Efficient biosynthesis of transglutaminase in Streptomyces mobaraensis via systematic engineering strategies.

Transglutaminases (TGases) have been widely used in food, pharmaceutical, biotechnology, and other industries because of their ability to catalyze deamidation, acyl transfer, and crosslinking reactions between Ƴ-carboxamide groups of peptides or protein-bound glutamine and the Ɛ-amino group of lysine. In this study, we demonstrated an efficient systematic engineering strategy to enhance the synthesis of TGase in a recombinant Streptomyces mobaraensis smL2020 strain in a 1000-L fermentor. Briefly, the enzymatic properties of the TGase TGL2020 from S. mobaraensis smL2020 and TGase TGLD from S. mobaraensis smLD were compared to obtain the TGase TGLD with perfected characteristics for heterologous expression in a recombinant S. mobaraensis smL2020ΔTG without the gene tgL 2020. Through multiple engineering strategies, including promoter engineering, optimizing the signal peptides and recombination sites, and increasing copies of the expression cassettes, the final TGLD activity in the recombinant S. mobaraensis smL2020ΔTG: (PL2020-spL2020-protgLD-tgLD)2 (tgL2020and BT1) reached 56.43 U/mL and 63.18 U/mL in shake flask and 1000-L fermentor, respectively, which was the highest reported to date. With the improvement of expression level, the application scope of TGLD in the food industry will continue to expand. Moreover, the genetic stability of the recombinant strain maintained at more than 20 generations. These findings proved the feasibility of multiple systematic engineering strategies in synthetic biology and provided an emerging solution to improve biosynthesis of industrial enzymes.

Isolation and Identification of Indole Alkaloids from Aspergillus amstelodami BSX001 and Optimization of Ultrasound-Assisted Extraction of Neoechinulin A.

This study aimed to investigate the alkaloid secondary metabolites of Aspergillus amstelodami BSX001, a fungus isolated from Anhua dark tea, and to improve the extraction yield of the active ingredients by optimizing the extraction process. The structural characterization of the compounds was investigated using mass spectrometry (MS) and nuclear magnetic resonance (NMR) spectroscopy. The antioxidant activity of echinulin-related alkaloids was evaluated by determining the total reducing power and DPPH radical scavenging capacity. The extraction process of the compound with optimum activity was optimized by a single-factor test and response surface methodology (RSM) combined with Box-Behnken design (BBD). The optimized result was validated. Finally, a new alkaloid 8-hydroxyechinulin (1), and four known alkaloids, variecolorin G (2), echinulin (3), neoechinulin A (4), and eurocristatine (5), were isolated. Echinulin-related compounds 1, 3, and 4 possessed certain antioxidant activities, with IC50 values of 0.587 mg/mL, 1.628 mg/mL, and 0.219 mg/mL, respectively, against DPPH radicals. Their total reducing power at a concentration of 0.5 mg/mL was 0.29 mmol/L, 0.17 mmol/L, and 4.25 mmol/L. The extraction process of neoechinulin A was optimized with the optimum extraction parameters of 72.76% methanol volume fraction, 25 mL/g solid-liquid ratio, and 50.8 °C soaking temperature. Under these conditions, the extraction yield of neoechinulin A was up to 1.500 mg/g.

Semi-rational design for enhancing thermostability of Culex pipiens acetylcholinesterase and sensitivity analysis of acephate.

Acetylcholinesterase (AChE) has emerged as a significant biological recognition element in the biosensor field, particularly for the detection of insecticides. Nevertheless, the weak thermostability of AChE restricts its utilization due to the complexities associated with production, storage, and application environments. By evaluating the binding affinity between representative AChE and insecticides, an AChE from Culex pipiens was screened out, which displayed a broad-spectrum and high sensitivity to insecticides. The C. pipiens AChE (CpA) was subsequently expressed in Escherichia coli (E. coli) as a soluble active protein. Furthermore, a three-point mutant, M4 (A340P/D390E/S581P), was obtained using a semi-rational design strategy that combined molecular dynamics (MD) simulation and computer-aided design, which exhibited a four-fold increase in half-life at 40 °C compared to the wild-type (WT) enzyme. The mutant M4 also demonstrated an optimal temperature of 50 °C and a melting temperature (Tm) of 51.2 °C. Additionally, the sensitivity of WT and M4 to acephate was examined, revealing a 50-fold decrease in the IC50 value of M4. The mechanism underlying the improvement in thermal performance was elucidated through secondary structure analysis and MD simulations, indicating an increase in the proportion of protein helices and local structural rigidity. MD analysis of the protein-ligand complexes suggested that the enhanced sensitivity of M4 could be attributed to frequent specific contacts between the organophosphorus (OP) group of acephate and the key active site residue Ser327. These findings have expanded the possibilities for the development of more reliable and effective industrial enzyme preparations and biosensors.

Surface-displayed phenolic acid decarboxylase for increased vinylphenolic pyranoanthocyanins in blueberry wine.

During the fruit wine production, phenolic acid decarboxylase (PAD) converts free hydroxycinnamic acid into 4-vinyl derivatives that can then react spontaneously with anthocyanins, generating more stable pyranoanthocyanins that are responsible for the color stability of fruit wine. Nevertheless, the low PAD activity in yeast under the winemaking conditions has largely limited the generation of 4-vinyl derivatives. To bridge this gap, we expressed PAD from Bacillus amyloliquefaciens in Pichia pastoris and surface-displayed it on Saccharomyces cerevisiae. As a result, S. cerevisiae surface-displayed PAD (SDPAD) exhibited an enhanced thermal stability and tolerance to acidic conditions. Fermentation experiments showed that SDPAD can significantly increase the content of vinylphenolic pyranoanthocyanins and thus maintain the color stability of blueberry wine. Our study demonstrated the feasibility of surface display technology for color stability enhancement during the production of blueberry wine, providing a new and effective solution to increase the content of vinylphenolic pyranoanthocyanins in the fruit-based wines.

Spatial and temporal differences and convergence analysis of multidimensional relative poverty in ethnic areas.

Reducing multidimensional relative poverty is one of the important issues in the current global poverty governance field. This article takes 12 ethnic regions in China as the research object and constructs a multidimensional relative poverty measurement system. The calculated multidimensional relative poverty index is decomposed according to provinces, cities, dimensions, and indicators. Then, the Dagum Gini coefficient and convergence analysis are used to analyze spatiotemporal heterogeneity and convergence characteristics. The results show that the multi-dimensional relative poverty situation of various provinces in ethnic minority areas has improved from 2012 to 2021, among which Tibet province is the most serious and Shaanxi is the best. According to the analysis of convergence, it was observed that there is no σ-convergence of multidimensional relative poverty in ethnic areas in general, and there is absolute ß-convergence in general and in the southwest and northwest regions, and there is no absolute ß-convergence in the northeast region. Based on this, policy recommendations for reducing multidimensional relative poverty are proposed at the end of the article. Compared with previous studies, this article focuses on ethnic regions that are easily overlooked. Starting from the dimensions of economy, social development, and ecological environment, the poverty measurement system has been enriched.

Metabolic Engineering of Escherichia coli for Production of 2,5-Dimethylpyrazine.

2,5-Dimethylpyrazine (2,5-DMP) is a high-value-added alkylpyrazine compound with important applications in both the food and pharmaceutical fields. In response to the increasing consumer preference for natural products over chemically synthesized ones, efforts have been made to develop efficient microbial cell factories for the production of 2,5-DMP. However, the previously reported recombinant strains have exhibited low yields and relied on expensive antibiotics and inducers. In this study, we employed metabolic engineering strategies to develop an Escherichia coli strain capable of producing 2,5-DMP at high levels without the need for inducers or antibiotics. Initially, the biosynthesis pathway of 2,5-DMP was constructed that realized 2,5-DMP production from glucose. Subsequently, efforts focused on enhancing 2,5-DMP production by improving the availability of the cofactor NAD+ and precursor l-threonine. Additionally, the supply and conversion of l-threonine were balanced by optimizing the copy number of the key gene tdh on the chromosome and by modifying the l-threonine transport system. The final engineering strain D19 produced 3.1 g/L of 2,5-DMP, which is the highest titer for fermentative production of 2,5-DMP using glucose as the carbon source up to date. The strategies used in this study lay a good foundation for the production of 2,5-DMP on a large scale.

Light irradiation changes the regulation pattern of BtCrgA on carotenogenesis in Blakeslea trispora.

CrgA has been shown to be a negative regulator of carotenogenesis in some filamentous fungi, while light irradiation is an inducible environmental factor for carotenoid biosynthesis. To clarify the relationship between CrgA and light-inducible carotenogenesis in Blakeslea trispora, the cis-acting elements of the btcrgA promoter region were investigated, followed by the analyses of correlation between the expression of btcrgA and carotenoid structural genes under different irradiation conditions. A variety of cis-acting elements associated with light response was observed in the promoter region of btcrgA, and transcription of btcrgA and carotenoid structural genes under different irradiation conditions was induced by white light with a clear correlation. Then, RNA interference and overexpression of btcrgA were performed to investigate their effects on carotenogenesis at different levels under irradiation and darkness. The analyses of transcription and enzyme activities of carotenoid structural gene, and accumulation of carotenoids among btcrgA-interfered, btcrgA-overexpressed, and wild-type strains under irradiation and darkness indicate that btcrgA negatively regulates the synthesis of carotenoid in darkness, while promotes the carotenogenesis under irradiation regardless of reduced or overexpression of btcrgA .

Effect of in vitro simulated digestion on the physicochemical properties and pancreatic lipase inhibitory activity of fucoidan.

BACKGROUND: Fucoidan has an anti-obesity effect. However, there are few studies on its mechanism. In this study, we investigated the in vitro and in silico inhibitory properties of fucoidan against pancreatic lipase for the first time. We examined the changes in composition, structure, and pancreatic lipase inhibition of fucoidan during in vitro digestion. RESULTS: Simulated saliva-gastrointestinal digestion resulted in a slight decrease in the molecular weight of fucoidan but no significant changes in the monosaccharide composition, sulfate content, and functional groups. Moreover, the digestion process significantly increased the inhibition of pancreatic lipase by fucoidan. The study on the type of inhibition showed that the inhibition of pancreatic lipase by fucoidan belonged to mixed inhibition with competitive inhibition. Molecular docking analysis showed that fucoidan could bind to the active site of pancreatic lipase. CONCLUSION: This study indicates that fucoidan can be a potential functional food for anti-obesity. © 2024 Society of Chemical Industry.

Enhancement of pyranoanthocyanin formation in blueberry wine with non-Saccharomyces yeasts.

The development of blueberry wine provides an alternative method for maintaining the nutritional value and extending the shelf life of blueberries. However, anthocyanin loss and off-flavor compound generation during fermentation impair blueberry wine color and quality. Hydroxycinnamate decarboxylase from yeast can catalyze the conversion of hydroxycinnamic acids to vinylphenols, which later may condense with anthocyanins to form more stable vinylphenolic pyranoanthocyanins. In this study, 10 non-Saccharomyces yeasts from Daqu that showed hydroxycinnamate decarboxylase activity were screened. Among the 10 strains, Wickerhamomyces anomalus Y5 showed the highest consumption (34.59%) of the total tested phenolic acids and almost no H2S production. Furthermore, Y5 seemed to produce four vinylphenol pyranoanthocyanins (cyanidin-3-O-galactoside/glucoside-4-vinylcatechol, cyanidin-3-O-galactoside/glucoside-4-vinylsyringol, malvidin-4-vinylguaiacol, and malvidin-4-vinylcatechol) during blueberry wine fermentation, which may improve the color stability of blueberry wine. These findings provide new insights for improving the quality of blueberry wine using non-Saccharomyces yeasts.

Preparation of Azoxystrobin-Zinc Metal-Organic Framework/Biomass Charcoal Composite Materials and Application in the Prevention and Control of Gray Mold in Tomato.

In order to reduce the use of fungicide and ensure food safety, it is necessary to develop fungicide with low toxicity and high efficiency to reduce residues. Azoxystrobin (AZOX), which is derived from mushrooms, is an excellent choice. However, conventional AZOX release is difficult to regulate. In this paper, a pH-responsive fungicide delivery system for the preparation of AZOX by impregnation method was reported. The Zinc metal-organic framework/Biomass charcoal (ZIF-8/BC) support was first prepared, and subsequently, the AZOX-ZIF-8/BC nano fungicide was prepared by adsorption of AZOX onto ZIF-8/BC by dipping. Gray mold, caused by Botrytis cinerea, is one of the most important crop diseases worldwide. AZOX-ZIF-8/BC could respond to oxalic acid produced by Botrytis cinerea to release loaded AZOX. When pH = 4.8, it was 48.42% faster than when pH = 8.2. The loading of AZOX on ZIF-8/BC was 19.83%. In vitro and pot experiments showed that AZOX-ZIF-8/BC had significant fungicidal activity, and 300 mg/L concentration of AZOX-ZIF-8-BC could be considered as a safe and effective control of Botrytis cinerea. The above results indicated that the prepared AZOX-ZIF-8/BC not only exhibited good drug efficacy but also demonstrated pH-responsive fungicide release.

Detection of insecticides by Tetronarce californica acetylcholinesterase via expression and in silico analysis.

The acetylcholinesterase (AChE) is involved in termination of synaptic transmission at cholinergic synapses and plays a vital role in the insecticide detection and inhibitor screening. Here, we report the heterologous expression of an AChE from Tetronarce californica (TcA) in Escherichia coli (E. coli) as a soluble active protein. TcA was immobilized in calcium alginate beads; the morphology, biochemical properties, and insecticide detection performance of free and immobilized TcA were characterized. Moreover, we used sequence, structure-based approaches, and molecular docking to investigate structural and functional characterization of TcA. The results showed that TcA exhibited a specific activity of 102 U/mg, with optimal activity at pH 8.0 and 30 °C. Immobilized TcA demonstrated superior thermal stability, pH stability, and storage stability compared to the free enzyme. The highest sensitivity of free TcA was observed with trichlorfon, whereas immobilized TcA showed reduced IC50 values towards tested insecticides by 3 to 180-fold. Molecular docking analysis revealed the interaction of trichlorfon, acephate, isoprocarb, λ-cyhalothrin, and fenpropathrin in the active site gorge of TcA, particularly mediated through the formation of hydrogen bonds and π-π stacking. Therefore, TcA expressed heterologously in E. coli is a promising candidate for applications in food safety and environmental analysis. KEY POINTS: • T. californica AChE was expressed solubly in prokaryotic system. • The biochemical properties of free/immobilized enzyme were characterized. • The sensitivity of enzyme to insecticides was evaluated in vitro and in silico.

Endoscopic Ruler for varix size measurement: A multicenter pilot study.

BACKGROUND: We invented Endoscopic Ruler, a new endoscopic device to measure the size of varices in patients with cirrhosis and portal hypertension. AIM: To assess the feasibility and safety of Endoscopic Ruler, and evaluate the agreement on identifying large oesophageal varices (OV) between Endoscopic Ruler and the endoscopists, as well as the interobserver agreement on diagnosing large OV using Endoscopic Ruler. METHODS: We prospectively and consecutively enrolled patients with cirrhosis from 11 hospitals, all of whom got esophagogastroduodenoscopy (EGD) with Endoscopic Ruler. The primary study outcome was a successful measurement of the size of varices using Endoscopic Ruler. The secondary outcomes included adverse events, operation time, the agreement of identifying large OV between the objective measurement of Endoscopic Ruler and the empirical reading of endoscopists, together with the interobserver agreement on diagnosing large OV by Endoscopic Ruler. RESULTS: From November 2020 to April 2022, a total of 120 eligible patients with cirrhosis were recruited and all of them underwent EGD examinations with Endoscopic Ruler successfully without any adverse event. The median operation time of Endoscopic Ruler was 3.00 min [interquartile range (IQR): 3.00 min]. The kappa value between Endoscopic Ruler and the endoscopists while detecting large OV was 0.52, demonstrating a moderate agreement. The kappa value for diagnosing large OV using Endoscopic Ruler among the six independent observers was 0.77, demonstrating a substantial agreement. CONCLUSION: The data demonstrates that Endoscopic Ruler is feasible and safe for measuring the size of varices in patients with cirrhosis and portal hypertension. Endoscopic Ruler is potential to promote the clinical practice of the two-grade classification system of OV.

Molecular mechanisms of TWIST1-regulated transcription in EMT and cancer metastasis.

TWIST1 induces epithelial-to-mesenchymal transition (EMT) to drive cancer metastasis. It is yet unclear what determines TWIST1 functions to activate or repress transcription. We found that the TWIST1 N-terminus antagonizes TWIST1-regulated gene expression, cancer growth and metastasis. TWIST1 interacts with both the NuRD complex and the NuA4/TIP60 complex (TIP60-Com) via its N-terminus. Non-acetylated TWIST1-K73/76 selectively interacts with and recruits NuRD to repress epithelial target gene transcription. Diacetylated TWIST1-acK73/76 binds BRD8, a component of TIP60-Com that also binds histone H4-acK5/8, to recruit TIP60-Com to activate mesenchymal target genes and MYC. Knockdown of BRD8 abolishes TWIST1 and TIP60-Com interaction and TIP60-Com recruitment to TWIST1-activated genes, resulting in decreasing TWIST1-activated target gene expression and cancer metastasis. Both TWIST1/NuRD and TWIST1/TIP60-Com complexes are required for TWIST1 to promote EMT, proliferation, and metastasis at full capacity. Therefore, the diacetylation status of TWIST1-K73/76 dictates whether TWIST1 interacts either with NuRD to repress epithelial genes, or with TIP60-Com to activate mesenchymal genes and MYC. Since BRD8 is essential for TWIST1-acK73/76 and TIP60-Com interaction, targeting BRD8 could be a means to inhibit TWIST1-activated gene expression.

A Review of the Development and Challenges of Cell Mechanical Models.

Cell models can express a variety of cell information, including mechanical properties, electrical properties, and chemical properties. Through the analysis of these properties, we can fully understand the physiological state of cells. As such, cell modeling has gradually become a topic of great interest, and a number of cell models have been established over the last few decades. In this paper, the development of various cell mechanical models has been systematically reviewed. First, continuum theoretical models, which were established by ignoring cell structures, are summarized, including the cortical membrane droplet model, solid model, power series structure damping model, multiphase model, and finite element model. Next, microstructural models based on the structure and function of cells are summarized, including the tension integration model, porous solid model, hinged cable net model, porous elastic model, energy dissipation model, and muscle model. What's more, from multiple viewpoints, the strengths and weaknesses of each cell mechanical model have been analyzed in detail. Finally, the potential challenges and applications in the development of cell mechanical models are discussed. This paper contributes to the development of different fields, such as biological cytology, drug therapy, and bio-syncretic robots.

[Protopanaxadiol-type ginsenoside hydrolases and their application in the preparation of ginsenoside Compound K: a review].

Ginsenoside Compound K (CK) has anti-cancer and anti-inflammatory pharmacological activities. It has not been isolated from natural ginseng and is mainly prepared by deglycosylation of protopanaxadiol. Compared with the traditional physicochemical preparation methods, the preparation of CK by hydrolysis with protopanaxadiol-type (PPD-type) ginsenoside hydrolases has the advantages of high specificity, environmental-friendliness, high efficiency and high stability. In this review, the PPD-type ginsenoside hydrolases were classified into three categories based on the differences in the glycosyl-linked carbon atoms of the hydrolase action. It was found that most of the hydrolases that could prepare CK were PPD-type ginsenoside hydrolase type Ⅲ. In addition, the applications of hydrolases in the preparation of CK were summarized and evaluated to facilitate large-scale preparation of CK and its development in the food and pharmaceutical industries.

High-Value Bioconversion of Ginseng Extracts in Betaine-Based Deep Eutectic Solvents for the Preparation of Deglycosylated Ginsenosides.

Deep eutectic solvents (DES), as a green alternative to traditional organic solvents in biocatalysis, not only activate proteins but even increase the efficiency of enzymatic reactions. Here, DES were used in a combinatorial enzyme-catalyzed system containing ß-glucosidase BGLAt and ß-galactosidase BGALAo to produce deglycosylated ginsenosides (De-g) from ginseng extracts (GE). The results showed that DES prepared with betaine and ethylene glycol (molar ratio, 1:2) could significantly stimulate the activity of the combinatorial enzymes as well as improve the acid resistance and temperature stability. The DES-based combinatorial enzyme-catalyzed system could convert 5 g of GE into 1.24 g of De-g (F1, F2, 20 (S)-PPT, and CK) at 24 h, which was 1.1 times that of the buffer sample. As confirmed by the spectral data, the changes in the conformations of the combinatorial enzymes were more favorable for the binding reaction with the substrates. Moreover, the constructed DES-based aqueous two-phase system enabled the recovery of substantial amounts of DES and De-g from the top phase. These results demonstrated that DES shows great application as a reaction solvent for the scale-up production of De-g and provide insights for the green extraction of natural products.

Combinatorial Enzymatic Catalysis for Bioproduction of Ginsenoside Compound K.

Ginsenoside compound K (CK) is an emerging functional food or pharmaceutical product. To date, there are still challenges to exploring effective catalytic enzymes for enzyme-catalyzed manufacturing processes and establishing enzyme-catalyzed processes. Herein, we identified three ginsenoside hydrolases BG07 (glucoamylase), BG19 (ß-glucosidase), and BG23 (ß-glucosidase) from Aspergillus tubingensis JE0609 by transcriptome analysis and peptide mass fingerprinting. Among them, BG23 was expressed in Komagataella phaffii with a high volumetric activity of 235.73 U mL-1 (pNPG). Enzymatic property studies have shown that BG23 is an acidic (pH adaptation range of 4.5-7.0) and mesophilic (thermostable < 50 °C) enzyme. Moreover, a one-pot combinatorial enzyme-catalyzed strategy based on BG23 and BGA35 (ß-galactosidase from Aspergillus oryzae) was established, with a high CK yield of 396.7 mg L-1 h-1. This study explored the ginsenoside hydrolases derived from A. tubingensis at the molecular level and provided a reference for the efficient production of CK.

High-level production of pullulan from high concentration of glucose by mutagenesis and adaptive laboratory evolution of Aureobasidium pullulans.

The cost of carbon sources and the low efficiency of the fermentation titer limit the industrial application of pullulan. In this study, a hypertonic-tolerant strain with efficient utilization of glucose was obtained using a double strategy. Initially, a strain for efficient synthesis of pullulan from glucose was generated by mutagenesis. Subsequently, the mutant was directionally evolved on the plate containing a high glucose concentration to enhance high osmotic resistance. The enzyme activities and the transcriptional levels involved in pullulan biosynthesis and high osmotic tolerance in mutants were increased. Nitrogen source and inorganic salts also significantly affected the production of pullulan by M233-20 from high concentration of glucose. The pullulan titer of 162.1 g/L was obtained using the response surface methodology in the flask. The strain M233-20 produced 162.3 g/L pullulan in a 30-L bioreactor with a yield of 0.82 g/g glucose. Hence, this work provides a potential industrial pullulan producer M233-20 and a strategy to develop excellent strain.

Interstitial Fluid Flows along Perivascular and Adventitial Clearances around Neurovascular Bundles.

This study reports new phenomena of the interstitial fluid (ISF) microflow along perivascular and adventitial clearances (PAC) around neurovascular bundles. The fluorescent tracing was used to observe the ISF flow along the PAC of neurovascular bundles in 8-10 week old BALB/c mice. The new results include: (1) the topologic structure of the PAC around the neurovascular bundles is revealed; (2) the heart-orientated ISF flow along the PAC is observed; (3) the double-belt ISF flow along the venous adventitial clearance of the PAC is recorded; (4) the waterfall-like ISF flow induced by the small branching vessel or torn fascia along the PAC is discovered. Based on the above new phenomena, this paper approached the following objectives: (1) the kinematic laws of the ISF flow along the PAC around neurovascular bundles are set up; (2) the applicability of the hypothesis on the PAC and its subspaces by numerical simulations are examined. The findings of this paper not only enriched the image of the ISF flow through the body but also explained the kernel structure of the ISF flow (i.e., the PAC). It helps to lay the foundation for the kinematics and dynamics of the ISF flow along the PAC around neurovascular bundles.