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Abstract 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

Abstract 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.

Abstract 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.

Abstract 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.

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.

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.

Deep-sea submersible is an important equipment for China to explore the far-reaching sea, the "Jiaolong" sea exploration project was proposed in the "13th Five-Year Plan", which is the overall layout and planning of the national marine cause. Through decades of technological leapfrog development, the "Jiao Long", "Qian Long" series, "Hai Long", "Hai Ma", "Hai Dou", "Hai Yan" and other deep-sea series equipment of different types and depths have been developed, and gradually developed to the lineage. In the future, according to the needs of marine scientific research, the development and utilization of marine resources, and the maintenance of marine rights and interests, more deep-sea equipment that meet the actual requirements will be developed. This paper draws lessons from other high-tech fields, carries out the exploration and research of submarine genealogy, and builds the genealogy framework.

Abstract 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.

Abstract 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-β-1,4-glucanase that preferentially hydrolyzed β-1,3-1,4-glucans and slightly hydrolyzed β-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 β-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.

Abstract Cloud energy storage is considered a promising application in future power systems. It focuses on optimally leveraging the capacity of centralized large‐scale energy storage compared with the requirements of small‐scale localized users. In this paper, to satisfy the small‐ and medium‐scale timely energy storage requirement from localized users, the concept of the cloud‐based location sharing energy storage is proposed. The modular mobile energy storage system is flexibly configured and deployed at different sites to fulfil the long‐term seasonally dynamic transformer capacity increment and short‐term daily energy arbitrage based on economic values. To optimize the overall incomes of the energy storage investment, a two‐step user potential identification algorithm is proposed to discover the most valuable users at different time scales from the regional power usage profile. Then, the number/capacity optimal planning algorithm is proposed to optimally share the mobile energy storage system among users seasonally and total profits are further increased through daily energy arbitrage at less valuable seasons. Finally, the structure and dispatching strategy of the cloud‐based location sharing energy storage management system is illustrated. Case study results prove that the proposed identification algorithm can excavate the most valuable users at different time scales and the optimal planning and operation strategy is able to guarantee the overall income benefits.

Abstract 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.

Abstract Trichoderma reesei (T. reesei) is well-known for its excellent ability to secret a large quantity of cellulase. However, unlike the endogenous proteins, little is known about the molecular mechanisms governing heterologous protein production. Herein, we focused on the integration loci and the secretory pathway, and investigated their combinatorial effects on heterologous gene expression in T. reesei using a glucose oxidase from Aspergillus niger as a model protein. Integration in the cel3c locus was more efficient than the cbh1 locus in expressing the AnGOx by increasing the transcription of AnGOx in the early stage. In addition, we discovered that interruption of the cel3c locus has an additional effect by increasing the expression of the secretory pathway component genes. Accordingly, overexpressing three secretory pathway component genes, that were snc1, sso2, and rho3, increased AnGOx expression in the cbh1 transformant but not in the cel3c transformant.

Abstract Postural abnormalities are common disabling motor complications affecting patients with Parkinson’s disease (PD). We proposed a summary index for postural abnormalities (IPA) based on Kinect depth camera and explored the clinical value of this indicator. Seventy individuals with PD and thirty age-matched healthy controls (HCs) were enrolled. All participants were tested using a Kinect-based system with IPA automatically obtained by algorithms. Significant correlations were detected between IPA and the Movement Disorder Society-Sponsored Revision of the Unified Parkinson’s Disease Rating Scale (MDS-UPDRS) total score (r s = 0.369, p = 0.002), MDS-UPDRS-III total score (r s = 0.431, p

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

Abstract Background The methylotrophic budding yeast Pichia pastoris GS115 is a powerful expression system and hundreds of heterologous proteins have been successfully expressed in this strain. Recently, P. pastoris has also been exploited as an attractive cell factory for the production of high-value biochemicals due to Generally Recognized as Safe (GRAS) status and high growth rate of this yeast strain. However, appropriate regulation of metabolic flux distribution between cell growth and product biosynthesis is still a cumbersome task for achieving efficient biochemical production. Results In this study, P. pastoris was exploited for high inositol production using an effective dynamic regulation strategy. Through enhancing native inositol biosynthesis pathway, knocking out inositol transporters, and slowing down carbon flux of glycolysis, an inositol-producing mutant was successfully developed and low inositol production of 0.71 g/L was obtained. The inositol production was further improved by 12.7% through introduction of heterologous inositol-3-phosphate synthase (IPS) and inositol monophosphatase (IMP) which catalyzed the rate-limiting steps for inositol biosynthesis. To control metabolic flux distribution between cell growth and inositol production, the promoters of glucose-6-phosphate dehydrogenase (ZWF), glucose-6-phosphate isomerase (PGI) and 6-phosphofructokinase (PFK1) genes were replaced with a glycerol inducible promoter. Consequently, the mutant strain could be switched from growth mode to production mode by supplementing glycerol and glucose sequentially, leading to an increase of about 4.9-fold in inositol formation. Ultimately, the dissolved oxygen condition in high-cell-density fermentation was optimized, resulting in a high production of 30.71 g/L inositol (~ 40-fold higher than the baseline strain). Conclusions The GRAS P. pastoris was engineered as an efficient inositol producer for the first time. Dynamic regulation of cell growth and inositol production was achieved via substrate-dependent modulation of glycolysis and pentose phosphate pathways and the highest inositol titer reported to date by a yeast cell factory was obtained. Results from this study provide valuable guidance for engineering of P. pastoris for the production of other high-value bioproducts.

Background: Chronic inflammatory demyelinating polyneuropathy (CIDP) is a rare and challenging demyelinating disorder. It is necessary to increase our understanding of potential connections between imaging, electromyography, and clinical characteristics. Objective: The aim of this study was to evaluate the relationships between multisequence magnetic resonance neurography (MRN) findings, electrophysiological parameters, and clinical characteristics in CIDP patients. Design: A cross-sectional study. Methods: Overall, 51 CIDP patients underwent MRN of the brachial and lumbosacral plexus, and nerve conduction studies. The inflammatory Rasch-built overall disability scale (I-RODS) questionnaire, CIDP disease activity status (CADS) scale, and muscle strength scores were evaluated by two neurologists. Electrophysiological parameters, clinical information, and multiparameter-MRN were analyzed for correlations. Multiparameter-MRN includes diameter, nerve-to-muscle T2 signal intensity ratio (nT2), contrast-enhanced ratio (CR), fractional anisotropy (FA), and apparent diffusion coefficient (ADC) of bilateral plexus nerve roots. Results: Electrophysiological parameters that were not elicited were significantly higher in the lower extremities than in the upper extremities, and those were higher in sensory nerve conduction than in motor. There were moderate correlations between motor nerve conduction velocity and distal motor latency in nerve diameter, nT2, FA, and ADC, respectively (| r |, 0.45–0.64, p

Abstract Background Automated and accurate assessment for postural abnormalities is necessary to monitor the clinical progress of Parkinson’s disease (PD). The combination of depth camera and machine learning makes this purpose possible. Methods Kinect was used to collect the postural images from 70 PD patients. The collected images were processed to extract three-dimensional body joints, which were then converted to two-dimensional body joints to obtain eight quantified coronal and sagittal features (F1-F8) of the trunk. The decision tree classifier was carried out over a data set established by the collected features and the corresponding doctors’ MDS-UPDRS-III 3.13 (the 13th item of the third part of Movement Disorder Society-Sponsored Revision of the Unified Parkinson’s Disease Rating Scale) scores. An objective function was implanted to further improve the human–machine consistency. Results The automated grading of postural abnormalities for PD patients was realized with only six selected features. The intraclass correlation coefficient (ICC) between the machine’s and doctors’ score was 0.940 (95%CI, 0.905–0.962), meaning the machine was highly consistent with the doctors’ judgement. Besides, the decision tree classifier performed outstandingly, reaching 90.0% of accuracy, 95.7% of specificity and 89.1% of sensitivity in rating postural severity. Conclusions We developed an intelligent evaluation system to provide accurate and automated assessment of trunk postural abnormalities in PD patients. This study demonstrates the practicability of our proposed method in the clinical scenario to help making the medical decision about PD.

Crop planting area and spatial distribution information have important practical significance for food security, global change, and sustainable agricultural development. How to efficiently and accurately identify crops in a timely manner by remote sensing in order to determine the crop planting area and its temporal–spatial dynamic change information is a core issue of monitoring crop growth and estimating regional crop yields. Based on hundreds of relevant documents from the past 25 years, in this paper, we summarize research progress in relation to farmland vegetation identification and classification by remote sensing. The classification and identification of farmland vegetation includes classification based on vegetation index, spectral bands, multi-source data fusion, artificial intelligence learning, and drone remote sensing. Representative studies of remote sensing methods are collated, the main content of each technology is summarized, and the advantages and disadvantages of each method are analyzed. Current problems related to crop remote sensing identification are then identified and future development directions are proposed.

Abstract Objective This study investigated the effects of aerobic exercise combined with resistance training on serum inflammatory factors and heart rate variability (HRV) in women with type 2 diabetes mellitus (T2DM). Methods A total of 30 patients with diabetic cardiovascular autonomic neuropathy (DCAN) were randomly divided into a control group (n = 15) and an exercise group (n = 15). The control group was treated with routine hypoglycemic drugs, while the exercise group was treated with routine hypoglycemic drugs + resistance training (AE + RT). The levels of fasting plasma glucose (FBG), two‐hour plasma glucose (2hPG), serum inflammatory factors C‐reactive protein (CRP), interleukin‐6 (IL‐6) and tumor necrosis factor alpha (TNF‐α) were measured before and after the intervention. The HRV was evaluated by 24‐h ambulatory electrocardiogram. Results After the intervention, the levels of FBG, 2hPG, serum inflammatory factors, IL‐6 and TNF‐α in the exercise group were significantly lower than those in the control group (p .05). After the intervention, the HRV time domain and frequency domain indexes in the two groups were significantly improved compared with those before the exercise experiment (p .05). The time‐domain indexes, i.e., SDNN and RMSSD, as well as the frequency domain index, i.e., (lnhf), were significantly higher in the exercise group than in the control group, whereas lnlf/lnhf were significantly lower than those in the control group (p

Abstract Background Humicola insolens is a filamentous fungus with high potential of producing neutral and heat- and alkali-resistant cellulase. However, the genetic engineering tools, particularly the genome-editing tool, are scarce, hindering the study of cellulase expression regulation in this organism. Results Herein, a CRISPR/Cas9 genome-editing system was established in H. insolens based on a hybrid 5S rRNA–tRNAGly promoter. This system is superior to the HDV (hepatitis delta virus) system in genome editing, allowing highly efficient single gene destruction in H. insolens with rates of deletion up to 84.1% (37/44). With this system, a putative pigment synthesis gene pks and the transcription factor xyr1 gene were disrupted with high efficiency. Moreover, the extracellular protein concentration and cellulase activity largely decreased when xyr1 was deleted, demonstrating for the first time that Xyr1 plays an important role in cellulase expression regulation. Conclusions The established CRISPR/Cas9 system is a powerful genetic operation tool for H. insolens, which will accelerate studies on the regulation mechanism of cellulase expression and engineering of H. insolens for higher cellulase production.

Abstract Background Gamma-aminobutyric acid (GABA) is an important bio-product used in pharmaceuticals and functional foods and as a precursor of the biodegradable plastic polyamide 4. Glutamate decarboxylase (GAD) converts l-glutamate (l-Glu) into GABA via decarboxylation. Compared with other methods, develop a bioconversion platform to produce GABA is of considerable interest for industrial use. Results Three GAD genes were identified from three Bacillus strains and heterologously expressed in Escherichia coli BL21 (DE3). The optimal reaction temperature and pH values for three enzymes were 40 °C and 5.0, respectively. Of the GADs, GADZ11 had the highest catalytic efficiency towards l-Glu (2.19 mM− 1 s− 1). The engineered E. coli strain that expressed GADZ11 was used as a whole-cell biocatalyst for the production of GABA. After repeated use 14 times, the cells produced GABA with an average molar conversion rate of 98.6% within 14 h. Conclusions Three recombinant GADs from Bacillus strains have been conducted functional identification. The engineered E. coli strain heterologous expressing GADZ1, GADZ11, and GADZ20 could accomplish the biosynthesis of l-Glu to GABA in a buffer-free reaction at a high l-Glu concentration. The novel engineered E. coli strain has the potential to be a cost-effective biotransformation platform for the industrial production of GABA.

BackgroundMultiple myeloma (MM) is a hematological malignancy, and intramedullary spinal cord metastasis is extremely rare.MethodsClinical and radiological data were collected from electronic medical records as well as a literature review of reported cases.ResultsWe report a rare case of IgA-LAM stage IIB MM with involvement of the spinal cord and peripheral nervous system. Laboratory studies showed elevated levels of serum β2-macroglobulin and cerebrospinal fluid protein. Electromyography revealed a demyelinating process with motor conduction blocks. On MRI, the lesions of MM bone marrow are characterized as a type of diffuse infiltration. MR neurography demonstrated an enhanced nodule in the thoracic segment with swelling of the cervicothoracic segments of the spinal cord. Moreover, swelling and hypertrophy of the entire nerve branchial, lumbosacral plexus, and cauda equina were detected, accompanied by myofascitis and denervated muscles. Ultimately, the condition of the patient deteriorated quickly and she died with a diagnosis of refractory MM.ConclusionMRI not only has the advantage of displaying the primary involved site of the bone marrow but also facilitates detecting extramedullary hematopoietic MM, such as infiltrating sites of the central and/or peripheral nervous system.

As a markerless motion capture system (MLS), the Microsoft Kinect sensor may be a low-cost and portable option for measuring shoulder joint angles. However, the system's concurrent validity to capture shoulder functional movement is unclear. The purpose of this study was to investigate the concurrent validity of MLS for capturing shoulder kinematics during functional movement by comparing the MLS measures to those of the marker-based motion capture system (MBS). Twenty-five healthy participants were included in this study. Using the Microsoft Kinect sensor as the MLS and Vicon as the MBS, six shoulder functional movements were measured in all three anatomical planes concurrently. The six movements included flexion to max, maximum extension, abduction to 90°, internal rotation at 90° abduction, external rotation at 0° abduction, and maximum horizontal adduction. The shoulder joint kinematics was measured with both systems. The MLS showed a good to excellent correlation (r ​> ​0.75) with MBS for abduction to 90°, maximum external rotation at 0° abduction and maximum internal rotation at 90° abduction. The results for maximum extension (r ​= ​0.727) and maximum horizontal adduction (r ​= ​0.619) showed a moderate to good correlation. For maximum flexion, a poor correlation was observed (r ​= ​0.479). The validity of the MLS to measure shoulder kinematics would be acceptable. The system demonstrates potential as a measurement tool of joint motions for a function assessment and rehabilitation purpose.

BackgroundAxial disturbances are the most disabling symptoms of Parkinson’s disease (PD). Kinect-based objective measures could extract motion characteristics with high reliability and validity.PurposeThe present research aimed to quantify the therapy–response of axial motor symptoms to daily medication regimen and to explore the correlates of the improvement rate (IR) of axial motor symptoms based on a Kinect camera.Materials and methodsWe enrolled 44 patients with PD and 21 healthy controls. All 65 participants performed the Movement Disorder Society-Sponsored Revision of the Unified Parkinson’s Disease Rating Scale part III and the Kinect-based kinematic evaluation to assess arising from a chair, gait, posture, and postural stability before and after medication. Spearman’s correlation analysis and multiple linear regression model were performed to explore the relationships between motor feature IR and clinical data.ResultsAll the features arising from a chair (P = 0.001), stride length (P = 0.001), velocity (P < 0.001), the height of foot lift (P < 0.001), and turning time (P = 0.001) improved significantly after a daily drug regimen in patients with PD. In addition, the anterior trunk flexion (lumbar level) exhibited significant improvement (P = 0.004). The IR of the axial motor symptoms score was significantly correlated with the IRs of kinematic features for gait velocity, stride length, foot lift height, and sitting speed (rs = 0.345, P = 0.022; rs = 0.382, P = 0.010; rs = 0.314, P = 0.038; rs = 0.518, P < 0.001, respectively). A multivariable regression analysis showed that the improvement in axial motor symptoms was associated with the IR of gait velocity only (β = 0.593, 95% CI = 0.023–1.164, P = 0.042).ConclusionAxial symptoms were not completely drug-resistant, and some kinematic features can be improved after the daily medication regimen of patients with PD.

The pathogenesis of Charcot–Marie–Tooth (CMT) disease, an inherited peripheral neuropathy, is associated with more than 60 nuclear genes. We reported a rare phenotype of the uncommon CMT genotype complicated with neuroinflammation, that is, an MPZ mutation, NC_000001.11 (NM_000530.6): c.308G > C detected by next-generation sequencing. Moreover, we present a case of the CMT type 1B, with atypical presentation as two patterns of hypertrophy in the brachial and lumbosacral plexus, as well as enhancement in the cauda equina and nerve roots on multimodal magnetic resonance neurography (MRN). MRN assessment facilitated the identification of coexisting neuroinflammation and provided more evidence, especially for patients with atypical symptoms in hereditary sensory and motor neuropathy, who could benefit from immunotherapy.

Polyneuropathy, organomegaly, endocrinopathy, monoclonal plasma cell disorder, skin changes (POEMS) syndrome is a multisystem disease associated with underlying plasma cell neoplasm. Here, we present two cases of POEMS syndrome that manifested on magnetic resonance neurography as an increasing bone mass with cortical disruption, direct invading nerve roots and lumbar gluteal muscles. These features have not been previously reported. We also report a case with diffuse hypertrophy and enhancement of the brachial and lumbosacral plexus, which mimics the most common chronic inflammatory demyelinating polyradiculoneuropathy. Moreover, we detected perineurium effusions in the plexus, coupled with a variety of myofascitis and atrophy in denervated muscle. The case series is of concern to atypical magnetic resonance imaging findings of POEMS syndrome in the bone and peripheral nerve system as critical attacked target organs, which would be facilitating diagnosis.

The entrepreneurial institutional environment is the external factor that entrepreneurial enterprises rely on for survival. Our interest is in how entrepreneurs cultivate entrepreneurial orientation in response to the highly uncertain entrepreneurial situation. Based on the cognitive appraisal theory of emotion, we analyzed the impact of the entrepreneurial institutional environment on entrepreneurial orientation through entrepreneurial passion. This study applied stepwise regression analyses to test the hypotheses on a sample of 197 entrepreneurs from the co-creation space in China. The output of the study showed that the entrepreneurial institutional environment had a positive effect on entrepreneurial orientation and that entrepreneurial passion played a mediating role between the entrepreneurial institutional environment and entrepreneurial orientation.

Family financing has become a powerful channel for entrepreneurs to obtain entrepreneurial funding. How do family members use intent and quality signals to select new ventures to provide lending support? Building on the signaling theory, this study provides the first quantitative evidence using a sample of 166 samples of family lenders in China. Our findings reveal that psychological capital can support entrepreneurs to obtain family lending. As an intent signal, psychological capital becomes more influential when quality signals, corporate competitive advantage, and firm performance perform more positively. This study emphasizes that family financing support is not only out of love or altruism and extends the literature concerning the influence of positive psychological capital in financial investment decisions.

Abstract Background Regarding plant cell wall polysaccharides degradation, multimodular glycoside hydrolases (GHs) with two catalytic domains separated by one or multiple carbohydrate-binding domains are rare in nature. This special mode of domain organization endows the Caldicellulosiruptor bescii CelA (GH9-CBM3c-CBM3b-CBM3b-GH48) remarkably high efficiency in hydrolyzing cellulose. CbXyn10C/Cel48B from the same bacterium is also such an enzyme which has, however, evolved to target both xylan and cellulose. Intriguingly, the GH10 endoxylanase and GH48 cellobiohydrolase domains are both dual functional, raising the question if they can act synergistically in hydrolyzing cellulose and xylan, the two major components of plant cell wall. Results In this study, we discovered that CbXyn10C and CbCel48B, which stood for the N- and C-terminal catalytic domains, respectively, cooperatively released much more cellobiose and cellotriose from cellulose. In addition, they displayed intramolecular synergy but only at the early stage of xylan hydrolysis by generating higher amounts of xylooligosaccharides including xylotriose, xylotetraose, and xylobiose. When complex lignocellulose corn straw was used as the substrate, the synergy was found only for cellulose but not xylan hydrolysis. Conclusion This is the first report to reveal the synergy between a GH10 and a GH48 domain. The synergy discovered in this study is helpful for understanding how C. bescii captures energy from these recalcitrant plant cell wall polysaccharides. The insight also sheds light on designing robust and multi-functional enzymes for plant cell wall polysaccharides degradation.

Citrinin (CIT) is a mycotoxin found in foods and feeds and most commonly discovered in red yeast rice, a food additive made from ordinary rice by fermentation with Monascus. Currently, no enzyme is known to be able to degrade CIT effectively. In this study, it was discovered that manganese peroxidase (MrMnP) from Moniliophthora roreri could degrade CIT. The degradation appeared to be fulfilled by a combination of direct and indirect actions of the MrMnP with the CIT. Pure CIT, at a final concentration of 10 mg/L, was completely degraded by MrMnP within 72 h. One degradation product was identified to be dihydrocitrinone. The toxicity of the CIT-degradation product decreased, as monitored by the increased survival rate of the Caco-2 cells incubated with MrMnP-treated CIT. In addition, MrMnP could degrade CIT (with a starting concentration of up to 4.6 mg/L) completely contaminated in red yeast rice. MrMnP serves as an excellent candidate enzyme for CIT detoxification.

Coronavirus disease 2019 (COVID-19) pandemic is caused by the novel coronavirus that has spread rapidly around the world, leading to high mortality because of multiple organ dysfunction; however, its underlying molecular mechanism is unknown. To determine the molecular mechanism of multiple organ dysfunction, a bioinformatics analysis method based on a time-order gene co-expression network (TO-GCN) was performed. First, gene expression profiles were downloaded from the gene expression omnibus database (GSE161200), and a TO-GCN was constructed using the breadth-first search (BFS) algorithm to infer the pattern of changes in the different organs over time. Second, Gene Ontology enrichment analysis was used to analyze the main biological processes related to COVID-19. The initial gene modules for the immune response of different organs were defined as the research object. The STRING database was used to construct a protein–protein interaction network of immune genes in different organs. The PageRank algorithm was used to identify five hub genes in each organ. Finally, the Comparative Toxicogenomics Database played an important role in exploring the potential compounds that target the hub genes. The results showed that there were two types of biological processes: the body’s stress response and cell-mediated immune response involving the lung, trachea, and olfactory bulb (olf) after being infected by COVID-19. However, a unique biological process related to the stress response is the regulation of neuronal signals in the brain. The stress response was heterogeneous among different organs. In the lung, the regulation of DNA morphology, angiogenesis, and mitochondrial-related energy metabolism are specific biological processes related to the stress response. In particular, an effect on tracheal stress response was made by the regulation of protein metabolism and rRNA metabolism-related biological processes, as biological processes. In the olf, the distinctive stress responses consist of neural signal transmission and brain behavior. In addition, myeloid leukocyte activation and myeloid leukocyte-mediated immunity in response to COVID-19 can lead to a cytokine storm. Immune genes such as SRC, RHOA, CD40LG, CSF1, TNFRSF1A, FCER1G, ICAM1, LAT, LCN2, PLAU, CXCL10, ICAM1, CD40, IRF7, and B2M were predicted to be the hub genes in the cytokine storm. Furthermore, we inferred that resveratrol, acetaminophen, dexamethasone, estradiol, statins, curcumin, and other compounds are potential target drugs in the treatment of COVID-19.

Background and PurposeChronic inflammatory demyelinating polyneuropathy (CIDP) is an uncommon demyelinating disorder. Although treatable, it is difficult to diagnose. The purpose of this study was to evaluate the diagnostic performance and abnormalities of plexus via quantitative multisequence magnetic resonance neurography (MRN) for CIDP.MethodsBrachial and lumbosacral (LS) plexus of 37 CIDP patients and 37 age- and gender-matched controls were examined by using multisequence MRN. Nerve diameter, nerve-to-muscle T2 signal intensity ratio (nT2), contrast-enhanced ratio (CR), fractional anisotropy (FA), and apparent diffusion coefficient (ADC) were determined in both plexus, and tractographies were performed. The disease histories and the Inflammatory Rasch-built Overall Disability Scale (I-RODS) questionnaire scores were documented before MRI scans.ResultsThe sizes of nerve roots were larger in CIDP (p < 0.01). CR, nT2, and ADC were significantly higher, while FA was lower in CIDP than in controls (p < 0.01). FA had the highest sensitivity (0.809) and area under the curve (AUC) (0.925), while the highest specificity was 0.961 for CR in single parameters. The combination of FA and CR has the highest sensitivity, specificity, accuracy, and AUC in the LS plexus. CR only had a weak correlation with nT2 (p < 0.05). ADC and diameter had a positive correlation with nT2, and the diameter and nT2 had a negative correlation with FA in CIDP (p < 0.05). FA had a negative correlation with the duration in the CIDP (r’s = −0.404, p < 0.05). There was no significant correlation between the I-RODS scores and MR multiparameters (p < 0.05).ConclusionMultisequence MRN possesses a high diagnostic performance in the LS plexus. Sampling perfection with application-optimized contrasts using different flip angle evolutions (SPACE) combined with DTI and contrast enhancement serves as a recommended composite protocol for CIDP.

Abstract Background CRISPR/Cas9 has wide application potentials in a variety of biological species including Trichoderma reesei, a filamentous fungus workhorse for cellulase production. However, expression of Cas9 heterologously in the host cell could be time-consuming and sometimes even troublesome. Results We tested two gene disruption methods in T. reesei using CRISPR/Cas9 in this study. The intracellularly expressed Cas9 led to unexpected off-target gene disruption in T. reesei QM9414, favoring inserting 9- or 12-bp at 70- and 100-bp downstream of the targeted ura5. An alternative method was, therefore, established by assembling Cas9 and gRNA in vitro, followed by transformation of the ribonucleoprotein complex with a plasmid containing the pyr4 marker gene into T. reesei TU-6. When the gRNA targeting cbh1 was used, eight among the twenty seven transformants were found to lose the ability to express CBH1, indicative of successful cbh1 disruption through genome editing. Large DNA fragments including the co-transformed plasmid, chromosomal genes, or a mixture of these nucleotides, were inserted in the disrupted cbh1 locus. Conclusions Direct transformation of Cas9/gRNA complex into the cell is a fast means to disrupt a gene in T. reesei and may find wide applications in strain improvement and functional genomics study.

The fungal secondary metabolite patulin is a mycotoxin widespread in foods and beverages which poses a serious threat to human health. However, no enzyme was known to be able to degrade this mycotoxin. For the first time, we discovered that a manganese peroxidase (MrMnP) from Moniliophthora roreri can efficiently degrade patulin. The MrMnP gene was cloned into pPICZα(A) and then the recombinant plasmid was transformed into Pichia pastoris X-33. The recombinant strain produced extracellular manganese peroxidase with an activity of up to 3659.5 U/L. The manganese peroxidase MrMnP was able to rapidly degrade patulin, with hydroascladiol appearing as a main degradation product. Five mg/L of pure patulin were completely degraded within 5 h. Moreover, up to 95% of the toxin was eliminated in a simulated patulin-contaminated apple juice after 24 h. Using Escherichia coli as a model, it was demonstrated that the deconstruction of patulin led to detoxification. Collectively, these traits make MrMnP an intriguing candidate useful in enzymatic detoxification of patulin in foods and beverages.

Abstract Background The white rot fungus Irpex lacteus exhibits a great potential in biopretreatment of lignocellulose as well as in biodegradation of xenobiotic compounds by extracellular ligninolytic enzymes. Among these enzymes, the possible involvement of dye-decolorizing peroxidase (DyP) in lignin degradation is not clear yet. Results Based on the extracellular enzyme activities and secretome analysis, I. lacteus CD2 produced DyPs as the main ligninolytic enzymes when grown in Kirk’s medium supplemented with lignin. Further transcriptome analysis revealed that induced transcription of genes encoding DyPs was accompanied by the increased expression of transcripts for H2O2-generating enzymes such as alcohol oxidase, pyranose 2-oxidase, and glyoxal oxidases. Meanwhile, accumulation of transcripts for glycoside hydrolase and protease was observed, in agreement with abundant proteins. Moreover, the biochemical analysis of IlDyP2 and IlDyP1 confirmed that DyPs were able to catalyze the oxidation of typical peroxidases substrates ABTS, phenolic lignin compounds DMP, and guaiacol as well as non-phenolic lignin compound, veratryl alcohol. More importantly, IlDyP1 enhanced catalytic activity for veratryl alcohol oxidation in the presence of mediator 1-hydroxybenzotriazole, which was similar to the laccase/1-hydroxybenzotriazole system. Conclusions The results proved for the first time that DyPs depolymerized lignin individually, combining catalytic features of different peroxidases on the functional level. Therefore, DyPs may be considered an important part of ligninolytic system in wood-decaying fungi.

Xylanase releases xylo-oligosaccharides from dietary xylan, which stimulate the growth of the gut bacteria lactobacilli. Many lactobacilli adhere to dietary fibers, which may facilitate the assimilation of xylo-oligosaccharides and help them gain competence in the gut, but the underlying mechanisms remain elusive. Herein we report, from the highly abundant transcripts of Lactobacillus brevis cultured in wheat arabinoxylan supplemented with a xylanase, the identification of genes encoding four putative cell-surface WxL proteins (Lb630, Lb631, Lb632, and Lb635) and one S-layer protein (Lb1325) with either cellulose- or xylan-binding ability. The repetitively occurring WxL proteins were encoded by a gene cluster, among which Lb630 was chosen for further mutational studies. The analysis revealed three aromatic residues (F30, W61, and W156) that might be involved in the interaction of the protein with cellulose. A homology search in the genome of Enterococcus faecium identified three WxL proteins with conserved counterparts of these three aromatic residues, and they were also found to be able to bind cellulose and xylan. The findings suggested a role of the cell-surface WxL and S-layer proteins in assisting the cellular adhesion of L. brevis to plant cell wall polysaccharides.

Background and Purpose: Many imaging studies have reported structure alterations in patients with type 1 diabetes mellitus (T1DM) by using voxel-based morphometry (VBM). Nevertheless, the results reported were inconsistent and had not been reviewed quantitatively. Accordingly, the quantitative meta-analysis which including VBM studies of patients with T1DM was conducted.Materials and Methods: The gray matter volume alterations in patients with T1DM was estimated by using the software seed-based d mapping. Meantime, the meta-regression was applied to detect the effects of some demographics and clinical characteristics.Results: Six studies were finally included, which with 6 datasets comprising 414 T1DM patients and 216 healthy controls. The pooled meta-analyses detected that patients with T1DM showed robustly increased gray matter volume in the left dorsolateral superior frontal gyrus and middle frontal gyrus and a decreased gray matter volume in the right lingual gyrus, cerebellum, precuneus, the left inferior temporal gyrus, and middle temporal gyrus. The meta-regression showed that the mean age, the female patient's ratio, duration of illness and HbAlc% for T1DM patients were not linearly related with gray matter alterations.Conclusion: This meta-analysis demonstrates that gray matter volume decreases in T1DM patients were mainly locates in the cortical regions and cerebellum.

N-acetylglucosamine (GlcNAc) is an amino sugar that has been widely used in the nutraceutical and pharmaceutical industries. Recently, microbial production of GlcNAc has been developed. One major challenge for efficient biosynthesis of GlcNAc is to achieve appropriate carbon flux distribution between growth and production. Here, a synergistic substrate co-utilization strategy was used to address this challenge. Specifically, glycerol was utilized to support cell growth and generate glutamine and acetyl-CoA, which are amino and acetyl donors, respectively, for GlcNAc biosynthesis, while glucose was retained for GlcNAc production. Thanks to deletion of the 6-phosphofructokinase (PfkA and PfkB) and glucose-6-phosphate dehydrogenase (ZWF) genes, the main glucose catabolism pathways of Escherichia coli were blocked. The resultant mutant showed a severe defect in glucose consumption. Then, the GlcNAc production module containing glucosamine-6-phosphate synthase (GlmS*), glucosamine-6-phosphate N-acetyltransferase (GNA1*) and GlcNAc-6-phosphate phosphatase (YqaB) expression cassettes was introduced into the mutant, to drive the carbon flux from glucose to GlcNAc. Furthermore, co-utilization of glucose and glycerol was achieved by overexpression of glycerol kinase (GlpK) gene. Using the optimized fermentation medium, the final strain produced GlcNAc with a high stoichiometric yield of 0.64 mol/mol glucose. This study offers a promising strategy to address the challenge of distributing carbon flux in GlcNAc production.

Thermoanaerobacterium bryantii strain mel9T is a thermophilic bacterium isolated from a waste pile of a corn-canning factory. The genome of T. bryantii mel9T was sequenced and a hemicellulase gene cluster was identified. The cluster encodes seven putative enzymes, which are likely an endoxylanase, an α-glucuronidase, two oxidoreductases, two β-xylosidases, and one acetyl xylan esterase. These genes were designated tbxyn10A, tbagu67A, tbheoA, tbheoB, tbxyl52A, tbxyl39A, and tbaxe1A, respectively. Only TbXyn10A released reducing sugars from birchwood xylan, as shown by thin-layer chromatography analysis. The five components of the hemicellulase cluster (TbXyn10A, TbXyl39A, TbXyl52A, TbAgu67A, and TbAxe1A) functioned in synergy to hydrolyze birchwood xylan. Surprisingly, the two putative oxidoreductases increased the enzymatic activities of the gene products from the xylanolytic gene cluster in the presence of NADH and manganese ions. The two oxidoreductases were therefore named Hemicellulase-Enhancing Oxidoreductases (HEOs). All seven enzymes were thermophilic and acted in synergy to degrade xylans at 60 °C. Except for TbXyn10A, the other enzymes encoded by the gene cluster were conserved with high amino acid identities (85–100%) in three other Thermoanaerobacterium species. The conservation of the gene cluster is, therefore, suggestive of an important role of these enzymes in xylan degradation by these bacteria. The mechanism for enhancement of hemicellulose degradation by the HEOs is under investigation. It is anticipated, however, that the discovery of these new actors in hemicellulose deconstruction will have a significant impact on plant cell wall deconstruction in the biofuel industry.

Abstract Background In the biofuel industry, cellulase plays an indispensable role in hydrolyzing cellulose into fermentable glucose. Trichoderma reesei is a popular filamentous fungus with prominent ability to produce cellulase. While classical mutagenesis and modern multiplex genome engineering are both effective ways to improve cellulase production, successful obtaining of strains with improved cellulase-producing ability requires screening a large number of strains, which is time-consuming and labor intensive. Results Herein, we developed a versatile method coupling expression of the red fluorescence protein (DsRed) in T. reesei and fluorescence-assisted cell sorting (FACS) of germinated spores. This method was first established by expressing DsRed intracellularly under the control of the major cellulase cbh1 promoter in T. reesei, which allowed us to rapidly isolate cellulase hyperproducers from T. reesei progenies transformed with a dedicated transcriptional activator ace3 and from an atmospheric and room temperature plasma-created mutant T. reesei library. Since intracellularly expressed DsRed was expected to isolate mutations mainly affecting cellulase transcription, this method was further improved by displaying DsRed on the T. reesei cell surface, enabling isolation of strains with beneficial genetic alterations (overexpressing hac1 and bip1) affecting regulatory stages beyond transcription. Using this method, T. reesei cellulase hyperproducers were also successfully isolated from an Agrobacterium-mediated random insertional mutant library. Conclusions The coupled DsRed-FACS high-throughput screening method proved to be an effective strategy for fast isolation of T. reesei cellulase hyperproducers and could also be applied in other industrially important filamentous fungi.

Abstract Pyranose oxidase (POx) is a homotetrameric flavoprotein that catalyzes the oxidation of pyranose-configured sugars at position C-2 to corresponding 2-ketoaldoses. The wide substrate specificity makes POx potential for application in various biotechnological industries. In the present study we reported the gene cloning and heterologous expression of a POx from the basidiomycete Trametes sp. and functionally expressed the gene in Escherichia coli BL21(DE3). Based on sequence alignment, three residues were chosen for site-directed mutagenesis to obtain two single mutants (K312E and E539K) and two double mutants (T166A/E539K and K312E/E539K). In comparison to the wild-type, K312E shifted its optimal pH to 5.5 while the optimal temperature of E539K and K312E/E539K increased by 10 °C. The mutants retained more activities over broader pH ranges and higher temperatures and catalyzed d-glucose at higher efficiency (5800‒12,667 M−1 s−1 for the mutants versus 5083 M−1 s−1 for the wild-type). The recombinant POx and its mutants were all useful in gluten agglomeration and enlarging the loaf volume, which depends on the amounts of enzymes added. Interestingly, adding the same amount (0.5 nkat/g of flour) of wild-type and mutant enzymes differed in the change of loaf volumes, pinpointing that the catalytic activity is not the sole determinant in applying POx in breadmaking.

Abstract Background Irpex lacteus is one of the most potent white rot fungi for biological pretreatment of lignocellulose for second biofuel production. To elucidate the underlying molecular mechanism involved in lignocellulose deconstruction, genomic and transcriptomic analyses were carried out for I. lacteus CD2 grown in submerged fermentation using ball-milled corn stover as the carbon source. Results Irpex lacteus CD2 efficiently decomposed 74.9% lignin, 86.3% cellulose, and 83.5% hemicellulose in corn stover within 9 days. Manganese peroxidases were rapidly induced, followed by accumulation of cellulase and hemicellulase. Genomic analysis revealed that I. lacteus CD2 possessed a complete set of lignocellulose-degrading enzyme system composed mainly of class II peroxidases, dye-decolorizing peroxidases, auxiliary enzymes, and 182 glycoside hydrolases. Comparative transcriptomic analysis substantiated the notion of a selection mode of degradation. These analyses also suggested that free radicals, derived either from MnP-organic acid interplay or from Fenton reaction involving Fe2+ and H2O2, could play an important role in lignocellulose degradation. Conclusions The selective strategy employed by I. lacteus CD2, in combination with low extracellular glycosidases cleaving plant cell wall polysaccharides into fermentable sugars, may account for high pretreatment efficiency of I. lacteus. Our study also hints the importance of free radicals for future designing of novel, robust lignocellulose-degrading enzyme cocktails.

Abstract Background Cellulases of glycosyl hydrolase (GH) family 5 share a (β/α)8 TIM-barrel fold structure with eight βα loops surrounding the catalytic pocket. These loops exposed on the surface play a vital role in protein functions, primarily due to the interactions of some key amino acids with solvent and ligand molecules. It has been reported that motions of these loops facilitate substrate access and product release, and loops 6 and 7 located at the substrate entrance of the binding pocket promote proton transfer reaction at the catalytic site motions. However, the role of these flexible loops in catalysis of GH5 cellulase remains to be explored. Results In the present study, an acidic, mesophilic GH5 cellulase (with optimal activity at pH 4.0 and 70 °C), GtCel5, was identified in Gloeophyllum trabeum CBS 900.73. The specific activities of GtCel5 toward CMC-Na, barley β-glucan, and lichenan were 1117 ± 43, 6257 ± 26 and 5318 ± 54 U/mg, respectively. Multiple sequence alignment indicates that one amino acid residue at position 233 on the loop 6 shows semi-conservativeness and might contribute to the great catalytic performance. Saturation mutagenesis at position 233 was then conducted to reveal the vital roles of this position in enzyme properties. In comparison to the wild type, variants N233A and N233G showed decreased optimal temperature (− 10 °C) but increased activities (27 and 70%) and catalytic efficiencies (k cat/K m; 45 and 52%), respectively. The similar roles of position 233 in catalytic performance were also verified in the other two GH5 homologs, TeEgl5A and PoCel5, by reverse mutation. Further molecular dynamics simulations suggested that the substitution of asparagine with alanine or glycine may introduce more hydrogen bonds, increase the flexibility of loop 6, enhance the interactions between enzyme and substrate, and thus improve the substrate affinity and catalytic efficiency. Conclusion This study proposed a novel cellulase with potentials for industrial application. A specific position was identified to play key roles in cellulase–substrate interactions and enzyme catalysis. It is of great importance for understanding the binding mechanism of GH5 cellulases, and provides an effective strategy to improve the catalytic performance of cellulases.

Abstract Trichoderma reesei is well known as an industrial workhorse fungus in cellulase production. The low dissolved oxygen supply in the highly viscous medium of T. reesei remains a major bottleneck that hampers growth and cellulase production in submerged fermentation. Vitreoscilla hemoglobin (VHb) has been demonstrated to improve metabolism and protein production in different heterologous hosts under hypoxic conditions, but the use of VHb in T. reesei remains uninvestigated. This study examines the effect of VHb in improving T. reesei performance in submerged fermentation. The VHb gene (vgb)-expressing cassette was successfully transformed into the TU-6 strain, integrated into the genome of T. reesei, and functionally expressed with biological activity, which was confirmed by carbon monoxide difference analysis. Compared to the parent strain, the expression of VHb increased the glucose consumption rate of the transformant. Moreover, in cellulase-inducing medium total protein secretion of the VHb expressing strain was 2.2-fold of the parental strain and the filter paper cellulase activity was increased by 58% under oxygen-limiting conditions. In summary, our results demonstrate that VHb has beneficial effects on improving total protein secretion and cellulase activity of T. reesei in submerged fermentation.

Multicopper oxidases (MCOs) are a diverse group of enzymes that could catalyze the oxidation of different xenobiotic compounds, with simultaneous reduction in oxygen to water. Aside from laccase, one member of the MCO superfamily has shown great potential in the biodegradation of mycotoxins; however, the mycotoxin degradation ability of other MCOs is uncertain. In this study, a novel MCO-encoding gene, StMCO, from Streptomyces thermocarboxydus, was identified, cloned, and heterologously expressed in Escherichia coli. The purified recombinant StMCO exhibited the characteristic blue color and bivalent copper ion-dependent enzyme activity. It was capable of oxidizing the model substrate ABTS, phenolic compound DMP, and azo dye RB5. Notably, StMCO could directly degrade aflatoxin B1 (AFB1) and zearalenone (ZEN) in the absence of mediators. Meanwhile, the presence of various lignin unit-derived natural mediators or ABTS could significantly accelerate the degradation of AFB1 and ZEN by StMCO. Furthermore, the biological toxicities of their corresponding degradation products, AFQ1 and 13-OH-ZEN-quinone, were remarkably decreased. Our findings suggested that efficient degradation of mycotoxins with mediators might be a common feature of the MCOs superfamily. In summary, the unique properties of MCOs make them good candidates for degrading multiple major mycotoxins in contaminated feed and food.

Ligninolytic enzymes, including laccase, manganese peroxidase, and dye-decolorizing peroxidase (DyP), have attracted much attention in the degradation of mycotoxins. Among these enzymes, the possible degradation pathway of mycotoxins catalyzed by DyP is not yet clear. Herein, a DyP-encoding gene, StDyP, from Streptomyces thermocarboxydus 41291 was identified, cloned, and expressed in Escherichia coli BL21/pG-Tf2. The recombinant StDyP was capable of catalyzing the oxidation of the peroxidase substrate 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), phenolic lignin compounds 2,6-dimethylphenol, and guaiacol, non-phenolic lignin compound veratryl alcohol, Mn2+, as well as anthraquinone dye reactive blue 19. Moreover, StDyP was able to slightly degrade zearalenone (ZEN). Most importantly, we found that StDyP combined the catalytic properties of manganese peroxidase and laccase, and could significantly accelerate the enzymatic degradation of ZEN in the presence of their corresponding substrates Mn2+ and 1-hydroxybenzotriazole. Furthermore, the biological toxicities of the main degradation products 15-OH-ZEN and 13-OH-ZEN-quinone might be remarkably removed. These findings suggested that DyP might be a promising candidate for the efficient degradation of mycotoxins in food and feed.

The co-occurrence of multiple mycotoxins, including aflatoxin B1 (AFB1), zearalenone (ZEN) and deoxynivalenol (DON), widely exists in cereal-based animal feed and food. At present, most reported mycotoxins degrading enzymes target only a certain type of mycotoxins. Therefore, it is of great significance for mining enzymes involved in the simultaneous degradation of different types of mycotoxins. In this study, a dye-decolorizing peroxidase-encoding gene BsDyP from Bacillus subtilis SCK6 was cloned and expressed in Escherichia coli BL21/pG-Tf2. The purified recombinant BsDyP was capable of oxidizing various substrates, including lignin phenolic model compounds 2,6-dimethylphenol and guaiacol, the substrate 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid), anthraquinone dye reactive blue 19 and azo dye reactive black 5, as well as Mn2+. In addition, BsDyP could efficiently degrade different types of mycotoxins, including AFB1, ZEN and DON, in presence of Mn2+. More important, the toxicities of their corresponding enzymatic degradation products AFB1-diol, 15-OH-ZEN and C15H18O8 were significantly lower than AFB1, ZEN and DON. In summary, these results proved that BsDyP was a promising candidate for the simultaneous degradation of multiple mycotoxins in animal feed and food.

Abstract Background Manganese peroxidase is one of the Class II fungal peroxidases that are able to oxidize the low redox potential phenolic lignin compounds. For high redox potential non-phenolic lignin degradation, mediators such as GSH and unsaturated fatty acids are required in the reaction. However, it is not known whether carboxylic acids are a mediator for non-phenolic lignin degradation. Results The white rot fungus Irpex lacteus is one of the most potent fungi in degradation of lignocellulose and xenobiotics. Two manganese peroxidases (IlMnP1 and IlMnP2) from I. lacteus CD2 were over-expressed in Escherichia coli and successfully refolded from inclusion bodies. Both IlMnP1 and IlMnP2 oxidized the phenolic compounds efficiently. Surprisingly, they could degrade veratryl alcohol, a non-phenolic lignin compound, in a Mn2+-dependent fashion. Malonate or oxalate was found to be also essential in this degradation. The oxidation of non-phenolic lignin was further confirmed by analysis of the reaction products using LC–MS/MS. We proved that Mn2+ and a certain carboxylate are indispensable in oxidation and that the radicals generated under this condition, specifically superoxide radical, are at least partially involved in lignin oxidative degradation. IlMnP1 and IlMnP2 can also efficiently decolorize dyes with different structures. Conclusions We provide evidence that a carboxylic acid may mediate oxidation of non-phenolic lignin through the action of radicals. MnPs, but not LiP, VP, or DyP, are predominant peroxidases secreted by some white rot fungi such as I. lacteus and the selective lignocellulose degrader Ceriporiopsis subvermispora. Our finding will help understand how these fungi can utilize MnPs and an excreted organic acid, which is usually a normal metabolite, to efficiently degrade the non-phenolic lignin. The unique properties of IlMnP1 and IlMnP2 make them good candidates for exploring molecular mechanisms underlying non-phenolic lignin compounds oxidation by MnPs and for applications in lignocellulose degradation and environmental remediation.

Objective To investigate the effects of parenteral nutrition (PN) including ω-3 fish-oil emulsion on nutritional state, inflammatory response, and prognosis in patients with acute paraquat poisoning. Methods Patients randomized to receive medium chain triglycerides (MCT)/long chain triglycerides (LCT)-based PN (control group) or MCT/LCT-based PN containing ω-3 fish-oil emulsion (intervention group) were compared for 90-day survival and short-term treatment efficacy. Results Tumour necrosis factor-α levels were significantly lower in the intervention group ( n = 101) versus controls ( n = 73) on treatment days 4 and 7. Intervention group C-reactive protein (CRP) levels were significantly increased on day 4, decreased to baseline (day 1) levels on day 7, and were significantly lower than baseline on day 10. Control group CRP levels were significantly increased on days 4 and 7 versus baseline, and returned to baseline levels on day 10. On day 7, retinol binding protein had recovered to baseline levels in the intervention group only. Intervention group mortality rate (36.6%) was significantly lower than controls (57.5%). ω-3 fish-oil PN was associated with reduced risk of death (hazard ratio 0.52; 95% confidence interval 0.33, 0.82). Conclusion In patients with acute paraquat poisoning, MCT/LCT with ω-3 fish-oil emulsion PN plus combination treatment advantageously attenuated the inflammatory response, modified the nutritional state, and was associated with significantly improved 90-day survival versus treatment without ω-3 fish oil.