The α-L-rhamnosidase (rha1) gene was homologously expressed in Aspergillus niger strains CCTCC 206047 and CCTCC 206047ΔpyrG, using hygromycin B and auxotrophic as selection markers. The engineered A. niger strains RHA001-1 and RHA003-1 were screened, yielding α-L-rhamnosidase activities of 20.81 ± 0.56 U/mL and 15.35 ± 0.87 U/mL, respectively. The copy numbers of the rha1 gene in strains RHA001-1 and RHA003-1 were found to be 18 and 14, respectively. Correlation analysis between copy number and enzyme activity in the A. niger strains revealed that α-L-rhamnosidase activity increased with the copy number of the rha1 gene. Recombinant α-L-rhamnosidase was utilized for the enzymatic debittering of Ougan juice, and its process conditions were optimized. Furthermore, the primary bitter substance neohesperidin (2.22 g/L) in Ougan juice was converted into hesperetin 7-O-glucoside (1.47 g/L) and hesperidin (0.143 g/L). This study presents a novel approach for the production of food-grade α-L-rhamnosidase and establishes a technical foundation for its application in the beverage industry.

Rotifers are small, ubiquitous invertebrate animals found throughout the world and have emerged as a promising model system for studying molecular mechanisms in the fields of experimental ecology, aquatic toxicology, and geroscience. However, the lack of efficient gene expression manipulation techniques has hindered the study of rotifers. In this study, we used the L4440 plasmid with two reverse-oriented T7 promoters, along with RNase-deficient E. coli HT115, to efficiently produce dsRNA and thereby present an efficient feeding-based RNAi method in Brachionus plicatilis. We targeted Bp-Ku70 & Ku80, key proteins in the DNA double-strand breaks repair pathway, and then subjected rotifers to UV radiation. We found that the mRNA expression, fecundity, as well as survival rate diminished significantly as a result of RNAi. Overall, our results demonstrate that the feeding-based RNAi method is a simple and efficient tool for gene knockdown in B. plicatilis, advancing their use as a model organism for biological research.

To assess microbial dynamics during anaerobic digestion (AD) of sewage sludge (SWS) from a municipal Wastewater Treatment Plant (WWTP), a Biochemical Methane Potential (BMP) assay at 37 °C under mono-digestion conditions was conducted. Utilizing the Illumina MiSeq platform, 16S ribosomal RNA (rRNA) gene sequencing unveiled a core bacterial community in the solid material, showcasing notable variations in profiles. The research investigates changes in microbial communities and metabolic pathways to understand their impact on the efficiency of the digestion process. Prior to AD, the relative abundance in SWS was as follows: Proteobacteria > Bacteroidota > Actinobacteriota. Post-AD, the relative abundance shifted to Firmicutes > Synergistota > Proteobacteria, with Sporanaerobacter and Clostridium emerging as dominant genera. Notably, the methanogenic community underwent a metabolic pathway shift from acetoclastic to hydrogenotrophic in the lab-scale reactors. At the genus level, Methanosaeta, Methanolinea, and Methanofastidiosum predominated initially, while post-AD, Methanobacterium, Methanosaeta, and Methanospirillum took precedence. This metabolic transition may be linked to the increased abundance of Firmicutes, particularly Clostridia, which harbor acetate-oxidizing bacteria facilitating the conversion of acetate to hydrogen.

The efficiency of triple-plasmid transfection in recombinant Adeno-Associated Virus (rAAV) production was analyzed by examining two distinct HEK-293 cells lines. These were categorized as high producer (HP) and low producer (LP) based on their differing levels of productivity under identical conditions. Analysis of RNA expression levels of viral genes revealed disparities in plasmid derived gene expression between the cell lines. Further assessment of transfection efficiency utilizing labeled plasmids revealed lower plasmid uptake and less efficient nuclear transport in LP cell line. Additionally, we observed inferior translation activity in LP, contributing to its shortcomings in overall productivity. In our attempt to optimize plasmid ratios to enhance fully packaged rAAV particle yield, we discovered cell-line-specific optimization potential. The findings highlight the transfection's complexity, urging tailored strategies for improved rAAV production based on each cell line's characteristics, enhancing understanding and guiding further efficiency optimization in rAAV production.

To enhance the de novo synthesis of SAM, the effects of several key genes on SAM synthesis were examined based on modular strategy, and the key genes were manipulated to obtain an engineered strain with high SAM production.

The current knowledge about Palestinian safflower landraces is relatively limited in terms of phenotypic and molecular characterization, however, the purpose of this investigation was to determine the amount of genetic diversity in eighteen local safflower landraces using seven DAMD markers. The banding patterns for each primer were scored and compiled into a data matrix. Subsequently, the data matrix was analyzed using UPGMA cluster analysis to identify distinct genetic groups among the landraces. In total, 88 DNA fragments were found, and there were an average of 12.6 loci per assay unit observed. Resolving Power (RP) revealed an average of 7.09 was determined, with the highest RP value at 13.3. The dendrogram obtained from DAMD data divided the landraces into three main clusters, denoted as I, II and III. The first cluster (I) consisted of one genotype (PTUK.SA16). The second cluster (II) consisted of two genotypes (PTUK.SA13 and PTUK.SA10). The third cluster (III) was later partitioned into two distinct sub-clusters, which are III.a and III.b. Sub-cluster III.a comprised seven genotypes (PTUK.SA4, PTUK.SA9, PTUK.SA8, PTUK.SA7, PTUK.SA6, PTUK.SA5 and PTUK.SA3). While Sub-cluster III.b consisted of eight genotypes (PTUK.SA15, PTUK.SA18, PTUK.SA17, PTUK.SA14, PTUK.SA12, PTUK.SA2, PTUK.SA11, and PTUK.SA1). This research assess the genetic diversity of Palestinian safflower landraces using PCR-based DAMD markers. The remarkable level of polymorphism detected using DAMD markers demonstrated their effectiveness in distinguishing between Palestinian safflower genotypes.

The cancer is one of the diseases of serious threat to people's health and life nowadays. But heterogeneity, drug resistance and treatment side effects of cancer, traditional treatments still have limitations. Tumor-targeting probiotics with a well-established Biosafety and efficient targeting as a delivery vectors to deliver anticancer genes or antitumor drugs to tumor microenvironment has attracted much attention in cancer therapies. In this study, E.coil Nissle 1917 (EcN) was utilized to deliver eukaryotic anti-tumor protein PTEN to tumor microenvironment and suppress tumor growth. Therefore, the EcN (PTEN) was developed. Our results demonstrated that EcN (PTEN) could colonize the tumor site accurately and inhibit the growth of colorectal cancer cells in tumor-bearing mice. It is worth noting that the tumor microenvironment of the treated mice showed significant recruitment of and M1 macrophages, neutrophils and T lymphocytes. No toxicity was observed in the normal tissues during the experiments. This research show the probiotic EcN(PTEN) holds the promise of becoming a powerful weapon against cancer and expected to provide more effective treatments for cancer patients.

Although online monitoring of dissolved O, pH, and dissolved CO is critical in bioprocesses, nearly all existing technologies require some level of direct contact with the cell culture environment, posing risks of contamination. This study addresses the need for an accurate, and completely noninvasive technique for simultaneous measurement of these analytes. A "non-contact" technique for simultaneous monitoring of dissolved O pH, and dissolved CO was developed. Instead of direct contact with the culture media, the measurements were made through permeable membranes via either a sampling port in the culture vessel wall or a flow cell. The efficacy of the "non-contact" technique was validated in Escherichia coli (E.coli), Chinese hamster ovary (CHO) culture processes, and dynamic environments created by sparging gases in cell culture medium. The measurements obtained through the developed techniques were comparable to those obtained through control methods. The noninvasive monitoring system can offer accurate, and contamination-minimized monitoring of critical process parameters including dissolved O, pH, and dissolved CO. These advancements will enhance the control and optimization of cell culture processes, promising improved cell culture performance.

Xylitol, as an important food additive and fine chemical, has a wide range of applications, including food, medicine, chemical, and feed. This review paper focuses on the research progress of xylitol biosynthesis, from overcoming the limitations of traditional chemical hydrogenation and xylose bioconversion, to the full biosynthesis of xylitol production using green and non-polluting glucose as substrate. In the review, the molecular strategies of wild strains to increase xylitol yield, as well as the optimization strategies and metabolic reconfiguration during xylitol biosynthesis are discussed. Subsequently, on the basis of existing studies, the paper further discusses the current status of research and future perspectives of xylitol production using glucose as a single substrate. The evolution of raw materials from xylose-based five-carbon sugars to glucose is not only cost-saving, but also safe and environmentally friendly, which brings new opportunities for the green industrial chain of xylitol.

This investigation probes the role of the electron mediator, neutral red (NR), in the electrosynthesis process, specifically examining its effect on the production of succinic acid by Actinobacillus succinogenes. Our findings reveal that NR, when integrated into the cell membrane, is pivotal for sustaining MEC efficiency. Nevertheless, it is susceptible to both intrinsic and MECs-induced degradation. Notably, during the exponential growth phase of the bacteria, NR is readily incorporated into the cell membrane. However, the supplemental addition of NR fails to significantly enhance the MEC's capacity for succinic acid synthesis, no matter what stage of bacterial growth. And significant depletion of membrane-associated NR is not adequately compensated by the NR present in the fermentation liquid. The ORP feedback-regulated MECs adeptly conserve the NR on the cell membrane, which is essential for maintaining the efficiency of long-term electrosynthesis. The presence of NR on the cell membrane is essential for the functionality of MECs, yet its external replenishment hard. Implementing precise electro-potential regulation strategies can effectively diminish the degradation of NR, thus maintaining the system's efficiency.

β-ionone, an apocarotenoid derived from a C40 terpenoid has an intense, woody smell and a low odor threshold that has been widely used in as an ingredient in food and cosmetics. Yarrowia lipolytica is a promising host for β-ionone production because of its oleaginous nature, its ability to produce high levels of acetyl-CoA (an important precursor for terpenoids), and the availability of synthetic biology tools to engineer the organism. In this study, β-carotene-producing Y. lipolytica strain XK17 was employed for β-ionone biosynthesis. First, we explored the effect of different sources of carotenoid cleavage dioxygenase (CCD) genes on β-ionone production. A high-yielding strain rUinO-D14 with 122 mg/L of β-ionone was obtained by screening promoters combined with rDNA mediated multi-round iterative transformations to optimize the expression of the CCD gene of Osmanthus fragrans. Second, to further develop a high-level production strain for β-ionone, we optimized key genes in the mevalonate pathway by multi-round iterative transformations mediated by non-homologous end joining, combined with a protein tagging strategy. Finally, the introduction of a heterologous oxidoreductase pathway enabled the engineered Y. lipolytica strain to use xylose as a sole carbon source and produce β-ionone. In addition, the potential for use of lignocellulosic hydrolysate as the carbon source for β-ionone production showed that the NHA-A31 strain had a high β-ionone productivity level. This study demonstrates that engineered Y. lipolytica can be used for the efficient, green and sustainable production of β-ionone.

The purpose of this study was to identify potential novel drug targets for Bacteroides fragilis infections using bioinformatics techniques, such as subtractive and comparative genomics. Bacteroides fragilis is a frequently isolated anaerobic pathogen, particularly in the human digestive tract, where its pathogenesis and persistence are influenced by various virulence factors. By understanding these factors, the study aims to explore alternative therapeutic strategies and provide insights for the development of treatments against B. fragilis infections, particularly as alternatives to antibiotic therapy. A comparative subtractive genomic analysis was performed against the B. fragilis (strain CL07T12C05) to identify unique drug targets. The analysis includes the identification of non-paralogous, non-homologous, essential, and drug target like proteins. Moreover, a comprehensive structural analysis of the protein was conducted utilizing structure modeling and validation techniques, along with network topology analysis. Furthermore, a library comprising approximately 9000 FDA-approved compounds accessible in the DrugBank database was employed to conduct virtual screenings for compounds effective against the designated drug target. The top shortlisted compounds were further studied by employing MD simulations using GROMACS. This approach was chosen due to the established safety, efficacy, pharmacokinetics, and toxicity profiles of these compounds. As a result, B. fragilis (strain CL07T12C05) was found to possess 4595 proteins. Among these, 3518 were identified as non-homologous, 1508 deemed essential for bacterial viability, 348 exhibited drug-like properties, 203 were implicated in virulence, and 135 displayed antibiotic resistance. Following an extensive literature review, the protein Sialic acid O-acetyltransferase was chosen through a hierarchical shortlisting process as a potential therapeutic target. The ongoing research facilitated the repurposing of drug compounds: DB12411, DB02112, DB03591, and DB00192, as cost-effective medications against B. fragilis related infections. MD simulations analysis showed that DB12411 may be a potential drug candidate against Sialic acid O-acetyltransferase from B. fragilis. Through subtractive and comparative genomic analysis, Sialic acid O-acetyltransferase was identified as a promising drug target against Bacteroides fragilis. The findings indicate that compounds targeting this protein could potentially be effective in treating B. fragilis infections. However, further experimental validation is required to conclusively confirm their efficacy.

Interleukin-37 is a cytokine with potent immunosuppressive properties that has been shown to have potential to treat autoimmune and chronic inflammatory diseases, as well as certain types of cancer. IL-37 is a 19 kDa protein which interacts with proteins in receptor-dependent and receptor-independent pathways. The expression of the IL-37 protein cloned into the pET-28a vector was optimized in Rosetta 2(DE3) after comparing its expression with Rosetta-gami 2(DE3) and Rosetta 2(DE3) pLysS, which was then used for the large-scale production of IL-37. IMAC purification of IL-37 yielded > 97% pure 0.9 mg/mL protein from auto-induced fermentation. The IC value of IL-37 was < 1 µM, which was similar to that of doxorubicin, and proliferation of > 80% of all cancer cells was inhibited by 100 µg/mL of IL-37 protein. IL-37 may be a promising theragnostic target for cancer due to its comparable IC value with that of doxorubicin.

To construct a derivative of the avirulent Pseudomonas aeruginosa ATCC 9027 that produces high levels of di-rhamnolipid, that has better physico-chemical characteristics for biotechnological applications than mono-rhamnolipid, which is the sole type produced by ATCC 9027. We used plasmids expressing the rhlC gene, which encodes for rhamnosyl transferase II that transforms mono- to di-rhamnolipids under different promoters and in combination with the gene coding for the RhlR quorum sensing regulator, or the mono-rhamnolipid biosynthetic rhlAB operon. The plasmids tested carrying the rhlC gene under the lac promoter were plasmid prhlC and prhlRC, while prhlAB-R-C expressed this gene from the rhlA promoter, forming part of the artificially constructed rhlAB-R-C operon. We measured rhamnolipds concentrations using the orcinol method and determined the proportion of mono-rhamnolipids and di-rhamnolipids by UPLC/MS/MS. We found that the expression of rhlC in P. aeruginosa ATCC 9027 caused the production of di-rhamnolipids and that the derivative carrying plasmid prhlAB-R-C gives the best results considering total rhamnolipids and a higher proportion of di-rhamnolipids. A P. aeruginosa ATCC 9027 derivative with increased di-rhamnolipids production was developed by expressing plasmid prhlAB-R-C, that produces similar rhamnolipids levels as PAO1 type-strain and presented a higher proportion of di-rhamnolipids than this type-strain.

Amides are an important type of synthetic intermediate used in the chemical, agrochemical, pharmaceutical, and nutraceutical industries. The traditional chemical process of converting nitriles into the corresponding amides is feasible but is restricted because of the harsh conditions required. In recent decades, nitrile hydratase (NHase, EC 4.2.1.84) has attracted considerable attention because of its application in nitrile transformation as a prominent biocatalyst. In this review, we provide a comprehensive survey of recent advances in NHase research in terms of natural distribution, enzyme screening, and molecular modification on the basis of its characteristics and catalytic mechanism. Additionally, industrial applications and recent significant biotechnology advances in NHase bioengineering and immobilization techniques are systematically summarized. Moreover, the current challenges and future perspectives for its further development in industrial applications for green chemistry were also discussed. This study contributes to the current state-of-the-art, providing important technical information for new NHase applications in manufacturing industries.

The RBD of SARS-CoV-2 mediates viral entry into host cells by binding to the host receptor ACE2. SARS-CoV-2 infection is linked to various health issues resembling amyloid-related problems, persuading us to investigate the amyloidogenicity of the SARS-CoV-2 spike RBD.

Precise identification of small extracellular vesicles (sEVs) is crucial for improving disease diagnosis and treatments, such as bladder cancer. However, accurate isolation and simultaneously quantification of sEVs remain a huge challenge. We have introduced a new technique that combines immobilization with aptamer-assisted dual cycle amplification to isolate and analyze sEVs with high sensitivity. In this method, the CD9 protein antibody is attached to the plate's surface for the initial identification of sEVs, while an aptamer probe is used to detect the exosomal surface protein CD63. We have created an sEVs-surface method that combines target recognition initiated signal recycling and rolling circle amplification (RCA) for signal amplification. This approach allows for the "AND" logic analysis of dual biomarkers, enabling both sEVs quantification and tracing. The proposed approach has a broad detection range and a low limit of detection. Moreover, the established method showed good stability in detecting sEVs with a low coefficient of variation. Our method can effectively isolate certain sEVs and accurately identify them, making it suitable for many uses in biological science, biomedical engineering, and personalized medicine.

Kojic acid derivatives are useful in the cosmetics and pharmaceutical industries. The current investigation focuses on the search for a safe and environmentally friendly newer whole-cell biocatalyst for the synthesis of kojic acid derivative especially 2-amino-6-(hydroxymethyl)-8-oxo-4-phenyl-4,8-dihydropyrano[3,2-b]pyran-3-carbonitrile (APhCN). In this context, a total of six cultures were isolated from fecal samples of infants and subjected to probiotic characterization followed by screening as whole cell biocatalyst (WCB). In this multicomponent reaction, benzaldehyde, malononitrile, and kojic acid were used to synthesize APhCN at room temperature under aqueous conditions. The screening of potent whole cell biocatalyst (WCB) from isolated cultures was done by comparing reaction time and percent yield. The potent WCB gave a good yield of 95% within 15 h of time and hence further characterized biochemically and identified as Lentilactobacillus farraginis by using 16S rRNA gene sequencing. Lactobacilli having GRAS (generally regarded as safe) status and being able to carry out this transformation under moderate reaction conditions with easy recovery of both product and biocatalyst, it has the potential to replace some of the chemical catalytic methods.

Inoculating heterotrophic nitrification-aerobic denitrification bacteria (HN-AD) to enhance membrane bioreactor (MBR) efficiency may result in the loss of functional bacteria. Therefore, this study compares the application results of enhancing MBR with a self-designed biological amplifier coupled with HN-AD against the performance of conventional MBR. After enhancement, the MBR achieved a removal efficiency of 96.7% for NH-N (100 mg/L) and 96.4% for COD (400 mg/L) in synthetic wastewater. There was a 33% increase in TN (100 mg/L) removal efficiency. The dominant bacteria in the MBR were Alcaligenes (48.4%) and Thauera (15.2%). Additionally, the abundance of denitrification genes (nirK, norB, nosZ) increased in the enhanced MBR, contributing to improved TN removal efficiency. The use of a biological amplifier effectively solved the problem of HN-AD loss in sewage treatment.

Ruminiclostridium papyrosolvens is an anaerobic, mesophilic, and cellulolytic clostridia, promising consolidated bioprocessing (CBP) candidate for producing renewable green chemicals from cellulose, but its genetic transformation has been severely impeded by extracellular biofilm. Here, we analyzed the effects of five different inhibitors with gradient concentrations on R. papyrosolvens growth and biofilm formation. Gallic acid was proved to be a potent inhibitor of biofilm synthesis of R. papyrosolvens. Furthermore, the transformation efficiency of R. papyrosolvens was significantly increased when the cells were treated by the gallic acid, and the mutant strain was successfully obtained by the improved transformation method. Thus, inhibition of biofilm formation of R. papyrosolvens by using gallic acid will contribute to its genetic transformation and efficient metabolic engineering.

This study aimed to produce an engineered recombinant laccase from extremophilic Halalkalibacterium halodurans C-125 (Lac-HhC-125) with higher protein yield, into a more active conformation and with properties that meet the fundamental needs of biotechnological application.