Estimation of multiple networks with common structures in heterogeneous subgroups.

Network estimation has been a critical component of high-dimensional data analysis and can provide an understanding of the underlying complex dependence structures. Among the existing studies, Gaussian graphical models have been highly popular. However, they still have limitations due to the homogeneous distribution assumption and the fact that they are only applicable to small-scale data. For example, cancers have various levels of unknown heterogeneity, and biological networks, which include thousands of molecular components, often differ across subgroups while also sharing some commonalities. In this article, we propose a new joint estimation approach for multiple networks with unknown sample heterogeneity, by decomposing the Gaussian graphical model (GGM) into a collection of sparse regression problems. A reparameterization technique and a composite minimax concave penalty are introduced to effectively accommodate the specific and common information across the networks of multiple subgroups, making the proposed estimator significantly advancing from the existing heterogeneity network analysis based on the regularized likelihood of GGM directly and enjoying scale-invariant, tuning-insensitive, and optimization convexity properties. The proposed analysis can be effectively realized using parallel computing. The estimation and selection consistency properties are rigorously established. The proposed approach allows the theoretical studies to focus on independent network estimation only and has the significant advantage of being both theoretically and computationally applicable to large-scale data. Extensive numerical experiments with simulated data and the TCGA breast cancer data demonstrate the prominent performance of the proposed approach in both subgroup and network identifications.

Elevated levels of alcohol dehydrogenase aggravate ethanol-evoked cardiac remodeling and contractile anomalies through FKBP5-yap-mediated regulation of ferroptosis and ER stress.

Alcohol intake provokes severe organ injuries including alcoholic cardiomyopathy with hallmarks of cardiac remodeling and contractile defects. This study examined the toxicity of facilitated ethanol metabolism in alcoholism-evoked changes in myocardial morphology and contractile function, insulin signaling and various cell death domains using cardiac-selective overexpression of alcohol dehydrogenase (ADH). WT and ADH mice were offered an alcohol liquid diet for 12 weeks prior to assessment of cardiac geometry, function, ER stress, apoptosis and ferroptosis. Alcohol intake provoked pronounced glucose intolerance, cardiac remodeling and contractile anomalies with apoptosis, ER stress, and ferroptosis, the effects were accentuated by ADH with the exception of global glucose intolerance. Hearts from alcohol ingesting mice displayed dampened insulin-stimulated phosphorylation of insulin receptor (tyr1146) and IRS-1 (tyrosine) along with elevated IRS-1 serine phosphorylation, the effect was augmented by ADH. Alcohol challenge dampened phosphorylation of Akt and GSK-3ß, and increased phosphorylation of c-Jun and JNK, the effects were accentuated by ADH. Alcohol challenge promoted ER stress, FK506 binding protein 5 (FKBP5), YAP, apoptosis and ferroptosis, the effects were exaggerated by ADH. Using a short-term ethanol challenge model (3 g/kg, i.p., twice in three days), we found that inhibition of FKBP5-YAP signaling or facilitated ethanol detoxification by Alda-1 alleviated ethanol cardiotoxicity. In vitro study revealed that the ethanol metabolite acetaldehyde evoked cardiac contractile anomalies, lipid peroxidation, and apoptosis, the effects of which were mitigated by Alda-1, inhibition of ER stress, FKBP5 and YAP. These data suggest that facilitated ethanol metabolism via ADH exacerbates alcohol-evoked myocardial remodeling, functional defects, and insulin insensitivity possibly through a FKBP5-YAP-associated regulation of ER stress and ferroptosis.

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.

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.

Sulfur vacancy-rich (α/ß-CdS)/SiO2 photocatalysts for enhanced visible-light-driven photocatalytic degradation of rhodamine B.

The development of highly efficient photocatalysts for visible-light-driven degradation of organic pollution is of great interest for wastewater purification. In this work, a sulfur vacancy-rich (α/ß-CdS)/SiO2 (α: hexagonal & ß: cubic) photocatalyst with a high catalytic activity was novelly synthesized on a nano-SiO2 carrier by the reaction of Cd2+ with a CS2 storage material (CS2SM) as sulfur source and crystalline modifiers. The dispersion of α/ß-CdS on the nano-SiO2 carrier significantly enhanced the visible-light-driven catalytic activity of (α/ß-CdS)/SiO2 photocatalyst, and 93.37 % rhodamine B (RhB) conversion was determined over 50 mg (α/ß-CdS)/SiO2 photocatalyst for 30 mL 400 mg/L RhB solution at light intensity of 150 mW/cm2 and 298.15 K. After five cycle tests, the (α/ß-CdS)/SiO2 photocatalyst still owned excellent visible-light-driven catalytic degradation stability (>90 %). The characterizations of morphology, functional groups, and photo-electrochemistry of (α/ß-CdS)/SiO2 photocatalyst demonstrated that nano-SiO2 as a carrier played meaningful role in dispersing α/ß-CdS and reducing agglomeration, thus increasing the active site of photocatalytic degradation reaction, and the presence of α/ß hetero-phase junctions and sulfur vacancies allows the rapid separation of photo-generated carriers and inhibits photo-generated electron-holes recombination. Meanwhile, the electron paramagnetic resonance (EPR) and free radical masking test have also proved that the main active species is ·O2- for the oxidation of RhB. Therefore, the work is providing a new reference to the visible-light-driven degradation of wastewater with high RhB concentration at room temperature.

FUNDC1 interacts with GPx4 to govern hepatic ferroptosis and fibrotic injury through a mitophagy-dependent manner.

INTRODUCTION: Liver fibrosis is a life-threatening pathological anomaly which usually evolves into advanced liver cirrhosis and hepatocellular carcinoma although limited therapeutic option is readily available. FUN14 domain containing 1 (FUNDC1) is a mitophagy receptor with little information in liver fibrosis. OBJECTIVE: This study was designed to examine the role for FUNDC1 in carbon tetrachloride (CCl4)-induced liver injury. METHODS: GEO database analysis and subsequent validation of biological processes including western blot, immunofluorescence, and co-immunoprecipitation were applied to clarify the regulatory role of FUNDC1 on mitophagy and ferroptosis. RESULTS: Our data revealed elevated FUNDC1 levels in liver tissues of patients with liver fibrotic injury and CCl4-challenged mice. FUNDC1 deletion protected against CCl4-induced hepatic anomalies in mice. Moreover, FUNDC1 deletion ameliorated CCl4-induced ferroptosis in vivo and in vitro. Mechanically, FUNDC1 interacted with glutathione peroxidase (GPx4), a selenoenzyme to neutralize lipid hydroperoxides and ferroptosis, via its 96-133 amino acid domain to facilitate GPx4 recruitment into mitochondria from cytoplasm. GPx4 entered mitochondria through mitochondrial protein import system-the translocase of outer membrane/translocase of inner membrane (TOM/TIM) complex, prior to degradation of GPx4 mainly through mitophagy along with ROS-induced damaged mitochondria, resulting in hepatocyte ferroptosis. CONCLUSION: Taken together, our data favored that FUNDC1 promoted hepatocyte injury through GPx4 binding to facilitate its mitochondrial translocation through TOM/TIM complex, where GPx4 was degraded by mitophagy to trigger ferroptosis. Targeting FUNDC1 may be a promising therapeutic approach for liver fibrosis.

Efficient production of γ-aminobutyric acid using engineered Escherichia coli whole-cell catalyst.

γ-Aminobutyric acid (GABA) has been widely used in the food, feed, pharmaceutical, and chemical industry fields. Previously, we developed a whole-cell catalyst capable of converting L-glutamate (L-Glu) into GABA by overexpressing the glutamate decarboxylase gene (gadz11) from Bacillus sp. Z11 in Escherichia coli BL21(DE3). However, to enhance cell permeability, a freeze-thaw treatment is required, and to enhance GADZ11 activity, pyridoxal 5'-phosphate (PLP) must be added to the reaction system. The aim of this study is to provide a more efficient approach for GABA production by engineering the recombinant E. coli above. First, the inducible expression conditions of the gadz11 in E. coli were optimized to 37 °C for 6 h. Next, an ideal engineered strain was produced via increasing cell permeability by overexpressing sulA and eliminating PLP dependence by constructing a self-sufficient system. Furthermore, an efficient whole-cell biocatalytic process was optimized. The optimal substrate concentration, cell density, and reaction temperature were 1.0 mol/L (the molecular ratio of L-Glu to L-monosodium glutamate (L-MSG) was 4:1), 15 and 37 °C, respectively. Finally, a whole-cell bioconversion procedure was performed in a 3-L bioreactor under optimal conditions. The strain could be reused for at least two cycles with GABA yield, productivity and conversion ratio of 206.2 g/L, 117.8 g/L/h and 100.0%, respectively. This is currently the highest GABA productivity from a mixture of L-Glu and L-MSG reported without the addition of cofactors or additional treatment of cells. This work demonstrates that the novel engineered E. coli strain has the potential for application in large-scale industrial GABA production.

Establishment of Biliary Atresia Prognostic Classification System via Survival-Based Forward Clustering - A New Biliary Atresia Classification.

OBJECTIVES: To develop a machine learning algorithm with prognosis data to identify different clinical phenotypes of biliary atresia (BA) and provide instructions for choosing treatment schemes. METHODS: Six hundred thirty-nine cases of type III BA were retrospectively collected from the Children's Hospital of Fudan University from Jan 1st, 2017 to Dec 1st, 2019 as a training dataset, and a survival-based forward clustering method, which can also be used to predict the subtype of a new patient was developed to identify BA subtypes. RESULTS: A total of 2 clusters were identified (cluster 1 = 324 and cluster 2 = 315), where cluster 2 had a lower 2 y native liver survival post-Kasai rate. The infant patients in cluster 2 have higher weight, liver, and spleen volume, wider portal vein width, and older operative age; worse coagulation and liver function results; higher grade of liver fibrosis and detection rate of hepatic portal fibrous mass, and higher recent infection detection rate of herpes simplex virus type I. With the proposed prognostic classification system, the authors predicted the subtypes of the 187 cases of type III BA in a testing dataset collected from the whole year of 2020. The p-value computed from the log-rank testing for the Kaplan-Meier survival curves of the predicted two testing groups was 0.0113. CONCLUSIONS: This classification system would be a convenient tool to choose appropriate treatment and accelerate the choice-making between clinicians and infant patients.

[Metabolic mechanism and intervention strategy of atrial fibrillation in the elderly].

Atrial fibrillation (AF) is a cardiovascular epidemic that occurs primarily in the elderly with primary cardiovascular diseases, leading to severe consequences such as stroke and heart failure. The heart is an energy-consuming organ, which requires a high degree of metabolic flexibility to ensure a quick switch of metabolic substrates to meet its energy needs in response to physiological and pathological stimulation. Metabolism is closely related to the occurrence of AF, and AF patients manifest metabolic inflexibility, such as insulin resistance and the metabolic shift from aerobic metabolism to anaerobic glycolysis. Moreover, our research group and the others have shown that metabolic inflexibility is a crucial pathologic mechanism for AF. Energy metabolism is closely linked to the aging process and aging-related diseases, and impaired metabolic flexibility is considered as an essential driver of aging. Therefore, this review focuses on the alteration of metabolic flexibility in the elderly and reveals that impaired metabolic flexibility may be an important driver for the high prevalence of AF in the elderly, hoping to provide intervention strategies for the prevention and treatment of AF in the elderly.

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.

Exploring the cellulolytic and hemicellulolytic activities of manganese peroxidase for lignocellulose deconstruction.

BACKGROUND: A cost-effective pretreatment and saccharification process is a necessary prerequisite for utilizing lignocellulosic biomass (LCB) in biofuel and biomaterials production. Utilizing a multifunctional enzyme with both pretreatment and saccharification functions in a single step for simultaneous biological pretreatment and saccharification process (SPS) will be a green method of low cost and high efficiency. Manganese peroxidase (MnP, EC 1.11.1.13), a well-known lignin-degrading peroxidase, is generally preferred for the biological pretreatment of biomass. However, exploring the role and performance of MnP in LCB conversion will promote the application of MnP for lignocellulose-based biorefineries. RESULTS: In this study, we explored the ability of an MnP from Moniliophthora roreri, MrMnP, in LCB degradation. With Mn2+ and H2O2, MrMnP decomposed 5.0 g/L carboxymethyl cellulose to 0.14 mM of reducing sugar with a conversion yield of 5.0 mg/g, including 40 µM cellobiose, 70 µM cellotriose, 20 µM cellotetraose, and 10 µM cellohexaose, and degraded 1.0 g/L mannohexaose to 0.33 µM mannose, 4.08 µM mannotriose, and 4.35 µM mannopentaose. Meanwhile, MrMnP decomposed 5.0 g/L lichenan to 0.85 mM of reducing sugar with a conversion yield of 30.6 mg/g, including 10 µM cellotriose, 20 µM cellotetraose, and 80 µM cellohexose independently of Mn2+ and H2O2. Moreover, the versatility of MrMnP in LCB deconstruction was further verified by decomposing locust bean gum and wheat bran into reducing sugars with a conversion yield of 54.4 mg/g and 29.5 mg/g, respectively, including oligosaccharides such as di- and tri-saccharides. The catalytic mechanism underlying MrMnP degraded lignocellulose was proposed as that with H2O2, MrMnP oxidizes Mn2+ to Mn3+. Subsequently, it forms a complex with malonate, facilitating the degradation of CMC and mannohexaose into reducing sugars. Without H2O2, MrMnP directly oxidizes malonate to hydroperoxyl acetic acid radical to form compound I, which then attacks the glucosidic bond of lichenan. CONCLUSION: This study identified a new function of MrMnP in the hydrolysis of cellulose and hemicellulose, suggesting that MrMnP exhibits its versatility in the pretreatment and saccharification of LCB. The results will lead to an in-depth understanding of biocatalytic saccharification and contribute to forming new enzymatic systems for using lignocellulose resources to produce sustainable and economically viable products and the long-term development of biorefinery, thereby increasing the productivity of LCB as a green resource.

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.

Improving the Activity and Stability of Serine Protease ThAPT3 by Alleviating Self-Cleavage and Its Application in Deproteinization of Shrimp Shells.

The self-cleavage properties of proteases result in low activity and instability, which limit their industrial application. In this study, the serine protease ThAPT3 from Torrubiella hemipterigena was successfully expressed in Komagataella phaffii. We investigated the self-degradation mechanism of ThAPT3 and presented a rational strategy to alleviate self-cleavage. A major self-degradation site (Leu238-Met239) and a primary autolysis region were identified. The autolysis regions (loop18, α8-helix, and loop19) were redesigned and optimized using loop transplantation, energy calculations, surface cavity optimization, and loop anchoring. A triple-superposition mutant, ThAPT3-M9 (M239GKDGAVAAGLC250 → M239TLNRTTAANAC250/A251E/A254Q/R259L/A267E/S280N), was obtained. Compared to the wild type, the autolysis of M9 was significantly alleviated, and its half-life at 60 °C was increased approximately 39-fold (from 1.6 to 62.4 min). The optimal temperature and specific activity of M9 increased by 5 °C (from 60 to 65 °C) and 62% (4985 vs 3078 U/mg), respectively. M9 showed significant advantages in shrimp shell deproteinization.

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.

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.

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..

Recognition of Abnormal-Laying Hens Based on Fast Continuous Wavelet and Deep Learning Using Hyperspectral Images.

The egg production of laying hens is crucial to breeding enterprises in the laying hen breeding industry. However, there is currently no systematic or accurate method to identify low-egg-production-laying hens in commercial farms, and the majority of these hens are identified by breeders based on their experience. In order to address this issue, we propose a method that is widely applicable and highly precise. First, breeders themselves separate low-egg-production-laying hens and normal-laying hens. Then, under a halogen lamp, hyperspectral images of the two different types of hens are captured via hyperspectral imaging equipment. The vertex component analysis (VCA) algorithm is used to extract the cockscomb end member spectrum to obtain the cockscomb spectral feature curves of low-egg-production-laying hens and normal ones. Next, fast continuous wavelet transform (FCWT) is employed to analyze the data of the feature curves in order to obtain the two-dimensional spectral feature image dataset. Finally, referring to the two-dimensional spectral image dataset of the low-egg-production-laying hens and normal ones, we developed a deep learning model based on a convolutional neural network (CNN). When we tested the model's accuracy by using the prepared dataset, we found that it was 0.975 percent accurate. This outcome demonstrates our identification method, which combines hyperspectral imaging technology, an FCWT data analysis method, and a CNN deep learning model, and is highly effective and precise in laying-hen breeding plants. Furthermore, the attempt to use FCWT for the analysis and processing of hyperspectral data will have a significant impact on the research and application of hyperspectral technology in other fields due to its high efficiency and resolution characteristics for data signal analysis and processing.

Identification and Characterization of the Determinants of Copper Resistance in the Acidophilic Fungus Acidomyces richmondensis MEY-1 Using the CRISPR/Cas9 System.

Copper (Cu) homeostasis has not been well documented in filamentous fungi, especially extremophiles. One of the main obstacles impeding their characterization is the lack of a powerful genome-editing tool. In this study, we applied a CRISPR/Cas9 system for efficient targeted gene disruption in the acidophilic fungus Acidomyces richmondensis MEY-1, formerly known as Bispora sp. strain MEY-1. Using this system, we investigated the basis of Cu tolerance in strain MEY-1. This strain has extremely high Cu tolerance among filamentous fungi, and the transcription factor ArAceA (A. richmondensis AceA) has been shown to be involved in this process. The ArAceA deletion mutant (ΔArAceA) exhibits specific growth defects at Cu concentrations of ≥10 mM and is transcriptionally more sensitive to Cu than the wild-type strain. In addition, the putative metallothionein ArCrdA was involved in Cu tolerance only under high Cu concentrations. MEY-1 has no Aspergillus nidulans CrpA homologs, which are targets of AceA-like transcription factors and play a role in Cu tolerance. Instead, we identified the Cu-transporting P-type ATPase ArYgA, homologous to A. nidulans YgA, which was involved in pigmentation rather than Cu tolerance. When the ΔArYgA mutant was grown on medium supplemented with Cu ions, the black color was completely restored. The lack of CrpA homologs in A. richmondensis MEY-1 and its high tolerance to Cu suggest that a novel Cu detoxification mechanism differing from the AceA-CrpA axis exists. IMPORTANCE Filamentous fungi are widely distributed worldwide and play an important ecological role as decomposers. However, the mechanisms of their adaptability to various environments are not fully understood. Various extremely acidophilic filamentous fungi have been isolated from acidic mine drainage (AMD) with extremely low pH and high heavy metal and sulfate concentrations, including A. richmondensis. The lack of genetic engineering tools, particularly genome-editing tools, hinders the study of these acidophilic and heavy metal-resistant fungi at the molecular level. Here, we first applied a CRISPR/Cas9-mediated gene-editing system to A. richmondensis MEY-1. Using this system, we identified and characterized the determinants of Cu resistance in A. richmondensis MEY-1. The conserved roles of the Cu-binding transcription factor ArAceA in Cu tolerance and the Cu-transporting P-type ATPase ArYgA in the Cu-dependent production of pigment were confirmed. Our findings provide insights into the molecular basis of Cu tolerance in the acidophilic fungus A. richmondensis MEY-1. Furthermore, the CRISPR/Cas9 system used here would be a powerful tool for studies of the mechanisms of adaptability of acidophilic fungi to extreme environments.

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.