Biotechnology Notes最新文献

Methylxanthines, including caffeine and theophylline, are a class of natural and synthetic compounds with important roles in food, cosmetics, and medicine. These compounds are metabolized by bacteria using five enzymes from the Rieske non-heme iron oxygenase family, NdmABCDE. The NdmCDE complex is responsible for the N₇-demethylation of 7-methylxanthine to xanthine and was originally described as being highly specific for 7-methylxanthine. Here, we report that the NdmCDE complex is also active toward theobromine, producing 3-methylxanthine due to N₇-demethylation. Minimal activity was observed when the enzyme complex was tested with caffeine or paraxanthine, indicating that the presence of the N₁-methyl group significantly inhibits N₇-demethylase activity by NdmCDE. We also demonstrated positional promiscuity in the N₃-demethylase, NdmB, which is able to carry out N₁-demethylation of paraxanthine. The N₁-demethylation by NdmB is limited to paraxanthine and was not observed when caffeine or theophylline were assayed. These newly discovered activities were observed when enzymes were overexpressed in E. coli and differ from results with purified enzymes assayed in vitro, indicating that they may behave differently in vivo. Furthermore, these results reveal promiscuity of bacterial N-demethylase enzymes that can be used to engineer new enzymes and bacterial strains for production of high-value methylxanthines.

Butyrate is a key microbial metabolite known to enhance host metabolic processes by reducing blood glucose levels and promoting energy metabolism, thereby potentially suppressing the risk of developing metabolic syndrome. In this study, we examined the activity of butyrate on modulating the endocannabinoid system, which regulates food intake and energy metabolism. Furthermore, we genetically engineered probiotics to produce butyrate to investigate their effects on the endocannabinoid system. Our study shows that the engineered probiotics exerted antagonistic effects on the endocannabinoid system by downregulating and upregulating the endocannabinoid-synthesizing and -degrading enzyme expression, respectively. Our results suggest that butyrate can modulate the endocannabinoid system, and incorporation of butyrate-producing bacteria into the gut microbiota can potentially aid in re-establishing homeostatic metabolic processes and alleviating metabolic syndrome.

Pearl millet [Pennisetum glaucum (L.) R. Br.] is a climate-hardy nutricereal and an essential staple for the people living in dry regions. Substantial improvement has been achieved for seed yield stability in pearl millet, the cultivable area under pearl millet reduces. Deployment of early-flowering, bold-seeded and dwarf genotypes in pearl millet is a vital breeding strategy to improve grain production and enhance the adaptability of pearl millet in low-input farms. Therefore, an experiment was performed for mapping agronomically important traits like flowering time (FT), plant height (PH), panicle length (PL), and 1000-grain weight (TGW) in 317 recombinant inbred line (RIL) population derived from ICMS 8511-S1-17-2-1-1-B-P03 × AIMP 92901-S1-183-2-2-B-08 cross. Broad-sense heritability estimates were high to very high, ranging from 0.52 (PL) to 0.86 (PH). FT showed a significant positive correlation with PH. A key QTL for FT was mapped on LG 1, 15 QTLs for PH scattered on 10 chromosomes, five QTLs for PL dispersed on four chromosomes, and two QTLs for TGW spanned linkage groups 3 and 7. One QTL on LG1 was common for FT and PH. Two major QTLs for PH, one each on LG4B/8 cM and LG7/110 cM were detected. The large effect QTL for TGW on LG7 had a phenotypic variance (R²) of 24.3%. The R² for PH and PL ranged between 5.2 - 24.5% and 5.0–11.5%, respectively. The QTLs mapped for FT and other agronomic traits in the current study can be exploited to develop elite hybrid parental genotypes/cultivars through marker-assisted breeding and genomic selection.

Public and private sector institutions share different goals and perspectives, but they can form a formidable innovation drive when they join forces. Private-Public Partnerships (PPPs), common in biomedical and pharmaceutical research, are entities where interactions and integrated innovation can take place to serve business and societal goals. In this perspective, we examine how PPPs function in the life sciences sector, and how they differ from other models of private-public cooperation. We examine two successful examples of PPPs in the Singaporean biomedical field. Finally, we look into the role PPPs can have in the development of future biotechnology applications, as one of the ways small(er) countries can develop significant bioengineering competencies and translate scientific research into real-life applications.

Biotechnology applications have contributed significantly to “factory in a lab” research. Although the largely adopted Design–Build–Test–Learn cycle has considerably improved synthetic biology and metabolic engineering capabilities, we are still far from achieving industrial efficiency. As we are now faced with the challenge of exponential population growth and drastic climatic changes affecting the traditional agriculture, there is an imminent need to optimize biotechnology applications, especially for the alternative food source initiative, which has received immense attention recently. Here, I highlight the importance of multi-disciplinary research, and the need to develop integrated systems biology methods, using high-throughput omics data, dynamic modelling and machine learning techniques, to further enhance the lab-based production process. Moving forward in this direction will likely reduce the overall cost and increase the output for the longer term future.

Cell-free protein synthesis system is emerging as a powerful tool for rapid expression of therapeutic proteins. However, one drawback of cell-free technology is the necessity to store the major components below freezing in bulky aqueous solutions. To preserve the cell-free synthesis system for a longer time, the lyophilized cell-free system was developed, and its stability under freeze-drying conditions was explored. The novelty and the difference of this study from the others lie in determining more detailed storage conditions, which could provide a more accurate storage strategy. The effects of the treatment time, the storage temperature, and the component-mixing mode on the freeze-dried cell-free system were investigated. This study explored a robust storage strategy for freeze-dried cell-free systems and could further open up a better opportunity for on-demand synthesis of therapeutic proteins.

Allosterically regulated transcription factors (aTFs) based biosensors from prokaryotes have been widely used to screen large gene libraries, stabilize engineered microbes from evolutionary drifting, and for detection of soil pollutants, among many other applications. However, even though aTF-based biosensors have been established as successful tools for bioengineering and remediation, rational engineering of aTF small molecule-specificity have so far not been demonstrated, highlighting the need for a deeper understanding of the sequence-function relationships that govern aTF allostery. Here, by combining directed evolution of a naïve library of VanR, a vanillic acid transcriptional regulator from Caulobacter crescentus in yeast, followed by saturation mutagenesis of selected positions we identify residues required for vanillic acid responsiveness, while at the same time maintaining responsiveness to vanillin. Selected single-position VanR mutants show both complete repression of transcription in the absence of any ligand, complete loss of vanillic acid responsiveness, while still maintaining high derepression in the presence of vanillin. By computational ligand docking analyses we also discuss the structure-function relationship single mutations can have on aTF specificity, an attribute potentially accounting for the wide-spread arise of aTF members belonging to the GntR superfamily of transcriptional regulators.

Bacterial endospores can be pathogenic to human beings. However, they are robust and thus difficult to kill due to their rigid structure. Conventional methods such as autoclaving for inactivating endospores are energy-intensive and time-consuming. In this study, we developed a copper-tripeptide complexes reagent composed of copper-tripeptide complexes, hydrogen peroxide, and cetyl trimethylammonium bromide (CTAB), to kill endospores. This copper-tripeptide complexes reagent can achieve a 10^7.8-fold reduction in viable endospore count after 60-min treatment at ambient conditions. As a cost-effective and stable sporicidal agent, this reagent may be applied as a general-purpose disinfectant and a replacement of standard sterilization procedures in the future.

The use of “Spinach”-based RNA sensors for the detection of small metabolites and proteins has received growing interest in the recent years. While this approach can be used in vivo for cell sensing and in vitro for microfluidic assays, their overall utility is limited as a result of a high magnesium ion dependence. Here, we alleviate this limitation through incorporating a human tRNA^Lys3 or an engineered viral F30 (a three-way junction RNA motif) scaffold to facilitate aptamer folding in vitro and improve the performance of selected streptavidin, tyrosine, and thrombin aptamers as exemplary cases. Furthermore, we demonstrate the use of a Broccoli aptamer scaffold in conjunction with the viral F30 to enable simultaneous sensing of two molecules in a logic gate type fashion. Our proof-of-concept results demonstrate the ability to redesign these aptamer sensors for improved brightness as well as signal stability without the need for high magnesium—both traits that can further enhance downstream screening applications.