-containing galacto-oligosaccharides (fGOS) from lactose and fucose. The production was optimized using experiment design and optimal conditions for a batch production in 3-liter scale. The reaction product was analyzed and the produced galactose-fucose disaccharides were purified. The structures of these disaccharides were determined using NMR and it was verified that one major product with the structure Galß1-3Fuc and two minor products with the structures Galß1-4Fuc and Galß1-2Fuc were formed. Additionally, the product composition was defined in more detail using several different analytical methods. It was concluded that the final product contained 42% total monosaccharides, 40% disaccharides and 18% of larger oligosaccharides. 290 µmol of fGOS was produced per gram of reaction mixture and 37% of the added fucose was bound to fGOS. The fraction of fGOS from total oligosaccharides was determined as 44%. This fGOS product could be used as a new putative route to deliver fucose to the intestine.

Understanding the metabolic burden of recombinant antibody production in Saccharomyces cerevisiae using a quantitative metabolomics approach.

The cellular changes induced by heterologous protein expression in the yeast Saccharomyces cerevisiae have been analysed on many levels and found to be significant. However, even though high-level protein production poses a metabolic burden, evaluation of the expression host at the level of the metabolome has often been neglected. We present a comparison of metabolite profiles of a wild-type strain with those of three strains producing recombinant antibody variants of increasing size and complexity: an scFv fragment, an scFv-Fc fusion protein and a full-length IgG molecule. Under producing conditions, all three recombinant strains showed a clear decrease in growth rate compared with the wild-type strain and the severity of the growth phenotype increased with size of the protein. The levels of 76 intracellular metabolites were determined using a targeted (semi) quantitative mass spectrometry based approach. Based on unsupervised and supervised multivariate analysis of metabolite profiles, together with pathway activity profiling, the recombinant strains were found to be significantly different from each other and from the wild-type strain. We observed the most prominent changes in metabolite levels for metabolites involved in amino acid and redox metabolism. Induction of the unfolded protein response was detected in all producing strains and is considered to be a contributing factor to the overall metabolic burden on the cells.

Relationship of SULT1A1 copy number variation with estrogen metabolism and human health.

Human cytosolic sulfotransferase 1A1 (SULT1A1) is considered to be one of the most important SULT isoforms for metabolism, detoxification, and carcinogenesis. This theory is driven by observations that SULT1A1 is widely expressed in multiple tissues and acts on a wide range of phenolic substrates. SULT1A1 is subject to functional common copy number variation (CNV) including deletions or duplications. However, it is less clear how SULT1A1 CNV impacts health and disease. To better understand the biological role of SULT1A1 in human health, we genotyped CNV in 14,275 Marshfield Clinic patients linked to an extensive electronic health record. Since SULT1A1 is linked to steroid metabolism, select serum steroid hormones were measured in 100 individuals with a wide spectrum of SULT1A1 CNV genotypes. Furthermore, comprehensive phenome-wide association studies (PheWAS) were conducted using diagnostic codes and clinical text data. For the first time, individuals homozygous null for SULT1A1 were identified in a human population. Thirty-six percent of the population carried >2 copies of SULT1A1 whereas 4% had ≤1 copy. Results indicate SULT1A1 CNV was negatively correlated with estrone-sulfate to estrone ratio predominantly in males (E1S/E1; p=0.03, r=-0.21) and may be associated with increased risk for common allergies. The effect of SULT1A1 CNV on circulating estrogen metabolites was opposite to the predicted CNV-metabolite trend based on enzymatic function. This finding, and the potential association with common allergies reported herein, warrants future studies.