Biotechnology Letters最新文献

Although online monitoring of dissolved O₂, pH, and dissolved CO₂ 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_2, pH, and dissolved CO₂ 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 O₂, pH, and dissolved CO₂. These advancements will enhance the control and optimization of cell culture processes, promising improved cell culture performance.

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.

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 IC₅₀ 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 IC₅₀ value with that of doxorubicin.

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.

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.

Objective: To introduce the Cre-loxP system for constructing marker-less multiple-gene deletion mutants in Pectobacterium, overcoming limitations of antibiotic markers and enhancing the understanding of pathogenic mechanisms.

Results: Firstly, a plasmid named pEX18-Cre, containing a sacB sucrose suicide gene, was constructed to express Cre recombinase in Pectobacterium. Secondly, a mutant in which the loxP-Km fragment replaced the target gene was obtained through homologous recombination double-crossover with the chromosome. Finally, pEX18-Cre was introduced into the mutant to excise the DNA between the loxP sites, thereby removing the markers and achieving multiple gene deletions. By utilizing the Cre-loxP system, we successfully constructed multiple marker-less gene deletion mutants in Pectobacterium strains.

Conclusions: The Cre-loxP system efficiently creates marker-less multiple-gene deletion mutants, enhancing the study of Pectobacterium pathogenic mechanisms by overcoming antibiotic marker limitations.

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.

Alginate lyases have the potential to be used as a therapeutic agent for P. aeruginosa infections. The present work was focused on the characterization of free and immobilized alginate lyase produced by marine bacteria, Enterobacter tabaci RAU2C isolated previously in the laboratory for alginate lyase production and exploring the potential of alginate lyase as an anti-biofilm agent against the P. aeruginosa biofilm. RAU2C alginate lyase was immobilized using an epoxy-activated curdlan matrix by three different methods. Further, the free and immobilized were characterized for its optimal pH and temperature. The effect of alginate concentration on alginate lyase activity was assessed and the kinetic parameters were evaluated. The anti-biofilm activity of the crude alginate lyase was studied using biofilm inhibition and disruption assays in microtiter plates with crystal violet. The biofilm disruption by RAU2C alginate lyase was also ascertained by microscopic analysis. The immobilization matrix prepared using method 3 had a better binding capacity compared to other methods. Both soluble and immobilized alginate lyase exhibited optimal activity at 37 °C and pH 7.0. K_m and V_max of soluble and immobilized alginate lyase were found to be 3.38 mg/mL, 22.98 mg/mL min and 3.67 mg/mL and 26.59 mg/mL min respectively. Both microtiter assay and microscopic analysis confirmed the prevention and dispersal of pre-existing biofilms by crude RAU2C alginate lyase, highlighting its potential as an anti-biofilm agent against P. aeruginosa. The study highlights the efficacy of RAU2C alginate lyase as an anti-biofilm agent in controlling P. aeruginosa biofilms.

The cyanobacterium Spirulina platensis was subjected to a fractionation process involving ultrasound-assisted extraction and membrane filtration to obtain a pure phycocyanin fraction and a clarified colorless protein fraction free of chlorophyll and carotenoids. The effects of pressure and power on total protein release were assessed. The retention of the extracted proteins was then assessed by ultrafiltration, with and without ammonium sulfate precipitation. Total protein recovery yields reached 97% in aqueous solution, at a low frequency (12 kHz), atmospheric pressure, and with an ultrasonic power of 200 Watts (W). Ammonium sulfate (25% w/v) precipitation was used to remove pigments and impurities from the crude protein extract. Finally, semi-frontal ultrafiltration resulted in high levels of C-phycocyanin recovery in the retentate: 95% and 91% with 10 and 100 kDa-cutoff membranes, respectively. However, the levels of total non-pigmented proteins in the permeate compartment did not exceed 67% with a 100 kDa-cutoff membrane. A fractionation process is proposed here for the valorization of two different protein fractions from Spirulina platensis.