Real-world effectiveness and predictors of super-responders to dupilumab in a Chinese uncontrolled asthma cohort.

Background: Dupilumab has been shown to be effective in clinical trials for moderate-to-severe uncontrolled asthma. However, the efficacy of dupilumab in the real world and the prediction of treatment response have not been well studied in patients with asthma. Objective: To investigate the efficacy of dupilumab and explore predictors of super-responders in a Chinese retrospective cohort. Methods: From January 2021 through December 2022, the patients with uncontrolled asthma who were treated with dupilumab for 4 months were included. Symptom control, type 2 inflammatory biomarkers, and lung function were collected at baseline and follow-up for efficacy assessment. Super-responders were defined as exacerbation-free, off maintenance of oral corticosteroids (mOCS), and with a score of the five-item Asthma Control Questionnaire (ACQ-5) of <0.5. The uni- and multivariable logistic regressions were used to construct predictive models for super-responders based on baseline features. Results: A total of 53 patients were included. After 4 months treatment, the median (interquartile range [IQR]) ACQ-5 score decreased from 1.8 (1.6-2.4) to 0.4 (0.2-0.8) (p < 0.001), the median (IQR) number of exacerbations, from 0.0 (0.0-1.0) to 0.0 (0.0-0.0) (p = 0.005). The median (IQR) dose of mOCS (prednisone equivalent) decreased from 15.0 mg/day (8.8-22.5 mg/day) to 2.5 mg/day (0.0-10.0 mg/day) (p = 0.008) in nine patients who were receiving mOCS. All efficacy assessment parameters, including sputum eosinophil were significantly improved, while blood eosinophil count did not decline (530 cells/mm³ [300-815 cells/mm³] versus 560 cells/mm³ [220-938 cells/mm³], p = 0.710). After taking dupilumab, 25 of 53 patients (47.2%) achieved a super-response. The age of onset < 42 years (odds ratio [OR] 7.471 [95% confidence interval {CI}, 1.286-43.394) and the baseline fractional exhaled nitric oxide (FeNO) of 25-50 ppb (OR 35.038 [95% CI, 3.104-395.553]) predicted super-responders, which showed a C-index of 0.822 (95% CI, 0.697-0.947). Conclusion: Dupilumab significantly improved symptom control, type 2 inflammatory markers, and lung function in Chinese patients with uncontrolled asthma. Airway eosinophils, rather than blood eosinophils, can be a reliable indicator of therapeutic efficacy. The early-onset asthma as well as the medium-high level of baseline FeNO contributed to the prediction of super-responders.

Progress in research on the biosynthesis of 1,2,4-butanetriol by engineered microbes.

1,2,4-butanetriol (BT) is a polyol with unique chemical properties, which has a stereocenter and can be divided into D-BT (the S-enantiomer) and L-BT (the R-enantiomer). BT can be used for the synthesis of 1,2,4-butanetriol trinitrate, 3-hydroxytetrahydrofuran, polyurethane, and other chemicals. It is widely used in the military industry, medicine, tobacco, polymer. At present, the BT is mainly synthesized by chemical methods, which are accompanied by harsh reaction conditions, poor selectivity, many by-products, and environmental pollution. Therefore, BT biosynthesis methods with the advantages of mild reaction conditions and green sustainability have become a current research hotspot. In this paper, the research status of microbial synthesis of BT was summarized from the following three aspects: (1) the biosynthetic pathway establishment for BT from xylose; (2) metabolic engineering strategies employed for improving BT production from xylose; (3) other substrates for BT production. Finally, the challenges and prospects of biosynthetic BT were discussed for future methods to improve competitiveness for industrial production.

Production and waste treatment of polyesters: application of bioresources and biotechniques.

Chemical resources and techniques have long been used in the history of bulk polyester production and still dominate today's chemical industry. The sustainable development of the polyester industry demands more renewable resources and environmentally benign polyester products. Accordingly, the rapid development of biotechnology has enabled the production of an extensive range of aliphatic and aromatic polyesters from renewable bio-feedstocks. This review addresses the production of representative commercial polyesters (polyhydroxyalkanoates, polylactic acid, poly ε-caprolactone, polybutylene succinate, polyethylene terephthalate, polybutylene terephthalate, polypropylene terephthalate, polyethylene furandicarboxylate, polypropylene furandicarboxylate, and polybutylene furandicarboxylate) or their monomers (lactic acid, succinic acid, 1,4-butanediol, ethylene glycol, terephthalic acid, 1,3-propanediol, and 2,5-furandicarboxylic acid) from renewable bioresources. In addition, this review summarizes advanced biotechniques in the treatment of polyester wastes, representing the near-term trends and future opportunities for waste-to-value recycling and the remediation of polyester wastes under sustainable models. For future prospects, it is essential to further expand: non-food bioresources, optimize bioprocesses and biotechniques in the preparation of bioderived or biodegradable polyesters with promising: material performance, biodegradability, and low production cost.

A fast and robust iterative genome-editing method based on a Rock-Paper-Scissors strategy.

The production of optimized strains of a specific phenotype requires the construction and testing of a large number of genome modifications and combinations thereof. Most bacterial iterative genome-editing methods include essential steps to eliminate selection markers, or to cure plasmids. Additionally, the presence of escapers leads to time-consuming separate single clone picking and subsequent cultivation steps. Herein, we report a genome-editing method based on a Rock-Paper-Scissors (RPS) strategy. Each of three constructed sgRNA plasmids can cure, or be cured by, the other two plasmids in the system; plasmids from a previous round of editing can be cured while the current round of editing takes place. Due to the enhanced curing efficiency and embedded double check mechanism, separate steps for plasmid curing or confirmation are not necessary, and only two times of cultivation are needed per genome-editing round. This method was successfully demonstrated in Escherichia coli and Klebsiella pneumoniae with both gene deletions and replacements. To the best of our knowledge, this is the fastest and most robust iterative genome-editing method, with the least times of cultivation decreasing the possibilities of spontaneous genome mutations.

Facile Fabrication of Energetic Nanocomposite Materials by Polydopamine.

Polydopamine-based materials have been widely investigated for incorporation in energetic nanocomposites due to their outstanding adherence. However, these materials are often prepared in alkaline environments, which negatively affects Al nanoparticles. In this study, a one-pot assembly was devised for the preparation of a polydopamine-based Al/CuO energetic nanocomposite material (Al/PDA/CuO) in a neutral environment. The CuO and Al nanoparticles of the Al/PDA/CuO nanothermite were uniformly dispersed and closely combined. Consequently, the Al/PDA/CuO nanothermite was able to release more heat (2069.7 J/g) than physically mixed Al/CuO (1438.9 J/g). Furthermore, the universality of using polydopamine in the assembly of different types of energetic nanocomposite materials was verified, including an organic energetic material-nanothermit (HMX/PDA/Al/CuO nanothermite) and an inorganic oxidant-metal nanocatalyst (AP/PDA/Fe2O3). This study provides a promising route for the preparation of polydopamine-based energetic nanocomposites in neutral aqueous solutions.

Engineering Cytochrome P450BM3 Enzymes for Direct Nitration of Unsaturated Hydrocarbons.

Applications of the peroxidase activity of cytochrome P450 enzymes in synthetic chemistry remain largely unexplored. We present herein a protein engineering strategy to increase cytochrome P450BM3 peroxidase activity for the direct nitration of aromatic compounds and terminal aryl-substituted olefins in the presence of a dual-functional small molecule (DFSM). Site-directed mutations of key active-site residues allowed the efficient regulation of steric effects to limit substrate access and, thus, a significant decrease in monooxygenation activity and increase in peroxidase activity. Nitration of several phenol and aniline compounds also yielded ortho- and para-nitration products with moderate-to-high total turnover numbers. Besides direct aromatic nitration by P450 variants using nitrite as a nitrating agent, we also demonstrated the use of the DFSM-facilitated P450 peroxidase system for the nitration of the vinyl group of styrene and its derivatives.

Identification and mechanistic analysis of a bifunctional enzyme involved in the C-N and N-N bond formation.

N-N bonded compounds are a relatively small but valuable group of natural products with a variety of important biological activities, including anti-inflammatory, anti-tumor, anti-bacterial and anti-oxidant activities. Currently, the synthesis of natural products containing N-N bonds is mainly based on chemical synthesis, but the chemical synthesis of N-N bonds may have safety issues and raw material sustainability problems. A variety of N-N bond biosynthetic mechanisms exist in nature, including comproportionation, rearrangement, and radical recombination reactions, which provide ideas for the biosynthesis of many N-N bond compounds. But at this stage, only a few N-N bond synthases have been reported, and difficulties exist in the biosynthesis of N-N bond-containing compounds. The new N-N-bond synthase PAI2, which was found to catalyze the synthesis of piperazinic acid, expands the family of N-N-bond synthases and provides better catalytic activity and different enzymatic properties for the substrate L-N5-hydroxyornithine than the known enzyme KtzT derived from the piperazinic acid biosynthetic gene cluster of Kutzneria sp. 744. Moreover, PAI2 was found to have the ability to catalyze the formation of C-N bonds in Pro.

Suppression function against environmental dust exposure after Dermatophagoides pteronyssinus immunotherapy is associated with production of specific and cross-reactive immunoglobulin G4.

BACKGROUND: Whether Dermatophagoides pteronyssinus (Der-p) allergen immunotherapy (AIT) can induce Dermatophagoides farina (Der-f)-specific immunoglobulin (sIg) G4 production, and tolerance to environmental allergens has not been fully investigated. OBJECTIVE: We aimed to determine serum Der-p-sIgG4 and Der-f-sIgG4 levels in asthma and/or rhinitis patients undergoing Der-p AIT and their ability to reduce immune responses triggered by indoor dust extracts. METHODS: We performed a real-world prospective trial and enrolled patients with allergic rhinitis and/or asthma in Guangzhou, China. These patients received either Der-p AIT (SCIT group) or routine medications (non-SCIT group) for 156 weeks. Clinical outcomes were assessed by the combined symptom medication score (SMS) and FEV1 % changes. House dust samples were collected to analyse allergen levels. Serum levels of Der-p-sIgG4 and Der-f-sIgG4, serum inhibitory capacity against Der-p, Der-f and indoor dust extract by sIgE-facilitated allergen binding to B cells (IgE-FAB), and serum blocking indoor dust extract-induced basophil activation inhibition assays (BATI) in peripheral blood monocytes were carried out at weeks 0, 4, 12, 16, 52, 104 and 156 after the initiations of the treatments. RESULTS: Our study enrolled a total of 60 participants, with 30 patients in each group. Patients in the SCIT group had significantly improved SMS when compared with the baseline and the patients in the non-SCIT group. Median levels of Der-p 1 and Der-f 1 in indoor dust extract were 1.86 µg/g and 4.74 µg/g, respectively. Serum Der-p-sIgG4 and Der-f-IgG4 levels in SCIT patients showed a significant increase from weeks 12 to 156. Serum in these SCIT patients could significantly block Der-p, Der-f and indoor dust extract formation of allergen-sIgE complex and reduced the threshold of IgE-FAB from 16 weeks after the initiation of the treatment. The capacity to inhibit Der-p, Der-f and indoor dust extract BATI was observed in SCIT serum after 12 weeks. Der-p-sIgG4 and Der-f-sIgG4 had a significant correlation with IgE-FAB and BATI in SCIT patients at all time points. CONCLUSION: Single Der-p immunotherapy induced both Der-p-sIgG4 and Der-f-sIgG4 production, which might cross-reactively induce tolerance against environmental allergen exposure in patients with asthma and/or rhinitis.

Allergic disease and sensitization disparity in urban and rural China: A EuroPrevall-INCO study.

BACKGROUND: Studies in comparison with allergic diseases and sensitization between rural and urban environments in westernized countries might be biased and not adequately reflect countries undergoing rapid transition. METHODS: A total of 5542 schoolchildren from urban area and 5139 from rural area were recruited for the EuroPrevall-INCO survey. A subsequent case-control sample with 196 children from urban area and 202 from rural area was recruited for a detailed face-to-face questionnaire and assessment of sensitization. Skin prick tests and serum-specific IgE measurements were used to assess sensitizations against food and aeroallergens. Logistic regression analysis was used to determine associations between risk/protective factors, food adverse reactions (FAR), allergic diseases, and sensitizations. RESULTS: Prevalence of self-reported allergic diseases, including asthma (6.6% vs.2.5%), rhinitis (23.2% vs.5.3%), and eczema (34.1% vs.25.9%), was higher in urban than in rural children. Urban children had a significantly higher prevalence of FAR and related allergic diseases, and lower food/inhalation allergen sensitization rate, than those of rural children. In urban children, frequent changing places of residency (odds ratio 2.85, 95% confidence interval: 1.45-5.81) and antibiotic usage (3.54, 1.77-7.32) in early life were risk factors for sensitization, while sensitization and family history of allergy were risk factors for allergic diseases. In rural children, exposure to rural environments in early life was protective against both allergen sensitizations (0.46, 0.21-0.96) and allergic diseases (0.03, 0.002-0.19). CONCLUSION: We observed a disparity in rates of allergic diseases and allergen sensitization between rural and urban children. In addition to family history, the development of allergic diseases and allergen sensitization were associated with specific urban/rural environmental exposures in early life.

Debottlenecking the biological hydrogen production pathway of dark fermentation: insight into the impact of strain improvement.

Confronted with the exhaustion of the earth's fossil fuel reservoirs, bio-based process to produce renewable energy is receiving significant interest. Hydrogen is considered as an attractive energy carrier that can replace fossil fuels in the future mainly due to its high energy content, recyclability and environment-friendly nature. Biological hydrogen production from renewable biomass or waste materials by dark fermentation is a promising alternative to conventional routes since it is energy-saving and reduces environmental pollution. However, the current yield and evolution rate of fermentative hydrogen production are still low. Strain improvement of the microorganisms employed for hydrogen production is required to make the process competitive with traditional production methods. The present review summarizes recent progresses on the screening for highly efficient hydrogen-producing strains using various strategies. As the metabolic pathways for fermentative hydrogen production have been largely resolved, it is now possible to engineer the hydrogen-producing strains by rational design. The hydrogen yields and production rates by different genetically modified microorganisms are discussed. The key limitations and challenges faced in present studies are also proposed. We hope that this review can provide useful information for scientists in the field of fermentative hydrogen production.

Blocking function of allergen-specific immunoglobulin G, F(ab')2, and Fab antibodies prepared from patients undergoing Dermatophagoides pteronyssinus immunotherapy.

BACKGROUND: The blocking function of allergen-specific F(ab')2 [sF(ab')2] and Fab (sFab) fragment antibodies prepared from allergen immunotherapy-induced specific immunoglobulin G (sIgG) has not been fully investigated. OBJECTIVE: To investigate the inhibitory function of sIgG, sF(ab')2, and sFab antibodies in patients undergoing Dermatophagoides pteronyssinus (Der-p) subcutaneous immunotherapy (SCIT). METHODS: This study involved 10 subjects (aged 18-42 years) with house dust mite allergic rhinitis or asthma who received a 156-week course of Der-p SCIT. Total IgG levels were purified from the serum of the participants at weeks 0 and 156 by protein A affinity chromatography. Der-p sIgG was purified by affinity chromatography with Der-p as a ligand at week 156. The sF(ab')2 and sFab antibodies were prepared from Der-p sIgG by treatment with pepsin and papain, respectively. Furthermore, IgE-facilitated allergen binding assay, basophil activation inhibition test, and cytokine release inhibition assay were used to assess the inhibitory function of Der-p sIgG, sF(ab')2, and sFab antibodies. RESULTS: We found that the Der-p sIgG, sF(ab')2, and sFab antibodies markedly blocked Der-p-allergen sIgE complex binding to B cells, inhibited basophil activation, and markedly reduced the production of interleukin (IL)-5, IL-13, IL-17, and tumor necrosis factor-α by peripheral blood mononuclear cells. CONCLUSION: SCIT-induced Der-p sIgG, sF(ab')2, and sFab antibodies may block the formation of Der-p-sIgE complexes and may serve as a potential allergen-targeted biologics candidate for the treatment of allergic asthma. CLINICAL TRIAL REGISTRATION: This study was approved by the Ethics Committee of the First Affiliated Hospital of Guangzhou Medical University and registered in the Chinese Clinical Trial Registry (ChiCTR-OOC-15006207, https://www.chictr.org.cn/enindex.aspx).

Peptide Assembly of Al/CuO Nanothermite for Enhanced Reactivity of Nanoaluminum Particles.

Biological self-assembly procedures, which are generally carried out in an aqueous solution, have been found to be the most promising method for directing the fabrication of diverse nanothermites, including Al/CuO nanothermite. However, the aqueous environment in which Al nanoparticles self-assemble has an impact on their stability. We show that using a peptide to self-assemble Al or CuO nanoparticles considerably improves their durability in phosphate buffer aqueous solution, with Al and CuO nanoparticles remaining intact in aqueous solution for over 2 weeks with minimal changes in the structure. When peptide-assembled Al/CuO nanothermite was compared with a physically mixed sample in phosphate buffer for 30 min, the energy release of the former was higher by 26%. Furthermore, the energy release of peptide-assembled Al/CuO nanocomposite in phosphate buffer showed a 6% reduction by Day 7, while that of the peptide-assembled Al/CuO nanocomposite in ultrapure water was reduced by 75%. Taken together, our study provides an easy method for keeping the thermal activity of Al/CuO nanothermite assembled in aqueous solution.

Distribution of ε-Poly-l-Lysine Synthetases in Coryneform Bacteria Isolated from Cheese and Human Skin.

ε-Poly-l-lysine is a potent antimicrobial produced through fermentation of Streptomyces and used in many Asian countries as a food preservative. It is synthesized and excreted by a special nonribosomal peptide synthetase (NRPS)-like enzyme called Pls. In this study, we discovered a gene from cheese bacterium Corynebacterium variabile that showed high similarity to the Pls from Streptomyces in terms of domain architecture and gene context. By cloning it into Streptomyces coelicolor with a Streptomyces albulus Pls promoter, we confirmed that its product is indeed ε-poly-l-lysine. A comprehensive sequence analysis suggested that Pls genes are widely spread among coryneform actinobacteria isolated from cheese and human skin; 14 out of 15 Brevibacterium isolates and 10 out of 12 Corynebacterium isolates contain it in their genomes. This finding raises the possibility that ε-poly-l-lysine as a bioactive secondary metabolite might be produced and play a role in the cheese and skin ecosystems.IMPORTANCE Every year, microbial contamination causes billions of tons of food wasted and millions of cases of illness. ε-Poly-l-lysine has potent, wide-spectrum inhibitory activity and is heat stable and biodegradable. It has been approved for food preservation by an increasing number of countries. ε-Poly-l-lysine is produced from soil bacteria of the genus Streptomyces, also producers of various antibiotic drugs and toxins and not considered to be a naturally occurring food component. The frequent finding of pls in cheese and skin bacteria suggests that ε-poly-l-lysine may naturally exist in cheese and on our skin, and ε-poly-l-lysine producers are not limited to filamentous actinobacteria.

Biosynthesis and metabolic engineering of 1-hydroxyphenazine in Pseudomonas chlororaphis H18.

BACKGROUND: 1-Hydroxyphenazine (1-OH-PHZ) is a phenazine microbial metabolite with broad-spectrum antibacterial activities against a lot of plant pathogens. However, its use is hampered by the low yield all along. Metabolic engineering of microorganisms is an increasingly powerful method for the production of valuable organisms at high levels. Pseudomonas chlororaphis is recognized as a safe and effective plant rhizosphere growth-promoting bacterium, and faster growth rate using glycerol or glucose as a renewable carbon source. Therefore, Pseudomonas chlororaphis is particularly suitable as the chassis cell for the modification and engineering of phenazines. RESULTS: In this study, enzyme PhzS (monooxygenase) was heterologously expressed in a phenazine-1-carboxylic acid (PCA) generating strain Pseudomonas chlororaphis H18, and 1-hydroxyphenazine was isolated, characterized in the genetically modified strain. Next, the yield of 1-hydroxyphenazine was systematically engineered by the strategies including (1) semi-rational design remodeling of crucial protein PhzS, (2) blocking intermediate PCA consumption branch pathway, (3) enhancing the precursor pool, (4) engineering regulatory genes, etc. Finally, the titer of 1-hydroxyphenazine reached 3.6 g/L in 5 L fermenter in 54 h. CONCLUSIONS: The 1-OH-PHZ production of Pseudomonas chlororaphis H18 was greatly improved through systematically engineering strategies, which is the highest, reported to date. This work provides a promising platform for 1-hydroxyphenazine engineering and production.

Whole-cell catalysis by surface display of fluorinase on Escherichia coli using N-terminal domain of ice nucleation protein.

BACKGROUND: Fluorinases play a unique role in the production of fluorine-containing organic molecules by biological methods. Whole-cell catalysis is a better choice in the large-scale fermentation processes, and over 60% of industrial biocatalysis uses this method. However, the in vivo catalytic efficiency of fluorinases is stuck with the mass transfer of the substrates. RESULTS: A gene sequence encoding a protein with fluorinase function was fused to the N-terminal of ice nucleation protein, and the fused fluorinase was expressed in Escherichia coli BL21(DE3) cells. SDS-PAGE and immunofluorescence microscopy were used to demonstrate the surface localization of the fusion protein. The fluorinase displayed on the surface showed good stability while retaining the catalytic activity. The engineered E.coli with surface-displayed fluorinase could be cultured to obtain a larger cell density, which was beneficial for industrial application. And 55% yield of 5'-fluorodeoxyadenosine (5'-FDA) from S-adenosyl-L-methionine (SAM) was achieved by using the whole-cell catalyst. CONCLUSIONS: Here, we created the fluorinase-containing surface display system on E.coli cells for the first time. The fluorinase was successfully displayed on the surface of E.coli and maintained its catalytic activity. The surface display provides a new solution for the industrial application of biological fluorination.

Study on the isoprene-producing co-culture system of Synechococcus elongates-Escherichia coli through omics analysis.

BACKGROUND: The majority of microbial fermentations are currently performed in the batch or fed-batch manner with the high process complexity and huge water consumption. The continuous microbial production can contribute to the green sustainable development of the fermentation industry. The co-culture systems of photo-autotrophic and heterotrophic species can play important roles in establishing the continuous fermentation mode for the bio-based chemicals production. RESULTS: In the present paper, the co-culture system of Synechococcus elongates-Escherichia coli was established and put into operation stably for isoprene production. Compared with the axenic culture, the fermentation period of time was extended from 100 to 400 h in the co-culture and the isoprene production was increased to eightfold. For in depth understanding this novel system, the differential omics profiles were analyzed. The responses of BL21(DE3) to S. elongatus PCC 7942 were triggered by the oxidative pressure through the Fenton reaction and all these changes were linked with one another at different spatial and temporal scales. The oxidative stress mitigation pathways might contribute to the long-lasting fermentation process. The performance of this co-culture system can be further improved according to the fundamental rules discovered by the omics analysis. CONCLUSIONS: The isoprene-producing co-culture system of S. elongates-E. coli was established and then analyzed by the omics methods. This study on the co-culture system of the model S. elongates-E. coli is of significance to reveal the common interactions between photo-autotrophic and heterotrophic species without natural symbiotic relation, which could provide the scientific basis for rational design of microbial community.

Hop bitter acids: resources, biosynthesis, and applications.

Diversified members of hop bitter acids (α- and ß-acids) have been found in hop (Humulus lupulus). Mixtures of hop bitter acids have been traditionally applied in brewing and food industries as bitterness flavors or food additives. Recent studies have discovered novel applications of hop bitter acids and their derivatives in medicinal and pharmaceutical fields. The increasing demands of purified hop bitter acid promoted biosynthesis efforts for the heterologous biosynthesis of objective hop bitter acids by engineered microbial factories. In this study, the updated information of hop bitter acids and their representative application in brewing, food, and medicine fields are reviewed. We also speculate future trends on the development of robust microbial cell factories and biotechnologies for the biosynthesis of hop bitter acids. KEY POINTS: • Structures and applications of hop bitter acids are summarized in this study. • Biosynthesis of hop bitter acids remains challenging. • We discuss potential strategies in the microbial production of hop bitter acids.

Recent advances of metabolic engineering strategies in natural isoprenoid production using cell factories.

Covering: up to 2019As abundant natural products, isoprenoids have many useful industrial applications in the manufacturing of drugs, fragrances, food additives, colorants, rubber and advanced biofuels. The microbial production of isoprenoids has received much attention in recent years. Metabolic engineering approaches and synthetic biology have been utilized to reconstruct and optimize the metabolic pathways for isoprenoid production in cell factories. In this review, the recent advances in isoprenoid production using microbes are summarized, with a focus on MEP and MVA pathway engineering, downstream isoprenoid pathway engineering and microbial host engineering, which mainly includes central carbon pathway engineering. Finally, future perspectives for the improvement of isoprenoid production are discussed.

Characterization and directed evolution of propionyl-CoA carboxylase and its application in succinate biosynthetic pathway with two CO2 fixation reactions.

Propionyl-CoA carboxylase (PCC) is a promising enzyme in the fields of biological CO2 utilization, synthesis of natrual products, and so on. The activity and substrate specificity of PCC are dependent on its key subunit carboxyltransferase (CT). To obtain PCC with high enzyme activity, seven pccB genes encoding CT subunit from diverse microorganisms were expressed in recombinant E. coli, and PccB from Bacillus subtilis showed the highest activity in vitro. To further optimize this protein using directed evolution, a genetic screening system based on oxaloacetate availability was designed to enrich the active variants of PccBBs. Four amino acid substitutions (D46G, L97Q, N220I and I391T) proved of great assistance in PccBBs activity improvement, and a double mutant of PccBBs (N220I/I391T) showed a 94-fold increase of overall catalytic efficiency indicated by kcat/Km. Moreover, this PccBBs double mutant was applied in construction of new succinate biosynthetic pathway. This new pathway produces succinate from acetyl-CoA with fixation of two CO2 molecules, which was confirmed by isotope labeling experiment with NaH13CO3. Compared with previous succinate production based on carboxylation of phosphoenolpyruvate or pyruvate, this new pathway showed some advantages including higher CO2 fixation potentiality and availability under aerobic conditions. In summary, this study developed a PCC with high enzyme activity which can be widely used in biotechnology field, and also demonstrated the feasibility of new succinate biosynthetic pathway with two CO2 fixation reactions.

Changes in CD4+CD25+FoxP3+ Regulatory T Cells and Serum Cytokines in Sublingual and Subcutaneous Immunotherapy in Allergic Rhinitis with or without Asthma.

BACKGROUND: Few studies have directly compared the immunologic responses to specific subcutaneous immunotherapy (SCIT) and sublingual immunotherapy (SLIT). OBJECTIVE: We aimed to directly compare clinical efficacy and immunological responses between SLIT and SCIT in allergic rhinitis (AR) sensitized to house dust mites. METHODS: Sixty-seven patients (age 5-55 years) with moderate-severe Dermatophagoides pteronyssinus (Der-p) and Dermatophagoides farinae AR with or without asthma were randomized (2:2:1) into SLIT (n = 27), SCIT (n = 26) and placebo (n = 14) groups. Symptom and medication scores, visual analogue score, serum Der-p specific immunoglobulin G4 (Der-p-sIgG4), CD4+CD25+FoxP3+ regulatory T cells (Tregs) and serum cytokines were measured. RESULTS: After 1-year treatment, a significant improvement of total rhinitis score (TRS), total rhinitis medication score (TRMS) and visual analogue score occurred in both SLIT and SCIT. There were no differences in clinical efficacy except for TRMS (p = 0.026) when SLIT and SCIT were directly compared. CD4+CD25+FoxP3+ Tregs had a trend towards upregulation in the 2 modes and inversely correlated with TRS (p = 0.024) only in SLIT. Der-p-sIgG4 significantly increased in SLIT and SCIT (p < 0.05), and it was 30 times higher in SCIT than SLIT after the treatment (p < 0.05). Serum interferon-γ significantly increased only in SCIT after 1 (p = 0.008), 6 (p = 0.007) and 12 (p = 0.008) months of treatment and inversely correlated with TRS (p = 0.032). CONCLUSION: While SCIT and SLIT have similar rates of clinical improvement, the 2 modes reveal heterogeneous changes of CD4+CD25+Foxp3+ Tregs, sIgG4 and cytokines.