Xylanase enhances gut microbiota-derived butyrate to exert immune-protective effects in a histone deacetylase-dependent manner.

BACKGROUND: Commensal bacteria in the intestine release enzymes to degrade and ferment dietary components, producing beneficial metabolites. However, the regulatory effects of microbial-derived enzymes on the intestinal microbiota composition and the influence on host health remain elusive. Xylanase can degrade xylan into oligosaccharides, showing wide application in feed industry. RESULTS: To validate the immune-protective effects of xylanase, Nile tilapia was used as the model and fed with xylanase. The results showed that dietary xylanase improved the survival rate of Nile tilapia when they were challenged with Aeromonas hydrophila. The transcriptome analysis showed significant enrichment of genes related to interleukin-17d (il-17d) signaling pathway in the xylanase treatment group. High-throughput sequencing revealed that dietary xylanase altered the composition of the intestinal microbiota and directly promoted the proliferation of Allobaculum stercoricanis which could produce butyrate in vitro. Consequently, dietary xylanase supplementation increased the butyrate level in fish gut. Further experiment verified that butyrate supplementation enhanced the expression of il-17d and regenerating islet-derived 3 gamma (reg3g) in the gut. The knockdown experiment of il-17d confirmed that il-17d is necessary for butyrate to protect Nile tilapia from pathogen resistance. Flow cytometry analysis indicated that butyrate increased the abundance of IL-17D+ intestinal epithelial cells in fish. Mechanistically, butyrate functions as an HDAC3 inhibitor, enhancing il-17d expression and playing a crucial role in pathogen resistance. CONCLUSION: Dietary xylanase significantly altered the composition of intestinal microbiota and increased the content of butyrate in the intestine. Butyrate activated the transcription of il-17d in intestinal epithelial cells by inhibiting histone deacetylase 3, thereby protecting the Nile tilapia from pathogen infection. This study elucidated how microbial-derived xylanase regulates host immune function, providing a theoretical basis for the development and application of functional enzymes. Video Abstract.

Engineering a non-oxidative glycolysis pathway in escherichia coli for high-level citramalate production.

BACKGROUND: Methyl methacrylate (MMA) is a key precursor of polymethyl methacrylate, extensively used as a transparent thermoplastic in various industries. Conventional MMA production poses health and environmental risks; hence, citramalate serves as an alternative bacterial compound precursor for MMA production. The highest citramalate titer was previously achieved by Escherichia coli BW25113. However, studies on further improving citramalate production through metabolic engineering are limited, and phage contamination is a persistent problem in E. coli fermentation. RESULTS: This study aimed to construct a phage-resistant E. coli BW25113 strain capable of producing high citramalate titers from glucose. First, promoters and heterologous cimA genes were screened, and an effective biosynthetic pathway for citramalate was established by overexpressing MjcimA3.7, a mutated cimA gene from Methanococcus jannaschii, regulated by the BBa_J23100 promoter in E. coli. Subsequently, a phage-resistant E. coli strain was engineered by integrating the Ssp defense system into the genome and mutating key components of the phage infection cycle. Then, the strain was engineered to include the non-oxidative glycolysis pathway while removing the acetate synthesis pathway to enhance the supply of acetyl-CoA. Furthermore, glucose utilization by the strain improved, thereby increasing citramalate production. Ultimately, 110.2 g/L of citramalate was obtained after 80 h fed-batch fermentation. The citramalate yield from glucose and productivity were 0.4 g/g glucose and 1.4 g/(L·h), respectively. CONCLUSION: This is the highest reported citramalate titer and productivity in E. coli without the addition of expensive yeast extract and additional induction in fed-bath fermentation, emphasizing its potential for practical applications in producing citramalate and its derivatives.

Sub-genomic RNAi-assisted strain evolution of filamentous fungi for enhanced protein production.

Genetic engineering at the genomic scale provides a rapid means to evolve microbes for desirable traits. However, in many filamentous fungi, such trials are daunted by low transformation efficiency. Differentially expressed genes under certain conditions may contain important regulatory factors. Accordingly, although manipulating these subsets of genes only can largely reduce the time and labor, engineering at such a sub-genomic level may also be able to improve the microbial performance. Herein, first using the industrially important cellulase-producing filamentous fungus Trichoderma reesei as a model organism, we constructed suppression subtractive hybridization (SSH) libraries enriched with differentially expressed genes under cellulase induction (MM-Avicel) and cellulase repression conditions (MM-Glucose). The libraries, in combination with RNA interference, enabled sub-genomic engineering of T. reesei for enhanced cellulase production. The ability of T. reesei to produce endoglucanase was improved by 2.8~3.3-fold. In addition, novel regulatory genes (tre49304, tre120391, and tre123541) were identified to affect cellulase expression in T. reesei. Iterative manipulation using the same strategy further increased the yield of endoglucanase activity to 75.6 U/mL, which was seven times as high as that of the wild type (10.8 U/mL). Moreover, using Humicola insolens as an example, such a sub-genomic RNAi-assisted strain evolution proved to be also useful in other industrially important filamentous fungi. H. insolens is a filamentous fungus commonly used to produce catalase, albeit with similarly low transformation efficiency and scarce knowledge underlying the regulation of catalase expression. By combining SSH and RNAi, a strain of H. insolens producing 28,500 ± 288 U/mL of catalase was obtained, which was 1.9 times as high as that of the parent strain.IMPORTANCEGenetic engineering at the genomic scale provides an unparalleled advantage in microbial strain improvement, which has previously been limited only to the organisms with high transformation efficiency such as Saccharomyces cerevisiae and Escherichia coli. Herein, using the filamentous fungus Trichoderma reesei as a model organism, we demonstrated that the advantage of suppression subtractive hybridization (SSH) to enrich differentially expressed genes and the convenience of RNA interference to manipulate a multitude of genes could be combined to overcome the inadequate transformation efficiency. With this sub-genomic evolution strategy, T. reesei could be iteratively engineered for higher cellulase production. Intriguingly, Humicola insolens, a fungus with even little knowledge in gene expression regulation, was also improved for catalase production. The same strategy may also be expanded to engineering other microorganisms for enhanced production of proteins, organic acids, and secondary metabolites.

Correlation between intestinal flora and GLP-1 receptor agonist dulaglutide in type 2 diabetes mellitus treatment-A preliminary longitudinal study.

GLP-1 receptor agonists (GLP-1 RA) are presently used as the first-line drugs for the clinical treatment of type 2 diabetes mellitus (T2DM). It can regulate blood glucose by stimulating insulin secretion and lowering glucagon levels. We used 16S rRNA amplicon sequencing to detect structural changes in the composition of the intestinal flora of newly diagnosed T2DM after 1 and 48 weeks of dulaglutide administration. Our research found no significant changes in the intestinal flora after the administration of dulaglutide for 1 week to subjects with newly diagnosed T2DM. Nevertheless, after 48 weeks of dulaglutide administration, the composition of the intestinal flora changed significantly, with a significant reduction in the abundance of intestinal flora. Furthermore, we found that fasting glucose levels, fasting c-peptide levels, HbA1c levels, and BMI are also closely associated with intestinal flora. This reveals that intestinal flora may be one of the mechanisms by which dulaglutide treats T2DM.

Recombinant production of SAG1 fused with xylanase in Pichia pastoris induced higher protective immunity against Eimeria tenella infection in chicken.

Chicken coccidiosis is an intestinal disease caused by the parasite Eimeria, which severely damages the growth of chickens and causes significant economic losses in the poultry industry. Improvement of the immune protective effect of antigens to develop high efficiency subunit vaccines is one of the hotspots in coccidiosis research. Sporozoite-specific surface antigen 1 (SAG1) of Eimeria tenella (E. tenella) is a well-known protective antigen and is one of the main target antigens for the development of subunit, DNA and vector vaccines. However, the production and immunoprotective effects of SAG1 need to be further improved. Here, we report that both SAG1 from E. tenella and its fusion protein with the xylanase XynCDBFV-SAG1 are recombinant expressed and produced in Pichia pastoris (P. pastoris). The substantial expression quantity of fusion protein XynCDBFV-SAG1 is achieved through fermentation in a 15-L bioreactor, reaching up to about 2 g/L. Moreover, chickens immunized with the fusion protein induced higher protective immunity as evidenced by a significant reduction in the shedding of oocysts after E. tenella challenge infection compared with immunized with recombinant SAG1. Our results indicate that the xylanase enhances the immunogenicity of subunit antigens and has the potential for developing novel molecular adjuvants. The high expression level of fusion protein XynCDBFV-SAG1 in P. pastoris holds promise for the development of effective recombinant anti-coccidial subunit vaccine.

Exploiting Systematic Engineering of the Expression Cassette as a Powerful Tool to Enhance Heterologous Gene Expression in Trichoderma reesei.

Many endeavors in expressing a heterologous gene in microbial hosts rely on simply placing the gene of interest between a selected pair of promoters and terminator. However, although the expression efficiency could be improved by engineering the host cell, how modifying the expression cassette itself systematically would affect heterologous gene expression remains largely unknown. As the promoter and terminator bear plentiful cis-elements, herein using the Aspergillus niger mannanase with high application value in animal feeds and the eukaryotic filamentous fungus workhorse Trichoderma reesei as a model gene/host, systematic engineering of an expression cassette was investigated to decipher the effect of its mutagenesis on heterologous gene expression. Modifying the promoter, signal peptide, the eukaryotic-specific Kozak sequence, and the 3'-UTR could stepwise improve extracellular mannanase production from 17 U/mL to an ultimate 471 U/mL, representing a 27.7-fold increase in expression. The strategies can be generally applied in improving the production of heterologous proteins in eukaryotic microbial hosts.

Targeting deoxynivalenol for degradation by a chimeric manganese peroxidase/glutathione system.

The manganese peroxidase (MnP) can degrade multiple mycotoxins including deoxynivalenol (DON) efficiently; however, the lignin components abundant in foods and feeds were discovered to interfere with DON catalysis. Herein, using MnP from Ceriporiopsis subvermispora (CsMnP) as a model, it was demonstrated that desired catalysis of DON, but not futile reactions with lignin, in the reaction systems containing feeds could be achieved by engineering MnP and supplementing with a boosting reactant. Specifically, two successive strategies (including the fusion of CsMnP to a DON-recognizing ScFv and identification of glutathione as a specific targeting enhancer) were combined to overcome the lignin competition, which together resulted into elevation of the degradation rate from 2.5% to as high as 82.7% in the feeds. The method to construct a targeting MnP and fortify it with an additional enhancer could be similarly applied to catalyze the many other mycotoxins with yet unknown responsive biocatalysts.

Theoretical insights into the mechanism underlying aflatoxin B1 transformation by the BsCotA-methyl syringate system.

Global concern exists regarding the contamination of food and animal feed with aflatoxin B1 (AFB1), which poses a threat to the health of both humans and animals. Previously, we found that a laccase from Bacillus subtilis (BsCotA) effectively detoxified AFB1 in a reaction mediated by methyl syringate (MS), although the underlying mechanism has not been determined. Therefore, our primary objective of this study was to explore the detoxification mechanism employed by BsCotA. First, the enzyme and mediator dependence of AFB1 transformation were studied using the BsCotA-MS system, which revealed the importance of MS radical formation during the oxidation process. Aflatoxin Q1 (AFQ1) resulting from the direct oxidation of AFB1 by BsCotA, was identified using ultra-high-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The results of UPLC-MS/MS and density functional theory calculations indicated that the products included AFQ1, AFB1-, and AFD1-MS-coupled products in the BsCotA-MS system. The toxicity evaluations revealed that the substances derived from the transformation of AFB1 through the BsCotA-MS mechanism exhibited markedly reduced toxicity compared to AFB1. Finally, we proposed a set of different AFB1-transformation pathways generated by the BsCotA-MS system based on the identified products. These findings greatly enhance the understanding of the AFB1-transformation mechanism of the laccase-mediator system.

Kinect-based objective assessment of the acute levodopa challenge test in parkinsonism: a feasibility study.

INTRODUCTION: The acute levodopa challenge test (ALCT) is an important and valuable examination but there are still some shortcomings with it. We aimed to objectively assess ALCT based on a depth camera and filter out the best indicators. METHODS: Fifty-nine individuals with parkinsonism completed ALCT and the improvement rate (IR, which indicates the change in value before and after levodopa administration) of the Movement Disorder Society-Sponsored Revision of the Unified Parkinson's Disease Rating Scale part III (MDS-UPDRS III) was calculated. The kinematic features of the patients' movements in both the OFF and ON states were collected with an Azure Kinect depth camera. RESULTS: The IR of MDS-UPDRS III was significantly correlated with the IRs of many kinematic features for arising from a chair, pronation-supination movements of the hand, finger tapping, toe tapping, leg agility, and gait (rs = - 0.277 ~ - 0.672, P < 0.05). Moderate to high discriminative values were found in the selected features in identifying a clinically significant response to levodopa with sensitivity, specificity, and area under the curve (AUC) in the range of 50-100%, 47.22%-97.22%, and 0.673-0.915, respectively. The resulting classifier combining kinematic features of toe tapping showed an excellent performance with an AUC of 0.966 (95% CI = 0.922-1.000, P < 0.001). The optimal cut-off value was 21.24% with sensitivity and specificity of 94.44% and 87.18%, respectively. CONCLUSION: This study demonstrated the feasibility of measuring the effect of levodopa and objectively assessing ALCT based on kinematic data derived from an Azure Kinect-based system.

Production of cello-oligosaccharides from corncob residue by degradation-synthesis reactions.

The cellulose-rich corncob residue (CCR) is an abundant and renewable agricultural biomass that has been under-exploited. In this study, two strategies were compared for their ability to transform CCR into cello-oligosaccharides (COS). The first strategy employed the use of endo-glucanases. Although selected endo-glucanases from GH9, GH12, GH45, and GH131 could release COS with degrees of polymerization from 2 to 4, the degrading efficiency was low. For the second strategy, first, CCR was efficiently depolymerized to glucose and cellobiose using the cellulase from Trichoderma reesei. Then, using these simple sugars and sucrose as the starting materials, phosphorylases from different microorganisms were combined to generate COS to a level up to 100.3 g/L with different patterns and degrees of polymerization. Using tomato as a model plant, the representative COS obtained from BaSP (a sucrose phosphorylase from Bifidobacterium adolescens), CuCbP (a cellobiose phosphorylase from Cellulomonas uda), and CcCdP (a cellodextrin phosphorylase from Clostridium cellulosi) were shown to be able to promote plant growth. The current study pointed to an approach to make use of CCR for production of the value-added COS. KEY POINTS: • Sequential use of cellulase and phosphorylases effectively generated cello-oligosaccharides from corncob residue. • Cello-oligosaccharides patterns varied in accordance to cellobiose/cellodextrin phosphorylases. • Spraying cello-oligosaccharides promoted tomato growth.

Development of an efficient protein expression system in the thermophilic fungus Myceliophthora thermophila.

BACKGROUND: Thermophilic fungus Myceliophthora thermophila has been widely used in industrial applications due to its ability to produce various enzymes. However, the lack of an efficient protein expression system has limited its biotechnological applications. RESULTS: In this study, using a laccase gene reporting system, we developed an efficient protein expression system in M. thermophila through the selection of strong constitutive promoters, 5'UTRs and signal peptides. The expression of the laccase was confirmed by enzyme activity assays. The results showed that the Mtpdc promoter (Ppdc) was able to drive high-level expression of the target protein in M. thermophila. Manipulation of the 5'UTR also has significant effects on protein expression and secretion. The best 5'UTR (NCA-7d) was identified. The transformant containing the laccase gene under the Mtpdc promoter, NCA-7d 5'UTR and its own signal peptide with the highest laccase activity (1708 U/L) was obtained. In addition, the expression system was stable and could be used for the production of various proteins, including homologous proteins like MtCbh-1, MtGh5-1, MtLPMO9B, and MtEpl1, as well as a glucoamylase from Trichoderma reesei. CONCLUSIONS: An efficient protein expression system was established in M. thermophila for the production of various proteins. This study provides a valuable tool for protein production in M. thermophila and expands its potential for biotechnological applications.

Production of capsaicinoid nonivamide from plant oil and vanillylamine via whole-cell biotransformation.

Capsaicinoids are mostly derived from chili peppers and have widespread applications in food, feed, and pharmacology. Compared with plant extraction, the use of microbial cell factories for capsaicinoids production is considered as a more efficient approach. Here, the biotransformation of renewable plant oil and vanillylamine into capsaicinoid nonivamide was investigated. Nonivamide biosynthesis using nonanoic acid and vanillylamine as substrates was achieved in Escherichia coli by heterologous expression of genes encoding amide-forming N-acyltransferase and CoA-ligase. Through increasing nonanoic acid tolerance of chassis cell, screening key enzymes involved in nonivamide biosynthesis and optimizing biotransformation conditions, the nonivamide titer reached 0.5 g/L. By further integrating a route for conversion of oleic acid to nonanoic acid, nonivamide biosynthesis was finally achieved using olive oil and vanillylamine as substrates, yielding a titer of approximately 10.7 mg/L. Results from this study provide valuable information for constructing highly efficient cell factories for the production of capsaicinoid compounds.

GLP-1 receptor agonists modulate blood glucose levels in T2DM by affecting Faecalibacterium prausnitzii abundance in the intestine.

BACKGROUND: Glucagon-like peptide 1 (GLP-1) receptor agonists are a class of medications used to treat type 2 diabetes, including metformin, which is considered first-line therapy for type 2 diabetes. In recent years, GLP-1 receptor agonists (GLP-1 RAs) have been found to alter the composition and structure of gut flora and also promote the production of gut probiotics. However, there have been few clinical studies regarding the effects of GLP-1 RAs on gut flora. In this study, we investigated changes in the abundance of Lactobacillus delbrueckii (L delbrueckii) and Faecalibacterium prausnitzii (F prausnitzii) 1 week after administration of a GLP-1 RA in the clinical treatment of type 2 diabetes. The association with glycemic and body mass index (BMI) correlations was also explored. METHODS: Twelve newly diagnosed patients with type 2 diabetes were examined for changes in the abundance of L delbrueckii and F prausnitzii by Fluorescence in Situ Hybridization 1 week after administration of GLP-1 RAs. Subjects BMI was measured and fasting glucose changes were detected using the glucose oxidase method, and Spearman correlation analysis was performed to explore their relevance. RESULTS: There was no significant change in the abundance of L delbrueckii in the intestine (P = .695) and no significant correlation with BMI and fasting glucose levels (R = 0.134, P = .534) after the use of GLP-1 RA (R = -0.098, P = .647); F prausnitzii on the other hand had a significantly higher abundance (P = .002) and a significant negative correlation with fasting glucose level (R = -0.689, P < .001), but no significant correlation with BMI (R = -0.056, P = .796). CONCLUSION: F prausnitzii may be one of the pathways through which glucose is regulated in the treatment of type 2 diabetes by GLP-1 RAs.

Xylanase improves the intestinal barrier function of Nile tilapia (Oreochromis niloticus) fed with soybean (Glycine max) meal.

BACKGROUND: Soybean (Glycine max) meal is one of the important protein sources for fish, but the non-starch polysaccharides (NSP) in soybean meal impair the intestinal barrier function. Here we aimed to investigate whether xylanase can alleviate the adverse effects on the gut barrier induced by soybean meal in Nile tilapia and to explore the possible mechanism. RESULTS: Nile tilapia (Oreochromis niloticus) (4.09 ± 0.02 g) were fed with two diets including SM (soybean meal) and SMC (soybean meal + 3,000 U/kg xylanase) for 8 weeks. We characterized the effects of xylanase on the gut barrier, and the transcriptome analysis was performed to investigate the underlying mechanism. Dietary xylanase improved intestinal morphology and decreased the concentration of lipopolysaccharide (LPS) in serum. The results of transcriptome and Western blotting showed that dietary xylanase up-regulated the expression level of mucin2 (MUC2) which may be related to the inhibition of protein kinase RNA-like endoplasmic reticulum kinase (perk)/activating transcription factor 4 (atf4) signaling pathways. Microbiome analysis showed that addition of xylanase in soybean meal altered the intestinal microbiota composition and increased the concentration of butyric acid in the gut. Notably, dietary sodium butyrate was supplemented into the soybean meal diet to feed Nile tilapia, and the data verified that sodium butyrate mirrored the beneficial effects of xylanase. CONCLUSIONS: Collectively, supplementation of xylanase in soybean meal altered the intestinal microbiota composition and increased the content of butyric acid which can repress the perk/atf4 signaling pathway and increase the expression of muc2 to enhance the gut barrier function of Nile tilapia. The present study reveals the mechanism by which xylanase improves the intestinal barrier, and it also provides a theoretical basis for the application of xylanase in aquaculture.

Recombinant expression of IGF-1 and LR3 IGF-1 fused with xylanase in Pichia pastoris.

Insulin-like growth factor-1 (IGF-1) is a pleiotropic protein hormone and has become an attractive therapeutic target because of its multiple roles in various physiological processes, including growth, development, and metabolism. However, its production is hindered by low heterogenous protein expression levels in various expression systems and hard to meet the needs of clinical and scientific research. Here, we report that human IGF-1 and its analog Long R3 IGF-1 (LR3 IGF-1) are recombinant expressed and produced in the Pichia pastoris (P. pastoris) expression system through being fused with highly expressed xylanase XynCDBFV. Furthermore, purified IGF-1 and LR3 IGF-1 display excellent bioactivity of cell proliferation compared to the standard IGF-1. Moreover, higher heterologous expression levels of the fusion proteins XynCDBFV-IGF-1 and XynCDBFV-LR3 IGF-1 are achieved by fermentation in a 15-L bioreactor, reaching up to about 0.5 g/L XynCDBFV-IGF-1 and 1 g/L XynCDBFV-TEV-LR3 IGF-1. Taken together, high recombinant expression of bioactive IGF-1 and LR3 IGF-1 is acquired with the assistance of xylanase as a fusion partner in P. pastoris, which could be used for both clinical and scientific applications. KEY POINTS: • Human IGF-1 and LR3 IGF-1 are produced in the P. pastoris expression system. • Purified IGF-1 and LR3 IGF-1 show bioactivity comparable to the standard IGF-1. • High heterologous expression of IGF-1 and LR3 IGF-1 is achieved by fermentation in a bioreactor.

Insights into the H2O2-Driven Lytic Polysaccharide Monooxygenase Activity on Efficient Cellulose Degradation in the White Rot Fungus Irpex lacteus.

In contrast to O2, H2O2 as the cosubstrate for lytic polysaccharide monooxygenases (LPMOs) exhibits great advantages in industrial settings for cellulose degradation. However, H2O2-driven LPMO reactions from natural microorganisms have not been fully explored and understood. Herein, secretome analysis unraveled the H2O2-driven LPMO reaction in the efficient lignocellulose-degrading fungus Irpex lacteus, including LPMOs with different oxidative regioselectivities and various H2O2-generating oxidases. Biochemical characterization of H2O2-driven LPMO catalysis showed orders of magnitude improvement in catalytic efficiency compared to that of O2-driven LPMO catalysis for cellulose degradation. Significantly, H2O2 tolerance of LPMO catalysis in I. lacteus was an order of magnitude higher than that in other filamentous fungi. In addition, natural reductants, gallic acid, in particular, presented in lignocellulosic biomass could sufficiently maintain LPMO catalytic reactions. Moreover, the H2O2-driven LPMO catalysis exhibited synergy with canonical endoglucanases for efficient cellulose degradation. Taken together, these findings demonstrate the great application potential of the H2O2-driven LPMO catalysis for upgrading cellulase cocktails to further improve cellulose degradation efficiency.

Heterologous expression and characterization of novel GH12 ß-glucanase and AA10 lytic polysaccharide monooxygenase from Streptomyces megaspores and their synergistic action in cellulose saccharification.

BACKGROUND: The combination of cellulase and lytic polysaccharide monooxygenase (LPMO) is known to boost enzymatic saccharification of cellulose. Although the synergy between cellulases (GH5, 6 or 7) and LPMOs (AA9) has been extensively studied, the interplay between other glycoside hydrolase and LPMO families remains poorly understood. RESULTS: In this study, two cellulolytic enzyme-encoding genes SmBglu12A and SmLpmo10A from Streptomyces megaspores were identified and heterologously expressed in Escherichia coli. The recombinant SmBglu12A is a non-typical endo-ß-1,4-glucanase that preferentially hydrolyzed ß-1,3-1,4-glucans and slightly hydrolyzed ß-1,4-glucans and belongs to GH12 family. The recombinant SmLpmo10A belongs to a C1-oxidizing cellulose-active LPMO that catalyzed the oxidation of phosphoric acid swollen cellulose to produce celloaldonic acids. Moreover, individual SmBglu12A and SmLpmo10A were both active on barley ß-1,3-1,4-glucan, lichenan, sodium carboxymethyl cellulose, phosphoric acid swollen cellulose, as well as Avicel. Furthermore, the combination of SmBglu12A and SmLpmo10A enhanced enzymatic saccharification of phosphoric acid swollen cellulose by improving the native and oxidized cello-oligosaccharides yields. CONCLUSIONS: These results proved for the first time that the AA10 LPMO was able to boost the catalytic efficiency of GH12 glycoside hydrolases on cellulosic substrates, providing another novel combination of glycoside hydrolase and LPMO for cellulose enzymatic saccharification.

Kinect-based objective evaluation of bradykinesia in patients with Parkinson's disease.

Objective: To quantify bradykinesia in Parkinson's disease (PD) with a Kinect depth camera-based motion analysis system and to compare PD and healthy control (HC) subjects. Methods: Fifty PD patients and twenty-five HCs were recruited. The Movement Disorder Society-Sponsored Revision of the Unified Parkinson's Disease Rating Scale part III (MDS-UPDRS III) was used to evaluate the motor symptoms of PD. Kinematic features of five bradykinesia-related motor tasks were collected using Kinect depth camera. Then, kinematic features were correlated with the clinical scales and compared between groups. Results: Significant correlations were found between kinematic features and clinical scales (P < 0.05). Compared with HCs, PD patients exhibited a significant decrease in the frequency of finger tapping (P < 0.001), hand movement (P < 0.001), hand pronation-supination movements (P = 0.005), and leg agility (P = 0.003). Meanwhile, PD patients had a significant decrease in the speed of hand movements (P = 0.003) and toe tapping (P < 0.001) compared with HCs. Several kinematic features exhibited potential diagnostic value in distinguishing PD from HCs with area under the curve (AUC) ranging from 0.684-0.894 (P < 0.05). Furthermore, the combination of motor tasks exhibited the best diagnostic value with the highest AUC of 0.955 (95% CI = 0.913-0.997, P < 0.001). Conclusion: The Kinect-based motion analysis system can be applied to evaluate bradykinesia in PD. Kinematic features can be used to differentiate PD patients from HCs and combining kinematic features from different motor tasks can significantly improve the diagnostic value.

Production of N-acetylglucosamine from carbon dioxide by engineering Cupriavidus necator H16.

The conversion of CO2 into valuable bioactive substances using synthetic biological techniques is a potential approach for mitigating the greenhouse effect. Here, the engineering of C. necator H16 to produce N-acetylglucosamine (GlcNAc) from CO2 is reported. First, GlcNAc importation and intracellular metabolic pathways were disrupted by the deletion of nagF, nagE, nagC, nagA and nagB genes. Second, the GlcNAc-6-phosphate N-acetyltransferase gene (gna1) was screened. A GlcNAc-producing strain was constructed by overexpressing a mutant gna1 from Caenorhabditis elegans. A further increase in GlcNAc production was achieved by disrupting poly(3-hydroxybutyrate) biosynthesis and the Entner-Doudoroff pathways. The maximum GlcNAc titers were 199.9 and 566.3 mg/L for fructose and glycerol, respectively. Finally, the best strain achieved a GlcNAc titer of 75.3 mg/L in autotrophic fermentation. This study demonstrated a conversion of CO2 to GlcNAc, thereby providing a feasible approach for the biosynthesis of various bioactive chemicals from CO2 under normal conditions..

Engineering Escherichia coli for efficient assembly of heme proteins.

BACKGROUND: Heme proteins, such as hemoglobin, horseradish peroxidase and cytochrome P450 (CYP) enzyme, are highly versatile and have widespread applications in the fields of food, healthcare, medical and biological analysis. As a cofactor, heme availability plays a pivotal role in proper folding and function of heme proteins. However, the functional production of heme proteins is usually challenging mainly due to the insufficient supply of intracellular heme. RESULTS: Here, a versatile high-heme-producing Escherichia coli chassis was constructed for the efficient production of various high-value heme proteins. Initially, a heme-producing Komagataella phaffii strain was developed by reinforcing the C4 pathway-based heme synthetic route. Nevertheless, the analytical results revealed that most of the red compounds generated by the engineered K. phaffii strain were intermediates of heme synthesis which were unable to activate heme proteins. Subsequently, E. coli strain was selected as the host to develop heme-producing chassis. To fine-tune the C5 pathway-based heme synthetic route in E. coli, fifty-two recombinant strains harboring different combinations of heme synthesis genes were constructed. A high-heme-producing mutant Ec-M13 was obtained with negligible accumulation of intermediates. Then, the functional expression of three types of heme proteins including one dye-decolorizing peroxidase (Dyp), six oxygen-transport proteins (hemoglobin, myoglobin and leghemoglobin) and three CYP153A subfamily CYP enzymes was evaluated in Ec-M13. As expected, the assembly efficiencies of heme-bound Dyp and oxygen-transport proteins expressed in Ec-M13 were increased by 42.3-107.0% compared to those expressed in wild-type strain. The activities of Dyp and CYP enzymes were also significantly improved when expressed in Ec-M13. Finally, the whole-cell biocatalysts harboring three CYP enzymes were employed for nonanedioic acid production. High supply of intracellular heme could enhance the nonanedioic acid production by 1.8- to 6.5-fold. CONCLUSION: High intracellular heme production was achieved in engineered E. coli without significant accumulation of heme synthesis intermediates. Functional expression of Dyp, hemoglobin, myoglobin, leghemoglobin and CYP enzymes was confirmed. Enhanced assembly efficiencies and activities of these heme proteins were observed. This work provides valuable guidance for constructing high-heme-producing cell factories. The developed mutant Ec-M13 could be employed as a versatile platform for the functional production of difficult-to-express heme proteins.