Enhanced production of ectoine from methane using metabolically engineered Methylomicrobium alcaliphilum 20Z

Background Ectoine (1,3,4,5-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) is an attractive compatible solute because of its wide industrial applications. Previous studies on the microbial production of ectoine have focused on sugar fermentation. Alternatively, methane can be used as an inexpensive and abundant resource for ectoine production by using the halophilic methanotroph, Methylomicrobium alcaliphilum 20Z. However, there are some limitations, including the low production of ectoine from methane and the limited tools for the genetic manipulation of methanotrophs to facilitate their use as industrial strains. Results We constructed M. alcaliphilum 20ZDP with a high conjugation efficiency and stability of the episomal plasmid by the removal of its native plasmid. To improve the ectoine production in M. alcaliphilum 20Z from methane, the ectD (encoding ectoine hydroxylase) and ectR (transcription repressor of the ectABC-ask operon) were deleted to reduce the formation of by-products (such as hydroxyectoine) and induce ectoine production. When the double mutant was batch cultured with methane, ectoine production was enhanced 1.6-fold compared to that obtained with M. alcaliphilum 20ZDP (45.58 mg/L vs. 27.26 mg/L) without growth inhibition. Notably, a maximum titer of 142.32 mg/L was reached by the use of an optimized medium for ectoine production containing 6% NaCl and 0.05 μM of tungsten without hydroxyectoine production. This result demonstrates the highest ectoine production from methane to date. Conclusions Ectoine production was significantly enhanced by the disruption of the ectD and ectR genes in M. alcaliphilum 20Z under optimized conditions favoring ectoine accumulation. We demonstrated effective genetic engineering in a methanotrophic bacterium, with enhanced production of ectoine from methane as the sole carbon source. This study suggests a potentially transformational path to commercial sugar-based ectoine production. Graphical Abstract

Engineering Escherichia coli for highly efficient production of lacto-N-triose II from N-acetylglucosamine, the monomer of chitin

Background Lacto-N-triose II (LNT II), an important backbone for the synthesis of different human milk oligosaccharides, such as lacto-N-neotetraose and lacto-N-tetraose, has recently received significant attention. The production of LNT II from renewable carbon sources has attracted worldwide attention from the perspective of sustainable development and green environmental protection. Results In this study, we first constructed an engineered E. coli cell factory for producing LNT II from N-acetylglucosamine (GlcNAc) feedstock, a monomer of chitin, by introducing heterologous β-1,3-acetylglucosaminyltransferase, resulting in a LNT II titer of 0.12 g L−1. Then, lacZ (lactose hydrolysis) and nanE (GlcNAc-6-P epimerization to ManNAc-6-P) were inactivated to further strengthen the synthesis of LNT II, and the titer of LNT II was increased to 0.41 g L−1. To increase the supply of UDP-GlcNAc, a precursor of LNT II, related pathway enzymes including GlcNAc-6-P deacetylase, glucosamine synthase, and UDP-N-acetylglucosamine pyrophosphorylase, were overexpressed in combination, optimized, and modulated. Finally, a maximum titer of 15.8 g L−1 of LNT II was obtained in a 3-L bioreactor with optimal enzyme expression levels and β-lactose and GlcNAc feeding strategy. Conclusions Metabolic engineering of E. coli is an effective strategy for LNT II production from GlcNAc feedstock. The titer of LNT II could be significantly increased by modulating the gene expression strength and blocking the bypass pathway, providing a new utilization for GlcNAc to produce high value-added products.

Seroresponse to SARS-CoV-2 vaccines among maintenance dialysis patients over six months

Background and Objectives: While most maintenance dialysis patients exhibit initial seroresponse to vaccination, concerns remain regarding the durability of this antibody response. This study evaluated immunity over time. Design, setting, participants, and measurements: This retrospective cohort study included maintenance dialysis patients from a midsize national dialysis provider who received a complete SARS-CoV-2 vaccine series and had at least one antibody titer checked after full vaccination. Immunoglobulin G spike antibodies (SAb-IgG) titers were assessed monthly with routine labs beginning after full vaccination and followed over time; the semiquantitative SAb-IgG titer reported a range between 0 and ≥20 U/L. Descriptive analyses compared trends over time by prior history of COVID-19 and type of vaccine received. Time-to-event analyses were conducted for the outcome of loss of seroresponse (SAb-IgG < 1 U/L or development of COVID-19). Cox proportional hazards regression was used to adjust for additional clinical characteristics of interest. Results: Among 1898 maintenance dialysis patients, 1567 (84%) had no prior history of COVID-19. Patients without a history of COVID-19 had declining titers over time. Among 441 BNT162b2/Pfizer recipients, median [IQR] SAb-IgG titer declined from 20 [5.99-20] U/L in month 1 to 1.30 [0.15-3.59] U/L by month 6. Among 779 mRNA-1273/Moderna recipients, median [IQR] SAb-IgG titer declined from 20 [20-20] in month 1 to 6.20 [1.74-20] by month 6. The 347 Ad26.COV2.S/Janssen recipients had a lower titer response than mRNA vaccine recipients over all time periods. In time-to-event analyses, Ad26.COV2.S/Janssen and mRNA-1273/Moderna recipients had the shortest and longest time to loss of seroresponse, respectively. The maximum titer reached in the first two months after full vaccination was predictive of the durability of the SAb-IgG seroresponse; patients with SAb-IgG titer 1-19.99 U/L were more likely to have loss of seroresponse compared to patients with SAb-IgG titer ≥20 U/L (HR 23.9 [95% CI: 16.1-35.5]). Conclusions: Vaccine-induced seroresponse wanes over time among maintenance dialysis patients across vaccine types. Early titers after full vaccination predict the durability of seroresponse.

Immunogenicity of SARS-CoV-2 Vaccine in Dialysis

Background: Patients receiving maintenance dialysis represent a high risk, immune-compromised population with 15-25% COVID-19 mortality rate who were unrepresented in clinical trials of mRNA vaccines. Method: All patients receiving maintenance dialysis who received two doses of SARS-CoV-2 mRNA vaccines with antibody test results drawn ≥14 days after the second dose, as documented in the electronic health record through March 18, 2021, were included. Response was based on levels of immunoglobulin-G against the receptor binding domain of the S1 subunit of SARS-CoV-2 spike-antigen (seropositive ≥2 U/L) using an FDA-approved semi-quantitative chemiluminescent assay (ADVIA Centaur® XP/XPT COV2G). Results: Among 186 dialysis patients from 30 clinics in 8 states tested 23±8 days after receiving 2 vaccine doses, there were 165 (88.7%) responders with 70% at maximum titer. There was no significant difference between BNT162b2/Pfizer (148/168, 88.1%) and mRNA-1273/Moderna (17/18, 94.4%), p=0.42. All 38 patients with COVID-19 history were responders, with 97% at maximum titer. Among patients without COVID-19, 127/148 (85.8%) were responders, comparable between BNT162b2/Pfizer (113/133) and mRNA-1273/Moderna (14/15) vaccines (85.0% vs. 93.3%, p=0.38). Conclusion: Most patients receiving maintenance dialysis responded after two doses of BNT162b2/Pfizer or mRNA-1273/Moderna vaccine, suggesting that the short-term development of anti-spike antibody is good, giving hope that most of these vulnerable patients, once immunized, will be protected from COVID-19. Longer-term evaluation is needed to determine antibody titer durability and if booster dose(s) are warranted. Further research to evaluate the approach to patients without a serologic response is needed, including benefits of additional dose(s) or administration of alternate options.

Immunogenicity of SARS-CoV-2 Vaccine in Dialysis

Importance: Patients receiving maintenance dialysis patients are at high risk for morbidity and mortality from COVID-19. The immunogenicity of SARS-CoV-2 mRNA vaccines is unknown in this vulnerable population where immune compromise is common. Objective: To determine seroresponse to vaccination against SARS-CoV-2 utilizing mRNA vaccines among patients receiving maintenance dialysis. Design: Retrospective observational study. Setting: Dialysis Clinic, Inc. (DCI) outpatient dialysis clinics in the United States. Participants: All patients receiving maintenance dialysis that received two doses of SARS-CoV-2 mRNA vaccines with SARS-CoV-2 spike-antibody test results drawn at least 14 days after the second dose, as documented in the electronic health record through March 18, 2021. Exposure: Two doses of BNT162b2/Pfizer or of mRNA-1273/Moderna vaccines administered per manufacturer recommendations. Main Outcomes and Measures: Levels of immunoglobulin-G against the receptor binding domain of the S1 subunit of SARS-CoV-2 spike antigen (seropositive: 2 or greater) using FDA-approved semi-quantitative chemiluminescent assay (ADVIA Centaur XP/XPT COV2G). The DCI clinical protocol for in-clinic administration included baseline and follow-up levels although initial administration of the vaccine occurred primarily elsewhere (e.g. long-term care facilities, hospitals, etc.) during the evaluation period. Hence, only post-vaccination antibody levels were reported. Results: Among 186 patients receiving maintenance dialysis from 32 clinics in 8 states tested an average of 23 days after receiving 2 vaccine doses, mean age was 68 years, with 47% women, 21% Black, 26% residents in long-term care facilities and 97% undergoing in-center hemodialysis. Overall seropositive rate was 165/186 (88.7%) with 70% at maximum titer and with no significant difference in seropositivity between BNT162b2/Pfizer (N=148) and mRNA-1273/Moderna (N=18) vaccines (88.1% vs. 94.4%, p=0.42). Among patients with COVID-19 history, seropositive rate was 38/38 (100%) with 97% at maximum titer. Conclusions and Relevance: Most patients receiving maintenance dialysis were seropositive after two doses of BNT162b2/Pfizer or mRNA-1273/Moderna vaccine. Early evidence suggests that vaccinated dialysis patients with prior COVID-19 develop robust antibody response. These results support an equitable and aggressive vaccination strategy for all eligible patients receiving maintenance dialysis, regardless of age, sex, race, ethnicity, or disability, to prevent the extremely high morbidity and mortality associated with COVID-19 in this high risk population.

Metabolic and Process Engineering for Microbial Production of Protocatechuate from Xylose with Corynebacterium glutamicum

3,4-Dihydroxybenzoate (protocatechuate, PCA) is a phenolic compound naturally found in edible vegetables and medicinal herbs. PCA is of interest in the chemical industry as a building block for novel polymers and has wide potential for pharmaceutical applications due to its antioxidant, anti-inflammatory, and antiviral properties. In the present study, we designed and constructed a novel Corynebacterium glutamicum strain to enable the efficient utilization of d-xylose for microbial production of PCA. The engineered strain showed a maximum PCA titer of 62.1 ± 12.1 mM (9.6 ± 1.9 g L−1) from d-xylose as the primary carbon and energy source. The corresponding yield was , which corresponds to 38 % of the maximum theoretical yield and is 14-fold higher compared to the parental producer strain on d-glucose. By establishing a one-pot bioreactor cultivation process followed by subsequent process optimization, the same maximum titer and a total amount of 16.5 ± 1.1 g was reached. Downstream processing of PCA from this fermentation broth was realized via electrochemically induced crystallization by taking advantage of the pH-dependent properties of PCA. Since PCA turned out to be electrochemically unstable in combination with several anode materials, a threechamber electrolysis setup was established to crystallize PCA and to avoid direct anode contact. This resulted in a maximum final purity of 95.4 %. In summary, the established PCA production process represents a highly sustainable approach, which will serve as a blueprint for the bio-based production of other hydroxybenzoic acids from alternative sugar feedstocks.

Heterologous Expression of the Core Genes in the Complex Fusarubin Gene Cluster of Fusarium Solani

Through stepwise recreation of the biosynthetic gene cluster containing PKS3 from Fusarium solani, it was possible to produce the core scaffold compound of bostrycoidin, a red aza-anthraquinone pigment in Saccharomyces cerevisiae. This was achieved through sequential transformation associated recombination (TAR) cloning of FvPPT, fsr1, fsr2, and fsr3 into the pESC-vector system, utilizing the inducible bidirectional galactose promoter for heterologous expression in S. cerevisiae. The production of the core metabolite bostrycoidin was investigated through triplicate growth cultures for 1–4 days, where the maximum titer of bostrycoidin was achieved after 2 days of induction, yielding 2.2 mg/L.

Analysis of the virus propagation profile of 14 dengue virus isolates in Aedes albopictus C6/36 cells

Objective The mosquito transmitted RNA virus dengue virus (DENV) shows significant variation as a consequence of the lack of proofreading activity of the RNA-dependent RNA polymerase that synthesizes new virus genomes. How this variation affects DENV replication, and how this in turn impacts drug development remains largely unknown. Given the technical limitations in working with large numbers of isolates few studies have sought to investigate this area. This study used a panel of 14 DENV isolates of different serotypes and origins to determine how much virus replication in Aedes albopictus C6/36 cells was affected by DENV variability. Results The results showed that there was considerable variation, with peak titers ranging from 6Log10 to 8Log10, and maximum titer being reached from day 3 to day 9 post infection. While strains from DENV 1 and 4 serotypes showed considerable uniformity, DENV 2 and 3 strains showed much greater variation. Overall, these results show that serotype specific strain variation can have a significant impact on DENV replication, suggesting that studies either investigating DENV pathogenesis or developing drug therapeutics should consider the contribution of DENV variability.

Polymorphism of clinical phenotypes and heterogeneity of autoimmune targets of myasthenia gravis

The aim of the study was to evaluate the presence of autoantibodies to different subunits of nAChR and nuclear antigens at different myasthenia phenotypes. The work has investigated the presence of antibodies to α1- and α7-subunits of nAChR, the repertoire and frequency of occurrence of antinuclear autoantibodies in different clinical phenotypes of myasthenia to understand the mechanisms of pathogenesis of various forms of the disease. Additional factors of autoimmunization were identified that affect certain mechanisms of pathogenesis in thymus-independent and thymus-dependent myasthenia gravis. Autoantibodies to α1 and α7 subunits of nAChR were detected in case of thymus-independent myasthenia gravis and thymus-dependent myasthenia gravis with thymus hyperplasia or locally spread thymoma, as well as the presence of antinuclear antibodies in case of thymoma on the background of myasthenia gravis. Autoantibodies to the α1 subunit of nAChR are available in all patients with thymus-independent and thymus-dependent myasthenia gravis; the maximum titer was in patients with myasthenia and thymus hyperplasia. The presence of autoimmune antibodies to another target – to the α7 subunit of nAChR was found in patients with thymus-independent myasthenia gravis and with myasthenia and thymus hyperplasia. Detected anti-nuclear antibodies (ANA) in patients with thymoma, are preferably antibodies to structures that are directly involved in mitotic cell division, that is to centromere, to centromeric protein F, to the centrosomal protein of achromatin spindle – NuMa and MSA-2 antigen mitotic spindle that affects the course of cell proliferation, reparative and regenerative processes in tissues. The selectivity of antibody damage by the subunit of NAHR in different myasthenia phenotypes and the presence of ANA in thymus-dependent myasthenia with thymoma has great diagnostic and prognostic value. The presence of specific autoantibodies to certain nuclear structures of the cell, along with other autoimmunization mechanisms, affects various metabolic mechanisms and can be used to select targeted therapy taking into account individual pathogenic targets of the autoimmune process.

Development and Optimization of the Biological Conversion of Ethane to Ethanol Using Whole-Cell Methanotrophs Possessing Methane Monooxygenase

The biological production of ethanol from ethane for the utilization of ethane in natural gas was investigated under ambient conditions using whole-cell methanotrophs possessing methane monooxygenase. Several independent variables including ethane concentration and biocatalyst amounts, among other factors, were optimized for the enhancement of ethane-to-ethanol bioconversion. We obtained 0.4 g/L/h of volumetric productivity and 0.52 g/L of maximum titer in optimum batch reaction conditions. In this study, we demonstrate that the biological gas-to-liquid conversion of ethane to ethanol has potent technical feasibility as a new application of ethane gas.